Publications by authors named "Franck Biet"

50 Publications

Engineering Synthetic Lipopeptide Antigen for Specific Detection of subsp. Infection.

Front Vet Sci 2021 23;8:637841. Epub 2021 Apr 23.

INRAE, Université de Tours, ISP, Nouzilly, France.

Unlike other MAC members, subsp. (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 or the more closely related species subsp. , the L5P did not cross react and therefore may be a valuable antigen to solve ambiguous results in other tests.
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http://dx.doi.org/10.3389/fvets.2021.637841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8103206PMC
April 2021

Interferon-γ Response of subsp. Infected Goats to Recombinant and Synthetic Mycobacterial Antigens.

Front Vet Sci 2021 26;8:645251. Epub 2021 Mar 26.

Animal and Plant Health Agency, Addlestone, United Kingdom.

Despite its potential for early diagnosis of subsp. (MAP) infection, the IFN-γ release assay is not used routinely, because of low specificity of the established crude antigen preparation Johnin (PPDj). Limited data are available assessing the potential of MAP-derived protein and lipopeptide antigens to replace PPDj in assays for goats, while cattle and sheep have been studied more extensively. Furthermore, MAP infection is claimed to interfere with the diagnosis of bovine tuberculosis when other crude antigen preparations (PPDb, PPDa) are applied. In this study, the diagnostic potential of MAP-derived recombinant protein antigens, synthetic MAP lipopentapeptides and of specific peptide cocktails was assessed compared to crude mycobacterial antigen preparations in experimentally infected goats. Goats were inoculated with MAP, or subsp. (MAH) as surrogate for environmental mycobacteria, non-exposed animals served as controls. Complex-specific antibody and PPDj-induced IFN-γ responses were monitored . Infection status was assessed by pathomorphological findings and bacteriological tissue culture at necropsy 1 year after inoculation. The IFN-γ response to 13 recombinant protein antigens of MAP, two synthetic MAP lipopentapeptides and three recombinant peptide cocktails of was investigated at three defined time points after infection. At necropsy, MAP or MAH infection was confirmed in all inoculated goats, no signs of infection were found in the controls. Antibody formation was first detected 3-6 weeks post infection (wpi) in MAH-inoculated and 11-14 wpi in the MAP-inoculated goats. Maximum PPDj-induced IFN-γ levels in MAH and MAP exposed animals were recorded 3-6 and 23-26 wpi, respectively. Positive responses continued with large individual variation. Antigens Map 0210c, Map 1693c, Map 2020, Map 3651cT(it), and Map 3651c stimulated increased whole blood IFN-γ levels in several MAP-inoculated goats compared to MAH inoculated and control animals. These IFN-γ levels correlated with the intensity of the PPDj-induced responses. The two synthetic lipopentapeptides and the other MAP-derived protein antigens had no discriminatory potential. Stimulation with peptide cocktails ESAT6-CFP10, Rv3020c, and Rv3615c did not elicit IFN-γ production. Further work is required to investigate if test sensitivity will increase when mixtures of the MAP-derived protein antigens are applied.
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http://dx.doi.org/10.3389/fvets.2021.645251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034290PMC
March 2021

Decrease of IL-5 Production by Naive T Cells Cocultured with IL-18-Producing BCG-Pulsed Dendritic Cells from Patients Allergic to House Dust Mite.

Vaccines (Basel) 2021 Mar 18;9(3). Epub 2021 Mar 18.

Department of Immunology and Infectious Biology, Institute of Microbiology, Biotechnology and Immunology, Faculty of Biology and Environmental Protection, University of Lodz, 90-237 Lodz, Poland.

The only currently available anti-tuberculosis vaccine, Bacillus Calmette-Guérin (BCG), has been reported to also protect against unrelated diseases, including inflammatory diseases such as allergic asthma. Recombinant BCG strains that produce IL-18 have been shown to enhance Th1 responses over non-recombinant BCG and to reduce IL-5 production and bronchoalveolar eosinophilia in mice. However, their ability to decrease the immune polarization of human Th2 cells is not known. Here, we show that BCG and recombinant BCG producing human IL-18 (rBCG-hIL-18) induced the maturation of Der p 1-stimulated monocyte-derived dendritic cells (MD-DCs) from healthy controls and from patients allergic to house dust mites. After incubation with mycobacteria and Der p 1, MD-DCs produced significantly more IL-23 and IP-10 but had no effect on IL-12p70 or IL-10 production compared to Der p 1-pulsed MD-DCs in the absence of mycobacteria. In the presence of Der p 1, BCG- and rBCG-hIL-18-pulsed MD-DCs cocultured with naive, but not with memory T cells from allergic patients, resulted in a decrease in IL-5 production compared to non-pulsed MD-DCs cultured in the presence of Der p 1. BCG, and especially rBCG-hIL-18, may thus be potential therapeutic tools to reduce exacerbated Th2 responses in patients with allergic asthma.
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http://dx.doi.org/10.3390/vaccines9030277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8003153PMC
March 2021

Complete Genome Sequence of a Type III Ovine Strain of Mycobacterium avium subsp. .

Microbiol Resour Announc 2021 Mar 11;10(10). Epub 2021 Mar 11.

INRAE, Université de Tours, ISP, Nouzilly, France.

The complete genome sequence of a type III strain of subsp. was determined. The genome size for this pathogen of sheep is 4,895,755 bp with no plasmid DNA. The chromosome contains 19 copies of the hallmark IS element, which is routinely used to identify this subspecies.
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http://dx.doi.org/10.1128/MRA.01480-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953304PMC
March 2021

Diagnostic Sequences That Distinguish Subspecies Strains.

Front Vet Sci 2020 28;7:620094. Epub 2021 Jan 28.

INRAE, Université de Tours, ISP, Nouzilly, France.

Over a decade ago subspecies () specific genes were initially identified in a whole genome context by comparing draft genome sequences of strain K-10 with subspecies () strain 104. This resulted in identification of 32 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 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 subspecies, including (), , and . 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 subspecies strains. This combined approach confirmed 86 genes as -specific, seven as -specific and three as -specific. A single-tube PCR reaction was conducted as a proof of concept method to quickly distinguish subspecies strains. With these novel data, researchers can classify isolates in their freezers, quickly characterize clinical samples, and functionally analyze these unique genes.
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http://dx.doi.org/10.3389/fvets.2020.620094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876471PMC
January 2021

Genetic Diversity Among Subspecies Revealed by Analysis of Complete Genome Sequences.

Front Microbiol 2020 7;11:1701. Epub 2020 Aug 7.

INRAE, Université de Tours, ISP, Nouzilly, France.

comprises four subspecies that contain both human and veterinary pathogens. At the inception of this study, twenty-eight 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 subspecies () 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 subspecies () and subspecies () strains (collectively termed non-) suggests horizontal gene transfer occurs in non-, but not in strains. Overall, subspecies could be divided into two major sub-divisions, with the type II (bovine strains) clustering tightly on one end of a phylogenetic spectrum and strains clustering more loosely together on the other end. The most evolutionarily distinct 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 strains as an intermediate between type II and . SNP analysis and genome organization analyses repeatedly demonstrated the conserved nature of versus the mosaic nature of non- strains. Finally, core and pangenomes were developed for and non- strains. A total of 80% genes belonged to the core genome, while only 40% of non- genes belonged to the non- 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.
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http://dx.doi.org/10.3389/fmicb.2020.01701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426613PMC
August 2020

Feature of Adhesins Produced by Human Clinical Isolates of , subsp. and Closely Related Species.

Microorganisms 2020 Jul 30;8(8). Epub 2020 Jul 30.

INRAE, Université de Tours, ISP, F-37390 Nouzilly, France.

The complex includes two closely related species, and . 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 ATCC13950 and examined clinical isolates from patients with different pathologies associated with infection for the presence and conservation of HBHA and LBP. Using a recombinant derivative strain of 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 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 isolates from patients with diverse disease manifestations, suggesting a conserved role of these adhesins in virulence in humans and their potential use as a diagnostic tool.
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http://dx.doi.org/10.3390/microorganisms8081154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465531PMC
July 2020

The complete genome sequence of Mycobacterium bovis Mb3601, a SB0120 spoligotype strain representative of a new clonal group.

Infect Genet Evol 2020 08 30;82:104309. Epub 2020 Mar 30.

Paris-Est University, French Agency for Food, Environmental and Occupational Health and Safety (Anses), Animal Health Laboratory, National reference Laboratory for Tuberculosis, 94701 Maisons-Alfort cedex, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.meegid.2020.104309DOI Listing
August 2020

Whole-Genome Sequencing Confirms the Coexistence of Different Colonizing Group B Isolates Underscored by CRISPR Typing.

Microbiol Resour Announc 2020 Jan 30;9(5). Epub 2020 Jan 30.

Université de Tours, INRAE, ISP, Tours, France

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.
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http://dx.doi.org/10.1128/MRA.01359-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992867PMC
January 2020

Transmission Network of Deer-Borne Infection Revealed by a WGS Approach.

Microorganisms 2019 Dec 12;7(12). Epub 2019 Dec 12.

Paris-Est University, National Reference Laboratory for Tuberculosis, Animal Health Laboratory, French Agency for Food, Environmental and Occupational Health and Safety (Anses), 94701 Maisons-Alfort CEDEX, France.

Bovine tuberculosis (TB) is a zoonotic disease, mainly caused by . 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.
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http://dx.doi.org/10.3390/microorganisms7120687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6955793PMC
December 2019

MAC-INMV-SSR: a web application dedicated to genotyping members of Mycobacterium avium complex (MAC) including Mycobacterium avium subsp. paratuberculosis strains.

Infect Genet Evol 2020 01 18;77:104075. Epub 2019 Oct 18.

ISP, INRA, Université de Tours, UMR 1282, 37380 Nouzilly, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.meegid.2019.104075DOI Listing
January 2020

Longitudinal study of Mycobacterium avium ssp. paratuberculosis fecal shedding patterns and concurrent serological patterns in naturally infected dairy cattle.

J Dairy Sci 2019 Oct 1;102(10):9117-9137. Epub 2019 Aug 1.

BIOEPAR, INRA, Oniris, 44307, Nantes, France. Electronic address:

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a disease that affects ruminants worldwide. Despite global interest in the control of this disease, gaps exist in our knowledge of fecal shedding patterns and concurrent serological patterns. This longitudinal study in dairy cattle herds with high MAP seroprevalence in France aimed at accurately describing fecal shedding patterns over 1 year; relating those shedding patterns to individual animal characteristics (age, breed, parity); and exploring the association between fecal shedding patterns and serological patterns. To describe temporal fecal shedding patterns and continuity of shedding, along with the standard quantitative PCR (qPCR) threshold cycle we used a cutoff value that related to low or nonculturable fecal shedding. We also defined a threshold cycle indicative of shedding in high quantities to describe infection progression patterns. Twenty-one herds completed the study, and 782 cows were tested 4 times each. We obtained 4 sets of paired fecal qPCR and serum ELISA results from 757 cows. Although we targeted highly likely infectious animals, we found a large diversity of shedding patterns, as well as high variability between herds in the proportion of animals showing a given pattern. The fecal qPCR results of almost 20% of the final study sample were positioned at least once in the range that indicated low or nonculturable fecal shedding (between the adjusted and the standard cutoff value). Although these animals would typically be classified as non-shedders, they could be important to infection dynamics on the farm. Animals that shed at least twice consecutively and animals that shed in high quantities rarely reverted to negativity. Repeated fecal qPCR can be used to detect temporal fecal shedding traits, and the decision to cull an animal could practically be based on temporal, semiquantitative results. Overall, we found a mismatch between fecal shedding and ELISA seropositivity (637 animals were ELISA-negative 4 times, but only 13% of those animals were qPCR-negative 4 times). We found that having more than 2 ELISA-positive samples was strongly related to persistent and continuous shedding. We suggest that although serological testing is much less sensitive than qPCR, it can also be used, particularly over the course of multiple testing events, to identify animals that are most likely to contribute to the contamination of the farm environment.
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http://dx.doi.org/10.3168/jds.2018-15897DOI Listing
October 2019

Sterile Lung Inflammation Induced by Silica Exacerbates Mycobacterium tuberculosis Infection via STING-Dependent Type 2 Immunity.

Cell Rep 2019 05;27(9):2649-2664.e5

CNRS, UMR7355, Orléans 45071, France; Experimental and Molecular Immunology and Neurogenetics, University of Orléans, Orléans 45071, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2019.04.110DOI Listing
May 2019

Accurate Phylogenetic Relationships Among Strains Circulating in France Based on Whole Genome Sequencing and Single Nucleotide Polymorphism Analysis.

Front Microbiol 2019 3;10:955. Epub 2019 May 3.

ISP, INRA, UMR 1282, Université de Tours, Nouzilly, France.

In recent years the diversity of the French 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 strains. Whole genome sequencing was carried out on a selection of 87 strains which reflect the French population's genetic diversity. Sequences were compared to the 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 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.
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http://dx.doi.org/10.3389/fmicb.2019.00955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509552PMC
May 2019

Tuberculin skin test reaction is related to memory, but not naive CD4 T cell responses to mycobacterial stimuli in BCG-vaccinated young adults.

Vaccine 2018 07 19;36(30):4566-4577. Epub 2018 Jun 19.

Department of Immunology and Infectious Biology, Institute of Microbiology, Biotechnology and Immunology, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland.

Bacillus Calmette-Guérin (BCG) is the only vaccine available against tuberculosis and the tuberculin skin test (TST) is the most widely used method to detect BCG take. However, subjects may remain TST-negative, even after several BCG administrations. To investigate some of the potential reasons underlying this inability of developing tuberculin sensitivity in response to BCG we compared the effect of different mycobacterial stimuli in the groups differently responding to tuberculin. TST was performed on 71 healthy adults aged 25-30 years, who had received BCG in their childhood, and considered TST-positive at ≥10 mm. Dendritic cells (DCs) were incubated with PPD, live BCG or rBCGhIL-18, producing human IL-18. The latter strain was used to investigate whether the production of IL-18 could overcome some of the immune read-out limitations in the TST-negative subjects. CD86, CD80, CD40, and DC-specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) expression was analysed by flow cytometry and IL-10, IL-23 and IP-10 secretion in culture supernatants by ELISA. In DCs-T cell co-cultures with naive and memory CD4 T cells, the IFN-γ and IL-10 levels were determined by ELISA. We found no difference in IL-10 and IFN-γ production by naive T cells between the TST-negative and TST-positive subjects. However, IFN-γ was produced in significantly higher amounts by memory T cells incubated with PPD, BCG or rBCGhIL-18-pulsed DCs in TST-positive than in TST-negative subjects, whereas the numbers of the IFN-γ-producing T cells were similar in both groups. This difference may be partially due to a decreased CD40 and enhanced reduction in DC-SIGN expression by DCs of TST-negative versus TST-positive subjects. A strong effect of IL-18 expression by rBCGhIL-18 on IL-23 production by the DC was seen in both groups, which likely was the reason for the increased IFN-γ production by naïve T cells upon incubation with mycobacteria-pulsed DC, regardless of the TST status.
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http://dx.doi.org/10.1016/j.vaccine.2018.05.068DOI Listing
July 2018

Evaluation of mycobacteria-specific gamma interferon and antibody responses before and after a single intradermal skin test in cattle naturally exposed to M. avium subsp. paratuberculosis and experimentally infected with M. bovis.

Vet Immunol Immunopathol 2018 Feb 12;196:35-47. Epub 2017 Dec 12.

Unit "Bacterial Zoonoses of livestock", Operational Direction Bacterial Diseases, Veterinary and Agrochemical Research Center (CODA-CERVA), Groeselenberg, Brussels, Belgium.

This study reports on the diagnostic potential of IFN-γ release assays and serology for Mycobacterium bovis in six naturally M. avium subsp. paratuberculosis (Map) exposed bulls of which four were intratracheally infected with a Belgian field strain of M. bovis. Heparinized blood, serum and fecal samples were collected at regular time intervals for mycobacteria-specific IFN-γ release assays, antibody analysis and for Map culture respectively. Single intradermal skin test (SIT) with bovine tuberculin (PPD-B) was performed on day 115 and animals were sacrificed on day 133 after M. bovis infection. Organs were collected and stored for histopathological examination, modified Ziehl-Neelsen staining and bacteriological analysis of M. bovis and Map by culture and RT-PCR. Prior to infection five animals showed positive IFN-γ responses to avian PPD (PPD-A) and four were positive in Map PCR (IS900) on faeces. Three M. bovis infected animals reacted as early as day 14 with sustained higher PPD-B than PPD-A specific IFN-γ responses, whereas the fourth animal (with the strongest PPD-A response prior to infection) showed sustained higher PPD-B specific IFN-γ levels only a day 56 after infection. Two of the infected animals had a sustained positive IFN-γ response to the ESAT-6/CFP-10/TB7.7 (QuantiFERON-TB Gold) peptide cocktail as early as day 14, among which the animal with the initial high PPD-A response. Later during infection, positive responses were found to ESAT-6 peptides in three infected bulls and to CFP-10 peptides in all four infected bulls. One of the control animals reacted intermittently to the ESAT-6/CFP10/TB7.7 cocktail. Prior to SIT, weak but positive MPB83/MBP70 specific antibody responses were detected in two of the infected bulls. All four M. bovis infected bulls reacted with a positive skin test and showed, as reported by others, increased mycobacteria specific IFN-γ production and increased positive responses in MPB83/MBP70 specific serology after SIT. At autopsy, M. bovis lesions were detected in all four experimentally infected bulls. Our results indicate that in Map exposed cattle, M. bovis diagnosis using IFN-γ assays needs a combination of PPD-B/A and ESAT-6/CFP10 for early and optimal sensitivity and that sensitivity of MPB83/MBP70 serodiagnosis is dramatically increased by prior skin testing. Map exposure did not interfere with the development of SIT in M. bovis infected animals, but resulted in a false positive M. bovis specific IFN-γ and antibody response after SIT in one of the two control animals (which remained negative in skin-test).
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http://dx.doi.org/10.1016/j.vetimm.2017.12.007DOI Listing
February 2018

Environmental subsp. Hosted by Free-Living Amoebae.

Front Cell Infect Microbiol 2018 9;8:28. Epub 2018 Feb 9.

Université de Poitiers, Laboratoire Ecologie et Biologie des Interactions, UMR Centre National de la Recherche Scientifique 7267, Equipe Microbiologie de l'Eau, Poitiers, France.

subsp. 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 subsp. 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 interactions between several strains of subsp. and . 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 subsp. in environmental amoebae, we sampled water from farms positive for paratuberculosis. A subsp. strain was detected within an environmental amoeba identified as related to the poorly described genus. The bacterial strain was genotyped, showing that it was similar to previous infectious strains isolated from cattle. In conclusion, we described that various subsp. 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 subsp. .
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http://dx.doi.org/10.3389/fcimb.2018.00028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5811464PMC
February 2019

Draft Genome Sequences of Three Strains Identified in Cattle and Wildlife in France.

Genome Announc 2017 Jul 6;5(27). Epub 2017 Jul 6.

INRA, Université de Tours, UMR 1282, Infectiologie et Santé Publique, Nouzilly, France

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 strains of spoligotypes SB0821 and SB0134, isolated from wildlife but circulating in wildlife-livestock multihost systems, and SB0121, circulating exclusively in cattle.
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http://dx.doi.org/10.1128/genomeA.00410-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5502845PMC
July 2017

Cell wall peptidolipids of Mycobacterium avium: from genetic prediction to exact structure of a nonribosomal peptide.

Mol Microbiol 2017 Aug 15;105(4):525-539. Epub 2017 Jun 15.

Infectiologie et Santé Publique, INRA, Université de Tours, UMR1282, Nouzilly, F-37380, France.

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.
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http://dx.doi.org/10.1111/mmi.13717DOI Listing
August 2017

Draft Genome Sequence of Mycobacterium bovis Strain D-10-02315 Isolated from Wild Boar.

Genome Announc 2016 Nov 10;4(6). Epub 2016 Nov 10.

INRA, Université de Tours, UMR1282, Infectiologie et Santé Publique, Nouzilly, France

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.
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http://dx.doi.org/10.1128/genomeA.01268-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5105107PMC
November 2016

MIRU-VNTR allelic variability depends on Mycobacterium bovis clonal group identity.

Infect Genet Evol 2016 11 1;45:165-169. Epub 2016 Sep 1.

Université Paris-Est, Laboratoire National de Référence Tuberculose, Unité Zoonoses Bactériennes, Laboratoire de Santé Animale, ANSES, 94706 Maisons-Alfort Cedex, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.meegid.2016.08.038DOI Listing
November 2016

Phylogenomic exploration of the relationships between strains of Mycobacterium avium subspecies paratuberculosis.

BMC Genomics 2016 Jan 26;17:79. Epub 2016 Jan 26.

Moredun Research Institute, Pentlands Science Park, Penicuik, EH26 0PZ, UK.

Background: Mycobacterium 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.

Results: Single 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.

Conclusions: This 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.
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http://dx.doi.org/10.1186/s12864-015-2234-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729121PMC
January 2016

Interferon gamma response to Mycobacterium avium subsp. paratuberculosis specific lipopentapeptide antigen L5P in cattle.

Res Vet Sci 2015 Oct 1;102:118-21. Epub 2015 Aug 1.

UMR1282, Infectiologie et Santé Publique (ISP-311), INRA Centre Val de Loire, F-37380 Nouzilly, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.rvsc.2015.07.017DOI Listing
October 2015

Dendritic Cell Activity Driven by Recombinant Mycobacterium bovis BCG Producing Human IL-18, in Healthy BCG Vaccinated Adults.

J Immunol Res 2015 3;2015:359153. Epub 2015 Aug 3.

Department of Immunology and Infectious Biology, Institute of Microbiology, Biotechnology and Immunology, University of Lodz, Banacha Street 12/19, 90-237 Lodz, Poland.

Tuberculosis remains an enormous global burden, despite wide vaccination coverage with the Bacillus Calmette-Guérin (BCG), the only vaccine available against this disease, indicating that BCG-driven immunity is insufficient to protect the human population against tuberculosis. In this study we constructed recombinant BCG producing human IL-18 (rBCGhIL-18) and investigated whether human IL-18 produced by rBCGhIL-18 modulates DC functions and enhances Th1 responses to mycobacterial antigens in humans. We found that the costimulatory CD86 and CD80 molecules were significantly upregulated on rBCGhIL-18-infected DCs, whereas the stimulation of DCs with nonrecombinant BCG was less effective. In contrast, both BCG strains decreased the DC-SIGN expression on human DCs. The rBCGhIL-18 increased IL-23, IL-10, and IP-10 production by DCs to a greater extent than nonrecombinant BCG. In a coculture system of CD4(+) T cells and loaded DCs, rBCGhIL-18 favoured strong IFN-γ but also IL-10 production by naive T cells but not by memory T cells. This was much less the case for nonrecombinant BCG. Thus the expression of IL-18 by recombinant BCG increases IL-23, IP-10, and IL-10 expression by human DCs and enhances their ability to induce IFN-γ and IL-10 expression by naive T cells, without affecting the maturation phenotype of the DCs.
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http://dx.doi.org/10.1155/2015/359153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539176PMC
May 2016

Genetic evolution of Mycobacterium bovis causing tuberculosis in livestock and wildlife in France since 1978.

PLoS One 2015 6;10(2):e0117103. Epub 2015 Feb 6.

Université Paris-Est, Laboratoire National de Référence de la Tuberculose, Unité de Zoonoses Bactériennes, Laboratoire de Santé Animale, ANSES, Maisons-Alfort Cedex, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0117103PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4319773PMC
January 2016

Non-tuberculous mycobacterial infections of veterinary relevance.

Res Vet Sci 2014 Oct 27;97 Suppl:S69-77. Epub 2014 Aug 27.

University Paris-Est, Anses, Laboratory for Animal Health, Bovine tuberculosis National Reference Laboratory, Bacterial Zoonoses Unit, 23, avenue du Général de Gaulle, 94706 Maisons-Alfort, France. Electronic address:

Mycobacteria play an important role in human and animal health fields. We here examine the place of non tuberculous mycobacteria (NTM) infections in the veterinary context. Relevant aspects of a reference laboratory experience and a literature review are presented in this article. Importance is given both to productivity and to economic losses due to misdiagnosis with bovine tuberculosis and paratuberculosis. The impact NTM may have is relative to geographical location, ecology, husbandry, extent of surveillance programs and bovine tuberculosis and paratuberculosis prevalence. The role of the most relevant NTM in animal disease is summarized with a special focus on Mycobacterium avium subsp. paratuberculosis, given its role as causative agent of paratuberculosis, a disease with huge economic consequences for ruminant livestock.
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http://dx.doi.org/10.1016/j.rvsc.2014.08.007DOI Listing
October 2014

Novel feature of Mycobacterium avium subsp. paratuberculosis, highlighted by characterization of the heparin-binding hemagglutinin adhesin.

J Bacteriol 2013 Nov 23;195(21):4844-53. Epub 2013 Aug 23.

INRA-Centre Val de Loire, UMR 1282, Infectiologie, Santé Publique (ISP-311), Nouzilly, 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.
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http://dx.doi.org/10.1128/JB.00671-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807500PMC
November 2013

Interaction of Mycobacterium leprae with human airway epithelial cells: adherence, entry, survival, and identification of potential adhesins by surface proteome analysis.

Infect Immun 2013 Jul 13;81(7):2645-59. Epub 2013 May 13.

Laboratory of Cellular Microbiology, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz-FIOCRUZ, Rio de Janeiro, RJ, Brazil.

This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.
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http://dx.doi.org/10.1128/IAI.00147-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3697615PMC
July 2013

Specific IgG response against Mycobacterium avium paratuberculosis in children and adults with Crohn's disease.

PLoS One 2013 2;8(5):e62780. Epub 2013 May 2.

Institut National de la Santé et de la Recherche Médicale (INSERM), UMR989, Paris, France.

Background And Aims: Presence of serum antibodies against Mycobacterium avium paratuberculosis (MAP) in Crohn's Disease (CD) as a disease characteristic remains controversial. In the present work, we assessed antibody reactivity of serum and intestinal fluid against four distinct MAP-antigens, including the recently identified MAP-specific lipopentapeptide (L5P).

Methods: Immunoglobulin concentrations and specificity against 3 non MAP-specific antigens: glycosyl-transferase-d (GSD), purified protein derivative from MAP (Johnin-PPD), heparin binding haemagglutinin (MAP-HBHA) and one MAP-specific antigen: synthetic L5P were determined by ELISA in gut lavage fluids from adult controls or patients with CD, and in sera of children or adult controls or patients with CD, ulcerative colitis or celiac disease.

Results: Total IgA and IgG concentrations were increased in sera of children with CD but were decreased in sera of adults with CD, thereof specificity against MAP antigens was assessed by normalizing immunoglobulin concentrations between samples. In CD patients, IgG reactivity was increased against the four MAP antigens, including L5P in gut lavage fluids but it was only increased against L5P in sera. By contrast, anti-L5P IgG were not increased in patients with ulcerative colitis or celiac disease.

Conclusions: A significant increase in anti-L5P IgG is observed in sera of children and adults with CD but not in patients with other intestinal inflammatory diseases. Anti-L5P antibodies may serve as serological marker for CD.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062780PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3642204PMC
December 2013

Purification of native HBHA from Mycobacterium avium subsp. paratuberculosis.

BMC Res Notes 2013 Feb 7;6:55. Epub 2013 Feb 7.

INRA, UMR ISP 1282 Infectiologie et Santé Publique, Nouzilly F-37380, France.

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.
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http://dx.doi.org/10.1186/1756-0500-6-55DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3586368PMC
February 2013