Growth curve of passaged mouse IECs in culture.

<p>The log phase started after day 2 of the lag phase with a sharper inclination.</p

Cultured mouse primary intestinal epithelial cells under an inverted phase-contrast microscope.

<p>(A) The single crypts obtained with collagenase-hyaluronidase dissociation of mouse small intestine. (B) A few cells gradually migrated out around the crypts after 24 h. (C) The epithelial cells started to grow rapidly after 48 h, and multiple large colonies were formed at day 5. (D) Cells continued to grow until confluence was reached after 9 days.</p

Observations on invasion of IECs by <i>Trichinella spiralis</i> infective larvae.

<p>(A) When <i>T. spiralis</i> infective larvae were inoculated onto monolayers of mouse IECs <i>in vitro</i>, the larvae invaded the cells, and their head and body resided in the cytoplasm of the syncytia composed of numerous IECs (showed as arrow). (B) After the removal of agarose, the cell monolayer was stained with propidium iodide (PI) and observed under a fluorescence microscope. Nuclei of the damaged cells were stained intensely and uniformly red, showing the serpentine trail left by the parasite. In contrast, nuclei of the live cells not invaded by the larvae were not stained. (C) After being stained with PI, the cell monolayer was fixed and reacted with rabbit immune sera against <i>T. spiralis</i> ES antigens and FITC-conjugated secondary antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027010#s2" target="_blank">Materials and Methods</a>. Green fluorescence was found in the cytoplasm of the damaged cells. (D) No fluorescence was found in the cells which were not invaded by the non-activated larvae.</p

Morphological characteristic and Immunofluorescence staining of fetal mouse IECs.

<p>(A) The IECs formed a tightly packed monolayer with typical cobblestone morphology (passage 8). (B) HE staining of IECs. (C) Immunofluorescence staining of IEC cytokeratins, cytokeratin 18 was clearly detected in the cytoplasm of IECs with a green color. (D) No green fluorescence in the cytoplasm of IECs was found and only the nucleus was stained red with propidium iodide (PI) in the negative controls.</p

Hematoxylin-and-eosin (HE) staining and immunofluorescence test (IFT) on the small intestines of mice infected with <i>Trichinella spiralis</i>.

<p>(A) The Kunming mice were orally infected with 300 <i>Trichinella spiralis</i> muscle larvae. After 18 hours, they were killed by anaesthetic inhalation with isoflurane, and their small intestines were fixed in 10% formaldehyde solution. HE staining showed that the larvae located at the crypt-villus junction (showed as arrow). (B and C) Different sections of the larvae at the crypt-villus junction were recognized by sera of the rabbits infected with <i>T. spiralis</i>, and exhibited green fluorescence (showed as arrows). The mouse small intestines exhibited only background red fluorescence (propidium iodide). (D) Longitudinal sections of the larvae (showed as arrows) were found in intestinal crypts (green fluorescence).</p

Relative expression by real-time PCR of the selected nine genes in <i>T.</i><i>spiralis</i> larvae.

<p>Total RNAs from muscle larvae (ML) and intestinal infective larvae (IIL) were subjected to real-time PCR as described in “Material and Methods.” Expression levels were normalized to the G3PDH gene and presented as relative expression to controls (mean ± SD, n = 9). Controls are arbitrarily assigned to a value of 1. *Significant difference of gene expression compared to controls.</p

Gene ontology categories for the up-regulated proteins of <i>T.</i><i>spiralis</i> larvae after their exposure to mouse intestine milieu.

<p>The identified proteins were classified into cellular component, molecular function, and biological process by WEGO according to their GO signatures. The number of genes denotes that of proteins with GO annotations.</p

The identified up-regulated genes of <i>T. spiralis</i> after exposure to mouse intestine milieu.

<p>Seq. name: code of EST sequence; Seq. length: EST length (base pair); Id (%): EST similarity; N: EST number.</p

Primers used in the real-time PCR assays.

<p>Primers used in the real-time PCR assays.</p

Genetic Structure Analysis of <i>Spirometra erinaceieuropaei</i> Isolates from Central and Southern China

<div><p>Background</p><p>Sparganosis caused by invasion of the plerocercoid larvae (spargana) of <i>Spirometra erinaceieuropaei</i> have increased in recent years in China. However, the population genetic structure regarding this parasite is still unclear. In this study, we used the sequences of two mitochondrial genes cytochrome <i>b</i> (<i>cytb</i>) and cytochrome <i>c</i> oxidase subunit I (<i>cox1</i>) to analyze genetic variation and phylogeographic structure of the <i>S</i>. <i>erinaceieuropaei</i> populations.</p><p>Methodology/Principal Findings</p><p>A total of 88 <i>S</i>. <i>erinaceieuropaei</i> isolates were collected from naturally infected frogs in 14 geographical locations of China. The complete <i>cytb</i> and <i>cox1</i> genes of each sample was amplified and sequenced. Total 61 haplotypes were found in these 88 concatenated sequences. Each sampled population and the total population have high haplotype diversity (Hd), accompanied by very low nucleotide diversity (Pi). Phylogenetic analyses of haplotypes revealed two distinct clades (HeN+HuN+GZ-AS clade and GX+HN+GZ-GY clade) corresponding two sub-networks yielded by the median-joining network. Pairwise <i>F</i>_ST values supported great genetic differentiation between <i>S</i>. <i>erinaceieuropaei</i> populations. Both negative Fu’s <i>F</i>_S value of neutrality tests and unimodal curve of mismatch distribution analyses supported demographic population expansion in the HeN+HuN+GZ-AS clade. The BEAST analysis showed that the divergence time between the two clades took place in the early Pleistocene (1.16 Myr), and by Bayesian skyline plot (BSP) an expansion occurred after about 0.3 Myr ago.</p><p>Conclusions</p><p><i>S</i>. <i>erinaceieuropaei</i> from central and southern China has significant phylogeographic structure, and climatic oscillations during glacial periods in the Quaternary may affect the demography and diversification of this species.</p></div