Genetic characterizations of Cryptosporidium spp. from children with or without diarrhea in Wenzhou, China: high probability of zoonotic transmission.

BACKGROUND: Cryptosporidium is a highly pathogenic parasite responsible for diarrhea in children worldwide. Here, the epidemiological status and genetic characteristics of Cryptosporidium in children with or without diarrhea were investigated with tracking of potential sources in Wenzhou City, China. METHODS: A total of 1032 children were recruited, 684 of whom had diarrhea and 348 without, from Yuying Children's Hospital in Wenzhou, China. Samples of stool were collected from each participant, followed by extraction of DNA, genotyping, and molecular identification of Cryptosporidium species and subtypes. RESULTS: Twenty-two of the 1032 (2.1%) children were infected with Cryptosporidium spp. with 2.5% (17/684) and 1.4% (5/348) in diarrhoeic and asymptomatic children, respectively. Four Cryptosporidium species were identified, including C. parvum (68.2%; 15/22), C. felis (13.6%; 3/22), C. viatorum (9.1%; 2/22), and C. baileyi (9.1%; 2/22). Two C. parvum subtypes named IIdA19G1 (n = 14) and IInA10 (n = 1), and one each of C. felis (XIXa) and C. viatorum (XVaA3g) subtype was found as well. CONCLUSIONS: This is the first research that identified Cryptosporidium in children of Wenzhou, China, using PCR. Identification of zoonotic C. parvum, C. felis, C. viatorum, and their subtypes indicate potential cross-species transmission of Cryptosporidium between children and animals. Additionally, the presence of C. baileyi in children suggests that this species has a wider host range than previously believed and that it possesses the capacity to infect humans.

Investigation of giardiasis in captive animals in zoological gardens with strain typing of assemblages in China.

Giardia duodenalis is a common zoonotic intestinal pathogen. It has been increasingly reported in humans and animals; however, genotyping information for G. duodenalis in captive animals is still limited. This study was conducted to assess the prevalence and multilocus genotyping of G. duodenalis in captive animals in zoological gardens in Shanghai, China. A total of 678 fresh fecal samples were randomly collected from captive animals including non-human primates (NHPs) (n = 190), herbivores (n = 190), carnivores (n = 151), birds (n = 138) and reptiles (n = 9) in a zoo and were examined for the presence of G. duodenalis using nested polymerase chain reaction (nested PCR). All G. duodenalis positive samples were assayed with PCR followed by sequencing at ß-giardin (bg), glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) genes. In this study, 42 specimens (6.2%) were tested G. duodenalis-positive of the 678 fecal samples examined based on a single locus. A total of 30 (4.4%), 30 (4.4%) and 22 (3.2%) specimens were successfully amplified and sequenced at gdh, tpi and bg loci, respectively. Assemblages A and B were identified with assemblage B dominating in NHPs. Sequence analysis demonstrated that one, two and five new isolates were identified at bg, gdh and tpi loci. DNA sequences and new assemblage-subtypes of zoonotic G. duodenalis assemblages A and B were identified in the current study. Our data indicate the occurrence and molecular diversity of G. duodenalis and the potential zoonotic transmission in captive animals in China.

First report of Cryptosporidium viatorum and Cryptosporidium occultus in humans in China, and of the unique novel C. viatorum subtype XVaA3h.

BACKGROUND: Cryptosporidium is a genus of common intestinal protozoa, members of which cause diarrhea in a wide variety of hosts. Previous studies on Cryptosporidium in China have mainly focused on diarrhea sufferers, children, and immunodeficient individuals such as HIV/AIDS patients. However, the epidemiological characteristics of Cryptosporidium in the population in rural areas remain unclear. Herein, we investigated the prevalence of, and risk factors for, Cryptosporidium in rural areas of Binyang County, Guangxi Zhuang Autonomous Region, China, and genetically characterized the Cryptosporidium isolates we obtained. METHODS: From August to December 2016, two villages in Binyang County, Guangxi, were sampled using a random cluster sampling method. Fresh fecal samples were collected from all eligible residents (residence time > 6 months). Molecular characterization of Cryptosporidium was carried out based on its SSU rRNA, gp60, actin and hsp70 gene sequences. Fisher's exact test were conducted to assess the risk factors for Cryptosporidium infection. RESULTS: A total of 400 fecal samples were collected from 195 males (48.8%) and 205 females (51.2%). Two samples (0.5%) were positive for Cryptosporidium and were identified as C. viatorum and C. occultus respectively. Moreover, a new C. viatorum subtype XVaA3h was identified based on the sequence of the gp 60 gene. CONCLUSIONS: To our knowledge, this is the first report of C. viatorum and C. occultus infections in humans in China and of C. viatorum subtype XVaA3h. The findings provide important information on the prevalence of Cryptosporidium in the Chinese population, and expand the range of Cryptosporidium species known to infect people in China.

Prevalence and multilocus genotyping of potentially zoonotic Giardia duodenalis in pigs in Shanghai, China.

Giardia duodenalis is a common intestinal parasite in humans and other mammals, and it causes major public and veterinary health problems worldwide. China is a major pig-raising country, and studies on Giardia in pigs have important public health significance. The present study was conducted to investigate the prevalence of Giardia and assess its genetic characterization. A total of 93 samples were collected from two farms in Shanghai. The presence of Giardia was determined using PCR and sequence analysis of glutamate dehydrogenase, beta-giardin and triose phosphate isomerase genes. The average prevalence of G. duodenalis infection was 26.88% (25/93) in the pigs, with 28.13% (18/64) in farm 1 vs 24.14% (7/29) in farm 2. All the PCR-positive products were successfully sequenced, and assemblage E was more prevalent. Zoonotic assemblages A and B and canine-specific assemblage C were identified in farm 1, whereas, only assemblage E was detected in farm 2. Interestingly, two pig isolates showed 100% homology with human-derived isolates from Australia and China at the bg and tpi loci respectively. Pigs infected with Giardia infect humans by polluting the environment; whether pigs are a potential environmental source of the human pathogen in China requires more epidemiological data.

Infection by and genotype characteristics of Enterocytozoon bieneusi in HIV/AIDS patients from Guangxi Zhuang autonomous region, China.

BACKGROUND: Enterocytozoon bieneusi has been increasingly reported to infect humans and various mammals. Microsporidia cause diarrhea in HIV-infected patients worldwide. PCR amplification and sequencing based on the internal transcribed spacer region have been used to describe the genotypes of E. bieneusi and transmission of microsporidiosis. METHODS: In this study, we examined E. bieneusi infection and genotypes in HIV-positive patients in Guangxi, China. Stool specimens were collected from 285 HIV-positive patients and 303 HIV-negative individuals. E. bieneusi genotypes were characterized using nested PCR and sequencing. RESULTS: Thirty-three (11.58%) HIV-positive patients were infected with microsporidia, and no infection was found in the 303 healthy controls. Three new genotypes were identified and named as GX25, GX456, and GX458; four known genotypes, PigEBITS7, Type IV/K, D, and Ebpc, were also identified. Our data showed that the positive rate for microsporidia was significantly higher in the rural patients than in the other occupation groups. In addition, the positive rate for microsporidia was significantly higher in the patients who drink unboiled water than in those with other drinking water sources. CONCLUSIONS: Our results will provide baseline data for preventing and controlling E. bieneusi infection in HIV/AIDS patients. Further studies are required to clarify the epidemiology and potential sources of microsporidia. Our study showed that microsporidium infection occurs in the HIV/AIDS patients in Guangxi, China.

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.

BACKGROUND: Cystic 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). RESULTS: Compared 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. CONCLUSIONS: We 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.

De novo assembly and transcriptome characterization: novel insights into the natural resistance mechanisms of Microtus fortis against Schistosoma japonicum.

BACKGROUND: Microtus fortis is a non-permissive host of Schistosoma japonicum. It has natural resistance against schistosomes, although the precise resistance mechanisms remain unclear. The paucity of genetic information for M. fortis limits the use of available immunological methods. Thus, studies based on high-throughput sequencing technologies are required to obtain information about resistance mechanisms against S. japonicum. RESULTS: Using Illumina single-end technology, a de novo assembly of the M. fortis transcriptome produced 67,751 unigenes with an average length of 868 nucleotides. Comparisons were made between M. fortis before and after infection with S. japonicum using RNA-seq quantification analysis. The highest number of differentially expressed genes (DEGs) occurred two weeks after infection, and the highest number of down-regulated DEGs occurred three weeks after infection. Simultaneously, the strongest pathological changes in the liver were observed at week two. Gene ontology terms and pathways related to the DEGs revealed that up-regulated transcripts were involved in metabolism, immunity and inflammatory responses. Quantitative real-time PCR analysis showed that patterns of gene expression were consistent with RNA-seq results. CONCLUSIONS: After infection with S. japonicum, a defensive reaction in M. fortis commenced rapidly, increasing dramatically in the second week, and gradually decreasing three weeks after infection. The obtained M. fortis transcriptome and DEGs profile data demonstrated that natural and adaptive immune responses, play an important role in M. fortis immunity to S. japonicum. These findings provide a better understanding of the natural resistance mechanisms of M. fortis against schistosomes.

Cryptosporidium andersoni as a novel predominant Cryptosporidium species in outpatients with diarrhea in Jiangsu Province, China.

BACKGROUND: Cryptosporidium hominis and C. parvum are usually considered to be the major pathogens responsible for human cryptosporidiosis. However, there have been few studies regarding the molecular epidemiology of Cryptosporidium in human infections in China. Here we investigated Cryptosporidium infection in patients with diarrhea, in Danyang Hospital of Jiangsu Province, China, at the genotype level. METHODS: A total of 232 stool specimens were collected from outpatients with diarrhea in Danyang Hospital of Jiangsu Province, China, from February 2012 to January 2013. Each specimen was stained from direct fecal smears and examined for Cryptosporidium using modified acid fast staining and microscopy. Moreover, genomic DNA of each fecal sample was screened for the presence of Cryptosporidium with nested PCR, which was genotyped by analyzing the DNA sequences of small subunit rRNA (SSU rRNA). RESULTS: The average infection rate of Cryptosporidium was 1.3% (3/232) by microscopy and subjected to PCR amplification of the SSU rRNA gene of Cryptosporidium, with 9.91% (23/232) being positive for Cryptosporidium with a significant peak in autumn. Based on the SSU rRNA gene, two Cryptosporidium spp. were identified, including C. andersoni (n =21) and C. hominis (n =2). Two types of C. andersoni, designated as A370 + and A370 - , were found in the SSU rRNA gene in our present study, which was 100% homologous to C. andersoni infections derived from dairy calves and goats, respectively. The clinical questionnaires showed no significant difference in age, gender and frequency of diarrhea, but duration of diarrhea was shorter for C. andersoni than that of C. hominis (mean, 2 vs. 4 days; p <0.01). CONCLUSIONS: C. andersoni is the dominant species in Danyang City of Jiangsu Province. The fact that SSU rRNA sequences of C. andersoni obtained from human stools exhibited 100% homologous to those derived from dairy calves and goats supported that C. andersoni infection might be attributable to animal origin. The difference in the duration of diarrhea of C. andersoni and C. hominis indicated that different Cryptosporidium species might cause different clinical manifestations.

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

BACKGROUND: Cryptosporidium 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. METHODS: Fecal 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. RESULTS: In 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. CONCLUSIONS: The 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.

Effects of Microtus fortis lymphocytes on Schistosoma japonicum in a bone marrow transplantation model.

Microtus fortis is a non-permissive host for Schistosoma japonicum. While M. fortis lymphocytes are known to provide natural resistance against S. japonicum, the specific mechanism remains unclear. A bone marrow transplantation (BMT) model was established using immunodeficient mice, either nude (experiment 1) or V(D)J recombination activation gene deficient mice (RAG-1(-/-)) (experiment 2) as recipients and M. fortis or C57BL/6 mice as donors. The growth and development of S. japonicum were evaluated in each group to assess the role of M. fortis lymphocytes in the response to infection. Lymphocyte ratios and S. japonicum-specific antibody production in transplanted groups increased significantly compared to those in non-transplanted group. Spleen indices and density of splenic lymphocytes in transplanted RAG-1(-/-) mice were higher than those in non-transplanted RAG-1(-/-) mice. No difference in the worm burden was observed among group A (transplants derived from M. fortis), B (transplants derived from C57BL/6 mouse) and C (non-transplanted mice), although worms in group A were shorter than those in other groups, except non-transplanted RAG-1(-/-) mice. Reproductive systems of worms in mice (nude or RAG-1(-/-)) transplanted from M. fortis were not as mature as those in mice (nude or RAG-1(-/-)) transplanted from C57BL/6 mouse and non-transplanted nude mice, but they were more mature than worms in non-transplanted RAG-1(-/-) mice. Therefore, the transplantation model using nude and RAG-1(-/-) mice was successfully established. The M. fortis lymphocytes did not appear to affect the S. japonicum worm burden, but they led to schistosome shortening and a significant reduction in parasite spawning. Thus, M. fortis cellular and humoral immunity provides a defense against schistosomes by negatively impacting the parasite growth and reproductive development.

[Effect of toll-like receptor (TLR) 7 deficiencies on the in vivo immune response against Schistosoma japonicum].

OBJECTIVE: To explore the toll-like receptor 7 knocked out (TLR7-/-) mice immune response against Schistosoma japonicum. METHODS: C57BL/6 mice (WT) and TLR7-/- mice (TLR7-/-) were infected with 20 S. japonicum cercariae via shaved abdomen. There were nine mice in each group. At 6 weeks post-infection, mice were sacrificed. Adult worms were harvested by perfusion of the portal venous system, and the number of adult worms was determined. At the time of perfusion, livers were collected, weighed, and digested overnight with 5% potassium hydroxide, and eggs were counted. In addition, spleens were aseptically harvested when WT and TLR7-/- mice were sacrificed at day zero and 6 weeks after S. japonicum infection. After 72 hours of the co-culture with or without S. japonicum eggs, the culture supernatants were collected for cytokine assays by ELISA assay. RESULTS: At 6 weeks after infection, there was no significant difference in number of worms [(10.5 +/- 3.3) vs (9.8 +/- 5.2)] and eggs per gram of liver tissue [(38 251.9 +/- 4 891.5) vs (38 160.9 +/- 3 341.0)] between WT and TLR7-/- mice. As for Th1/Th2 cytokine secretion from spleen cells, the levels of TNF-alpha [(43.7 +/- 9.8) pg/ml] and INF-gamma [(215.2 +/- 35.4) pg/ml] from TLR7-/- infected mice were lower than those of WT infected mice[(63.4 +/- 22.9) pg/ml, (383.5 +/- 253.3) pg/ml]. For Th2 cytokines detection, the production of IL-10 [(1702.6 +/- 572.3) pg/ml] and IL-4 [(59.5 +/- 10.1) pg/ml] from TLR7-/- mice were higher than those of WT mice [(595.2 +/- 386.3) pg/ml, (8.3 +/- 0.9) pg/ml] (P < 0.05, P < 0.01), while IL-4 level [(63.9 +/- 33.9) pg/ml] from TLR7-/- infected mice was higher than those of WT infected mice [(23.3 +/- 11.5) pg/ml]. CONCLUSION: TLR7-/- mice has a dominant Th2 response under the normal state. The absence of TLR7 does not influence the immune response against S. japonicum infection at 6 weeks post-infection.

[Accumulation of myeloid-derived suppressor cells in the spleen and peripheral blood of Schistosoma japonicum-infected mice].

OBJECTIVE: To determine the accumulation of CD11b+ Gr-1+ myeloid-derived suppressor cells (MDSC) in Schistosorna japonicum-infected mice. METHODS: Twenty-four C57BL/6 mice were infected cutaneously with S. japonicum cercariae. Peripheral blood samples were collected at 1, 2, 6 and 8 weeks post-infection (6 mice for each group). At 6 and 8 weeks post-infection, spleens were removed and a single-cell suspension was prepared. At the same time, 6 healthy mice each served as control. During the different stages of infection, the levels of MDSC, Gr-1+ cells, CD11b+ cells in murine peripheral blood and spleen were detected by flow cytometry. The possible function of MDSC on T cells was evaluated by using a CCK-8 method and CFSE proliferation assay. RESULTS: At 6 and 8 weeks post-infection, the levels of MDSC (38.2%-57.8% and 47.1-77.6%, respectively), Gr-1+ cells (28.9%-44.6%, 40.4%-72.9%), and CD11b+ cells (36.0%-48.1%, 40.3%-68.3%) in infection group were significantly higher than that of the controls (15.1%-20.4%, 8.4%-17.3%, 9.8%-22.6%), and that of infection group at 1 week (16.2%-19.8%, 13.0%-16.8%, 17.6%-19.4%) and 2 weeks (19.8%-29.5%, 17.2%-22.2%, 20.9%-33.3%) post-infection (P < 0.01). No significant difference was found in the levels of MDSC, Gr-1+ cells, CD11b+ cells among infection group at 1 and 2 weeks post-infection and control group. Moreover, the fluctuation trends of these cells in the spleens of infected mice were similar to those cells in peripheral blood (P > 0.05). Strikingly, the proliferation index of normal CD4 T cells was significantly lower after co-culture with Gr-1+ cells isolated from infected mice. CONCLUSION: Schistosoma japonicum infection induces higher level of MDSC in mice, and Gr-1+ cells isolated from the infected mice can significantly inhibit the proliferation of the normal CD4+ T cells.

Cross-species transmission of Cryptosporidium in wild rodents from the southern region of Zhejiang Province of China and its possible impact on public health.

Wild rodents serve as reservoirs for Cryptosporidium and are overpopulated globally. However, genetic data regarding Cryptosporidium in these animals from China are limited. Here, we have determined the prevalence and genetic characteristics of Cryptosporidium among 370 wild rodents captured from three distinct locations in the southern region of Zhejiang Province, China. Fresh feces were collected from the rectum of each rodent, and DNA was extracted from them. The rodent species was identified by PCR amplifying the vertebrate cytochrome b gene. Cryptosporidium was detected by PCR amplification and amplicon sequencing the small subunit of ribosomal RNA gene. Positive samples of C. viatorum and C. parvum were further subtyped by analyzing the 60-kDa glycoprotein gene. A positive Cryptosporidium result was found in 7% (26/370) of samples, involving five rodent species: Apodemus agrarius (36), Niviventer niviventer (75), Rattus losea (18), R. norvegicus (155), and R. tanezumi (86). Their respective Cryptosporidium positive rates were 8.3%, 5.3%, 11.1%, 7.1%, and 7.0%. Sequence analysis confirmed the presence of three Cryptosporidium species: C. parvum (4), C. viatorum (1), and C. muris (1), and two genotypes: Cryptosporidium rat genotype IV (16) and C. mortiferum-like (4). Additionally, two subtypes of C. parvum (IIdA15G1 and IIpA19) and one subtype of C. viatorum (XVdA3) were detected. These results demonstrate that various wild rodent species in Zhejiang were concurrently infected with rodent-adapted and zoonotic species/genotypes of Cryptosporidium, indicating that these rodents can play a role in maintaining and dispersing this parasite into the environment and other hosts, including humans.

Title: Transmission interspécifique de Cryptosporidium chez les rongeurs sauvages de la région sud de la province chinoise du Zhejiang et son impact possible sur la santé publique. Abstract: Les rongeurs sauvages servent de réservoirs à Cryptosporidium et ont des grandes populations à l'échelle mondiale. Cependant, les données génétiques concernant Cryptosporidium chez ces animaux en Chine sont limitées. Ici, nous avons déterminé la prévalence et les caractéristiques génétiques de Cryptosporidium parmi 370 rongeurs sauvages capturés dans trois endroits distincts de la région sud de la province du Zhejiang, en Chine. Des excréments frais ont été collectés dans le rectum de chaque rongeur et l'ADN en a été extrait. L'espèce de rongeur a été identifiée par amplification par PCR du gène du cytochrome b des vertébrés. Cryptosporidium a été détecté par amplification PCR et séquençage d'amplicons de la petite sous-unité du gène de l'ARN ribosomal. Les échantillons positifs de C. viatorum et C. parvum ont ensuite été sous-typés en analysant le gène de la glycoprotéine de 60 kDa. Un résultat positif pour Cryptosporidium a été trouvé dans 7 % (26/370) des échantillons, impliquant cinq espèces de rongeurs : Apodemus agrarius (36), Niviventer niviventer (75), Rattus losea (18), R. norvegicus (155) et R. tanezumi (86). Leurs taux respectifs de positivité pour Cryptosporidium étaient de 8,3 %, 5,3 %, 11,1 %, 7,1 % et 7,0 %. L'analyse des séquences a confirmé la présence de trois espèces de Cryptosporidium : C. parvum (4), C. viatorum (1) et C. muris (1), et de deux génotypes : Cryptosporidium génotype IV de rat (16) et C. mortiferum-like (4). De plus, deux sous-types de C. parvum (IIdA15G1 et IIpA19) et un sous-type de C. viatorum (XVdA3) ont été détectés. Ces résultats démontrent que diverses espèces de rongeurs sauvages du Zhejiang sont simultanément infectées par des espèces/génotypes de Cryptosporidium zoonotiques et adaptés aux rongeurs, ce qui indique que ces rongeurs peuvent jouer un rôle dans le maintien et la dispersion de ce parasite dans l'environnement et d'autres hôtes, y compris les humains.

Molecular identification and genetic characteristics of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in human immunodeficiency virus/acquired immunodeficiency syndrome patients in Shanghai, China.

BACKGROUND: Opportunistic infections are a ubiquitous complication in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients. Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common opportunistic intestinal pathogens in humans. In China, despite the number of HIV/AIDS patients being extremely large, only a few studies have investigated opportunistic infections caused by intestinal pathogens in this patient population. The aims of this study were to elucidate the occurrence and genetic characteristics of Cryptosporidium spp., G. duodenalis, and E. bieneusi in HIV/AIDS patients. METHODS: We collected fecal specimens from 155 HIV/AIDS patients (one from each patient). All of the specimens were examined for the presence of the pathogens by genotyping using polymerase chain reaction and sequencing of the small subunit ribosomal RNA gene for Cryptosporidium spp.; the triosephosphate isomerase, ß-giardin and glutamate dehydrogenase genes for G. duodenalis; and the internal transcribed spacer region of the rRNA gene for E. bieneusi. The Cryptosporidium-positive specimens were further subtyped by polymerase chain reacion and sequencing of the 60-kDa glycoprotein gene. RESULTS: Six (3.9%), three (1.9%), and eight (5.2%) HIV/AIDS patients were positive for Cryptosporidium spp., G. duodenalis, and E. bieneusi, respectively. No statistical differences were observed in occurrence rate between the groups by gender, clinical symptom (diarrhea), and CD4+ cell count. Four Cryptosporidium species were identified: Cryptosporidium hominis (n = 2), Cryptosporidium parvum (n = 1), Cryptosporidium meleagridis (n = 1), and Cryptosporidium andersoni (n = 2). Furthermore, two C. hominis subtypes (IeA12G3T3 and IaA28R4) were detected. Three G. duodenalis-positive specimens were successfully amplified and sequenced at the triosephosphate isomerase and ß-giardin loci, which led to the identification of assemblages C and B, respectively. Seven genotypes (D, Type IV, EbpC, Peru11, EbpD, A, and I) were identified in E. bieneusi-positive specimens. CONCLUSIONS: Our findings should increase awareness of AIDS-related opportunistic intestinal pathogens, and indicate the need for routine examination in clinical practice for the detection of Cryptosporidium spp., G. duodenalis, and E. bieneusi. Homology analyses of the three intestinal pathogens at the nucleotide and/or amino acid levels indicated their zoonotic potential.

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.

Intestinal Protozoan Infections in Patients with Diarrhea - Shanghai Municipality, Zhenjiang City, and Danyang City, China, 2011-2015 and 2019-2021.

What is already known about this topic?: Intestinal protozoa are common pathogens of diarrhea globally. However, the etiology of diarrhea due to intestinal protozoan infections in China is not known. What is added by this report?: Based on active syndromic surveillance in Shanghai, Zhenjiang, and Danyang during 2011-2015 and 2019-2021, 89 (1.67%) patients were infected with intestinal protozoa (Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Cyclospora cayetanensis), and positivity rates statistically differed by region and age. What are the implications for public health practice?: This was the most comprehensive data collection in investigating parasitic diarrheal diseases in humans. Identification of these protozoa in diarrhea will provide new perspectives for detecting hidden etiological agents of diarrhea as early as possible.

The identification of the Cryptosporidium ubiquitum in pre-weaned Ovines from Aba Tibetan and Qiang autonomous prefecture in China.

OBJECTIVE: Cryptosporidium spp. are prevalent globally and sheep are an important zoonotic reservoir. Little data regarding the rates of Cryptosporidium infections in ovines in China are available. This study assessed the prevalence of Cryptosporidium spp. in pre-weaned ovines from Aba Tibetan and Qiang Autonomous Prefecture in the Sichuan province of China. METHODS: A total of 213 fecal samples were collected from pre-weaned ovines and were examined microscopically (following modified acid fast staining). In addition, 18S rRNA genetic sequences were amplified from fecal samples by nested PCR and phylogenetically analyzed. RESULTS: The prevalence of Cryptosporidium in the collected samples was at 14.6% (31/213) and four isolates identified by PCR belonged to the Cryptosporidium cervine genotype (Cryptosporidium ubiquitum) demonstrating that this species was the primary sheep species found in sheep in China. CONCLUSION: The present study suggested that the high incidence of Cryptosporidium in sheep poses a significant public health threat and that surveillance practices must be established to prevent zoonotic disease of humans.

[Cloning, expression and immunodiagnostic evaluation of antigen EPC1 from Echinococcus granulosus].

OBJECTIVE: To clone and express EPCl gene of Echinococcus granulosus, and investigate its immunogenicity and diagnostic value. METHODS: Total RNA was extracted from hydatid cyst protoscoleces and EPC1 gene of Echinococcus granulosus was amplified by RT-PCR. The PCR product was cloned into pGEM-T vector, and then subcloned into the prokaryotic expression vector PET28a(+). The positive recombinants were transformed into Escherichia coli BL21 (DE3), and followed by expression of the protein induced by IPTG. The recombinant protein was identified by SDS-PAGE and Western blotting, and used to establish ELISA. Serum samples from patients with cystic echinococcosis (60 cases), alve-olar echinococcosis (37 cases), cysticercosis (16 cases), clonorchiasis sinensis (7 cases), schistosomiasis japonica (4 cases) and healthy persons (33 cases) were examined. RESULTS: The recombinant plasmid PET28a-EgEPC1 was identified by restriction enzyme digestion and sequencing. SDS-PAGE result showed that the recombinant containing recombinant plasmid PET28a-EgEPC1 expressed a soluble fission protein of EgEPC1 (about M, 11 000). The protein was recognized by pool sera of cystic echinococcosis patients. The overall sensitivity and specificity of diagnosis by ELISA for cystic echinococcosis were 78.3% (47/60), and 98.3% (59/60), respectively. The cross reaction with sera of alveolar echinococcosis was 40.5% (15/37). CONCLUSION: The recombinant EgEPC1 antigen has diagnostic value in cystic echinococcosis.

[Preliminary study on the immunological characteristics of permissive and non-permissive hosts infected with Schistosoma japonicum].

OBJECTIVE: To study the difference among immune responses of three kinds of experimental animals with different susceptibility to the infection of Schistosoma japonicum, and preliminarily explore the mechanism of the immune response in permissive and non-permissive hosts. METHODS: Twelve animals of each kind of rodents, C57BL/6 mice, Sprague Dawley (SD) rats and Microtus fortis, were randomly divided into the infected group and uninfected group each with 6 animals. In infected groups of C57BL/6 mice, SD rats, and M. fortis, each animal was infected with 20, 200 and 1000 cercariae of S. japonicum, respectively. 42 d later, all rodents were sacrificed. Adult worms in portal vein and granulomas in liver were observed and the sera were collected. The levels of cytokines IL-10 and IFN-gamma as well as serum IgG, IgG2a, and IgG1 were detected by ELISA. RESULTS: At the 42th day post infection, worms in portal vein and liver granulomas were observed in C57BL/6 mice and SD rats, but not in M. fortis. The level of IL-10 in the sera of SD rats [(2.21 +/- 0.12) pg/ml] was significantly higher than that in the sera of M. fortis [(1.64 +/- 0.39) pg/ml] and C57BL/6 mice [(0.10 +/- 0.04) pg/ml] (P<0.01). IL-10 in the sera of M. fortis was also significantly higher than that in the sera of C57BL/6 mice (P<0.01). IFN-gamma in the sera of SD rats [(0.21 +/- 0.11) pg/ml] was significantly higher than that in the sera of M. fortis [(0.11 +/- 0.03) pg/ml] and C57BL/6 mice [(0.09 +/- 0.02) pg/ml] (P<0.05), but no difference between M. fortis and C57BL/6 mice (P>0.05). The levels of IgG (1.53 +/- 0.31), IgG1 (1.48 +/- 0.44) and IgG2a (0.41 +/- 0.11) in SD rats were significantly higher than that in the sera of M. fortis (0.48 +/- 0.14, 0.15 +/- 0.03 and 0.12 +/- 0.061) (P<0.01). The levels of IgG (1.21 +/- 0.16), IgG1 (0.88 +/- 0.31) in C57BL/6 mice were significantly higher than that in the sera of M. fortis (P<0.01). IgG1 antibody is the predominant subclass in the three kinds of rodents. The levels of IL-10, IFN-gamma and antibody subclass IgG, IgG1, IgG2a in all non-infected rodents were not detected. CONCLUSION: IL-10 in non-permissive hosts, which is an essential agent in the regulation of Th2 immune response, is higher than that in permissive host It may play an important role in the resistance to schistosome in the non-permissive hosts.

Alteration of the fecal microbiota in Chinese patients with Schistosoma japonicum infection.

Schistosoma japonicum infection causes pathological injury to the host. Multiple studies have shown that intestinal helminth infection causes dysbiosis for the gut microbial community and impacts host immunology. However, the effect of acute S. japonicum infection on the gut microbiome structure (abundance and diversity) is still unclear. We collected fecal samples from healthy and infected patients from a single hospital in Hunan Province, China. The bacterial community was analyzed using 16S ribosomal RNA gene sequencing of the V4 hypervariable region using the HiSeq platform. Compared with healthy subjects, infected patients exhibited an increase in relative abundance of the TM7 phylum. At the genus level, there were seven differentially abundant genera between groups. The most significant finding was a Bacteroides enterotype in patients with acute schistosomiasis. These results suggest that S. japonicum infection has a significant effect on microbiome composition characterized by a higher abundance of the TM7 phylum and development of a Bacteroides enterotype.

TITLE: Altération du microbiote fécal chez les patients chinois atteints d'une infection à Schistosoma japonicum. ABSTRACT: L'infection à Schistosoma japonicum provoque des lésions pathologiques chez l'hôte. Plusieurs études ont montré qu'une infection intestinale par les helminthes provoque une dysbiose de la communauté microbienne intestinale et a un impact sur l'immunologie de l'hôte. Cependant, l'effet de l'infection aiguë à S. japonicum sur la structure du microbiome intestinal (abondance et diversité) n'est toujours pas clair. Nous avons collecté des échantillons fécaux de patients sains et infectés dans un hôpital de la province du Hunan, en Chine. La communauté bactérienne a été analysée par séquençage du gène de l'ARN ribosomal 16S de la région hypervariable V4 en utilisant la plateforme HiSeq. Par rapport aux sujets sains, les patients infectés ont présenté une augmentation de l'abondance relative du phylum TM7. Au niveau du genre, il y avait sept genres différentiellement abondants entre les groupes. La découverte la plus significative était un entérotype Bacteroides chez les patients atteints de schistosomiase aiguë. Ces résultats suggèrent que l'infection à S. japonicum a un effet significatif sur la composition du microbiome caractérisé par une plus grande abondance du phylum TM7 et le développement d'un entérotype Bacteroides.