Comparative analysis of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> isolates from cattle,sheep and goats by short sequence repeat and pulsed-field gel electrophoresis typing

Background  

Mycobacterium avium subsp. paratuberculosis (Map) causes the chronic enteritis called paratuberculosis mainly in cattle, sheep and goats. Evidences that point out an association between Map and Crohn's Disease in humans are increasing. Strain differentiation among Map isolates has proved to be difficult and has limited the study of the molecular epidemiology of paratuberculosis. In order to asses the usefulness of the PCR based short sequence repeat (SSR) analysis of locus 1 and locus 8 in the epidemiological tracing of paratuberculosis strains we here compare for the first time the results of SSR and SnaBI-SpeI pulsed-field gel electrophoresis (PFGE) typing methods in a set of 268 Map isolates from different hosts (cattle, sheep, goats, bison, deer and wild boar).     

Immunoreactivity of the <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> 19-kDa lipoprotein

Background  

The Mycobacterium tuberculosis 19-kDa lipoprotein has been reported to stimulate both T and B cell responses as well as induce a number of Th1 cytokines. In order to evaluate the Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis) 19-kDa lipoprotein as an immunomodulator in cattle with Johne's disease, the gene encoding the 19-kDa protein (MAP0261c) was analyzed.     

Prevalence of avian influenza viruses, <Emphasis Type="Italic">Mycobacterium avium</Emphasis>, and <Emphasis Type="Italic">Mycobacterium avium</Emphasis>, subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> in marsh-dwelling passerines in Slovakia, 2008

Prevalence of the infectious respiratory agens, avian influenza virus (AIV), Mycobacterium avium (M. avium), and Mycobacterium avium subspecies paratuberculosis (MAP), was studied in migratory marsh-dwelling passerines captured in the Parížske močiare wetlands in Western Slovakia during 2008. Surveillance of 650 birds revealed a lower prevalence of AIV in spring (13.6%) than in summer (17.5%). A total of 14 different subtypes were detected in samples obtained from birds captured during the spring, with the most prevalent subtypes being H8N3, H6N4, H11N6 and H12N6. Subtypes H12N6, H6N6 and H2N5 were predominant in passerines captured during summer months. In eight cases, different AIV infections were detected in the oropharyngeal and cloacal samples originating from a single bird (H1N1 and H8N3; H1N3 and H9N3; H2N3 and H12N6; H2N1 and H8N1; H4N2 and H9N6; H5N5 and H11N6; H6N4 and H11N6; H7N1 and H10N3 in the oropharynx and cloaca, respectively). M. avium was detected in 9.2% and 0.8% of marsh-dwelling passerines captured during spring and summer, respectively. Only two birds were co-infected with AIV and M. avium. All birds were negative for MAP.     

Production of and applications for a polyclonal IgY diagnostic reagent specific for <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis>

Antibodies specific to the cell surface antigens of Mycobacterium avium subsp. paratuberculosis (MAP) have multiple useful applications, e.g. organism detection, immunoconcentration, and cell visualization. The aim of this study was to produce and compare polyclonal antibodies for such research and diagnostic purposes. Three polyclonal antibodies to MAP were produced using sera from immunized rabbits and chickens plus naturally infected cows. Cross-reactive antibodies in each MAP antibody preparation were removed by absorption with heterologous mycobacterial and non-mycobacterial cells. The specificity of each resulting polyclonal antibody preparation was evaluated by ELISA to multiple bacterial cell wall extract antigens. After absorption, chicken anti-MAP IgY had the highest specificity of the three antibody preparations. FITC-la-beled anti-MAP IgY was used to effectively locate MAP in macrophages 12 h post-infection. Also, immunomagnetic beads coated with anti-MAP IgY enhanced recovery of MAP from bacterial suspensions in comparison with non-antibody coated beads. Anti-MAP IgY provides a novel new reagent with broad diagnostic and research applications requiring specific concentration, detection, and quantification of MAP.     

IS<Emphasis Type="Italic">900</Emphasis> Restriction Fragment Length Polymorphism (RFLP) Analysis of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis>Isolates from Goats and Cattle in Norway

In Norway, paratuberculosis has been frequently diagnosed in goats, while cattle have been almost free of the infection. This difference in prevalence between goats and cattle has led to speculations about the existence of a Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) isolate that is non-pathogenic for cattle. There is little information available on genotypic variation of M. a. paratuberculosis isolated from animals in Norway. In the present study, genotypic information on 51 isolates from goats and four isolates from cattle in Norway was obtained by use of IS 900 restriction fragment length polymorphism (RFLP) analysis. All isolates from cattle and 84% of the isolates from goats had the same RFLP pattern (B-C1). Five RFLP patterns not previously detected were found. No genotypic variation that could explain a difference in host origin was found between the isolates from cattle and the majority of the Norwegian goat isolates. This lack of difference indicates that the most common M. a. paratuberculosis isolates in Norway may infect both cattle and goats.     

Localization of proteins in the cell wall of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> K10 by proteomic analysis

Mycobacterium avium subsp. paratuberculosis is a pathogen which causes a debilitating chronic enteritis in ruminants. Unfortunately, the mechanisms that control M. avium subsp. paratuberculosis persistence during infection are poorly understood and the key steps for developing Johne's disease remain elusive. A proteomic analysis approach, based on one dimensional polyacrylamide gel electrophoresis (SDS-PAGE) followed by LC-MS/MS, was used to identify and characterize the cell wall associated proteins of M. avium subsp. paratuberculosis K10 and an cell surface enzymatic shaving method was used to determine the surface-exposed proteins. 309 different proteins were identified, which included 101 proteins previously annotated as hypothetical or conserved hypothetical. 38 proteins were identified as surface-exposed by trypsin treatment. To categorize and analyze these proteomic data on the proteins identified within cell wall of M. avium subsp. paratuberculosis K10, a rational bioinformatic approach was followed. The analyses of the 309 cell wall proteins provided theoretical molecular mass and p I distributions and determined that 18 proteins are shared with the cell surface-exposed proteome. In short, a comprehensive profile of the M. avium subsp. paratuberculosis K10 cell wall subproteome was created. The resulting proteomic profile might become the foundation for the design of new preventive, diagnostic and therapeutic strategies against mycobacterial diseases in general and M. avium subsp. paratuberculosis in particular.     

Virulence-related <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp <Emphasis Type="Italic">hominissuis</Emphasis> MAV_2928 gene is associated with vacuole remodeling in macrophages

Background  

Mycobacterium avium subsp hominissuis (previously Mycobacterium avium subsp avium) is an environmental organism associated with opportunistic infections in humans. Mycobacterium hominissuis infects and replicates within mononuclear phagocytes. Previous study characterized an attenuated mutant in which the PPE gene (MAV_2928) homologous to Rv1787 was inactivated. This mutant, in contrast to the wild-type bacterium, was shown both to have impaired the ability to replicate within macrophages and to have prevented phagosome/lysosome fusion.     

<Emphasis Type="Italic">Mycobacterium avium</Emphasis> disease in wild red-legged partridges (<Emphasis Type="Italic">Alectoris rufa</Emphasis>)

Three wild red-legged partridges (Alectoris rufa) from intensively managed hunting areas in Spain were received for necropsy. They showed granulomatous lesions in different parts of the body, mainly in liver and spleen. Microscope examination of the granulomas showed central caseous necrosis and large amounts of acid-fast bacilli, surrounded by epitheloid cells, giant cells, and lymphocytes. Attempts to isolate and culture the bacillus in Colestsos medium were unsuccessful, but the polymerase chain reaction technique revealed the presence of microorganisms belonging to the Mycobacterium avium complex in one of the partridges. This is the first report of avian tuberculosis in free-living red-legged partridges.     

Common genomic features of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> subsp. <Emphasis Type="Italic">doylei</Emphasis> strains distinguish them from <Emphasis Type="Italic">C. jejuni</Emphasis> subsp. <Emphasis Type="Italic">jejuni</Emphasis>

Background  

Campylobacter jejuni has been divided into two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Nearly all of the C. jejuni strains isolated are Cjj; nevertheless, although Cjd strains are isolated infrequently, they differ from Cjj in two key aspects: they are obtained primarily from human clinical samples and are associated often with bacteremia, in addition to gastroenteritis. In this study, we utilized multilocus sequence typing (MLST) and a DNA microarray-based comparative genomic indexing (CGI) approach to examine the genomic diversity and gene content of Cjd strains.     

Characterization and identification of distinct <Emphasis Type="Italic">Mycobacterium massiliense</Emphasis> extracellular proteins from those of <Emphasis Type="Italic">Mycobacterium abscessus</Emphasis>

Mycobacterium massiliense is an emerging pathogen and very similar to Mycobacterium abscessus of rapidly growing mycobacteria in the phenotype and genotype. Pathogenic bacteria secrete a diversity of factors into extracellular medium which contribute to the bacterial pathogenicity. In the present study, we performed the comparative proteome analysis of culture filtrate proteins from a clinical isolate of M. massiliense and M. abscessus strains using two-dimensional gel electrophoresis and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). Interestingly, 9 proteins of M. massiliense were distinctly expressed from those of M. abscessus. Bioinformatic analysis of the identified proteins revealed that 3 unique proteins corresponded to serine/arginine rich protein, membrane protein from Streptomyces coelicolor, and one hypothetical protein from Corynebacterium efficiens YS-314, respectively. Culture filtrate proteins from M. massiliense induced the release of pro-inflammatory cytokines from macrophages in a dose-dependent manner but not that from M. abscessus. Taken together, the functional study on the identified proteins uniquely produced from M. massiliense may provide not only the clues for the different pathogensis, but also help develop the diagnostic tools for the differentiation between two mycobacterial species.     

Genome-wide association study of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subspecies <Emphasis Type="Italic">Paratuberculosis</Emphasis> infection in Chinese Holstein

Background

Paratuberculosis is a contagious, chronic and enteric disease in ruminants, which is caused by Mycobacterium avium subspecies paratuberculosis (MAP) infection, resulting in enormous economic losses worldwide. There is currently no effective cure for MAP infection or a vaccine, it is thus important to explore the genetic variants that contribute to host susceptibility to infection by MAP, which may provide a better understanding of the mechanisms of paratuberculosis and benefit animal genetic improvement. Herein we performed a genome-wide association study (GWAS) to identify genomic regions and candidate genes associated with susceptibility to MAP infection in dairy cattle.

Results

Using Illumina Bovine 50?K (54,609 SNPs) and GeneSeek HD (138,893 SNPs) chips, two analytical approaches were performed, GRAMMAR-GC and ROADTRIPS in 937 Chinese Holstein cows, among which individuals genotyped by the 50?K chip were imputed to HD SNPs with Beagle software. Consequently, 15 and 11 significant SNPs (P?<?5?×?10^??5) were identified with GRAMMAR-GC and ROADTDRIPS, respectively. A total of 10 functional genes were in proximity to (i.e., within 1?Mb) these SNPs, including IL4, IL5, IL13, IRF1, MyD88, PACSIN1, DEF6, TDP2, ZAP70 and CSF2. Functional enrichment analysis showed that these genes were involved in immune related pathways, such as interleukin, T cell receptor signaling pathways and inflammatory bowel disease (IBD), implying their potential associations with susceptibility to MAP infection. In addition, by examining the publicly available cattle QTLdb, a previous QTL for MAP was found to be overlapped with one of regions detected currently at 32.5?Mb on BTA23, where the TDP2 gene was anchored.

Conclusions

In conclusion, we identified 26 SNPs located on 15 chromosomes in the Chinese Holstein population using two GWAS strategies with high density SNPs. Integrated analysis of GWAS, biological functions and the reported QTL information helps to detect positional candidate genes and the identification of regions associated with susceptibility to MAP traits in dairy cattle.

     

Comparison of the Arylamine <Emphasis Type="Italic">N-</Emphasis>Acetyltransferase from <Emphasis Type="Italic">Mycobacterium marinum</Emphasis> and <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Arylamine N-acetyltansferase (NAT) from Mycobacterium tuberculosis (TBNAT) is a potential drug target for anti-tubercular therapy. Recombinant TBNAT is much less soluble and is produced in lower yields than the closely related NAT from Mycobacterium marinum (MMNAT). In order to explore MMNAT as a model for TBNAT in drug discovery, we compare the two mycobacterial NAT enzymes. Two site-directed mutants of MMNAT have been prepared and characterised: MMNAT71, Tyr → Phe and MMNAT209, Met → Thr, in which residues within 6 Å of the active-site cysteine have been replaced with the corresponding residue from TBNAT. Two chimeric proteins have also been produced in which the third domain of MMNAT has been replaced by the third domain of TBNAT and vice versa. The activity profile of the chimeric proteins suggests a role for the third domain in the evolutionary divergence of NAT between these closely related mycobacterial species.     

Molecular Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis>

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.