Th2-related cytokines are associated with Fasciola gigantica infection and evasion in the natural host, swamp buffalo.

The infection of ruminants by Fasciola spp. always induces a non-protective Th2-type immune response. However, little is known about changes in the local and systemic immune environment during F. gigantica migration in buffalo. In this study, native swamp buffaloes were each infected with 500 viable F. gigantica metacercariae. Mesenteric lymph node (MLN), hepatic lymph node (HLN), spleen, and serum samples were collected from control and infected buffaloes at 3, 10, 28, 42, 70, and 98 days post-infection (DPI). The mRNA expression levels of the Th1- and Th2-related cytokines IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p40, IFN-γ, TNF-α, and CD4 were measured during different infection stages in the MLNs, spleens, and HLNs using quantitative real-time PCR (qRT-PCR). Levels of the specific anti-ESP isotype antibodies IgG, IgG1, and IgG2 were used to reflect changes in humoral immunity. The results of this study indicated that swamp buffaloes were susceptible to F. gigantica infection, and that susceptibility to this infection was closely related to the cytokine environment associated with the Th2-type immune response. The MLNs showed a mixed Th1- and Th2-type immune response during the acute infection stages, after which the production of these cytokines returned to normal. Cytokine expression in the HLNs also expressed a mixed Th1- and Th2-type immune response during the early infection stages. When the infection became chronic, the typical Th2 immune response was induced in the HLNs. At the acute infection stages, the spleen exhibited a Th2 immune response. Nevertheless, cytokines associated with the Th1 and Th2 immune responses were upregulated at 98 DPI. In addition, the total IgG and IgG1 of the parasite-specific antibodies increased. This suggested that the Th2-related cytokines and IgG1 induced by F. gigantica infection might mediate successful F. gigantica infection in the natural host, swamp buffalo.

Immune responses induced by co-infection with Capillaria hepatica in Clonorchis sinensis-infected rats.

Clonorchis sinensis and Capillaria hepatica are zoonotic parasites that mainly infect the liver and cause serious liver disorders. However, immunological parameters induced by co-infection with these parasites remain unknown. In this study, for the first time, we investigated immunological profiles induced by co-infection with C. hepatica (CH) in C. sinensis (CS)-infected rats (Sprague-Dawley). Rats were infected primarily with 50 metacercariae of C. sinensis; 4 weeks later, they were subsequently infected with 1000 infective C. hepatica eggs. Significantly higher levels of C. sinensis- or C. hepatica-specific IgG antibodies were found in the sera of rats. Interestingly, no cross-reacting antibody was observed between C. sinensis and C. hepatica infections. Significantly raised eosinophil levels were found in the blood of C. sinensis/C. hepatica co-infected rats (CS + CH) compared to the blood of rats infected singly with C. sinensis. Co-infected rats showed significantly higher levels of lymphocyte proliferation and cytokine production compared to a single C. sinensis infection. The worm burden of C. sinensis was significantly reduced in co-infected rats compared to the single C. sinensis infection. These results indicate that the eosinophils, lymphocyte proliferation and cytokine production induced by subsequent infection with C. hepatica in C. sinensis-infected rats might contribute to the observed C. sinensis worm reduction.

Immunization and challenge shown by hamsters infected with Opisthorchis viverrini following exposure to gamma-irradiated metacercariae of this carcinogenic liver fluke.

Here we report findings to optimize and standardize conditions to attenuate metacercariae of Opisthorchis viverrini by ionizing radiation to elicit protective immune responses to challenge infection. Metacercariae were gamma-irradiated and the ability of irradiated metacercariae to prevent patent infection of challenge metacercariae in hamsters was determined, as well as their ability to induce a host antibody response. Metacercariae irradiated in a dose-dependent manner, with 3, 5, 10, 12, 20, 25 and 50 Gray, were used to infect Syrian golden hamsters by stomach gavage to ascertain the effect of irradiation on ability of the worms to establish infection. In addition, other hamsters were infected with metacercariae irradiated with 20-50 Gray, followed by challenge with intact/wild-type (non-irradiated) metacercariae to determine the protective effect as established by the numbers of adult flukes, eggs of O. viverrini in hamster faeces and anti-O. viverrini antibody titres. Significantly fewer worms were recovered from hamsters immunized with metacercariae irradiated at 20, 25 and 50 Gray than from control hamsters infected with intact metacercariae or 0 Gray, and the worms showed damaged reproductive organs. Faecal egg numbers were decreased significantly in hamsters immunized with 25 and 50 Gray metacercariae of O. viverrini. Moreover, hamsters administered metacercariae that were protected elicited a robust, specific anti-fluke immunoglobulin G response compared to control hamsters, suggesting a role for antibody in protection elicited by radiation-attenuated metacercariae.

Production and characterization of a monoclonal antibody against recombinant saposin-like protein 2 of Fasciola gigantica.

A monoclonal antibody (MoAb) against recombinant Fasciola gigantica saposin-like protein 2 (rFgSAP-2) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with rFgSAP-2. This MoAb is an IgG1, κ light chain isotype. By immunoblotting and indirect ELISA, the MoAb reacted specifically with rFgSAP-2, the natural FgSAP-2 at 10kDa in whole body (WB) and excretory-secretory (ES) fractions of F. gigantica. It did not cross react with antigens in WB fractions from other parasites, including Opisthorchis viverrini, Schistosoma mansoni which are human parasites, Haemonchus placei, Setaria labiato-papillosa, Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gigantocotyle explanatum, Gastrothylax crumenifer, and Paramphistomum cervi which are ruminant parasites. By immunohistochemistry, the FgSAP-2 protein was localized only in the cytoplasm of caecal epithelial cells of 4-week-old juvenile and adult stages, but not in metacercariae, newly excysted juvenile (NEJ), 2- and 3-week-old juveniles. This finding indicated that FgSAP-2 is an abundantly expressed parasite protein that is released into the ES, hence SAP-2 and its MoAb may be used for immunodiagnosis of ruminant and human fasciolosis.

Stage-specific expression and antigenicity of glycoprotein glycans isolated from the human liver fluke, Opisthorchis viverrini.

Infection by Opisthorchis viverrini (liver fluke) is a major public health problem in southeastern Asia, resulting in hepatobiliary disease and cholangiocarcinoma. Fluke surface glycoconjugates are prominently presented to the host, thereby constituting a crucial immunological interface that can determine the parasite's success in establishing infection. Therefore, N- and O-linked glycoprotein glycan profiles of the infective metacercarial stage and of the mature adult were investigated by nanospray ionisation-linear ion trap mass spectrometry (NSI-MS(n)). Glycan immunogenicity was investigated by immunoblotting with serum from infected humans. Metacercariae and adult parasites exhibit similar glycan diversity, although the prevalence of individual glycans and glycan classes varies by stage. The N-glycans of the metacercaria are mostly high mannose and monofucosylated, truncated-type oligosaccharides (62.7%), with the remainder processed to complex and hybrid type glycans (37.3%). The N-linked glycan profile of the adult is also dominated by high mannose and monofucosylated, truncated-type oligosaccharides (80.0%), with a smaller contribution from complex and hybrid type glycans (20.0%). At both stages, complex and hybrid type glycans are detected as mono-, bi-, tri-, or tetra-antennary structures. In metacercariae and adults, O-linked glycans are detected as mono- to pentasaccharides. The mucin type core 1 structure, Galß1-3GalNAc, predominates in both stages but is less prevalent in the adult than in the metacercaria. Immunogenic recognition of liver fluke glycoproteins is reduced after deglycosylation but infected human serum was unable to recognise glycans released from peptides. Therefore, the most potent liver fluke antigenic epitopes are mixed determinants, comprised of glycan and polypeptide elements.

Molecular identification and characterization of leucine aminopeptidase 2, an excretory-secretory product of Clonorchis sinensis.

Aminopeptidases serve vital roles in metabolism of hormones, neurotransmission, turnover of proteins and immunological regulations. Leucine aminopeptidases catalyze the hydrolysis of amino-acid residues from the N-terminus of proteins and peptides. In the present study, leucine aminopeptidase 2 (LAP2) gene of Clonorchis sinensis (C. sinensis) was isolated and identified from an adult cDNA library of C. sinensis. Recombinant CsLAP2 was expressed and purified in Escherichia coli BL21. The open reading frame of LAP2 contains 1,560 bp equivalent to 519 amino acids, a similarity analysis showed a relatively low homology with Homo sapiens (19.0 %), Trypanosoma cruzi (18.0 %), Mus musculus (19.3 %), and relatively high homology with Schistosoma mansoni (65.6 %). The optimum condition of rCsLAP2 enzyme activity was investigated using a fluorescent substrate of Leu-MCA at 37 °C and pH 7.5. The K (m) and V (max) values of rCsLAP2 were 18.2 µM and 10.7 µM/min, respectively. CsLAP2 gene expression can be detected at the stages of the adult worm, metacercaria, excysted metacercaria and egg of C. sinensis using real-time PCR, no difference was observed at the stages of the adult worm, metacercaria and egg. However, CsLAP2 showed a higher expression level at the stage of excysted metacercaria than the adult worm (3.90-fold), metacercaria (4.60-fold) and egg (4.59-fold). Histochemistry analysis showed that CsLAP2 was located at the tegument and excretory vesicle of metacercaria, and the tegument and intestine of adult worm. The immune response specific to rCsLAP2 was characterized by a mixed response patterns of Th1 and Th2, indicating a compounded humoral and cellular immune response. The combined results from the present study indicate that CsLAP2 was an important antigen exposed to host immune system, and probably implicated as potential role in interaction with host cells in clonorchiasis.

Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice.

Mice have shown various susceptibility to infection by Clonorchis sinensis. To compare the intra-specific variation in the host-parasite relationship of C. sinensis, 6 strains of mice (ICR, BALB/c, C57BL/6, DDY, CBA/N, and C3H/HeN) with 3 different haplotypes were evaluated on their susceptibility. The worm recovery rate and immunological responses were observed after 4 and 8 weeks of infection with 30 metacercariae. The highest worm recovery rate was observed as 20.7% in the C3H/HeN strain after 4 weeks of infection along with histopathological changes. The rate was 10.0% in C57BL/6 mice after 8 weeks. ICR, BALB/c, and CBA/N showed elevated levels of IgE at both time points when compared to the rest of the strains. The serum IgG1 and IgG2a levels were elevated in most of the strains; however, the C57BL/6 strain showed a lower level of IgG2a that indicated the IgG1 predominance over IgG2a. The production of IL-4 after concanavalin-A stimulation of splenocytes slightly increased among the mouse strains except C3H/HeN after 4 or 8 weeks of infection, but each strain produced high levels of IFN-γ after 8 weeks, which implied mixed Th1/Th2 responses. ICR, DDY, CBA/N, and C3H/HeN strains showed a significantly increased level of IL-10 after 8 weeks as compared to C57BL/6. All of the strains showed an increased level of IL-13 and suggested fibrotic changes in the mice. In conclusion, mice are insusceptible to infection with C. sinensis; however, the C57BL/6, BALB/c and ICR strains are relatively susceptible after 8 weeks of infection among the six strains. Worm expulsion may be one of the causes of low susceptibility of C3H/HeN mice strain at the 8th week. Elevated IgE, IFN-γ, and IL-13 of infected mice suggest both Th1 and Th2 responses that may be related to the low host susceptibility.

Identification and molecular characterization of a novel signaling molecule 14-3-3 epsilon in Clonorchis sinensis excretory/secretory products.

Increasing evidence shows that 14-3-3 proteins are involved in many biology events in addition to signal transduction. Extensive investigations on structural and biochemical features of these signaling molecules have implied their importance in the biological process. In the present study, we have identified and characterized the 14-3-3 epsilon (Cs14-3-3) in Clonorchis sinensis that causes human clonorchiasis. Recombinant protein was expressed in Escherichia coli (E. coli) and identified by MALDI-TOF/TOF. Immunoblot results revealed that Cs14-3-3 was a component of excretory/secretory products. Ligand blot assay indicated that 14-3-3 epsilon could bind C. sinensis MAPKAPK 2 in a nonphosphorylation-dependent manner. This protein could be detected at four stages of the life cycle by RT-PCR experiments and immunolocalization showed that Cs14-3-3 was extensively distributed in C. sinensis, especially at the outer surface and the sucker of adult worm and cyst wall of metacercaria. Taken together, 14-3-3 epsilon might play some roles in the development of the parasites. In addition, Cs14-3-3 epsilon should be addressed for the diagnostic value in C. sinensis infection in consideration of high sensitivity and specificity. As an immune stimulus, C. sinensis 14-3-3 epsilon was found to provoke a Th1/Th2 balanced immune response by inducing high levels of both IgG1 and IgG2a. Recombinant Cs14-3-3 conferred effective protection both in worm reduction rate and egg reduction rate, suggesting that the signaling molecule Cs14-3-3 was a promising vaccine candidate against C. sinensis infection.

Coproantigen capture ELISA for detection of Clonorchis sinensis infection in experimentally infected rats.

The present study investigated the diagnostic value of an ELISA for the detection of Clonorchis sinensis antigen in the feces of experimentally infected rats. A mouse polyclonal IgG antibody against adult C. sinensis crude antigen (CsAg) was used to capture the C. sinensis coproantigen. The detection limit for pure CsAg was 20 ng/ml in sample buffer and 40 ng/ml in uninfected fecal extract. The test was evaluated using a follow-up of five groups of rats experimentally infected with 100, 50, 10, 5 and 1 metacercariae of C. sinensis and an uninfected control group. Coproantigen was detected in all infected groups of rats from 2 weeks of infection, whereas fecal eggs were not observed until 3 weeks of infection. As the infection period progressed, the fecal CsAg concentration increased in all groups of infected rats, even those infected with a single metacercaria. The fecal CsAg concentration was correlated positively with fecal egg counts and worm burden. This coproantigen capture ELISA is highly sensitive for the detection of CsAg in rat feces, and with further development, should be useful for mass screening of human subjects in clonorchiasis-endemic areas.

Secreted Opisthorchis viverrini glutathione S-transferase regulates cell proliferation through AKT and ERK pathways in cholangiocarcinoma.

Opisthorchis viverrini can develop mitogenic substances into the excretory/secretory product (ESP) that may play an important role in promoting the genesis of cholangiocarcinoma (CCA). In the present study, glutathione S-transferase (GST) is identified as being secreted into Ov-ESP and acting as one of the parasitic mitogens. Its proliferative effect and possible mechanism were explored and its association with the tumor development is proposed. Ov-ESP was concentrated and purified by gel filtration chromatography. SDS-PAGE, 2-DE, and LC-MS/MS identified GST predominantly expressed in the proliferative ESP fraction. The recombinant OvGST (rOvGST) was produced by wheat germ cell-free expression and confirmed by an MTS assay to have a proliferative function on NIH-3T3 murine fibroblasts and MMNK1 non-tumorigenic human bile duct epithelial cells in a dose dependent manner with different optimal doses. The cell surface binding of rOvGST was confirmed in vitro and the activation of both pAKT and pERK was revealed as the mechanism of OvGST-mediated cell proliferation. With support from the observation of secreted OvGST on the biliary cells surrounding the parasites, it is suggested that OvGST can promote cell proliferation that consequently may accelerate the genesis of CCA.

Human paragonimiasis in Viet Nam: epidemiological survey and identification of the responsible species by DNA sequencing of eggs in patients' sputum.

Parasitological and sero-epidemiological surveys for human paragonimiasis were conducted in three provinces of Viet Nam. A total of 590 participants from two known endemic areas of human paragonimiasis (Sinho district of Laichau province and Lucyen district of Yenbai province) and from Dakrong district of Quangtri province where we recently found crab hosts heavily infected with Paragonimus westermani metacercariae. By multiple dot-ELISA screening, 28 (12.7%) out of 220 participants in Sinho district of Laichau province and 4 (3.3%) out of 120 participants in Lucyen district of Yenbai province were proven to be antibody-positive against the Paragonimus antigen. None of the 250 sera of the residents in Dakrong, Quangtri province, gave sero-positivity. Among a total of 32 sero-positive patients Paragonimus eggs were found in 6 cases. ITS2 sequences were successfully determined from a single Paragonimus egg from each patient. The results of homology search by BLAST and alignment clearly confirmed that Paragonimus eggs collected from 6 patients were all of Paragonimus heterotremus. The pathogenicity of P. westermani for human paragonimiasis in Viet Nam is still questionable and needs to be explored in the future.