Analysis of long non-coding RNA expression profile of bovine monocyte-macrophage infected by Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis. As a potential zoonotic pathogen, MAP also seriously threatens human health and social security. At present, long non-coding RNA (lncRNA) has attracted wide attention as an useful biomarker in various diseases. Therefore, our study analyzed the lncRNA expression profiles and lncRNA-mRNA regulatory network of MAP infected bovine monocytes-macrophages and uninfected bovine cells by high-throughput sequencing. A total of 4641 differentially expressed lncRNAs genes were identified, including 3111 up-regulated genes and 1530 down-regulated genes. In addition, lncRNA-mRNA interaction analysis was performed to predict the target genes of lncRNA. Among them, after MAP infection, 86 lncRNAs targeted to mRNA, of which only 6 genes were significantly different. The results of Gene Ontology (GO) enrichment analysis showed that the differentially expressed genes significantly enriched in functional groups were related to immune regulation. Multiple signal pathways including NF-κB, NOD-like receptor, Cytokine-cytokine receptor, Toll-like receptor signaling pathway, Chemokine signaling pathway, and other important biochemical, metabolic and signal transduction pathways were enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG). In this study, analysis of macrophage transcriptomes in response to MAP infection is expected to provide key information to deeply understand role of the pathogen in initiating an inappropriate and persistent infection in susceptible hosts and molecular mechanisms that might underlie the early phases of paratuberculosis.

Analysis of the microRNA Expression Profile of Bovine Monocyte-derived Macrophages Infected with Mycobacterium avium subsp. Paratuberculosis Reveals that miR-150 Suppresses Cell Apoptosis by Targeting PDCD4.

M. avium subsp. paratuberculosis (MAP) is the causative pathogen of Johne's disease, a chronic granulomatous enteritis that principally affects ruminants and can survive, proliferate and disseminate in macrophages. MicroRNAs (miRNAs) are important regulators of gene expression and can impact the processes of cells. To investigate the role of miRNAs in monocyte-derived macrophages (MDMs) during MAP infection, we used high-throughput sequencing technology to analyze small RNA libraries of MAP-infected and control MDMs. The results showed that a total of 21 miRNAs were differentially expressed in MDMs after MAP infection, and 8864 target genes were predicted. A functional analysis showed that the target genes were mainly involved in the MAPK signaling pathway, Toll-like receptor signaling pathway, NF-kappa B signaling pathway and apoptosis. In addition, using a dual-luciferase reporter assay, flow cytometry, and a small interfering (si)RNA knockdown assay, the role of miR-150 in regulating macrophage apoptosis by targeting the programmed cell death protein-4 (PDCD4) was demonstrated. These results provide an experimental basis to reveal the regulatory mechanism of MAP infection and suggest the potential of miRNAs as biomarkers for the diagnosis of Johne's disease in bovines.

First morphological and molecular isolation of Talaromyces marneffei in beech marten (Martes foina) in Portugal.

Talaromyces marneffei is a zoonotic fungus that mostly infects immunocompromised individuals. For the first time, this fungus was isolated in an adult beech marten (Martes foina) hit by a car, found dead in Penamacor, Portugal. During the necropsy, different samples (skin, fur, lymph nodes, lung, spleen, kidneys, and brain) were collected and processed for microbiology (including mycology) and molecular biology. T. marneffei was identified through its mycological characteristics and confirmed by PCR in hair samples. No other lesions or alterations were reported, except a concomitant presence of M. avium subsp. paratuberculosis in lung, kidney and brain samples. To the authors' knowledge, this is the first description of this fungus beech marten, as well as the first case of co-infection with M. avium subsp. paratuberculosis in wildlife fauna. These results suggest a sylvatic life-cycle of T. marneffei, involving beech martens, in Portugal.

Analysis of mRNA and circRNA Expression Profiles of Bovine Monocyte-Derived Macrophages Infected With Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the pathogen of Johne's disease (paratuberculosis), which mainly causes chronic infectious granulomatous enteritis in ruminants and has brought huge economic losses to animal husbandry. As a specific intracellular pathogen, when MAP invades the body, it is internalized by macrophages where it is able to replicate by inhibition of the phagosome maturation, escaping the host immune system and surviving, which leads to the spread of the disease. More recent studies have shown that circRNA is involved in many pathological and physiological processes of the body as the molecular sponge of miRNA, the scaffold of RNA binding protein and having the characteristic of being able to translate into protein. In this study, the mRNA and circRNA expression profiles of MAP-infected bovine monocyte-macrophages and uninfected bovine cells were analyzed by high-throughput sequencing. A total of 618 differentially expressed mRNA were screened out, including 322 upregulated mRNA and 296 downregulated mRNA. In addition, the analysis of circRNA differential expression profile showed 39 differentially expressed genes including 12 upregulated and 27 downregulated genes. Moreover, differential genes belonging to cytokine activity, chemokine activity, inflammatory reaction, apoptosis, and other functional groups related to macrophage immune response were significantly enriched in Gene Ontology (GO). Multiple signal pathways including NF-κB, MAPK, Toll-like receptor, IL-17, JAK-STAT, and other signaling pathways related to activating macrophage immune response were significantly enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG). In addition, RT-qPCR technology verified the accuracy of the mRNA sequencing results. In this study, we have obtained the transcriptome information of mRNA and circRNA of bovine monocyte-macrophage infected with MAP. These results will provide data support for the further study of mRNA-miRNA-circRNA network and immune escape mechanism of MAP and will enrich the knowledge of the molecular immune mechanisms of Johne's disease as well.

Acanthamoeba castellanii as a Screening Tool for Mycobacterium avium Subspecies paratuberculosis Virulence Factors with Relevance in Macrophage Infection.

The high prevalence of Johne's disease has driven a continuous effort to more readily understand the pathogenesis of the etiological causative bacterium, Mycobacterium avium subsp. paratuberculosis (MAP), and to develop effective preventative measures for infection spread. In this study, we aimed to create an in vivo MAP infection model employing an environmental protozoan host and used it as a tool for selection of bacterial virulence determinants potentially contributing to MAP survival in mammalian host macrophages. We utilized Acanthamoeba castellanii (amoeba) to explore metabolic consequences of the MAP-host interaction and established a correlation between metabolic changes of this phagocytic host and MAP virulence. Using the library of gene knockout mutants, we identified MAP clones that can either enhance or inhibit amoeba metabolism and we discovered that, for most part, it mirrors the pattern of MAP attenuation or survival during infection of macrophages. It was found that MAP mutants that induced an increase in amoeba metabolism were defective in intracellular growth in macrophages. However, MAP clones that exhibited low metabolic alteration in amoeba were able to survive at a greater rate within mammalian cells, highlighting importance of both category of genes in bacterial pathogenesis. Sequencing of MAP mutants has identified several virulence factors previously shown to have a biological relevance in mycobacterial survival and intracellular growth in phagocytic cells. In addition, we uncovered new genetic determinants potentially contributing to MAP pathogenicity. Results of this study support the use of the amoeba model system as a quick initial screening tool for selection of virulence factors of extremely slow-grower MAP that is challenging to study.

Examination of Mycobacterium avium subspecies paratuberculosis mixed genotype infections in dairy animals using a whole genome sequencing approach.

Many pathogenic mycobacteria are known to cause severe disease in humans and animals. M. avium subspecies paratuberculosis (Map) is the causative agent of Johne's disease-a chronic wasting disease affecting ruminants such as cattle and sheep, responsible for significant economic losses in the dairy and beef industries. Due to the lack of treatment options or effective vaccines, mitigating losses can be difficult. In addition, the early stages of Map infection may occur in asymptomatic hosts that continue to shed viable bacteria in their faeces, leading to the infection of other healthy animals. Using multi-locus short sequence repeat (ML-SSR) analysis we previously reported that individual Johne's positive dairy cattle from farms across the island of Newfoundland were infected by Map with multiple SSR-types simultaneously. The occurrence of multiple mixed genotype infections has the potential to change pathogen and disease dynamics as well as reduce the efficacy of treatments and vaccines. Therefore, we conducted whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis on a subset of these isolates for a more in-depth examination. We also implemented a PCR assay using two discriminatory SNPs and demonstrated the incidence of a mixed infection by three genotypically diverse Map isolates in a single animal. In addition, results show that WGS and SNP analysis can provide a better understanding of the relationship between Map isolates from individual and different animals. In the future such studies on the occurrence of mixed genotype infections could potentially lead to the identification of variable pathogenicity of different genotypes and allow for better tracking of Map isolates for epidemiological studies.

Genetic diversity and phylogeny of Mycobacterium avium.

Mycobacterium avium, one of the species of the M. avium complex (MAC), includes 4 subspecies, i.e., M. avium subsp. hominissuis (MAH), M. avium subsp. avium (MAA), M. avium subsp. silvaticum (MAS) and M. avium subsp. paratuberculosis (MAP), in turn classified into the S (sheep) and C (cattle) types. These subspecies, although closely related, represent distinct organisms, each endowed with specific pathogenetic and host range characteristics, ranging from environmental opportunistic bacteria that cause infections in swine and immunocompromised patients to pathogens of birds and ruminants. The present review summarizes the basic epidemiological and pathological features of the M. avium subspecies, describes the major genomic events responsible of M. avium subspecies diversity (insertion sequences, sequence variations in specific chromosome loci or genes, deletions, duplications and insertions of large genomic regions) and then reconstructs the phylogenetic relationships among the M. avium subspecies.