Yuxin Xu

Prevalence and genetic characterization of Cryptosporidium, Enterocytozoon, Giardia and Cyclospora in diarrheal outpatients in China.

BackgroundCryptosporidium spp., Enterocytozoon spp., Giardia spp. and Cyclospora spp. are important intestinal protozoan parasites causing diarrhea in humans, livestocks and wildlife and have a significant impact on public health. No reports exist about simultaneous prevalence rates or genotyping data of these four parasites in outpatients from China.MethodsFecal specimens from 252 diarrhea patients in a pediatric clinic (n = 169) and an intestinal clinic (n = 83) of a hospital in Shanghai, China, were collected between October 2012 and March 2013. All samples were examined for the presence of the four parasites by using molecular methods.ResultsIn total, 76/252 (30.16%) patients were positive for at least one intestinal parasite, of which Cryptosporidium spp., Enterocytozoon bieneusi and Giardia intestinalis were detected by nested PCR in 34 (13.49%), 34 (13.49%) and 17 (6.75%) of the fecal specimens, respectively. Sequence analysis showed that all Cryptosporidium-positive specimens were C. andersoni and that most G. intestinalis- positive patients were infected by assemblage C, which is usually found in canids, while only one sample was from assemblage B. Eight patients were co-infected with Cryptosporidium spp. and Enterocytozoon, while one was co-infected with Cryptosporidium and Giardia.ConclusionsThe patients infected with Cryptosporidium and Enterocytozoon bieneusi had higher infection rates in winter than in spring in this area. Data indicated that C. andersoni is the fourth major Cryptosporidium species infecting humans in addition to C. hominis, C. parvum and C. meleagridis. Our study also revealed a short-term outbreak of cryptosporidiosis and microsporidiosis and sporadic cases of giardiasis that occurred among humans in Shanghai, China.

Surveillance on the Status of Immune Cells after Echinnococcus granulosus Protoscoleces Infection in Balb/c Mice

Background Cystic echinococcosis is a global parasitic disease caused by infection with Echinococcus granulosus larvae with potentially life-threatening complications in humans. To date, the status of the immune cells believed to be associated with the pathogenicity of E. granulosus infection has not been demonstrated clearly. Methodology/Principal Findings In this study, we developed a multiplex flow cytometry assay to investigate the systemic immune status of innate and adaptive immunity at 30, 180, 360 days post-infection (dpi) in mice infected with E. granulousus. At 30 dpi, an increase in the number of CD11b+ and CD11c+ antigen-presenting cells (APCs) was observed. This was accompanied by the slight down-regulated expression of the co-stimulatory molecule MHC-II, indicating the impairment of APCs in early infection through the release of secretory-excretory products. In all infected groups, we observed a significant increase in innate immune cells, including APCs and GR-1+ cells, and a dramatic increase in the myeloid-derived suppressor cells (MDSC) expressing CD11b+/GR-1+. Moreover, the upregulation of the activated markers CD69, CD44, CD40L, and the downregulation of CD62L were observed in the CD4+ and CD8+ T cells following infection. Regulatory T cells expressing CD4+/CD25+/FoxP3 + increased significantly over the course of infection. Conclusions Our findings demonstrate that the microenvironment in the peripheral immune system after E. granulosus infection changes in subtle but detectably ways, especially during the persistent period of infection. We found that T cells were activated following infection, but observed that the significant increase of immunosuppressive cells such as MDSC and Treg cells could inhibit T cell response to E. granulosus antigens. We suggest these cells may play a neglected but key role in the downregulation of the immune response in long-term parasitic infection. Understanding the basic functions and temporal interactions of these immunosuppressive cells will pave the way for new strategies of parasite vaccine design.

Impairment of dendritic cell function and induction of CD4(+)CD25(+)Foxp3(+) T cells by excretory-secretory products: a potential mechanism of immune evasion adopted by Echinococcus granulosus.

BackgroundCystic echinococcosis, caused by infection with Echinococcus granulosus, is one of the most widespread zoonotic helminth diseases. Modulation of host responses is an important strategy used by helminth parasites to promote infection. To better understand the mechanisms adopted by E. granulosus to escape host immune responses, we investigated the effects of excretory–secretory products (ES) and adult worm antigen (AWA) derived from adult E. granulosus on murine bone marrow-derived dendritic cells (BMDC).ResultsCompared with lipopolysaccharide (LPS), AWA, but not ES, induced BMDC maturation or stimulated BMDC cytokine production and co-stimulatory molecule expression (CD40, CD80 and MHC class II). Furthermore, ES-treated BMDCs pulsed with ovalbumin exhibited reduced co-stimulatory molecule expression in comparison with untreated BMDC, even in the presence of the strong Th1 inducer, CpG. Moreover, we detected the effects of ES-treated DC on T cell activation by an in vitro T cell priming assay. We observed that ES-treated BMDC co-cultured with DO11.10 transgenic CD4+ T cells induced the generation of CD4+CD25+Foxp3+ T cells. In addition, in contrast to AWA-treated BMDCs, which had markedly induced IFN-γ secretion and reduced of IL-4 levels in co-cultured T cells, ES-treated BMDCs did not modify their capacity to stimulate IFN-γ or IL-4 production by T cells.ConclusionsWe conclude that ES of adult E. granulosus inhibited DC function, impaired the development of Th1 cells induced by CpG, and induced CD4+CD25+Foxp3+ regulatory T cells in an IL-10-independent manner.

Evidence That Rhesus Macaques Self-Cure from a Schistosoma japonicum Infection by Disrupting Worm Esophageal Function: A New Route to an Effective Vaccine?

Background Rhesus macaques are unusual among schistosome hosts, self-curing from an established infection and thereafter manifesting solid immunity against a challenge, an ideal model for vaccine development. Previously, the immunological basis of self-cure was confirmed; surviving worms had ceased feeding but how immunological pressure achieved this was unclear. The schistosome esophagus is not simply a conduit for blood but plays a central role in its processing. Secretions from the anterior and posterior esophageal glands mix with incoming blood causing erythrocyte lysis and tethering and killing of leucocytes. Methodology/Principal Findings We have analysed the self-cure process in rhesus macaques infected with Schistosoma japonicum. Faecal egg output and circulating antigen levels were used to chart the establishment of a mature worm population and its subsequent demise. The physiological stress of surviving females at perfusion was especially evident from their pale, shrunken appearance, while changes in the structure and function of the esophagus were observed in both sexes. In the anterior region electron microscopy revealed that the vesicle secretory process was disrupted, the tips of lining corrugations being swollen by greatly enlarged vesicles and the putative sites of vesicle release obscured by intense deposits of IgG. The lumen of the posterior esophagus in starving worms was occluded by cellular debris and the lining cytoplasmic plates were closely adherent, also potentially preventing secretion. Seven proteins secreted by the posterior gland were identified and IgG responses were detected to some or all of them. Intrinsic rhesus IgG colocalized with secreted SjMEGs 4.1, 8.2, 9, 11 and VAL-7 on cryosections, suggesting they are potential targets for disruption of function. Conclusions/Significance Our data suggest that rhesus macaques self-cure by blocking esophagus function with antibody; the protein products of the glands provide a new class of potential vaccine targets.

Prevalence of the Cryptosporidium pig genotype II in pigs from the Yangtze River Delta, China.

Background Cryptosporidium spp. is prevalent globally, pigs are an important Cryptosporidium reservoir. In China, little data regarding rates of Cryptosporidium infections in pigs are available. The present study was therefore aimed at characterizing the distribution of Cryptosporidium species in pigs from two different cities, Shaoxing and Shanghai, from the Yangtze River delta. Methodology/Principal Findings Nested PCR to amplify the 18S rRNA locus on DNA extracted from fecal samples (n = 94) revealed the positive rate of Cryptosporidium in pigs from two cities was approximately 17.0%. The positive rates in Shanghai and Shaoxing were 14.3% and 25.0% respectively. Amplified sequences were verified by sequencing. The identified strain belonged to the C. pig genotype II using BLAST analysis in the NCBI database. Conclusion/Significance Our finding of Cryptosporidium pig genotype II in pigs in the Yangtze River delta area suggests that pig farms in this region must be considered a public health threat and proper control measures be introduced.

Transcriptome Profiles of the Protoscoleces of Echinococcus granulosus Reveal that Excretory-Secretory Products Are Essential to Metabolic Adaptation

Background Cystic hydatid disease (CHD) is caused by the larval stages of the cestode and affects humans and domestic animals worldwide. Protoscoleces (PSCs) are one component of the larval stages that can interact with both definitive and intermediate hosts. Previous genomic and transcriptomic data have provided an overall snapshot of the genomics of the growth and development of this parasite. However, our understanding of how PSCs subvert the immune response of hosts and maintains metabolic adaptation remains unclear. In this study, we used Roche 454 sequencing technology and in silico secretome analysis to explore the transcriptome profiles of the PSCs from E. granulosus and elucidate the potential functions of the excretory-secretory proteins (ESPs) released by the parasite. Methodology/Principal Findings A large number of nonredundant sequences as unigenes were generated (26,514), of which 22,910 (86.4%) were mapped to the newly published E. granulosus genome and 17,705 (66.8%) were distributed within the coding sequence (CDS) regions. Of the 2,280 ESPs predicted from the transcriptome, 138 ESPs were inferred to be involved in the metabolism of carbohydrates, while 124 ESPs were inferred to be involved in the metabolism of protein. Eleven ESPs were identified as intracellular enzymes that regulate glycolysis/gluconeogenesis (GL/GN) pathways, while a further 44 antigenic proteins, 25 molecular chaperones and four proteases were highly represented. Many proteins were also found to be significantly enriched in development-related signaling pathways, such as the TGF-β receptor pathways and insulin pathways. Conclusions/Significance This study provides valuable information on the metabolic adaptation of parasites to their hosts that can be used to aid the development of novel intervention targets for hydatid treatment and control.

The formation of egg granulomas in the spleens of mice with late Schistosoma japonicum infection alters splenic morphology.

BackgroundSplenomegaly is a characteristic symptom of schistosome infection. Unlike the well known hepatic pathology of schistosomiasis, splenomegaly has received little scientific research and is generally considered to be a non-specific congestion caused by increased blood pressure within the venous sinuses. Moreover, to date, few studies have reported the deposition of schistosome eggs in the spleen. In a previous study, however, we observed that prolonged S. japonicum infections destroyed the structure of the lymphoid follicles in the spleen of mice at 8 weeks post-infection and found that eggs were frequently deposited in the spleen. These prior observations suggested a relationship between granulomas and splenic morphology which we investigate further in this study.MethodsC57BL/6 mice were infected percutaneously with twenty cercariae of S. japonicum and sacrificed at different times post-infection. The number of eggs present in the homogenates of spleens and livers was quantified by light microscopy. Splenic pathology was observed by immunohistochemistry staining of paraffin-embedded sections. At 18 weeks post-infection the infected mice were divided into two groups (granulomatous spleens and non-granulomatous spleens). Serum antibodies and cytokines in the antigen- or mitogen-stimulated lymphocyte cultures were then determined by ELISA.ResultsWe found that eggs deposition in the spleens of infected mice occurred frequently but only occasionally led to granulomas formation. The lymphoid follicles within the granulomatous spleens maintained their structural integrity until 20 weeks post-infection, unlike the lymphoid follicles in spleens without egg granulomas. Mice with granulomatous spleens accompanied by lymphoid follicles exhibited a germinal center (GC)-like structure and had enhanced humoral immune responses. Splenocytes from granulomatous spleens also showed significantly elevated levels of Th2 cytokines during late infection stages.ConclusionsOur results highlight that lymphoid follicles, which are not completely destroyed or are re-established in the spleen, can change the local immune environment and lead to changes in the splenic morphology of mice with chronic schistosomiasis.

Immune changes of Schistosoma japonicum infections in various rodent disease models

Rodent models for Schistosoma japonicum infections have demonstrated that these animals possess a degree of resistance to schistosome infections that may be both T and B lymphocyte-mediated. However, their exact role is not well-defined and other immune mechanisms are likely to also play a role in protecting against infection. Immunosuppressed and immunocompetent reed voles (Microtus fortis, Mf), rats and mice (n=24/group) were infected with S. japonicum, and animals were sacrificed 42 days later under anesthesia. Neither worms nor eggs were observed in infected immunosuppressed Mf or rats, with the exception of one rat that presented with few eggs. In immunosuppressed mice, changes in the number and size of the worms were not significantly different compared to immunocompetent mice, but worm fecundity was affected. The size and number of granulomas in immunosuppressed animals was also reduced. Analysis of serum antibodies specific to schistosome adult worm antigen at 3 weeks post-infection demonstrated that the levels of antibodies in the sera of rats were significantly higher than in Mf and mice. In addition, Mf serum levels of IL-4 and IL-12 were significantly higher than levels observed in rats and mice. Antibodies and cytokines in the sera of Mf peaked 3 weeks post-infection and then began to decrease, while antibody responses in rats and mice increased gradually between weeks 3-7 post-infection. It is possible that T and B cells have a dual role in both mediating protection and exacerbating disease outcomes.

Higher Frequency of Circulating PD-1high CXCR5+CD4+ Tfh Cells in Patients with Chronic Schistosomiasis

The current knowledge of immunological responses to schistosomiasis is insufficient for the development of vaccine and therapies. The role of T follicular helper (Tfh) cells in schistosome infections is not fully defined. The frequency of circulating Tfh cells and serum cytokine levels were analyzed in 11 patients with chronic schistosomiasis and 10 healthy controls (HC), who reside in an endemic area for Schistosomiasis japonicum. Significantly higher frequencies of circulating CXCR5+ CD4+ Tfh cells and higher expression levels of ICOS and PD-1 in CXCR5+ CD4+ Tfh cells were observed in patients with chronic schistosomiasis compared to HC. The levels of IL-21 in serum and the expression of IL-21 mRNA were higher in chronic schistosomiasis patients than in HC. Moreover, the frequency of circulating PD-1high CXCR5+ CD4+ Tfh cells positively correlated with the levels of IL-21 in serum from patients with chronic schistosomiasis. A positive correlation was also found between the frequency of PD-1high CXCR5+ CD4+ Tfh cells and the levels of soluble egg antigen (SEA)-specific antibodies in serum samples from the patient group. Our study is the first regarding Tfh cells in chronic human schistosomiasis and the finding indicate that PD-1high CXCR5+ CD4+Tfh cells might play an important role in the production of specific antibodies in schistosomiasis. This study contributes to the understanding of immune response to schistosomiasis and may provide helpful support in vaccine development.

High throughput data analyses of the immune characteristics of Microtus fortis infected with Schistosoma japonicum

Microtus fortis exhibits natural resistance against Schistosoma japonicum, and the parasite cannot grow and develop in M. fortis. Extensive research has been carried out, however, the associated mechanism remains unclear. In the present study, we analysed the combined data obtained from a cytokine chip assay, transcriptome, and metabolome. The cytokine profile from C57BL/6 and M. fortis mice was assessed before and after infection. Several cytokines increased during the second and third week post-infection. Some transcripts related to cytokine genes and associated proteins were also highly expressed (i.e., Hgf, C3, and Lbp). The liver metabolism of M. fortis following infection with S. japonicum was assessed. We identified 25 different metabolites between the uninfected and infected M. fortis, and 22 different metabolites between infected M. fortis and C57BL/6 mice. The metabolomic pathways of these differential metabolites were then analysed with MetPA, revealing that they were involved in histidine metabolism, valine, leucine, and isoleucine biosyntheses, and lysine degradation. Thus, the elevated expression of these metabolites and pathways may promote the phagocytic function of the neutrophils and natural killer cell activity following TLR activation. These results provide novel insight into the resistance mechanism of M. fortis against S. japonicum.