In Trichinella spiralis infection, type 2 helper T (Th2) cell-related and regulatory T (T(reg)) cell-related immune responses are the most important immune events. In order to clarify which Toll-like receptors (TLRs) are closely associated with these responses, we analyzed the expression of mouse TLR genes in the small intestine and muscle tissue during T. spiralis infection. In addition, the expression of several chemokine- and cytokine-encoding genes, which are related to Th2 and T(reg) cell mediated immune responses, were analyzed in mouse embryonic fibroblasts (MEFs) isolated from myeloid differentiation factor 88 (MyD88)/TIR-associated proteins (TIRAP) and Toll receptor-associated activator of interferons (TRIF) adapter protein deficient and wild type (WT) mice. The results showed significantly increased TLR4 and TLR9 gene expression in the small intestine after 2 weeks of T. spiralis infection. In the muscle, TLR1, TLR2, TLR5, and TLR9 gene expression significantly increased after 4 weeks of infection. Only the expression of the TLR4 and TLR9 genes was significantly elevated in WT MEF cells after treatment with excretory-secretory (ES) proteins. Gene expression for Th2 chemokine genes were highly enhanced by ES proteins in WT MEF cells, while this elevation was slightly reduced in MyD88/TIRAP-/- MEF cells, and quite substantially decreased in TRIF-/- MEF cells. In contrast, IL-10 and TGF-beta expression levels were not elevated in MyD88/TIRAP-/- MEF cells. In conclusion, we suggest that TLR4 and TLR9 might be closely linked to Th2 cell and T(reg) cell mediated immune responses, although additional data are needed to convincingly prove this observation.
Animals ; Gene Expression ; Humans ; Interleukin-10/genetics ; Mice ; Mice, Knockout ; Th2 Cells/metabolism ; Toll-Like Receptors/*genetics/metabolism ; Trichinella spiralis/genetics/*physiology ; Trichinellosis/genetics/metabolism/*parasitology

In order to obtain greater insight into the relevant genomic expression patterns of Trichinella spiralis, 992 expressed sequence tags (ESTs) were collected from a cDNA library of T. spiralis muscle stage larvae and assembled into 60 clusters and 385 singletons. Of them, 445 (44.7%) ESTs were annotated to their homologous genes, and small fractions were matched to known genes of nematodes. The annotated ESTs were classified into 25 eukaryotic orthologous groups (KOG). Cytochrome C oxidase (34 clones) was found to be most frequent species.
Animals ; *Expressed Sequence Tags ; Gene Library ; Helminth Proteins/genetics ; Larva/*genetics ; Muscle, Skeletal/*parasitology ; Rats ; Trichinella spiralis/*genetics/isolation & purification ; Trichinosis/*parasitology

We have studied the genetic differences among four isolates of Trichinella including a new strain of Trichinella spiralis (ISS 623) recently found from a human case who took a badger in Korea. Because they have a different host origin and came from geographically separated regions, we supposed the genetic pattern of the isolates might be different as had been previously reported. It was analysed by PCR-RFLP analysis of the rDNA repeat that can readily distinguish a species or strain from others. Isolated genomic DNA of each isolate of Trichinella larvae was amplified with ITS1 specific primers and digested with restriction endonucleases. The PCR product of ITS1 was confirmed using Southern blot analysis to be a 910 bp fragment. The restriction fragments of each isolate had variable patterns when it was digested with Rsa 1 only. According to the RFLP patterns, the estimated genetic divergence between each isolate was different. In conclusion, four isolates of Trichinella including a new strain of T. spiralis obtained from a Korean patient may have genetic differences in the ITS1 region and the Shanghai isolate was genetically more similar to the Japanese unknown isolate than others in the ITS1 region.
Animals ; *DNA, Helminth ; *DNA, Ribosomal ; Human ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Repetitive Sequences, Nucleic Acid ; Trichinella spiralis/*genetics/isolation & purification

Muscle larvae of Trichinella isolates from two outbreaks in Korea were analyzed by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiple-PCR. All of the muscle larvae showed a band similar to that of T. spiralis larvae of the reference strain. The two Korean Trichinella isolates (isolate code ISS623 and ISS1078) might be classifiable to Trichinella spiralis.
Animals ; DNA Fingerprinting ; DNA, Helminth/chemistry/genetics ; *Disease Outbreaks ; Human ; Korea/epidemiology ; Mice ; Mice, Inbred ICR ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Trichinella spiralis/*genetics ; Trichinosis/epidemiology/*parasitology

The first human case with trichinellosis was reported in 1964 in Tibet, China. However, up to the present, the etiological agent of trichinellosis has been unclear. The aim of this study was to identify a Tibet Trichinella isolate at a species level by PCR-based methods. Multiplex PCR revealed amplicon of the expected size (173 bp) for Trichinella spiralis in assays containing larval DNA from Tibet Trichinella isolate from a naturally infected pig. The Tibet Trichinella isolate was also identified by PCR amplification of the 5S ribosomal DNA intergenic spacer region (5S ISR) and mitochondrial large-subunit ribosomal RNA (mt-lsrDNA) gene sequences. The results showed that 2 DNA fragments (749 bp and 445 bp) of the Tibet Trichinella isolate were identical to that of the reference isolates of T. spiralis. The Tibet Trichinella isolate might be classifiable to T. spiralis. This is the first report on T. spiralis in southwestern China.
Animals ; DNA, Helminth/chemistry/genetics ; DNA, Mitochondrial/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; DNA, Ribosomal Spacer/genetics ; Genotype ; Humans ; Multiplex Polymerase Chain Reaction ; RNA, Ribosomal, 5S/genetics ; Sequence Analysis, DNA ; Swine ; Swine Diseases/*parasitology ; Tibet ; Trichinella spiralis/*classification/genetics/isolation & purification ; Trichinellosis/parasitology/*veterinary

The 5th outbreak of trichinosis occurred in a mountainous area of North Vietnam in 2012, involving 24 patients among 27 people who consumed raw pork together. Six of these patients visited several hospitals in Hanoi for treatment. Similar clinical symptoms appeared in these patients within 5-8 days after eating infected raw pork, which consisted of fever, muscle pain, difficult moving, edema, difficult swallowing, and difficult breathing. ELISA revealed all (6/6) positive reactions against Trichinella spiralis antigen and all cases showed positive biopsy results for Trichinella sp. larvae in the muscle. The larvae detected in the patients were identified as T. spiralis (Vietnamese strain) by the molecular analysis of the mitochondrial cytochrome c oxidase subunit III (cox3) gene.
Adult ; Animals ; Antigens, Helminth/analysis/immunology ; *Disease Outbreaks ; Electron Transport Complex IV/genetics ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Larva ; Male ; Meat/*parasitology ; Mitochondria/genetics ; Muscles/parasitology/pathology ; Swine ; Trichinella spiralis/genetics/immunology/*isolation & purification ; Trichinellosis/*epidemiology/parasitology/pathology ; Vietnam/epidemiology

In order to know the effect of pre-existing Trichinella spiralis infection on experimentally induced intestinal inflammation and immune responses, we induced colitis in T. spiralis-infected mice and observed the severity of colitis and the levels of Th1, Th2, and regulatory cytokines and recruitment of CD4+CD25+Foxp3+ T (regulatory T; Treg) cells. Female C57BL/6 mice were infected with 250 muscle larvae; after 4 weeks, induction of experimental colitis was performed using 3% dextran sulfate sodium (DSS). During the induction period, we observed severity of colitis, including weight loss and status of stool, and evaluated the disease activity index (DAI). A significantly low DAI and degree of weight loss were observed in infected mice, compared with uninfected mice. In addition, colon length in infected mice was not contracted, compared with uninfected mice. We also observed a significant increase in production of pro-inflammatory cytokines, IL-6 and IFN-gamma, in spleen lymphocytes treated with DSS; however, such an increase was not observed in infected mice treated with DSS. Of particular interest, production of regulatory cytokines, IL-10 and transforming growth factor (TGF)-beta, in spleen lymphocytes showed a significant increase in mice infected with T. spiralis. A similar result was observed in mesenteric lymph nodes (MLN). Subsets of the population of Treg cells in MLN and spleen showed significant increases in mice infected with T. spiralis. In conclusion, T. spiralis infection can inhibit the DSS-induced colitis in mice by enhancing the regulatory cytokine and Treg cells recruitment.
Animals ; Colitis/chemically induced/*immunology/pathology ; Cytokines/genetics/*immunology/metabolism ; Dextran Sulfate/adverse effects ; Disease Models, Animal ; Female ; Forkhead Transcription Factors/immunology/metabolism ; *Gene Expression Regulation ; Larva ; Mice ; Mice, Inbred C57BL ; Spleen/immunology ; T-Lymphocytes, Regulatory/*immunology ; Trichinella spiralis/*immunology ; Trichinellosis/*immunology/parasitology