Early antibody response against <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subspecies <Emphasis Type="Italic">paratuberculosis</Emphasis> antigens in subclinical cattle

Background  

Our laboratories have previously reported on the experimental infection of cattle with Mycobacterium avium subsp paratuberculosis (M. paratuberculosis) using an intratonsillar infection model. In addition, we have recently developed a partial protein array representing 92 M. paratuberculosis coding sequences. These combined tools have enabled a unique look at the temporal analysis of M. paratuberculosis antigens within the native host. The primary objective of this study was to identify M. paratuberculosis antigens detected by cattle early during infection. A secondary objective was to evaluate the humoral immune response in cattle during the initial year of infection.     

Cytokine responses of bovine macrophages to diverse clinical Mycobacterium avium subspecies paratuberculosis strains

Background  

Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease (JD) persistently infects and survives within the host macrophages. While it is established that substantial genotypic variation exists among MAP, evidence for the correlates that associate specific MAP genotypes with clinical or sub-clinical disease phenotypes is presently unknown. Thus we studied strain differences in intracellular MAP survival and host responses in a bovine monocyte derived macrophage (MDM) system.     

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.     

Identification of two Mycobacterium tuberculosis H37Rv ORFs involved in resistance to killing by human macrophages

Background  

The ability of Mycobacterium tuberculosis to survive and replicate in macrophages is crucial for the mycobacterium's ability to infect the host and cause tuberculosis. To identify Mycobacterium tuberculosis genes involved in survival in macrophages, a library of non-pathogenic Mycobacterium smegmatis bacteria, each carrying an individual integrated cosmid containing M. tuberculosis H37Rv genomic DNA, was passed through THP-1 human macrophages three times.     

Investigation of Mannheimia haemolytica bacteriophages relative to host diversity

Aims

This study aimed to characterize the impact of lytic and temperate bacteriophages on the genetic and phenotypic diversity of Mannheimia haemolytica from feedlot cattle.

Methods and Results

Strictly lytic phages were not detected from bovine nasopharyngeal (n = 689) or water trough (n = 30) samples, but Myoviridae‐ or Siphoviridae‐like phages were induced from 54 of 72 M. haemolytica strains by mitomycin C, occasionally from the same strain. Phages with similar restriction fragment length polymorphism profiles (RFLP ≥70% relatedness) shared common host serotypes 1 or 2 (P < 0·000 1). Likewise, phages with similar RFLP tended to occur in genetically related host bacteria (70–79% similarity). Host range assays showed that seven phages from host serotypes 1, 2 and 6 lysed representative strains of serotypes 1, 2 or 8. The genome of vB_MhM_1152AP from serotype 6 was found to be collinear with P2‐like phage φMhaA1‐PHL101.

Conclusions

Prophages are a significant component of the genome of M. haemolytica and contribute significantly to host diversity. Further characterization of the role of prophage in virulence and persistence of M. haemolytica in cattle could provide insight into approaches to control this potential respiratory pathogen.

Significance and Impact of the Study

This study demonstrated that prophages are widespread within the genome of M. haemolytica isolates and emphasized the challenge of isolating lytic phage as a therapeutic against this pathogen.     

Comparative genomics between human and animal associated subspecies of the Mycobacterium avium complex: a basis for pathogenicity

Background

A human isolate of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis 43525) was sequenced and compared genomically to other mycobacterial pathogens. M. paratuberculosis 43525 was recently isolated from a patient with ulcerative colitis and belongs to the M. avium complex, a group known to infect both humans and animals. While M. paratuberculosis is a known pathogen of livestock, there are only 20 human isolates from the last 20 years, therefore we took the opportunity to perform a whole genome comparison between human and animal mycobacterial pathogens. We also compared virulence determinants such as the mycobactin cluster, PE/PPE genes and mammalian cell entry (mce) operons between MAC subspecies that infect animals and those that infect humans. M. tuberculosis was also included in these analyses given its predominant role as a human pathogen.

Results

This genome comparison showed the PE/PPE profile of M. paratuberculosis 43525 to be largely the same as other M. paratuberculosis isolates, except that it had one PPE and one PE_PGRS protein that are only present in human MAC strains and M. tuberculosis. PE/PPE proteins that were unique to M. paratuberculosis 43525, M. avium subsp. hominissuis and a caprine M. paratuberculosis isolate, were also identified. In addition, the mycobactin cluster differed between human and animal isolates and a unique mce operon flanked by two mycobactin genes, mbtA and mbtJ, was identified in all available M. paratuberculosis genomes.

Conclusions

Despite the whole genome comparison placing M. paratuberculosis 43525 as closely related to bovine M. paratuberculosis, key virulence factors were similar to human mycobacterial pathogens. This study highlights key factors of mycobacterial pathogenesis in humans and forms the basis for future functional studies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1889-2) contains supplementary material, which is available to authorized users.     

Interaction between Mycobacterium avium subsp. paratuberculosis and environmental protozoa

Background  

Interactions between Mycobacterium avium subsp. paratuberculosis (Map) and free-living protozoa in water are likely to occur in nature. The potential impact of ingestion of Map by two naturally occurring Acanthamoeba spp. on this pathogen's survival and chlorine resistance was investigated.     

New probes used for IS1245 and IS1311 restriction fragment length polymorphism of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis isolates of human and animal origin in Norway

Background  

Mycobacterium avium is an environmental mycobacterium that can be divided into the subspecies avium, hominissuis, paratuberculosis and silvaticum. Some M. avium subspecies are opportunistic pathogens for animals and humans. They are ubiquitous in nature and can be isolated from natural sources of water, soil, plants and bedding material. Isolates of M. avium originating from humans (n = 37), pigs (n = 51) and wild birds (n = 10) in Norway were examined by IS1245 and IS1311 RFLP using new and specific probes and for the presence of IS901 and ISMpa1 by PCR. Analysis and generation of a dendrogram were performed with the software BioNumerics.     

Superoxide dismutase from Helicobacter pylori suppresses the production of pro‐inflammatory cytokines during in vivo infection

Background

Helicobacter pylori has undergone considerable adaptation to allow chronic persistence within the gastric environment. While H. pylori‐associated diseases are driven by an excessive inflammation, severe gastritis is detrimental to colonization by this pathogen. Hence, H. pylori has developed strategies to minimize the severity of gastritis it triggers in its host. Superoxide dismutase (SOD) is well known for its role in protecting against oxidative attack; less recognized is its ability to inhibit immunity, shown for SOD from mammalian sources and those of some bacterial species. This study examined whether H. pylori SOD (HpSOD) has the ability to inhibit the host immune response to these bacteria.

Materials and Methods

The ability of recombinant HpSOD to modify the response to LPS was measured using mouse macrophages. A monoclonal antibody against HpSOD was generated and injected into H. pylori‐infected mice.

Results

Addition of HpSOD to cultures of mouse macrophages significantly inhibited the pro‐inflammatory cytokine response to LPS stimulation. A monoclonal antibody was generated that was specific for SOD from H. pylori. When injected into mice infected with H. pylori for 3 months, this antibody was readily detected in both sera and gastric tissues 5 days later. While treatment with anti‐HpSOD had no effect on H. pylori colonization at this time point, it significantly increased the levels of a range of pro‐inflammatory cytokines in the gastric tissues. This did not occur with antibodies against other antioxidant enzymes.

Conclusions

SOD from H. pylori can inhibit the production of pro‐inflammatory cytokine during in vivo infection.     

Downregulation of protein kinase C-α enhances intracellular survival of Mycobacteria: role of PknG

Background  

Intracellular trafficking of mycobacteria is comprehensively dependent on the unusual regulation of host proteins. Recently, we have reported that infection of macrophages by Mycobacterium tuberculosis H37Rv (Rv) selectively downregulates the expression of PKCα while infection by Mycobacterium smegmatis (MS) does not.     

Involvement of SPI-2-encoded SpiC in flagellum synthesis in Salmonella enterica serovar Typhimurium

Background  

SpiC encoded within Salmonella pathogeniCity island 2 on the Salmonella enterica serovar Typhimurium chromosome is required for survival within macrophages and systemic infection in mice. Additionally, SpiC contributes to Salmonella-induced activation of the signal transduction pathways in macrophages by affecting the expression of FliC, a component of flagella filaments. Here, we show the contribution of SpiC in flagellum synthesis.     

Identification of transposon insertion mutants of Francisella tularensis tularensis strain Schu S4 deficient in intracellular replication in the hepatic cell line HepG2

Background  

Francisella tularensis is a zoonotic intracellular bacterial pathogen that causes tularemia. The subspecies tularensis is highly virulent and is classified as a category A agent of biological warfare because of its low infectious dose by an aerosol route, and its ability to cause severe disease. In macrophages F. tularensis exhibits a rather novel intracellular lifestyle; after invasion it remains in a phagosome for three to six hours before escaping to, and replicating in the cytoplasm. The molecular mechanisms that allow F. tularensis to invade and replicate within a host cell have not been well defined.     

Survival of Mycobacterium avium subsp. paratuberculosis in the intermediate and final digestion products of biogas plants

Aims

To evaluate the survival of Mycobacterium avium subsp. paratuberculosis (MAP) during anaerobic digestion (AD), we studied two different biogas plants loaded with manure and slurry from paratuberculosis‐infected dairy herds.

Methods and Results

Both plants were operating under mesophilic conditions, the first with a single digester and the second with a double digester. Mycobacterium avium subsp. paratuberculosis detection was performed by sampling each stage of the process, specifically the prefermenter, fermenter, liquid digestate and solid digestate stages, for 11 months. In both plants, MAP was isolated from the prefermenter stage. Only the final products, the solid and liquid digestates, of the one‐stage plant showed viable MAP, while no viable MAP was detected in the digestates of the two‐stage plant.

Conclusions

Mycobacterium avium subsp. paratuberculosis showed a significant decrease during subsequent steps of the AD process, particularly in the two‐stage plant. We suggest that the second digester maintained the digestate under anaerobic conditions for a longer period of time, thus reducing MAP survival and MAP load under the culture detection limit.

Significance and Impact of the Study

Our data are unable to exclude the presence of MAP in the final products of the biogas plants, particularly those products from the single digester; therefore, the use of digestates as fertilizers is a real concern related to the possible environmental contamination with MAP.     

Biomarker Discovery in Subclinical Mycobacterial Infections of Cattle

Background

Bovine tuberculosis is a highly prevalent infectious disease of cattle worldwide; however, infection in the United States is limited to 0.01% of dairy herds. Thus detection of bovine TB is confounded by high background infection with M. avium subsp. paratuberculosis. The present study addresses variations in the circulating peptidome based on the pathogenesis of two biologically similar mycobacterial diseases of cattle.

Methodology/Principal Findings

We hypothesized that serum proteomes of animals in response to either M. bovis or M. paratuberculosis infection will display several commonalities and differences. Sera prospectively collected from animals experimentally infected with either M. bovis or M. paratuberculosis were analyzed using high-resolution proteomics approaches. iTRAQ, a liquid chromatography and tandem mass spectrometry approach, was used to simultaneously identify and quantify peptides from multiple infections and contemporaneous uninfected control groups. Four comparisons were performed: 1) M. bovis infection versus uninfected controls, 2) M. bovis versus M. paratuberculosis infection, 3) early, and 4) advanced M. paratuberculosis infection versus uninfected controls. One hundred and ten differentially elevated proteins (P≤0.05) were identified. Vitamin D binding protein precursor (DBP), alpha-1 acid glycoprotein, alpha-1B glycoprotein, fetuin, and serine proteinase inhibitor were identified in both infections. Transthyretin, retinol binding proteins, and cathelicidin were identified exclusively in M. paratuberculosis infection, while the serum levels of alpha-1-microglobulin/bikunin precursor (AMBP) protein, alpha-1 acid glycoprotein, fetuin, and alpha-1B glycoprotein were elevated exclusively in M. bovis infected animals.

Conclusions/Significance

The discovery of these biomarkers has significant impact on the elucidation of pathogenesis of two mycobacterial diseases at the cellular and the molecular level and can be applied in the development of mycobacterium-specific diagnostic tools for the monitoring progression of disease, response to therapy, and/or vaccine based interventions.     

Pulsed-field gel electrophoresis profile homogeneity of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> isolates from cattle and heterogeneity of those from sheep and goats

Background  

Mycobacterium avium subsp. paratuberculosis (Map) causes paratuberculosis in animals and is suspected of causing Crohn's Disease in humans. Characterization of strains led to classify paratuberculosis isolates in two main types, cattle type strains, found affecting all host species, and sheep type strains, reported affecting mainly sheep. In order to get a better understanding of the epidemiology of paratuberculosis a large set of Map isolates obtained from different species over the last 25 years have been characterized. Five-hundred and twenty isolates from different hosts (cattle, sheep, goats, bison, deer and wild boar) and origins had been cultured and typed by IS1311 restriction-endonuclease-analysis. Two-hundred and sixty-nine isolates were further characterized by pulsed-field gel electrophoresis (PFGE) using SnaBI and SpeI endonucleases. Differences in strain isolation upon various media conditions were also studied.     

Exosomal Hsp70 Induces a Pro-Inflammatory Response to Foreign Particles Including Mycobacteria

Background

Exosomes are endosome-derived vesicles that are released when multi-vesicular bodies (MVBs) fuse with the plasma membrane. Exosomes released from mycobacteria-infected cells have recently been shown to be pro-inflammatory. A prominent host molecule that is found within these exosomes is Hsp70, a member of the heat-shock family of proteins.

Methodology/Principal Findings

We first characterized the exosomes purified from control and mycobacteria-infected cells. We found that relative to uninfected cells, macrophages infected with M. smegmatis and M. avium release more exosomes and the exosomes they released had more Hsp70 on their surface. Both exosomes and exogenous Hsp70 treatment of macrophages led to NF-κB activation and TNFα release in uninfected macrophages; Hsp70 levels were elevated in mycobacteria-infected cells. Macrophage treatment with Hsp70 also led to increase in the phagocytosis and maturation of latex-bead phagosomes. Finally, Hsp70 pre-incubation of M. smegmatis- and M. avium-infected cells led to increased phago-lysosome fusion, as well as more killing of mycobacteria within macrophages.

Conclusions/Significance

Our results fit into an emerging concept whereby exosomes-containing Hsp70 are effective inducers of inflammation, also in response to mycobacterial infection.     

Biofilm formation by Mycobacterium avium isolates originating from humans, swine and birds

Background  

Mycobacterium avium includes the subspecies avium, silvaticum, paratuberculosis and hominissuis, and M. avium subspecies has been isolated from various environments all over the world including from biofilms in water distribution systems. The aim of this study was to examine isolates of M. avium subsp. avium and M. avium subsp. hominissuis of different origin for biofilm formation and to look for correlations between biofilm formation and RFLP-types, and to standardise the method to test for biofilm formation. In order to determine the best screening method, a panel of 14 isolates of M. avium subsp. avium and M. avium subsp. hominissuis, were tested for their ability to form biofilm in microtiter plates under different conditions. Subsequently, 83 additional isolates from humans, swine and birds were tested for biofilm formation. The isolates were tested for the presence of selected genes involved in the synthesis of glycopeptidolipids (GPLs) in the cell wall of M. avium, which is believed to be important for biofilm formation. Colony morphology and hsp65 sequvar were also determined.     

Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

Background  

During inhalational anthrax, internalization of Bacillus anthracis spores by host cells within the lung is believed to be a key step for initiating the transition from the localized to disseminated stages of infection. Despite compelling in vivo evidence that spores remain dormant within the bronchioalveolar spaces of the lungs, and germinate only after uptake into host cells, most in vitro studies of infection have been conducted under conditions that promote rapid germination of spores within the culture medium.     

Environmental amoebae do not support the long‐term survival of virulent mycobacteria

Aims

To test the hypothesis that Mycobacterium bovis can persist in the environment within protozoa.

Methods and Results

In this study, we used a novel approach to detect internalized mycobacteria in environmental protozoa from badger latrines. Acid‐fast micro‐organisms were visualized in isolated amoebae, although we were unable to identify them to species level as no mycobacteria were grown from these samples nor was M. bovis detected by IS6110 PCR. Co‐incubation of Acanthamoeba castellanii with virulent M. bovis substantially reduced levels of bacilli, indicating that the amoebae have a negative effect on the persistence of M. bovis.

Conclusions

The internalization of mycobacteria in protozoa might be a rare event under environmental conditions. The results suggest that amoebae might contribute to the inactivation of M. bovis rather than representing a potential environmental reservoir.

Significance and Impact of the Study

Protozoa have been suggested to act as an environmental reservoir for M. bovis. The current study suggests that environmental amoebae play at most a minor role as potential reservoirs of M. bovis and that protozoa might inhibit persistence of M. bovis in the environment.