Comparative Transcriptional Analysis of Homologous Pathogenic and Non-Pathogenic <em>Lawsonia intracellularis</em> Isolates in Infected Porcine Cells

<div><p><em>Lawsonia intracellularis</em> is the causative agent of proliferative enteropathy. This disease affects various animal species, including nonhuman primates, has been endemic in pigs, and is an emerging concern in horses. Non-pathogenic variants obtained through multiple passages in vitro do not induce disease, but bacterial isolates at low passage induce clinical and pathological changes. We hypothesize that genes differentially expressed between pathogenic (passage 10) and non-pathogenic (passage 60) <em>L. intracellularis</em> isolates encode potential bacterial virulence factors. The present study used high-throughput sequencing technology to characterize the transcriptional profiling of a pathogenic and a non-pathogenic homologous <em>L. intracellularis</em> variant during <em>in vitro</em> infection. A total of 401 genes were exclusively expressed by the pathogenic variant. Plasmid-encoded genes and those involved in membrane transporter (e.g. ATP-binding cassette), adaptation and stress response (e.g. transcriptional regulators) were the categories mostly responsible for this wider transcriptional landscape. The entire gene repertoire of plasmid A was repressed in the non-pathogenic variant suggesting its relevant role in the virulence phenotype of the pathogenic variant. Of the 319 genes which were commonly expressed in both pathogenic and non-pathogenic variants, no significant difference was observed by comparing their normalized transcription levels (fold change±2; p<0.05). Unexpectedly, these genes demonstrated a positive correlation (r² = 0.81; p<0.05), indicating the involvement of gene silencing (switching off) mechanisms to attenuate virulence properties of the pathogenic variant during multiple cell passages. Following the validation of these results by reverse transcriptase-quantitative PCR using ten selected genes, the present study represents the first report characterizing the transcriptional profile of <em>L. intracellularis</em>. The complexity of the virulence phenotype was demonstrated by the diversity of genes exclusively expressed in the pathogenic isolate. The results support our hypothesis and provide the basis for prospective mechanistic studies regarding specific roles of target genes involved in the pathogenesis, diagnosis and control of proliferative enteropathy.</p> </div

Functional categories of genes expressed by the pathogenic and non-pathogenic homologous <i>L. intracellularis</i> isolate PHE/MN1-00.

<p>Black and gray bars represent the number of genes expressed by the pathogenic and non-pathogenic variants, respectively.</p

A novel RNA-based <i>in situ</i> hybridization to detect Seneca Valley virus in neonatal piglets and sows affected with vesicular disease

<div><p>Seneca Valley virus (SVV) is the causative agent of an emerging vesicular disease in swine, which is clinically indistinguishable from other vesicular diseases such as foot-and-mouth disease. In addition, SVV has been associated with neonatal mortality in piglets. While a commercial SVV qRT-PCR is available, commercial antibodies are lacking to diagnose SVV infections by immunohistochemistry (IHC). Thus, a novel <i>in situ</i> hybridization technique—RNAscope (ISH) was developed to detect SVVRNA in infected tissues. From a total of 78 samples evaluated, 30 were positive by qRT-PCR and ISH-RNA, including vesicular lesions of affected sows, ulcerative lesions in the tongue of piglets and various other tissues with no evidence of histological lesions. Nineteen samples were negative for SVV by qRT-PCR and ISH-RNA. The Ct values of the qRT-PCR from ISH-RNA positive tissues varied from 12.0 to 32.6 (5.12 x 10⁶ to 5.31 RNA copies/g, respectively). The ISH-RNA technique is an important tool in diagnosing and investigating the pathogenesis of SVV and other emerging pathogens.</p></div

Schematic representation of the <i>L. intracellularis</i> genome.

<p>Distribution of genes expressed by the pathogenic (black circles) and the non-pathogenic (gray circles) variants. Overlapping zones represent genes expressed in both variants.</p

Chromosomal genes showing highest transcript levels exclusively expressed by the pathogenic variant of the <i>L.</i><i>intracellularis</i> isolate PHE/MN1-00 according to the putative biological function.

<p>Chromosomal genes showing highest transcript levels exclusively expressed by the pathogenic variant of the <i>L.</i><i>intracellularis</i> isolate PHE/MN1-00 according to the putative biological function.</p

Genes exclusively expressed by the non-pathogenic variant of the <i>L. intracellularis</i> isolate PHE/MN1-00.

<p>Genes exclusively expressed by the non-pathogenic variant of the <i>L. intracellularis</i> isolate PHE/MN1-00.</p

Average log-transformed RPKM (log₂ [RPKM]) of the 319 genes commonly expressed by the pathogenic (y-axis) and non-pathogenic (x-axis) variant.

<p>The trend line represent a linear regression model (<i>p</i>-value <0.05; r² = 0.809).</p

SVV distribution in tissues without evidence of histological lesions.

<p>Swine, ISH-RNA. a) Piglet, spleen (central arteriole). Strong SVV positive staining diffusely distributed throughout the splenic parenchyma. ISH-RNA, 20x.; b) Piglet, spleen. Negative control. ISH-RNA, 20x; c) Piglet, small intestine. SVV mRNA was multifocally distributed within enterocytes (black arrows) and lamina propria. ISH-RNA, 20x; d) Piglet, lung, SVV positive signals were found in alveolar septum. ISH-RNA, 20x.</p

Plasmid-encoded genes with highest transcript levels exclusively expressed by the pathogenic variant of the <i>L.</i><i>intracellularis</i> isolate PHE/MN1-00.

<p>Plasmid-encoded genes with highest transcript levels exclusively expressed by the pathogenic variant of the <i>L.</i><i>intracellularis</i> isolate PHE/MN1-00.</p