Characterisation of autophagy disruption in the ileum of pigs infected with Lawsonia intracellularis

Lawsonia intracellularis is the aetiological agent of proliferative enteropathy, an enteric disease endemic in swine. Survival in its intracellular niche of the ileum epithelial lining requires the capacity to subvert, repress or exploit the host immune response to create an environment conducive to bacterial propagation. To better understand how L. intracellularis survives in its intracellular niche, we have performed an investigation into the dynamic relationship between infection and the host autophagy response by immunohistochemistry in experimentally infected porcine ileum samples. Beclin1, a protein required early in the autophagy pathway was observed to be distributed with a basal to apical concentration gradient in the crypts of healthy piglets, whilst infected piglets were observed to have no gradient of distribution and an increase in the presence of Beclin1 in crypts with histological characteristics of L. intracellularis residence. Detecting microtubule-associated protein light chain 3 (LC3) is used as a method for monitoring autophagy progression as it associates with mature autophagosomes. For LC3 there was no notable change in signal intensity between crypts with characteristic L. intracellularis infection and healthy crypts of uninfected pigs. Finally, as p62 is degraded with the internal substrate of an autophagosome it was used to measure autophagic flux. There was no observed reduction or redistribution of p62. These preliminary results of the autophagy response in the ileum suggest that L. intracellularis affects autophagy. This disruption to host ileum homeostasis may provide a mechanism that assists in bacterial propagation and contributes to pathogenesis

A fast and robust method for full genome sequencing of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Type 1 and Type 2

PRRSV is a positive-sense RNA virus with a high degree of genetic variability among isolates. For diagnostic sensitivity and vaccine design it is essential to monitor PRRSV genetic diversity. However, to date only a few full genome sequences of PRRSV isolates have been made publicly available. In the present study, fast and robust methods for long range RT-PCR amplification and subsequent next generation sequencing (NGS) were developed and validated on nine Type 1 and nine Type 2 PRRSV viruses. The methods generated robust and reliable sequences both on primary material and cell culture adapted viruses and the protocols performed well on all three NGS platforms tested (Roche 454 FLX, Illumina HiSeq2000, and Ion Torrent PGM™ Sequencer). These methods will greatly facilitate the generation of more full genome PRRSV sequences globally

Pig immune response to general stimulus and to porcine reproductive and respiratory syndrome virus infection: a meta-analysis approach

BACKGROUND: The availability of gene expression data that corresponds to pig immune response challenges provides compelling material for the understanding of the host immune system. Meta-analysis offers the opportunity to confirm and expand our knowledge by combining and studying at one time a vast set of independent studies creating large datasets with increased statistical power. In this study, we performed two meta-analyses of porcine transcriptomic data: i) scrutinized the global immune response to different challenges, and ii) determined the specific response to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection. To gain an in-depth knowledge of the pig response to PRRSV infection, we used an original approach comparing and eliminating the common genes from both meta-analyses in order to identify genes and pathways specifically involved in the PRRSV immune response. The software Pointillist was used to cope with the highly disparate data, circumventing the biases generated by the specific responses linked to single studies. Next, we used the Ingenuity Pathways Analysis (IPA) software to survey the canonical pathways, biological functions and transcription factors found to be significantly involved in the pig immune response. We used 779 chips corresponding to 29 datasets for the pig global immune response and 279 chips obtained from 6 datasets for the pig response to PRRSV infection, respectively. RESULTS: The pig global immune response analysis showed interconnected canonical pathways involved in the regulation of translation and mitochondrial energy metabolism. Biological functions revealed in this meta-analysis were centred around translation regulation, which included protein synthesis, RNA-post transcriptional gene expression and cellular growth and proliferation. Furthermore, the oxidative phosphorylation and mitochondria dysfunctions, associated with stress signalling, were highly regulated. Transcription factors such as MYCN, MYC and NFE2L2 were found in this analysis to be potentially involved in the regulation of the immune response. The host specific response to PRRSV infection engendered the activation of well-defined canonical pathways in response to pathogen challenge such as TREM1, toll-like receptor and hyper-cytokinemia/ hyper-chemokinemia signalling. Furthermore, this analysis brought forth the central role of the crosstalk between innate and adaptive immune response and the regulation of anti-inflammatory response. The most significant transcription factor potentially involved in this analysis was HMGB1, which is required for the innate recognition of viral nucleic acids. Other transcription factors like interferon regulatory factors IRF1, IRF3, IRF5 and IRF8 were also involved in the pig specific response to PRRSV infection. CONCLUSIONS: This work reveals key genes, canonical pathways and biological functions involved in the pig global immune response to diverse challenges, including PRRSV infection. The powerful statistical approach led us to consolidate previous findings as well as to gain new insights into the pig immune response either to common stimuli or specifically to PRRSV infection

Metagenomic sequencing of clinical samples reveals a single widespread clone of Lawsonia intracellularis responsible for porcine proliferative enteropathy.

Lawsonia intracellularis is a Gram-negative obligate intracellular bacterium that is the aetiological agent of proliferative enteropathy (PE), a common intestinal disease of major economic importance in pigs and other animal species. To date, progress in understanding the biology of L. intracellularis for improved disease control has been hampered by the inability to culture the organism in vitro. In particular, our understanding of the genomic diversity and population structure of clinical L. intercellularis is very limited. Here, we utilized a metagenomic shotgun approach to directly sequence and assemble 21 L. intracellularis genomes from faecal and ileum samples of infected pigs and horses across three continents. Phylogenetic analysis revealed a genetically monomorphic clonal lineage responsible for infections in pigs, with distinct subtypes associated with infections in horses. The genome was highly conserved, with 94 % of genes shared by all isolates and a very small accessory genome made up of only 84 genes across all sequenced strains. In part, the accessory genome was represented by regions with a high density of SNPs, indicative of recombination events importing novel gene alleles. In summary, our analysis provides the first view of the population structure for L. intracellularis, revealing a single major lineage associated with disease of pigs. The limited diversity and broad geographical distribution suggest the recent emergence and clonal expansion of an important livestock pathogen

Down-regulation of mechanisms involved in cell transport and maintenance of mucosal integrity in pigs infected with Lawsonia intracellularis

Lawsonia intracellularis is an obligate intracellular bacterium, responsible for the disease complex known as proliferative enteropathy (PE). L. intracellularis is associated with intestinal crypt epithelial cell proliferation but the mechanisms responsible are yet to be defined. Microarray analysis was used to investigate the host-pathogen interaction in experimentally infected pigs to identify pathways that may be involved. Ileal samples originating from twenty-eight weaner pigs experimentally challenged with a pure culture of L. intracellularis (strain LR189/5/83) were subjected to microarray analysis. Microarray transcriptional signatures were validated using immunohistochemistry and quantitative real time PCR of selected genes at various time points post challenge. At peak of infection (14 days post challenge) 86% of altered transcripts were down regulated, particularly those involved in maintenance of mucosal integrity and regulation of cell transport. Among the up-regulated transcripts, CD163 and CDK1 were novel findings and considered to be important, due to their respective roles in innate immunity and cellular proliferation. Overall, targeted cellular mechanisms included those that are important in epithelial restitution, migration and protection; maintenance of stable inter-epithelial cell relationships; cell transport of nutrients and electrolytes; innate immunity; and cell cycle