A p85α-osteopontin axis couples the receptor ICOS to sustained Bcl-6 expression by follicular helper and regulatory T cells.

Follicular helper T cells (TFH cells) and follicular regulatory T cells (TFR cells) regulate the quantity and quality of humoral immunity. Although both cell types express the costimulatory receptor ICOS and require the transcription factor Bcl-6 for their differentiation, the ICOS-dependent pathways that coordinate their responses are not well understood. Here we report that activation of ICOS in CD4(+) T cells promoted interaction of the p85α regulatory subunit of the signaling kinase PI(3)K and intracellular osteopontin (OPN-i), followed by translocation of OPN-i to the nucleus, its interaction with Bcl-6 and protection of Bcl-6 from ubiquitin-dependent proteasome degradation. Post-translational protection of Bcl-6 by OPN-i was essential for sustained responses of TFH cells and TFR cells and regulation of the germinal center B cell response to antigen. Thus, the p85α-OPN-i axis represents a molecular bridge that couples activation of ICOS to Bcl-6-dependent functional differentiation of TFH cells and TFR cells; this suggests new therapeutic avenues to manipulate the responses of these cells.

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.

Transgenic RFP-RPS-30UbL strain of the nematode Caenorhabditis elegans as a biomonitor for environmental pollutants.

Environmental pollutants are recognized as one of the major concerns for public health. The free-living nematode Caenorhabditis elegans are widely used to evaluate the toxicity of environmental contaminants in biomonitoring researches. In the present study, a new transgenic strain, rps-30-/- ;RFP-RPS-30UbL was generated, with constitutively active rps-30 promoter used to control the expression of RFP-RPS-30UbL fusion protein. We found RFP-RPS-30UbL would accumulate to form 'rod-like' structures, when worms were exposed to environmental contaminants, including Cd, Hg, Pb, As, Paraquat and Dichlorvos. The number of the 'rod-like' structures was induced by environmental contaminants in a concentration- and time-dependent manner. The 'rod-like' structure formation could be detectable in response to the concentration of each contaminant as low as 24-h LC50 × 10-7 , and the detectable time could be within 2 h. Detecting the transcription and expression levels of RFP-RPS-30UbL in worms exposed to different kinds of environmental contaminants showed that the expression level of RFP-RPS-30UbL was not regulated by environmental contaminants, and the number differences of 'rod-like' structures were just due to the morphological change of RFP-RPS-30UbL from dispersion to accumulation induced by environmental contaminants. In addition, this transgenic strain was developed in rps-30-/- homozygous worm, which was a longevity strain. Detection of lifespan and brood size showed that rps-30-/- ;RFP-RPS-30UbL transgenic worm was more suitable to be cultured and used further than N2;GFP-RPS-30UbL , for expressing RPS-30UbL in wild type N2 worms shortened the lifespan and deceased the brood size. Therefore, rps-30-/- ;RFP-RPS-30UbL transgenic worm might play a potential role in versatile environmental biomonitoring, with the advantage of not only the convenient and quick fluorescence-based reporter assay, but also the quantificational evaluation of the toxicities of environmental contaminants using 'rod-like' structures with high sensitivity, off-limited the expression level of the reporter protein.

Occurrence and potentially zoonotic genotypes of Enterocytozoon bieneusi in wild rhesus macaques (Macaca mulatta) living in Nanwan Monkey Island, Hainan, China: a public health concern.

BACKGROUND: Enterocytozoon bieneusi, a microsporidian species, is a zoonotic pathogen found in both humans and animals. Here, we determined the prevalence, explored the different genotypes of E. bieneusi in wild rhesus macaques (Macaca mulatta) (Hainan Island of China), and assessed their zoonotic potential. METHODS: We collected 173 fecal specimens from wild rhesus macaques living in Nanwan Monkey Island, Hainan, China. Subsequently, we identified and genotyped E. bieneusi using nested PCR analysis amplification of the internal transcribed spacer region (ITS) of the rRNA gene. Lastly, a neighbor-joining tree was built based on gene sequences from the ITS region of E. bieneusi. RESULTS: Of the 173 specimens from wild rhesus macaques, 26 (15%) were infected with E. bieneusi. We identified six genotypes of E. bieneusi, of which five were known: PigEBITS7 (n = 20), D (n = 2), Type IV (n = 1), Peru6 (n = 1), Henan-III (n = 1), and a novel genotype: HNM-IX (n = 1). From the phylogenetic analysis, the six genotypes identified here were all clustered into zoonotic group 1. CONCLUSION: This study is the first report to detect E. bieneusi infection in wild rhesus macaques from Hainan, China. Human-pathogenic genotypes D, Henan-III, Peru6, PigEbITS7, and Type IV in the wild rhesus macaques support these animals infected with E. bieneusi have a public health significance.

[Cloning of galectin-1 gene of Angiostrongylus cantonensis and testing the agglutination property of the galectin-1 protein].

Objective: To clone and express the galectin-1 gene of Angiostrongylus cantonensis, and test the agglutination property of its protein. Methods: The three-dimensional structure of galectin-1 was analyzed with Swiss Model. Total RNA was extracted from male worms of A. cantonensis. Primers were designed for galectin-1 based on its coding region (GenBank Accession No. JN133961.1). RT-PCR was performed, and the PCR products were subcloned to pCold Ⅲ plasmid and transduced into Escherichia coli BL21 strain. The recombinant plasmid was extracted from positive clones on LB plate containing 100 µg/ml Kanamycin, and validated with double digestion, PCR identification and sequencing. The confirmed positive clones of E. coli BL21 with the recombinant plasmid were grown in LB medium containing ampicillin (100 µg/ml, 100 µl). IPTG was added to induce expression of the plasmid. The galectin-1 recombinant protein was purified with Ni-NTA beads, and analyzed with SDS-PAGE and Western blotting using anti-serum of mouse immunized with whole worms of A. cantonensis. The agglutination reaction with red blood cells in fresh blood of ICR mouse was observed for the 10-fold serial dilutions of recombinant proteins (5.55 × 10(-1)-5.55 × 10(-5) ng/µl). Results: The Swiss Model analysis showed that the functional galectin-1 had a non-dimeric form. As was expected, the RT-PCR products had a size of 850 bp. Results of double digestion, PCR and sequencing showed successful construction of the pCold Ⅲ-galectin-1 plasmid. SDS-PAGE revealed expression of soluble recombinant fusion protein with molecular weight of ~36 000. Western blotting showed that the galectin-1 protein was recognized by mouse anti-serum. In addition, the minimun concentration of galectin-1 that showed significant agglutination reactions with mouse red blood cells was 5.55 × 10(-4) ng/µl. Conclusion: The galectin-1 clone can be expressed in the pCold Ⅲ plasmid, and its protein product has agglutination property.

[Analysis and identification of peptide epitope of the fifth stage larvae of Angiostrongylus cantonensis].

OBJECTIVE: To identify the specific antigens of the fifth stage larvae (larvae V) of Angiostrongylus cantonensis in soluble proteins. METHODS: The techniques of two-dimensional electrophoresis (2-DE) and immunoblot were employed to analyze the soluble components of larvae V antigens. The resulting protein spots were identified by mass spectrometry and the specific antigen and immunogenic peptide segment profiles determined.Larvae V was identified by immunoblot of peptide immune murine sera. RESULTS: Immunoblot of 2-DE showed that there were 8 specific antigen spots in females and 4 in males of larvae V. The 12 protein spots had a pH range of 5-7 and a molecular weight of 24-40 kD. Among 12 spot, actin-1 and galectin-1 were identified successfully. And a peptide (NH2-KNGDIALHFNPRFDEK) was predicted as epitope and synthesized. The binding capacity of murine immune sera could identify the 32 000 of larvae V. CONCLUSIONS: Galectin and actin are common antigens of female and male larvae V of A.cantonensis. And a peptide from galectin-1 may become a potential target for immunologic diagnosis and immunological mediator.

Enterocytozoon bieneusi and Cryptosporidium bat genotype XXI and bat genotype XXII in fruit bats (Rousettus leschenaultii) inhabiting a tropical park in Hainan Province, China.

Bats stand as one of the most diverse groups in the animal kingdom and are key players in the global transmission of emerging pathogens. However, their role in transmitting Enterocytozoon bieneusi and Cryptosporidium spp. remains unclear. This study aimed to evaluate the occurrence and genetic diversity of the two pathogens in fruit bats (Rousettus leschenaultii) in Hainan, China. Ten fresh fecal specimens of fruit bats were collected from Wanlvyuan Gardens, Haikou, China. The fecal samples were tested for E. bieneusi and Cryptosporidium spp. using Polymerase Chain Reaction (PCR) analysis and sequencing the internal transcribed spacer (ITS) region and partial small subunit of ribosomal RNA (SSU rRNA) gene, respectively. Genetic heterogeneity across Cryptosporidium spp. isolates was assessed by sequencing 4 microsatellite/minisatellite loci (MS1, MS2, MS3, and MS16). The findings showed that out of the ten specimens analyzed, 2 (20 %) and seven (70.0 %) were tested positive for E. bieneusi and Cryptosporidium spp., respectively. DNA sequence analysis revealed the presence of two novel Cryptosporidium genotypes with 94.4 to 98.6 % sequence similarity to C. andersoni, named as Cryptosporidium bat-genotype-XXI and bat-genotype-XXII. Three novel sequences of MS1, MS2 and MS16 loci identified here had 95.4 to 96.9 % similarity to the known sequences, which were deposited in the GenBank. Two genotypes of E. bieneusi were identified, including a novel genotype named HNB-I and a zoonotic genotype PigEbITS7. The discovery of these novel sequences provides meaningful data for epidemiological studies of the both pathogens. Meanwhile our results are also presented that the fruit bats infected with E. bieneusi, but not with Cryptosporidium, should be considered potential public health threats.

Occurrence and multilocus genotyping of Giardia duodenalis in diarrheic and asymptomatic children from South of Zhejiang province in China.

Giardia duodenalis is an intestinal pathogen that is found globally. Children are more susceptible and often suffer severe consequences after infection. Despite this, the health effects of this pathogen continue to be poorly understood and neglected. In Wenzhou, Zhejiang province, China, stool samples were obtained from 1032 children who were admitted to Yuying Children's Hospital. Out of these, 684 presented with diarrhea, while 348 were asymptomatic. The stool samples were screened for G. duodenali by targeting the small subunit of the ribosomal RNA (SSU rRNA) gene. Subtypes of G. duodenalis were identified via amplification of the glutamate dehydrogenase (gdh), beta-giardin (bg), and triosephosphate isomerase (tpi) genes in samples positive for the G. duodenalis. The findings indicated the presence of G. duodenalis in 0.9 % (9/1032) of the samples, with 9/684 (1.3 %) of the samples originating from children with diarrhea and none from the asymptomatic samples. All 9 samples that tested positive for G. duodenalis were determined to be of assemblage A. Of these, 6 samples were effectively genotyped at all 3 loci, resulting in the identification of 3 distinct MLGs: MLG-AII1 (n = 1), MLG-AII2 (n = 4), and MLG-AII2 (n = 1), all belonging to G. duodenalis assemblage AII. This was the first study that confirmed G. duodenalis infections in children residing in southern Zhejiang, China, with comparatively low rates of infection. The detection of G. duodenalis assemblage AII indicates a possibility of transfer from one human to another. The parasite's effect on the health of young children requires special attention and consideration.

Molecular prevalence, subtype distribution, and zoonotic potential of Blastocystis sp. in wild rodents and shrews inhabiting Zhejiang province of China.

Introduction: Globally, rodents and shrew populations constitute crucial elements of diverse environments and animal communities. It is imperative to study their population dynamics to mitigate any potential negative impact on humans, as they can be involved in the transmission of critical zoonotic agents, such as Blastocystis. Therefore, this study aimed to identify the prevalence and genetic composition of Blastocystis in wild rodents and shrews residing in the Zhejiang provinces of China. Methods: A total of 652 wild rodents and and shrews were captured from three different regions in Zhejiang Province from April 1st to October 31, 2023. The DNA was isolated by collecting fresh feces from the intestines of each rodent or and shrew. Rodent and shrew species were examined by vertebrate cytochrome b (cytb) analysis and PCR amplification. Blastocystis was also found in all fecal samples using PCR analysis and sequencing of the partial small subunit of ribosomal RNA (SSU rRNA) gene. Results: Among all the samples, 6.6% (43/652) showed a positive result for Blastocystis. In the results, 6 species of rodent and shrew were identified with Blastocystis, including Apodemus agrarius (n = 36) (2.8%), Niviventer confucianus (n = 75) (17.3%), Rattus losea (n = 18) (5.6%), R. norvegicus (n = 155) (2.6%), R. tanezumi (n = 86) (3.5%), and Suncus murinus (n = 282) (7.4%). The existence of 6 Blastocystis subtypes, ST4 (n = 33), ST1 (4), ST7 (n = 3), ST2 (n = 1), ST3 (n = 1), and ST5 (n = 1), were confirmed by sequence analysis. Discussion: Based on the molecular data obtained, the wild rodents and shrews under investigation were found to be concurrently infected with zoonotic subtypes of Blastocystis, including ST1 to ST5 and ST7. This suggests that these animals could potentially pose a zoonotic threat to humans and other animals susceptible to Blastocystis infection.

Molecular detection and genetic variability of Cryptosporidium spp. in wild Asian house shrews (Suncus murinus) from southern Zhejiang province, China.

Shrews play a crucial role as repositories for diverse pathogens linked to zoonotic infectious diseases. However, the genetic information regarding Cryptosporidium in Chinese shrews remains unexplored. The objectives of this study were twofold: to determine the occurrence rate of Cryptosporidium spp. in wild shrews residing in the southern part of Zhejiang Province, China, and to investigate their genetic characteristics. A total of 282 wild shrews were captured between April and October of 2023. The detection of Cryptosporidium in fecal samples, collected from each animal's rectum, was performed using PCR and sequencing of the partial small subunit of ribosomal RNA (SSU rRNA) gene. The 60-kDa glycoprotein (gp60) gene was utilized to further subtype the positive samples of C. viatorum and C. parvum. All animals were identified as Suncus murinus, and a positive result for Cryptosporidium was obtained in 14.2 % (40/282) of the samples. The following species and genotypes were identified: C. ratti (n = 19), C. parvum (n = 2), C. viatorum (n = 1), Cryptosporidium rat genotype IV (n = 13), and Cryptosporidium skunk genotype (n = 5). Furthermore, the subtypes IIdA15G1 and XVdA3 were detected within C. parvum and C. viatorum, respectively. Molecular evidence indicates that S. murinus is concurrently infected with rodent-adapted and zoonotic species/genotypes, actively contributing to the dissemination of cryptosporidiosis.

Enterocytozoon bieneusi in Wild Rats and Shrews from Zhejiang Province, China: Occurrence, Genetic Characterization, and Potential for Zoonotic Transmission.

Globally, Enterocytozoon bieneusi has been detected in humans and various animal hosts. Wild rats and shrews have the potential to act as carriers of E. bieneusi, facilitating the parasite's transmission to humans and domestic animals. We aimed to investigate the prevalence of E. bieneusi in 652 wild rats and shrews from Zhejiang Province, China, by amplifying the internal transcribed spacer (ITS) region of rDNA through polymerase chain reaction (PCR). To determine animal species, we amplified the Cytochrome b (Cyt-b) gene in their fecal DNA using PCR. Furthermore, we determined the genotype of E. bieneusi by amplifying the ITS region of rDNA through PCR. Genetic traits and zoonotic potential were evaluated using similarity and phylogenetic analyses. Suncus murinus (n = 282) and five rat species, Rattus losea (n = 18), Apodemus agrarius (n = 36), Rattus tanezumi (n = 86), Rattus norvegicus (n = 155), and Niviventer niviventer (n = 75), were identified. The average infection rate of E. bieneusi was 14.1% (92/652) with 18.1% (51/282) in S. murinus and 11.1% (41/370) in rats (27.8% in R. losea, 22.2% in A. agrarius, 10.5% in R. tanezumi, 8.4% in R. norvegicus, and 8.0% in N. niviventer). Thirty-three genotypes were identified, including 16 known genotypes. The most commonly known genotypes were HNR-VI (n = 47) and Peru11 (n = 6). Type IV, KIN-1, SHW7, and HNPL-II were each found in two samples, while Macaque4, CH5, K, Henan-III, Henan-V, HNP-II, HNPL-I, HNPL-III, HNHZ-II, and HNHZ-III were each found in one sample. Additionally, 17 novel genotypes were discovered: WZR-VIII (n = 5), WZR-I to WZR-VII, WZR-IX to WZR-XII, and WZSH-I to WZSH-V (n = 1 each). Those 33 genotypes were divided into three groups: Group 1 (n = 25), Group 2 (n = 3), and Group 13 (n = 5). The initial report underscores the extensive occurrence and notable genetic diversity of E. bieneusi in wild rats and shrews from Zhejiang province, China. These results suggest that these animals play a pivotal role in the transmission of E. bieneusi. Furthermore, animals carrying the zoonotic genotypes of E. bieneusi pose a serious threat to residents.

Molecular prevalence and subtype distribution of Blastocystis spp. among children who have diarrheia or are asymptomatic in Wenzhou, Zhejiang Province, China.

Blastocystis sp., a significant zoonotic parasite with a global distribution, was the focus of this study, which aimed to investigate its prevalence and genetic diversity among diarrheic and asymptomatic children in Wenzhou, China. We collected 1,032 fecal samples from Yuying Children's Hospital, Wenzhou, China, comprising 684 from children with diarrhea and 348 from asymptomatic children. Genomic DNA extracted from these samples was used to detect Blastocystis spp. by PCR, targeting the small subunit ribosomal RNA gene. Subsequently, a phylogenetic tree was constructed, applying the maximum likelihood method. Blastocystis spp. were detected in 67 (6.5%) of the fecal samples. The prevalence rate of Blastocystis spp. in diarrheic children (8.8%; 60/684) was significantly higher than that in asymptomatic children (2.0%; 7/348) (χ 2 = 17.3, p < 0.001). Sequence analysis of the SSU rRNA gene identified five known Blastocystis spp. subtypes, ST1 (n = 12), ST2 (n = 5), ST3 (n = 35), ST4 (n = 12), and ST7 (n = 3). ST1 and ST3 were present in both diarrheic and asymptomatic children, while ST2, ST4, and ST7 were exclusive to diarrheic children. Intra-subtype genetic polymorphisms were identified, comprising four variations in ST1 (ST1-1 to ST1-4), five in ST3 (ST3-1 to ST3-5), two in ST4 (ST4-1 and ST4-2), and two in ST7 (ST7-1 and ST7-2). Notably, ST1-2 to ST1-4, ST3-3 to ST3-5, and ST7-1 and ST7-2 represent newly identified variations. The composition and genetic characteristics of subtypes among children in this region suggest various sources of infection, including human-to-human and animal-to-human transmission.

Title: Prévalence moléculaire et distribution des sous-types de Blastocystis spp. parmi les enfants diarrhéiques et asymptomatiques à Wenzhou, Province du Zhejiang, Chine. Abstract: Blastocystis sp., un parasite zoonotique important avec une distribution mondiale, était au centre de cette étude, qui visait à étudier sa prévalence et sa diversité génétique parmi les enfants diarrhéiques et asymptomatiques de Wenzhou, en Chine. Nous avons collecté 1 032 échantillons fécaux à l'hôpital pour enfants Yuying de Wenzhou, en Chine, dont 684 provenant d'enfants souffrant de diarrhée et 348 d'enfants asymptomatiques. L'ADN génomique extrait de ces échantillons a été utilisé pour détecter Blastocystis sp. par PCR, ciblant le gène de la petite sous-unité de l'ARN ribosomal. Par la suite, un arbre phylogénétique a été construit, en appliquant la méthode du maximum de vraisemblance. Blastocystis sp. a été détecté dans 67 (6,5 %) des échantillons fécaux. Le taux de prévalence de Blastocystis spp. chez les enfants diarrhéiques (8,8 % ; 60 / 684) était significativement plus élevé que chez les enfants asymptomatiques (2,0 % ; 7 / 348) (χ2 = 17,3, p < 0,001). L'analyse de la séquence du gène de l'ARNr SSU a identifié cinq sous-types de Blastocystis spp., ST1 (n = 12), ST2 (n = 5), ST3 (n = 35), ST4 (n = 12) et ST7 (n = 3). Les sous-types ST1 et ST3 étaient présents chez les enfants diarrhéiques et asymptomatiques, tandis que ST2, ST4 et ST7 étaient exclusifs aux enfants diarrhéiques. Des polymorphismes génétiques intra-sous-types ont été identifiés, comprenant quatre variations dans ST1 (ST1-1 à ST1-4), cinq dans ST3 (ST3-1 à ST3-5), deux dans ST4 (ST4-1 et ST4-2) et deux dans ST7 (ST7-1 et ST7-2). Notamment, ST1-2 à ST1-4, ST3-3 à ST3-5, ST7-1 et ST7-2 représentent des variations nouvellement identifiées. La composition et les caractéristiques génétiques des sous-types chez les enfants de cette région suggèrent diverses sources d'infection, notamment la transmission interhumaine et animale.

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 detection of Enterocytozoon bieneusi in farmed Asiatic brush-tailed porcupines (Atherurus macrourus) and bamboo rats (Rhizomys pruinosus) from Hainan Province, China: Common occurrence, wide genetic variation and high zoonotic potential.

We investigated the occurrence and genotypic diversity of E. bieneusi in farmed Asiatic brush-tailed porcupines and bamboo rats from Hainan Province, China. Four hundred and sixty-seven fresh feces were collected from 164 Asiatic brush-tailed porcupines and 303 bamboo rats. DNA extraction from the feces and genotyping of E. bieneusi were performed by the amplification of the internal transcribed spacer (ITS) region of rDNA of E. bieneusi using PCR. A neighbor-joining tree was constructed based on the sequences obtained here and other sequences of E. bieneusi genotypes stored in Genbank. The total rate of infection with E. bieneusi was 32.5% (152/467), with 14.6% (24/164) in Asiatic brush-tailed porcupines and 42.2% (128/303) in bamboo rats infected. Seventeen genotypes of E. bieneusi were identified including 12 known genotypes, i.e., D (n = 78), Henan-III (n = 21), SHW7 (n = 19), KIN-1 (n = 11), ETMK5 (n = 7), TypeIV (n = 4), EbpD (n = 2), EbpA (n = 1), EbpC (n = 1), S7 (n = 1), HNPL-III (n = 1), HNR-VII (n = 1), and five novel genotypes named as HNZS-I (n = 1) and HNHZ-I to HNHZ-IV (n = 1 per genotype). Phylogenetic analysis revealed that all the genotypes found here except genotype S7 fell into Group 1. The present study demonstrated a relatively high prevalence of E. bieneusi infection (32.5%) and a large genetic variation of E. bieneusi (seventeen genotypes) in farmed Asiatic brush-tailed porcupines and bamboo rats in Hainan, China. The high proportion (78.3%) of zoonotic genotypes identified in the animals investigated here suggests that there is the potential for zoonotic or cross-species transmission which may pose a serious public health threat in the area. Public education on the management of Asiatic brush-tailed porcupines and bamboo rats should be implemented in the investigated areas.

Prevalence and distribution of subtypes of Blastocystis in Asiatic brush-tailed porcupines (Atherurus macrourus), bamboo rats (Rhizomys pruinosus), and masked palm civets (Paguma larvata) farmed in Hainan, China.

Blastocystis sp. is an important gastrointestinal parasite with global distribution, prevalent in humans, farmed animals, and wildlife. Therefore, this study aimed to investigate the prevalence and genetic diversity of Blastocystis sp. in Asiatic brush-tailed porcupines (Atherurus macrourus), bamboo rats (Rhizomys pruinosus), and masked palm civets (Paguma larvata) in Hainan Province, China. A total of 900 fecal samples were collected from three farmed animal species including 257 porcupines, 360 rats, and 283 civets. Genomic DNA was extracted from each fecal sample and Blastocystis sp. was detected by PCR at the small subunit ribosomal RNA (SSU rRNA) gene. A phylogenetic tree was constructed using the maximum likelihood method. Blastocystis sp. was detected in 47 (5.2%) fecal samples: 12 (4.7%) Asiatic brush-tailed porcupines, 8 (2.2%) bamboo rats, and 27 (9.5%) masked palm civets. Three known Blastocystis sp. subtypes, including ST1, ST4, ST5, and one unnamed subtype (unST), were found in one, 19, 26, and one animal, respectively. Subtypes ST4 and unST were detected in porcupines, ST4 in rats, and ST1 and ST5 in civets. Our results suggest that the three farmed animal species reported in this study could serve as reservoirs for potentially zoonotic Blastocystis sp. subtypes and transmit this parasite to humans, other farmed animals, and wildlife.

Title: Prévalence et répartition des sous-types de Blastocystis chez les athérures à longue queue (Atherurus macrourus), les rats des bambous (Rhizomys pruinosus) et les civettes masquées (Paguma larvata) élevés en Chine dans le Hainan. Abstract: Blastocystis sp. est un parasite gastro-intestinal important avec une distribution mondiale, répandu chez les humains, les animaux d'élevage et la faune. Par conséquent, cette étude visait à étudier la prévalence et la diversité génétique de Blastocystis sp. chez les athérures à longue queue (Atherurus macrourus), les rats des bambous (Rhizomys pruinosus) et les civettes masquées (Paguma larvata) dans la province de Hainan, en Chine. Au total, 900 échantillons fécaux ont été collectés sur ces trois espèces animales d'élevage dont 257 athérures, 360 rats et 283 civettes. L'ADN génomique a été extrait de chaque échantillon fécal et Blastocystis sp. a été détecté par PCR au niveau du gène de la petite sous-unité de l'ARN ribosomal. Un arbre phylogénétique a été construit en utilisant la méthode du maximum de vraisemblance. Blastocystis sp. a été détecté dans 47 (5,2 %) échantillons fécaux : 12 (4,7 %) athérures, 8 (2,2 %) rats et 27 (9,5 %) civettes. Trois sous-types de Blastocystis sp., dont ST1, ST4, ST5 et un sous-type sans nom (unST), ont été trouvés respectivement chez 1, 19, 26 et 1 animal. Les sous-types ST4 et unST ont été détectés chez les athérures, ST4 chez les rats et ST1 et ST5 chez les civettes. Nos résultats suggèrent que les trois espèces animales d'élevage concernées par cette étude pourraient servir de réservoirs à des sous-types potentiellement zoonotiques de Blastocystis sp. et transmettre ce parasite aux humains, à d'autres animaux d'élevage et à la faune.

Differential proteomics analysis of female and male adults of Angiostrongylus cantonensis.

In this study, we identified the differentially expressed proteins of female and male adults of Angiostrongylus cantonensis through differential proteomics. We extracted and purified total proteins from male and female adults, separated proteins by two-dimensional difference gel electrophoresis (2D-DIGE) in pH 4-7, analyzed the gel images by DeCyder 7.0 software, and sacrificed the infected rats to count the number of male and female adults. It was found 28 protein spots that were differentially expressed; seven protein spots were then identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Five proteins were up-regulated and two proteins down-regulated in male adults compared with female adults. Three of the five up-regulated proteins with known functions ascribed to them were identified as galectin-1, proteasome alpha subunit and peroxiredoxin. The two down-regulated proteins were identified as indoleamine dioxygenase like-myoglobin and galectin. Furthermore, the female was significantly greater than male adults (P<0.01) in the rats. The findings demonstrate the differences in protein expression profiles and the ability to survive in the final host between female and male adults of A. cantonensis, and may provide a theoretical basis to study their developmental biology further.

Upregulated galectin-1 in Angiostrongylus cantonensis L5 reduces body fat and increases oxidative stress tolerance.

BACKGROUND: Angiostrongylus cantonensis L5, parasitizing human cerebrospinal fluid, causes eosinophilic meningitis, which is attributed to tissue inflammatory responses caused primarily by the high percentage of eosinophils. Eosinophils are also involved in killing helminths, using the peroxidative oxidation and hydrogen peroxide (H2O2) generated by dismutation of superoxide produced during respiratory burst. In contrast, helminthic worms have evolved to attenuate eosinophil-mediated tissue inflammatory responses for their survival. In previous study, we demonstrated the extracellular function of Acan-Gal-1 in inducing the apoptosis of macrophages. Here, the intracellular functions of Acan-Gal-1 were investigated, aiming to further reveal the mechanism involved in A. cantonensis L5 worms surviving inflammatory responses in the human central nervous system. METHODS: In this study, a model organism, Caenorhabditis elegans, was used as a surrogate to investigate the intracellular functions of Acan-Gal-1 in protecting the worm from its host's immune attacks. First, structural characterization of Acan-Gal-1 was analyzed using bioinformatics; second, qRT-PCR was used to monitor the stage specificity of Acan-gal-1 expression in A. cantonensis. Microinjections were performed to detect the tissue specificity of lec-1 expression, the homolog of Acan-gal-1 in C. elegans. Third, microinjection was performed to develop Acan-gal-1::rfp transgenic worms. Then, oxidative stress assay and Oil Red O fat staining were used to determine the functions of Acan-Gal-1 in C. elegans. RESULTS: The results of detecting the stage specificity of Acan-gal-1 expression showed that Acan-Gal-1 was upregulated in both L5 and adult worms. Detection of the tissue specificity showed that the homolog of Acan-gal-1 in C. elegans, lec-1 was expressed ubiquitously and mainly localized in cuticle. Investigating the intracellular functions of Acan-Gal-1 in the surrogate C. elegans showed that N2 worms expressing pCe-lec-1::Acan-gal-1::rfp, with lipid deposition reduced, were significantly resistant to oxidative stress; lec-1 mutant worms, where lipid deposition increased, showed susceptible to oxidative stress, and this phenotype could be rescued by expressing pCe-lec-1::Acan-gal-1::rfp. Expressing pCe-lec-1::Acan-gal-1::rfp or lec-1 RNAi in fat-6;fat-7 double-mutant worms, where fat stores were reduced, had no significant effect on the oxidative stress tolerance. CONCLUSION: In C. elegans worms, upregulated Acan-Gal-1 plays a defensive role against damage due to oxidative stress for worm survival by reducing fat deposition. This might indicate the mechanism by which A. cantonensis L5 worms, with upregulated Acan-Gal-1, survive the immune attack of eosinophils in the human central nervous system.

Molecular-Based Detection of Enterocytozoon bieneusi in Farmed Masked Palm Civets (Paguma larvata) in Hainan, China: A High-Prevalence, Specificity, and Zoonotic Potential of ITS Genotypes.

Enterocytozoon bieneusi is a microsporidian and zoonotic species. This study investigated the prevalence and distribution of E. bieneusi genotypes in farmed masked palm civets using nested PCR, as well as assessed their zoonotic potential by phylogenetic analysis of the ITS region of the rRNA region. Here, we collected 251 fecal specimens from farmed masked palm civets (Paguma larvata) from the Hainan Island, China. In total, 128 of 251 samples were positive for E. bieneusi, with an average infection rate of 51.0%. Seventeen genotypes were identified including 12 known genotypes-HNR-VI (n = 56), SHR1 (n = 45), SHW7 (n = 6), KIN-1 (n = 3), D (n = 3), New1 (n = 3), EbpC (n = 2), CHC5 (n = 1), CHG19 (n = 1), CHN4 (n = 1), EbpA (n = 1), and Henan-III (n = 1)-and five novel genotypes (HNPL-I to HNPL-II; one each). Phylogenetic analysis categorized these genotypes into two groups. Thirteen of them were members of the zoonotic group 1, and the remaining four genotypes were in group 12. This study has shown that the infection rates of E. bieneusi in masked palm civets from Hainan were relatively high and provide baseline data to control and prevent microsporidiosis in farm-related communities. Therefore, infections in masked palm civets with zoonotic genotypes D, EbpC, CHN4, EbpA, KIN-1, and Henan-III should be considered potential threats to public health.

[Bacterial 16S rRNA genes and expression of IL-1ß, TNF-α and IgA in prostate tissues].

OBJECTIVE: To investigate the role of bacteria in the etiology of chronic prostatitis. METHODS: Complete prostate specimens were obtained at autopsy from 192 organ donors (aged 20 - 38 years old) during 2002 to 2008 who died of non-prostatic diseases. One tissue taken from the peripheral prostatic zone according to McNeal was divided into two pieces. One piece of tissue was taken for routine pathological examinations and immunohistochemical studies of interleukin (IL)-1ß, tumor necrosis factor-α (TNF-α) and IgA. Another one was taken for PCR assay to detect the bacterial 16S rRNA genes (16S rDNA). RESULTS: Of 192 prostate specimens, 64 (33.3%) had pathological changes of chronic prostatitis and 38 (19.8%) specimens was positive for bacterial 16S rDNA. Positive rates of 16S rDNA in chronic prostatitis and non-prostatitis specimens were 50.0% (32/64) and 4.6% (6/128) respectively (χ(2) = 55.185, P < 0.001). Expressions of IL-1ß, TNF-α and IgA in specimens of chronic prostatitis were significantly higher than those in non-prostatitis specimens (P < 0.001). A positive correlation could be found among three immunohistochemical indicators (P < 0.01). In 64 specimens with chronic prostatitis, a significant expression of IL-1ß, TNF-α and IgA was more often demonstrated in 16S rDNA positive group than in 16S rDNA negative group (P < 0.001). CONCLUSIONS: The up-regulations of bacterial 16S rDNA, cytokines and immunoglobulin A are involved in inflammatory response of chronic prostatitis. Bacterial infection may be an important cause of chronic prostatitis.

[Expressions of bacterial 16S rRNA, IL-1beta, TNF-alpha and NGF in prostate tissues].

OBJECTIVE: To investigate the role of bacteria in the etiology of chronic prostatitis. METHODS: A total of 162 complete prostate specimens were obtained at autopsy from organ donors (aged 20 -38 yr) who died of non-prostatic diseases. Each of the samples from the peripheral zone of the prostate was divided into two parts, one for routine pathological examination and immunohistochemical studies of interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha) and the nerve growth factor (NGF), and the other for PCR assay to detect the bacterial 16S rRNA gene (16S rDNA). RESULTS: Fifty-one (31.5%) of the total specimens presented pathological changes of chronic prostatitis, of which 44 had mild focal stromal, 5 mild focal stromal and periglandular and 2 mild focal periglandular inflammation. The positive rate of 16S rDNA was 19.1% (31/162), 51.0% (26/51) in the chronic prostatitis and 4.5% (5/111) in the non-prostatitis specimens (chi2 = 29.783, P < 0.01). In the specimens with chronic prostatitis, the expressions of IL-1beta, TNF-alpha and NGF were significantly higher in the 16S rDNA positive than in the 16S rDNA negative group (P < 0.01). CONCLUSION: Bacterial inflammation may play an important role in the etiology of chronic prostatitis.