OBJECTIVETo observe the effect of Trichinella spiralis and its worm-derived proteins on cecal ligation and puncture (CLP)-induced sepsis in mice.

METHODSEighty male BALB/c mice were randomly divided into sham-operated group, CLP group, Trichinella spiralis muscle larvae (ML) pre-infection group (ML+CLP group), soluble muscle larvae proteins (SMP) treatment group (SMP+CLP group) and excretory-secretory proteins (MES) treatment group (MES+CLP group). In ML+CLP group, the mice were orally infected with 300 Trichinella spiralis muscle larvae at 28 days before CLP and those in the other groups were intraperitioneally injected with PBS or SMP (25 µg/mice) or MES (25µg/mice) 30 min after CLP. The general condition and 72-h survival after CLP of the mice were observed. The levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), creatinine (Cr), TNF-α, IL-6, IL-1β, IL-10 and TGF-β were measured at 12 h after the operation, and the pathological changes of the liver and kidney were observed.

RESULTSs Compared with the sham-operated mice, the mice in CLP group showed decreased 72-h survival, obviously increased ALT, AST, BUN, Cr, TNF-α, IL-6, IL-1β, IL-10, and TGF-β with hepatic cords disorder, hepatocytes swelling, glomerulus shrinkage, and renal tubular cell edema. Compared with CLP group, the mice in ML+CLP group showed lowered levels of ALT, AST, Cr, TNF-α and IL-1β and increased levels of IL-10 and TGF-β; in SMP+CLP group, the levels of ALT, AST, Cr, TNF-α and IL-1β were decreased and TGF-β increased. In MES+CLP group, the mice showed obviously increased 72-h survival with lowered levels of ALT, AST, BUN, Cr, TNF-α, IL-6 and IL-1β, increased levels of IL-10 and TGF-β, and alleviated liver and kidney damages.

CONCLUSIONTrichinella spiralis and its worm-derived proteins can decrease the levels of pro-inflammatory cytokines and increase immunomodulatory cytokines, and MES has more potent effect to reduce structural and functional damages of the liver and kidney.

Alanine Transaminase ; blood ; Animals ; Antigens, Helminth ; pharmacology ; Aspartate Aminotransferases ; blood ; Blood Urea Nitrogen ; Cecum ; Creatinine ; blood ; Cytokines ; blood ; Helminth Proteins ; pharmacology ; Kidney ; physiopathology ; Kidney Diseases ; therapy ; Ligation ; Liver ; physiopathology ; Liver Diseases ; therapy ; Male ; Mice ; Mice, Inbred BALB C ; Sepsis ; therapy ; Trichinella spiralis

A total 7 outbreaks of trichinellosis have occurred in Korea, mostly as a result of consumption of raw wild boar (Sus scrofa) meat. Since only 1 serological survey on wild boars had yet been performed in Korea, the present study aimed to estimate the prevalence of trichinellosis in wild boars and some species of rodents by artificial digestion and serological examinations in Yanggu-gun, Gangwon-do, the endemic area of trichinellosis. Both the wild boar and rodent muscle samples revealed no Trichinella larvae by direct examination and artificial digestion method. However, serological examinations revealed that 4 wild boar sera samples out of 118 (3.4%) were positive to Trichinella antigen. Although the recovery of Trichinella larvae ended in a failure, it is proved for the first time that the sylvatic cycle of Trichinella has been maintained in wild boars of Gangwon-do, Korea.
Animals ; Antibodies, Helminth/*blood ; Antigens, Helminth/blood ; Female ; Male ; Republic of Korea/epidemiology ; Seroepidemiologic Studies ; Sus scrofa ; Swine ; Swine Diseases/*blood/diagnosis/epidemiology/parasitology ; Trichinella/classification/genetics/immunology/*isolation & purification

Roundworms of Toxocara canis and Toxascaris leonina are common gastrointestinal helminths of canids over the world. Humans are infected with T. canis larvae through ingestion of infective eggs in contaminated environments or larvae by consumption of raw or uncooked meat or livers. Recently, patients of clinically diagnosed toxocariasis are increasing and require correct diagnosis in Korea. The present study investigated serological cross-reactivity between crude antigens of T. canis (TCLA) and T. leonina (TLLA) larvae. We collected serum specimens from 177 toxocariasis patients who were clinically suspected in the Seoul National University Hospital and 115 healthy controls. An ELISA method for toxocariasis was used to evaluate diagnostic efficacy of TLLA for serodiagnosis of human toxocariasis. The IgG ELISA using TLLA gave 14 (14.3%) positives of 98 TCLA positive specimens among 177 suspected toxocariasis patients. Most of them showed high absorbances with TCLA. In conclusion, there is a partial cross reaction between serum specimens of toxocariasis and TLLA.
Animals ; Antibodies, Helminth/blood ; Antigens, Helminth/*immunology ; Cross Reactions ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin G/blood ; Larva/immunology/metabolism ; Toxascaris/growth & development/*immunology/isolation & purification ; Toxocara canis/growth & development/*immunology/isolation & purification ; Toxocariasis/*diagnosis/parasitology

OBJECTIVETo characterize the biological function of calmodulin (CaM) from Clonorchis sinensis (C. sinensis, Cs) and investigate its role in clonorchiasis-associated hepatic fibrosis.

METHODSThe full-length sequence of CsCaM gene was isolated from Cs cDNA library and its homologues were searched using BLASTx for comparison. Bioinformatics analysis was performed to compare the homologues and predict the physiochemical characteristics and functional domains. The gene was cloned in a prokaryotic plasmid and expressed in E. coli, and the recombinant protein was purified by affinity chromatography for immunizing rats to produce polyclonal antibodies, whose titer was determined using ELISA analysis. Immunoblotting analysis was carried out to determine of the purity and antibody recognition of CsCaM. Immunofluorescence assay was employed to analyze the tissue location of the protein. A rat model of liver fibrosis was established by introperitoneal injection of the recombinant protein.

RESULTSThe recombinant CsCaM protein obtained contained 150 amino acids with a theoretical molecular mass of 23.4 kD. CsCaM homologue had EF hand motifs. The recombinant pET-30a-CsCaM plasmid expressed in BL21 E. coli was about 23.4 kD. The total IgG antibody titer in the immunized mice reached the peak level (over 1: 51200) 2 to 4 weeks after the first injection. Immunohistochemistry showed that CsCaM located in the testis of adult C. sinensis. The rats receiving intraperitoneal injection of CsCaM showed severe liver inflammation with mild to moderate liver fibrosis.

CONCLUSIONThe pro-inflammation and pro-fibrosis effects of CsCaM in rat liver suggest its involvement in clonorchiasis- associated hepatic fibrosis.

Animals ; Antibodies, Helminth ; blood ; Antigens, Helminth ; immunology ; Calmodulin ; immunology ; Clonorchiasis ; immunology ; Clonorchis sinensis ; immunology ; Enzyme-Linked Immunosorbent Assay ; Gene Library ; Immunoglobulin G ; blood ; Inflammation ; Liver Cirrhosis ; parasitology ; Male ; Mice ; Rats ; Recombinant Proteins ; immunology

Dirofilaria immitis (heartworm) infections affect domestic dogs, cats, and various wild mammals with increasing incidence in temperate and tropical areas. More sensitive antibody detection methodologies are required to diagnose asymptomatic dirofilariasis with low worm burdens. Applying current transcriptomic technologies would be useful to discover potential diagnostic markers for D. immitis infection. A filarial homologue of the mammalian translationally controlled tumor protein (TCTP) was initially identified by screening the assembled transcriptome of D. immitis (DiTCTP). A BLAST analysis suggested that the DiTCTP gene shared the highest similarity with TCTP from Loa loa at protein level (97%). A histidine-tagged recombinant DiTCTP protein (rDiTCTP) of 40 kDa expressed in Escherichia coli BL21 (DE3) showed immunoreactivity with serum from a dog experimentally infected with heartworms. Localization studies illustrated the ubiquitous presence of rDiTCTP protein in the lateral hypodermal chords, dorsal hypodermal chord, muscle, intestine, and uterus in female adult worms. Further studies on D. immitis-derived TCTP are warranted to assess whether this filarial protein could be used for a diagnostic purpose.
Animal Structures/chemistry ; Animals ; Antibodies, Helminth/blood ; Antigens, Helminth/chemistry/*genetics/immunology/*isolation & purification ; Cloning, Molecular ; Dirofilaria immitis/chemistry/*genetics/immunology ; Disease Models, Animal ; Dogs ; Escherichia coli/genetics ; Gene Expression ; Molecular Sequence Data ; Molecular Weight ; Recombinant Fusion Proteins/chemistry/genetics/immunology/isolation & purification ; Sequence Analysis, DNA ; Tumor Markers, Biological/chemistry/*genetics/immunology/*isolation & purification

A synthetic peptide was prepared based on the antigenic region of Paragonimus westermani pre-procathepsin L, and its applicability for immunodiagnosis for human paragonimiasis (due to Paragonimus heterotremus) was tested using an ELISA to detect IgG4 antibodies in the sera of patients. Sera from other helminthiases, tuberculosis, and healthy volunteers were used as the references. This peptide-based assay system gave sensitivity, specificity, accuracy, and positive and negative predictive values of 100%, 94.6%, 96.2%, 100%, and 88.9%, respectively. Cross reactivity was frequently seen against the sera of fascioliasis (75%) and hookworm infections (50%). Since differential diagnosis between paragonimiasis and fascioliasis can be easily done by clinical presentation and fascioliasis serology, this cross reaction is not a serious problem. Sera from patients with other parasitoses (0-25%) rarely responded to this synthetic antigen. This synthetic peptide antigen seems to be useful for development of a standardized diagnostic system for paragonimiasis.
Adult ; Animals ; Antibodies, Helminth/*blood ; Antigens, Helminth/*diagnostic use ; Clinical Laboratory Techniques/*methods ; Cross Reactions ; Enzyme-Linked Immunosorbent Assay/methods ; Humans ; Immunoglobulin G/*blood ; Paragonimiasis/*diagnosis ; Paragonimus/*immunology ; Parasitology/*methods ; Predictive Value of Tests ; Sensitivity and Specificity

Gnathostoma spinigerum can cause subarachnoid hemorrhage (SAH). The detection of specific antibodies in serum against G. spinigerum antigen is helpful for diagnosis of neurognathostomiasis. There is limited data on the frequency of G. spinigerum infection in non-traumatic SAH. A series of patients diagnosed as non-traumatic SAH at the Srinagarind Hospital, Khon Kaen University, Thailand between January 2011 and January 2013 were studied. CT or MR imaging of the brain was used for diagnosis of SAH. Patients were categorized as aneurysmal subarachnoid hemorrhage (A-SAH) or non-aneurysmal subarachnoid hemorrhage (NA-SAH) according to the results of cerebral angiograms. The presence of specific antibodies in serum against 21- or 24-kDa G. spinigerum antigen was determined using the immunoblot technique. The detection rate of antibodies was compared between the 2 groups. Of the 118 non-traumatic SAH patients for whom cerebral angiogram and immunoblot data were available, 80 (67.8%) patients had A-SAH, whereas 38 (32.2%) had NA-SAH. Overall, 23.7% were positive for specific antibodies against 21- and/or 24-kDa G. spinigerum antigen. No significant differences were found in the positive rate of specific antibodies against G. spinigerum in both groups (P-value=0.350).
Adult ; Aged ; Animals ; Antibodies, Helminth/*blood ; Antigens, Helminth/diagnostic use ; Brain/radiography ; Female ; Gnathostoma/immunology/*isolation & purification ; Gnathostomiasis/*diagnosis/*parasitology ; Humans ; Immunoblotting ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Serum/immunology ; Subarachnoid Hemorrhage/*diagnosis/*etiology ; Thailand ; Tomography, X-Ray Computed

Neurognathostomiasis is a severe form of human gnathostomiasis which can lead to disease and death. Diagnosis of neurognathostomiasis is made presumptively by using clinical manifestations. Immunoblotting, which recognizes antigenic components of molecular mass 21 kDa and 24 kDa in larval extracts of Gnathostoma spinigerum (Gs 21/24), has high sensitivity and specificity for diagnosis of neurognathostomiasis. However, only very small amounts of the Gs 21/24 antigens can be prepared from parasites harvested from natural or experimental animals. To overcome this problem, we recently produced a recombinant matrix metalloproteinase (rMMP) protein from G. spinigerum. In this study, we evaluated this rMMP alongside the Gs 21/24 antigens for serodiagnosis of human neurognathostomiasis. We studied sera from 40 patients from Srinagarind Hospital, Khon Kaen University, Thailand, with clinical criteria consistent with those of neurognathostomiasis, and sera from 30 healthy control adults from Thailand. All sera were tested for specific IgG antibodies against both G. spinigerum crude larval extract and rMMP protein using immunoblot analysis. The sensitivity and specificity for both antigenic preparations were all 100%. These results show that G. spinigerum rMMP protein can be used as an alternative diagnostic antigen, in place of larval extract, for serodiagnosis of neurognathostomiasis.
Adult ; Animals ; Antibodies, Helminth/blood ; Antigens, Helminth/*diagnostic use/genetics/isolation & purification ; Central Nervous System Parasitic Infections/*diagnosis/parasitology ; Gnathostoma/enzymology/immunology/*isolation & purification ; Gnathostomiasis/*diagnosis/parasitology ; Healthy Volunteers ; Humans ; Immunoblotting/methods ; Immunoglobulin G/blood ; Matrix Metalloproteinases/*diagnostic use/genetics/isolation & purification ; Parasitology/*methods ; Prospective Studies ; Recombinant Proteins/diagnostic use/genetics/isolation & purification ; Sensitivity and Specificity ; Serologic Tests/methods ; Thailand

In December 2011, we reported an autochthonous case of Echinococcus multilocularis infection in a 42-year-old woman in Korea. The diagnosis was based on histopathological findings of the surgically resected liver cyst. In the present study, we evaluated the serological and molecular characteristics of this Korean E. multilocularis case. The patient's serum strongly reacted with affinity-purified native Em18 and recombinant Em18 antigens (specific for E. multilocularis) but negative for recombinant antigen B8/1 (reactive for Echinococcus granulosus). In immunoaffinity chromatography, the serum also strongly reacted with E. multilocularis and only weakly positive for E. granulosus. We determined the whole nucleotide sequence of cox1 (1,608 bp) using the paraffin-embedded cystic tissue which was compared with E. multilocularis isolates from China, Japan, Kazakhstan, Austria, France, and Slovakia. The Korean case showed 99.8-99.9% similarity with isolates from Asia (the highest similarity with an isolate from Sichuan, China), whereas the similarity with European isolates ranged from 99.5 to 99.6%.
Adult ; Animals ; Antibodies, Helminth/*blood ; Antigens, Helminth/genetics/*immunology/metabolism ; Base Sequence ; Echinococcosis, Hepatic/*immunology/parasitology ; Echinococcosis, Pulmonary/diagnosis/genetics/immunology ; Echinococcus granulosus/genetics/immunology ; Echinococcus multilocularis/genetics/*immunology/isolation & purification ; Electron Transport Complex IV/genetics ; Female ; Humans ; Mitochondria/genetics ; Molecular Sequence Data ; Republic of Korea ; Sequence Analysis, DNA

This study aimed to investigate the antibody responses in mice immunized with Gnathostoma spinigerum crude antigen (GsAg) incorporated with the combined adjuvant, a synthetic oligonucleotide containing unmethylated CpG motif (CpG ODN 1826) and a stable water in oil emulsion (Montanide ISA720). Mice immunized with GsAg and combined adjuvant produced all antibody classes and subclasses to GsAg except IgA. IgG2a/2b/3 but not IgG1 subclasses were enhanced by immunization with CpG ODN 1826 when compared with the control groups immunized with non-CpG ODN and Montanide ISA or only with Montanide ISA, suggesting a biased induction of a Th1-type response by CpG ODN. After challenge infection with live G. spinigerum larvae, the levels of IgG2a/2b/3 antibody subclasses decreased immediately and continuously, while the IgG1 subclass remained at high levels. This also corresponded to a continuous decrease of the IgG2a/IgG1 ratio after infection. Only IgM and IgG1 antibodies, but not IgG2a/2b/3, were significantly produced in adjuvant control groups after infection. These findings suggest that G. spinigerum infection potently induces a Th2-type biased response.
Adjuvants, Immunologic/*administration & dosage ; Animals ; Antibodies, Helminth/*blood ; Antigens, Helminth/*administration & dosage/*immunology ; Gnathostoma/*immunology ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Male ; Mannitol/administration & dosage/*analogs & derivatives ; Mice ; Oleic Acids/*administration & dosage ; Oligodeoxyribonucleotides/*administration & dosage ; Th1 Cells/immunology ; Th2 Cells/immunology