Hong Ling

Distribution and Genetic Characterizations of Cryptosporidium spp. in Pre-Weaned Dairy Calves in Northeastern China’s Heilongjiang Province

Background Cryptosporidium spp. are common parasites of humans and animals. Farm animals, especially pre-weaned calves, are considered to be one of main animal reservoir hosts of Cryptosporidium in the transmission of human cryptosporidiosis. The aim of this study was to determine the distribution and genotypes of Cryptosporidium spp. in pre-weaned calves using molecular tools and to assess zoonotic transmission and elucidate the public health significance in northeastern China. Methodology/Principal Findings A total of 151 fecal specimens from pre-weaned calves were collected in Heilongjiang Province and were screened for Cryptosporidium by PCR. The average prevalence of Cryptosporidium was 47.68% (72/151). Cryptosporidium spp. were characterized by DNA sequencing of the small subunit (SSU) rRNA gene and the 60-kDa glycoprotein (gp60) gene. Based on the SSU rRNA gene, five Cryptosporidium spp. were identified, including C. bovis (n = 34), C. andersoni (n = 26), C. ryanae (n = 5), C. meleagridis (n = 5) and C. parvum (n = 2). The SSU rRNA nucleotide sequences were identical to each other, respectively, within C. ryanae, C. parvum, C. meleagridis and C. andersoni. Four types of C. bovis were found in the SSU rRNA gene, with two novel types. The gp60 gene was successfully sequenced in one C. parvum isolate and three C. meleagridis isolates, with IIdA19G1 for C. parvum and IIIeA22G2R1 for C. meleagridis. Conclusion/Significance Molecular analysis indicates that Cryptosporidium spp. are endemic in pre-weaned calves in Heilongjiang Province. The findings of C. parvum and C. meleagridis suggested the possibility of zoonotic transmission and public health significance. The transmission dynamics of C. parvum and C. meleagridis needed to be clarified by further molecular epidemiologic studies from humans and animals. Whether calves could act as the natural reservoirs of C. meleagridis needed to be confirmed by more systematic experimental infection studies.

Genetic characterizations of Giardia duodenalis in sheep and goats in Heilongjiang Province, China and possibility of zoonotic transmission.

Background Giardia duodenalis is a widespread intestinal protozoan of both humans and mammals. To date, few epidemiological studies have assessed the potential and importance of zoonotic transmission; and the human giardiasis burden attributable to G. duodenalis of animal origin is unclear. No information about occurrence and genotyping data of sheep and goat giardiasis is available in China. The aim of the present study was to determine prevalence and distribution of G. duodenalis in sheep and goats in Heilongjiang Province, China, and to characterize G. duodenalis isolates and assess the possibility of zoonotic transmission. Methodology/Principal Findings A total of 678 fecal specimens were collected from sheep and goats on six farms ranging in age from one month to four years in Heilongjiang Province, China. The average prevalence of G. duodenalis infection was 5.0% (34/678) by microscopy after Lugols iodine staining, with 5.6% (30/539) for the sheep versus 2.9% (4/139) for the goats. Molecular analysis was conducted on 34 G. duodenalis isolates based on the triosephosphate isomerase (tpi) gene. 29 tpi gene sequences were successfully obtained and identified as assemblages A (n = 4), B (n = 2) and E (n = 23). High heterogeneity was observed within assemblage E at the tpi locus, with five novel subtypes found out of seven subtypes. Two subtypes of assemblage A were detected, including subtype AI (n = 3) and a novel subtype (designated as subtype AIV) (n = 1). Two assemblage B isolates were identical to each other in the tpi gene sequences. Conclusions/Significance This is the first report of G. duodenalis infections in sheep and goats in China. The present data revealed the unique endemicity on prevalence, distribution and genetic characterization of G. duodenalis in sheep and goats in Heilongjiang Province. The findings of assemblages A and B in sheep and goats implied the potential of zoonotic transmission.

MiR-122 modulates type I interferon expression through blocking suppressor of cytokine signaling 1

MiR-122 is a liver-specific miRNA. Recent studies demonstrated that the interferon (IFN) therapy efficacy is poor in the hepatitis C virus (HCV)-infected patients with lower miR-122 abundance in the livers. The hepatocarcinoma patients also have low miR-122 levels in their livers. We previously found that the IFN expression was reduced when miR-122 was knocked down in human oligodendrocytes. The mechanism is unclear. In this study, the miR-122-abundant cell Huh7 was used to explore the regulatory mechanism of miR-122 on type I IFN expression. We found that miR-122 significantly increased the type I IFN expression in Huh7 cells, while knocking down miR-122 decreased the type I IFN expression. By screening potential miR-122 targets among the negative regulators in IFN signaling pathways, we found that there were putative miR-122 targets in the suppressor of cytokine signaling 1 (SOCS1) mRNA. Over-expressing miR-122 decreased the SOCS1 expression by 50.55% in Huh7 cells, while knocking down miR-122 increased SOCS1 expression by 62.56%. Using a green fluorescence protein (EGFP) fused SOCS1-expressing plasmid, the SOCS1-EGFP fluorescence intensity and protein were lower in miR-122 mimic-treated cells than those in mock-miRNA-treated cells, while miR-122 knockdown significantly increased the SOCS1-EGFP fluorescence intensity and protein expression. Mutations in the nt359-nt375 region abandoned the impact of miR-122 on SOCS1-EGFP expression. Taken together, SOCS1 is a target of miR-122. MiR-122 can regulate the type I IFN expression through modulating the SOCS1 expression.

Molecular identification and distribution of Cryptosporidium and Giardia duodenalis in raw urban wastewater in Harbin, China.

Contamination of the water supply by protozoa often causes outbreaks of cryptosporidiosis and giardiasis. The goals of the present study was to investigate the level of Cryptosporidium and Giardia duodenalis in wastewater from wastewater treatment plants in Harbin, China, and to understand the endemic transmission characteristics of cryptosporidiosis and giardiasis. Forty-eight domestic wastewater specimens from the two wastewater treatment plants in Harbin City were collected from April 2009 to March 2010. Cryptosporidium spp. and G. duodenalis assemblages were identified by PCR and sequencing of the 18S ribosomal RNA and the triosephosphate isomerase genes, respectively. In total, 15 wastewater specimens were PCR positive for Cryptosporidium and 23 were PCR positive for G. duodenalis. The prevalence of contamination with G. duodenalis (47.9%) was higher than that of Cryptosporidium (31.3%). Molecular identification showed the presence of two Cryptosporidium spp. (14 belonging to Cryptosporidium andersoni and one belonging to Cryptosporidium ubiquitum) and two G. duodenalis assemblages (18 belonging to assemblage AII and six belonging to assemblage B). In addition, eight specimens contained both Cryptosporidium and G. duodenalis, and one specimen contained G. duodenalis assemblages AII and B. These results suggested humans might be the primary source of G. duodenalis contamination in wastewater in the studied area. In contrast, a low prevalence of C. ubiquitum suggested a reduced risk of human cryptosporidiosis caused by C. ubiquitum via waterborne route. This work provides basic experimental data needed for local wastewater treatment plants to develop protective strategies for water safety and to eliminate waterborne parasites.

Modulation of miR-122 on persistently Borna disease virus infected human oligodendroglial cells

Using RNAhybrid software we found the predicted binding of complementary sequences between miR-122 and viral mRNAs, may be important for the antiviral effect of miR-122 on Borna disease virus (BDV). A moderate expression of miR-122 was identified in human oligodendroglial cells (OL), but with a much lower level of miR-122 in BDV persistent infection (OL/BDV) and cells transfected with BDV gene expression vectors. Over-expression of miR-122 and specific blocking experiments demonstrated that miR-122 was able to specifically inhibit BDV protein synthesis, viral gene replication and transcription, and induce the secretion/synthesis of interferon (IFN) in OL and OL/BDV cells. The abolishment of miR-122 by AMO-122 inhibited endogenous IFN induction by IFN-beta. These results indicate that miR-122 can exert direct antiviral function by inhibiting BDV translation and replication on one hand, while acting indirectly through IFN to increase the host innate immunity to modulate the virus-host interactions on the other hand.

Genotyping of Enterocytozoon bieneusi in Farmed Blue Foxes (Alopex lagopus) and Raccoon Dogs (Nyctereutes procyonoides) in China

Enterocytozoon bieneusi is the most common species of microsporidia found both in humans and animals. Farmed animals, particularly closely associated to humans, may play an important role of zoonotic reservoir in transmitting this disease to humans. The fur industry is a major economic component in some parts of China. To understand the prevalence, genotype variety and zoonotic risk of E. bieneusi in farmed foxes and raccoon dogs, two species of fur animals, fecal specimens of 110 blue foxes and 49 raccoon dogs from Heilongjiang and Jilin provinces in China were examined by internal transcribed spacer (ITS)-based PCR. E. bieneusi was detected in 16.4% (18/110) blue foxes and 4.1% (2/49) raccoon dogs. Altogether, four genotypes of E. bieneusi were identified, including two known genotypes D (n = 13) and EbpC (n = 5), and two novel genotypes named as CHN-F1 (n = 1) in a fox and CHN-R1 (n = 1) in a raccoon dog. Phylogenetic analysis revealed that all the four genotypes were the members of zoonotic group 1. Genotypes D and EbpC were found in humans previously. The findings of zoonotic genotypes of E. bieneusi in the foxes and raccoon dogs suggest these animals infected with E. bieneusi may pose a threat to human health.

Borna disease virus encoded phosphoprotein inhibits host innate immunity by regulating miR-155

It has been reported that the Borna disease virus (BDV) encoded phosphoprotein (P protein) can inhibit the activity of Traf family member-associated NF-kappaB activator (TANK)-binding kinase 1 (TBK-1), thus preventing the induction of type I interferon (IFN). However, the effects of microRNA on the regulation of BDV infection and the hosts immune response have not been characterized. miR-155 was predicted to be complementary to the BDV P mRNA by RNAhybrid software. Here, we showed that miR-155 was down-regulated in BDV persistently infected human oligodendroglial (OL/BDV) cells and that the BDV P protein, but not the X protein, directly inhibited miR-155 expression in cells. When miR-155 was over-expressed, the inhibition of type I IFNs by BDV in cells was reversed, and the expression of type I IFNs was increased. When miR-155 expression was specifically blocked, cellular IFN expression and the induction of IFN by poly I:C treatment were suppressed. Furthermore, miR-155 promoted type I IFN production by targeting suppressor of cytokine signaling 1 (SOCS1) and SOCS3. Mutations in the nt1138-nt1158 region of SOCS3 abandoned the impact of miR-155 on the expression of SOCS3-enhanced green fluorescent protein (EGFP). The levels of BDV P mRNA and protein were significantly decreased in OL/BDV cells when miR-155 was over-expressed; however, miR-155-mutation did not affect the expression of BDV P-EGFP. Thus, BDV persistent infection inhibited the expression of type I IFNs through the suppression of miR-155, and miR-155 played an important immune regulatory role in BDV persistent infection.

A Combination of the Kato-Katz Methods and ELISA to Improve the Diagnosis of Clonorchiasis in an Endemic Area, China

Background Examination of feces by light microscopy is widely used for specific parasitological diagnosis of clonorchiasis. However, the true incidence of infection is underestimated owing to the high missing diagnosis rate of this method. The enzyme-linked immunosorbent assay (ELISA) is widely used for the detection and control of clonorchiasis but the practicality of this method is unclear. The purpose of this study was to evaluate the effect of ELISA as a supplementary method for the diagnosis of clonorchiasis. Methodology/Principal Findings The present study recruited 2,359 clinically suspected patients from Heilongjiang Province, China. In all, 954 cases were identified as antibody-positive by immunoglobulin (IgG)-ELISA and 495 individuals were diagnosed as egg-positive by the Kato-Katz (KK) method. The seropositive and egg-negative individuals were re-examined by repeated egg counts and/or the number of KK slides and 18 (18.18%) cases were confirmed as clonorchiasis. The 40.44%, antibody-positive rate determined by IgG-ELISA was significantly higher (P<0.05) than the 21.75% egg-positive rate found by examination of feces. A Bayesian approach indicated that the prevalence of clonorchiasis in this region was 22.27% and that the sensitivity, specificity, positive predictive value and negative predictive value of IgG-ELISA were 98.7%, 76.53%, 54.66% and 99.52%, respectively. The agreement between the two methods was moderate (kappa value = 0.564). The clonorchiasis patients lived mainly along the Songhua River. The risk factors, except for ethnic factors, were estimated effectively by both methods. Conclusions/Significance The present study suggested that clonorchiasis was widely distributed in Heilongjiang Province, China. The missing diagnosis rate was high using the KK technique alone. The combination of immunological methods and parasitological techniques could improve diagnostic accuracy and reduce the missing diagnosis rate. ELISA used as an auxiliary diagnostic method was realistic and practical for a large-scale screening test, monitoring the prevalence and assessing the risk factors of clonorchiasis.

Genetic Analysis of the Gdh and Bg Genes of Animal-Derived Giardia duodenalis Isolates in Northeastern China and Evaluation of Zoonotic Transmission Potential

Background Giardia duodenalis is a common intestinal parasite that infects humans and many other mammals, mainly distributing in some areas with poor sanitation. The proportion of the human giardiasis burden attributable to G. duodenalis of animal origin differs in different geographical areas. In Mainland China, genetic data of the gdh and bg genes of G. duodenalis from animals are only limited in dogs and cats. The aim of the study was to provide information on the genetic characterizations of animal-derived G. duodenalis isolates (from rabbits, sheep and cattle) at both loci in Heilongjiang Province, Northeastern China, and to assess the potential for zoonotic transmission. Methodology/Principal Findings 61 G. duodenalis isolates from animal feces (dairy and beef cattle, sheep and rabbits) in Heilongjiang Province were characterized at the gdh and bg loci in the present study. The gdh and bg gene sequences of sheep-derived G. duodenalis assemblage AI, and the gdh sequences of rabbit-derived G. duodenalis assemblage B had 100% similarity with those from humans, respectively. Novel subtypes of G. duodenalis were identified, with one and seven subtypes for assemblages A and E at the gdh locus, and two and three subtypes for assemblages B and E at the bg locus, respectively. Three pairs of the same bg sequences of assemblage E were observed in sheep and cattle. Conclusions/Significance This is the first description of genetic characterizations of the gdh and bg genes of G. duodenalis from rabbits, sheep and cattle in Mainland China. Homology analysis of assemblages AI and B implied the possibility of zoonotic transmission. The novel subtypes of assemblages of G. duodenalis may represent the endemic genetic characteristics of G. duodenalis in Heilongjiang Province, China.

Subtyping of Cryptosporidium cuniculus and genotyping of Enterocytozoon bieneusi in rabbits in two farms in Heilongjiang Province, China

Cryptosporidium spp. and Enterocytozoon bieneusi are two prevalent opportunistic pathogens in humans and animals. Currently, few data are available on genetic characterization of both pathogens in rabbits in China. The aim of the present study was to understand prevalence and genetic characterization of Cryptosporidium spp. and E. bieneusi in rabbits. We collected 215 fecal samples from 150 Rex rabbits and 65 New Zealand White rabbits on two different farms in Heilongjiang Province, China. Cryptosporidium spp. and E. bieneusi were tested by polymerase chain reaction (PCR) and sequencing the partial small subunit of ribosomal DNA (SSU rDNA) and the internal transcribed spacer (ITS) region of rDNA, respectively. Cryptosporidium was detected in 3.3% (5/150) of Rex rabbits and 29.2% (19/65) of New Zealand White rabbits. All the 24 Cryptosporidium isolates were identified as C. cuniculus. Enterocytozoon bieneusi was only found in 14.7% (22/150) of Rex rabbits. Five known genotypes: CHN-RD1 (n = 12), D (n = 3), Type IV (n = 2), Peru6 (n = 1), and I (n = 1), and three novel ones CHN-RR1 to CHN-RR3 (one each) were detected. By analyzing the 60-kDa glycoprotein (gp60) gene sequences of C. cuniculus isolates, three subtypes were obtained: VbA28 (n = 2), VbA29 (n = 16), and VbA32 (n = 3). All these three C. cuniculus subtypes were reported previously in humans. Four known E. bieneusi genotypes have been found to be present in humans. The three novel ones fell into zoonotic group 1. The results suggest zoonotic potential of C. cuniculus and E. bieneusi isolates in rabbits.