Understanding the Molecular Epidemiology and Global Relationships of <i>Brachyspira hyodysenteriae</i> from Swine Herds in the United States: A Multi-Locus Sequence Typing Approach

<div><p>Outbreaks of mucohemorrhagic diarrhea in pigs caused by <i>Brachyspira hyodysenteriae</i> in the late 2000s indicated the re-emergence of Swine Dysentery (SD) in the U.S. Although the clinical disease was absent in the U.S. since the early 1990s, it continued to cause significant economic losses to other swine rearing countries worldwide. This study aims to fill the gap in knowledge pertaining to the re-emergence and epidemiology of <i>B. hyodysenteriae</i> in the U.S. and its global relationships using a multi-locus sequence typing (MLST) approach. Fifty-nine post re-emergent isolates originating from a variety of sources in the U.S. were characterized by MLST, analyzed for epidemiological relationships (within and between multiple sites of swine systems), and were compared with pre re-emergent isolates from the U.S. Information for an additional 272 global isolates from the MLST database was utilized for international comparisons. Thirteen nucleotide sequence types (STs) including a predominant genotype (ST93) were identified in the post re-emergent U.S. isolates; some of which showed genetic similarity to the pre re-emergent STs thereby suggesting its likely role in the re-emergence of SD. In the U.S., in general, no more than one ST was found on a site; multiple sites of a common system shared a ST; and STs found in the U.S. were distinct from those identified globally. Of the 110 STs characterized from ten countries, only two were found in more than one country. The U.S. and global populations, identified as clonal and heterogeneous based on STs, showed close relatedness based on amino acid types (AATs). One predicted founder type (AAT9) and multiple predicted subgroup founder types identified for both the U.S. and the global population indicate the potential microevolution of this pathogen. This study elucidates the strain diversity and microevolution of <i>B. hyodysenteriae</i>, and highlights the utility of MLST for epidemiological and surveillance studies.</p></div

MST analysis of 110 STs representing 341 global <i>B. hyodysenteriae</i> isolates showing geographical distribution.

<p>In the Minimum Spanning Tree (MST) analysis, each circle represents a different sequence type (ST) as labeled, its size reflects the number of isolates and the color indicates the country of origin. The width of the lines reflects the genetic difference between two STs, wherein dark/heavy lines connect single locus variants (SLVs), light/thin lines connect double locus variants (DLVs) and dotted lines indicate the most likely connection between two STs differing by three or more loci. STs which were DLVs of at least one other ST were grouped together (shaded in grey).</p

Population snapshot from 17 AATs of 69 North American isolates using the BURST algorithm.

<p>In the Based Upon Related Sequence Types (BURST) algorithm of eBURST, the labeled dots represent the amino acid type (AAT), the size of the dot denotes the number of isolates represented by that AAT, and the solid lines between two dots indicate those which are single locus variants (SLVs) of each other. The position and the distance between the dots are arbitrary. AATs that represented isolates that were classified either as pre-, post- or both (pre- and post-) re-emergence were labelled in black, green or pink, respectively. Predicted primary founder types and subgroup founder types are denoted by blue and yellow dots respectively.</p

UPGMA depicting molecular relatedness of 69 North American (pre and post re-emergence) <i>B. hyodysenteriae</i> isolates.

<p><b>A: Bootstrap consensus tree (1000 replicates) based on nucleotide sequences:</b> The Tamura 3-parameter model of Unweighted Pair Group Method with Arithmetic Mean (UPGMA) was used to compute the genetic similarity of isolates using concatenated nucleotide sequences of the seven <i>B. hyodysenteriae</i> multi-locus sequence typing loci. Bootstrap values greater than 50% are shown at the nodes, and the total length of the scale represents 30 substitutions per 10,000 base pairs of nucleotide sequence. Isolate information includes details of the ST and the state (or country), decade and host species of origin. <b>B: Bootstrap consensus tree (1000 replicates) based on amino acid sequences:</b> The dendogram was constructed and depicted as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107176#pone-0107176-g001" target="_blank">Figure 1A</a>; however, here amino acid sequences (AATs) were the unit of analysis. The total length of the scale represents 14 substitutions per 10,000 amino acids sequence.</p

MST analysis of 67 AATs representing 341 global <i>B. hyodysenteriae</i> isolates showing geographical distribution.

<p>In the Minimum Spanning Tree (MST) analysis, each circle represents a different Amino Acid Type (AAT) as labeled, the size of the circle and the partition lines within a circle represent the relative and the absolute number of isolates represented by the AAT, and the color indicates the country of origin. The width of the lines reflects the allelic differences between two AATs, wherein dark/heavy lines connect single locus variants (SLVs) and light/thin lines connect double locus variants (DLVs).</p

Summary numerical values of <i>B. hyodysenteriae</i> isolates and STs on a country and global level.

<p>n/a: not applicable.</p><p>Abbreviations: I_A Index of association values; mean dN/dS mean ratio of non-synonymous to synonymous substitutions; DI Simpson's Index of Diversity; ST Sequence Type.</p><p>Summary numerical values of <i>B. hyodysenteriae</i> isolates and STs on a country and global level.</p

UPGMA dendograms depicting molecular relatedness of 341 global <i>B. hyodysenteriae</i> isolates.

<p><b>A: Bootstrap consensus tree (1000 replicates) based on nucleotide sequences of 110 STs:</b> The Tamura 3-parameter model of Unweighted Pair Group Method with Arithmetic Mean (UPGMA) was used to compute the genetic similarity of isolates using concatenated nucleotide sequences of the seven <i>B. hyodysenteriae</i> multi-locus sequence typing loci. Bootstrap values greater than 50% are shown at the nodes, and the total length of the scale represents 140 substitutions per 10,000 base pairs of nucleotide sequence. ST information includes details of the host species and country (AU Australia; BE Belgium; CA Canada; DE Germany; ES Spain; IT Italy; PT Portugal; SE Sweden; UK United Kingdom; US United States of America) of origin. <b>B: Bootstrap consensus tree (1000 replicates) based on amino acid sequences of 67 AATs:</b> The dendogram was constructed and depicted as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107176#pone-0107176-g003" target="_blank">Figure 3A</a>, however amino acid sequences (AATs) were the unit of analysis. The total length of the scale represents 60 substitutions per 10,000 amino acid sequence.</p

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

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

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