Morphological and molecular characteristics of a Trypanosoma sp. from triatomines (Triatoma rubrofasciata) in China.

BACKGROUND: Triatomines (kissing bugs) are natural vectors of trypanosomes, which are single-celled parasitic protozoans, such as Trypanosoma cruzi, T. conorhini and T. rangeli. The understanding of the transmission cycle of T. conorhini and Triatoma rubrofasciata in China is not fully known. METHODS: The parasites in the faeces and intestinal contents of the Tr. rubrofasciata were collected, and morphology indices were measured under a microscope to determine the species. DNA was extracted from the samples, and fragments of 18S rRNA, heat shock protein 70 (HSP70) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) were amplified and sequenced. The obtained sequences were then identified using the BLAST search engine, followed by several phylogenetic analyses. Finally, laboratory infections were conducted to test whether Tr. rubrofasciata transmit the parasite to rats (or mice) through bites. Moreover, 135 Tr. rubrofasciata samples were collected from the Guangxi region and were used in assays to investigate the prevalence of trypanosome infection. RESULTS: Trypanosoma sp. were found in the faeces and intestinal contents of Tr. rubrofasciata, which were collected in the Guangxi region of southern China and mostly exhibited characteristics typical of epimastigotes, such as the presence of a nucleus, a free flagellum and a kinetoplast. The body length ranged from 6.3 to 33.9 µm, the flagellum length ranged from 8.7 to 29.8 µm, the nucleus index was 0.6 and the kinetoplast length was -4.6. BLAST analysis revealed that the 18S rRNA, HSP70 and gGAPDH sequences of Trypanosoma sp. exhibited the highest degree of similarity with those of T. conorhini (99.7%, 99.0% and 99.0%, respectively) and formed a well-supported clade close to T. conorhini and T. vespertilionis but were distinct from those of T. rangeli and T. cruzi. Laboratory experiments revealed that both rats and mice developed low parasitaemia after inoculation with Trypanosoma sp. and laboratory-fed Tr. rubrofasciata became infected after feeding on trypanosome-positive rats and mice. However, the infected Tr. rubrofasciata did not transmit Trypanosoma sp. to their offspring. Moreover, our investigation revealed a high prevalence of Trypanosoma sp. infection in Tr. rubrofasciata, with up to 36.3% of specimens tested in the field being infected. CONCLUSIONS: Our study is the first to provide a solid record of T. conorhini from Tr. rubrofasciata in China with morphological and molecular evidence. This Chinese T. conorhini is unlikely to have spread through transovarial transmission in Tr. rubrofasciata, but instead, it is more likely that the parasite is transmitted between Tr. rubrofasciata and mice (or rats). However, there was a high prevalence of T. conorhini in the Tr. rubrofasciata from our collection sites and numerous human cases of Tr. rubrofasciata bites were recorded. Moreover, whether these T. conorhini strains are pathogenic to humans has not been investigated.

New Variant of Vibrio parahaemolyticus, Sequence Type 3, Serotype O10:K4, China, 2020.

In 2020, a new serotype of Vibrio parahaemolyticus O10:K4 emerged and caused several outbreaks and sporadic cases in Guangxi, China. Phylogenetic analysis indicated that those strains are new variants of the sequence type 3 pandemic clone. The new serotype may become dominant, warranting enhanced investigations and surveillance.

Analysis of entire hepatitis B virus genomes reveals reversion of mutations to wild type in natural infection, a 15 year follow-up study.

It has been reported that some mutations in the genome of hepatitis B virus (HBV) may predict the outcome of the virus infection. However, evolutionary data derived from long-term longitudinal analysis of entire HBV genomes using next generation sequencing (NGS) remain rare. In this study, serum samples were collected from asymptomatic hepatitis B surface antigen (HBsAg) carriers from a long-term prospective cohort. The entire HBV genome was amplified by polymerase chain reaction (PCR) and sequenced using NGS. Twenty-eight time series serum samples from nine subjects were successfully analysed. The Shannon entropy (Sn) ranged from 0 to 0.89, with a median value of 0.76, and the genetic diversity (D) ranged from 0 to 0.013, with a median value of 0.004. Intrahost HBV viral evolutionary rates ranged from 2.39E-04 to 3.11E-03. Double mutations at nt1762(A â†’ T) and 1764(G â†’ A) and a stop mutation at nt1896(G â†’ A) were seen in all sequences from subject BO129 in 2007. However, in 2019, most sequences were wild type at these positions. Deletions between nt 2920-3040 were seen in all sequences from subject TS115 in 2007 and 2013 but these were not present in 2004 or 2019. Some sequences from subject CC246 had predicted escape substitutions (T123N, G145R) in the surface protein in 2004, 2013 and 2019 but none of the sequences from 2007 had these changes. In conclusion, HBV mutations may revert to wild type in natural infection. Clinicians should be wary of predicting long-term prognoses on the basis of the presence of mutations.

Modelling the Ecological Suitability of the Chagas Disease Vector Triatoma rubrofasciata- China.

WHAT IS ALREADY KNOWN ON THIS TOPIC?: Triatoma rubrofasciata is a potential vector that can transmit American trypanosomiasis and was widely recorded in South of China. WHAT IS ADDED BY THIS REPORT?: Because of the low density of the triatomines, more habitats have not been discovered. This study mainly focused on predicting the geographical distribution of T. rubrofasciata under current and future climatic conditions in China using the MaxEnt model. WHAT ARE THE IMPLICATIONS FOR PUBLIC HEALTH PRACTICE?: The result showed that the distribution of T. rubrofasciata was largely affected by annual mean temperature and possessed a high potential for expansion in southern China in the future. Our predictions are useful for targeting surveillance efforts in high-risk areas and increasing the efficiency and accuracy of public health investigations and vector control efforts in China.

High prevalence of Clonorchis sinensis infection in Guangxi, Southern China.

BACKGROUND: Soil-transmitted helminths (STHs), such as hookworm, roundworm and whipworm, and food-borne trematodiases, including Clonorchis sinensis, remain a public health problem worldwide, especially in tropical and subtropical regions. OBJECTIVE: We aimed to determine the current prevalence of these parasites in Guangxi, China, which is located in a subtropical region. METHODS: A cross-sectional study and a 4-year longitudinal surveillance study were carried out. Stool samples were collected and examined microscopically for parasite eggs using the modified Kato-Katz thick smear method. RESULTS: The study subjects selected using stratified random cluster sampling for the cross-sectional study and longitudinal surveillance study numbered 15,683 and 24,429, respectively. In the cross-sectional study, hookworm, roundworm, whipworm, pinworm, C. sinensis, and tapeworm were found. The total prevalence of soil-transmitted helminths (STHs) was 6.4% (95% CI, 6.0-6.8). The prevalences of C. sinensis, hookworm, roundworm, whipworm, and pinworm were 10.6%, 4.2%, 0.3%, 0.3%, and 1.8%, respectively. The prevalence of C. sinensis in males (14.0%, 95% CI, 13.3-14.8) was significantly higher than in females (7.2%, 95% CI, 6.7-7.8) (P = 0.0001). The prevalence also was significantly higher in the medical worker group (20.8%, 95% CI, 12.9-28.7) than in all other occupational groups (10.5%, 95% CI, 10.0-11.0) (P = 0.0001). The prevalence of hookworm in females (5.3%, 95% CI, 4.8-5.8) was significantly higher than in males (3.0%, 95% CI, 2.6-3.3) (P = 0.0001). In the longitudinal surveillance study, the prevalence of C. sinensis and STHs in 2016, 2017, 2018, and 2019 were 12.0%, 6.0%, 11.0%, and 10.0% and 2.6%, 2.8%, 1.5%, and 1.5%, respectively. CONCLUSIONS: Adult male and occupation of and medical workers are risk factors for infection with C. sinensis and hookworm. The prevalence rate of C. sinensis remains high while those of the other STHs are decreasing, suggesting that enhanced health education should be focused on C. sinensis in Guangxi.

Identification and Analysis of the Tegument Protein and Excretory-Secretory Products of the Carcinogenic Liver Fluke Clonorchis sinensis.

Liver fluke proteins, including excretory-secretory products (ESPs) and tegument proteins, are critical for the pathogenesis, nutrient metabolism, etiology and immune response of liver cancer. To understand the functions of various proteins in Clonorchis sinensis physiology and human clonorchiasis, the ESPs and tegument proteins of C. sinensis were identified. Supernatants containing ESPs from adult C. sinensis after culture for 6 h were harvested and concentrated. The tegument was detached using a freeze/thaw method and successively extracted using various extraction buffers. The outer surface proteins of C. sinensis were labeled with biotin, and the biotinylated proteins were purified. The ESP, tegument and labeled outer surface proteins were identified and analyzed by high-resolution LC-MS/MS. The identified proteins were compared with those of other flukes, and the protein functions associated with pathogenesis, carcinogenesis and potential vaccine antigens and drug targets were predicted and analyzed. A total of 175 proteins were identified after the 6-h culture of C. sinensis ESPs. A total of 352 tegument proteins were identified through sequential solubilization of the isolated teguments, and a subset of these proteins were localized to the surface membrane of the tegument by labeling with biotin. Thirty identified proteins, including annexins, actin and tetraspanins, were identified as potential immunomodulators and promising vaccine antigens. Interestingly, among the 352 tegument proteins, as many as 155 were enzymes, and most were oxidoreductases, hydrolases or transferases. A comparison of the outer surface proteins of C. sinensis with those of other flukes indicated that flukes have some common outer surface proteins, such as actin, tetraspanin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and annexin. Granulin, thioredoxin peroxiredoxin, carbonyl reductase 1 and cystatin were identified in the C. sinensis proteome and predicted to be related to liver disease and cancer. The analysis of the C. sinensis proteome could contribute to a more in-depth understanding of complex parasite-host relationships, improve the diagnosis of clonorchiasis and benefit research on the pathogenesis and development of novel interventions, drugs and vaccines to control C. sinensis infection.

Determination of the Transportation Limits of Heavy Crude Oil Using Three Combined Methods of Heating, Water Blending, and Dilution.

Conventional methods for pipeline transportation of heavy or extraheavy crude oils adopt heating, water blending, and dilution, and several methods are generally required to be used simultaneously to ensure normal transportation. However, how to determine the optimal transport boundary conditions for heavy oils is still one of the technical challenges. In this paper, the circulating piping experiment at different water contents (0-90 wt % with an interval of 10 wt %) and temperatures (65-90 °C with an interval of 5 °C) of three heavy oils from the Xinjiang oilfield is carried out. The apparent viscosity calculated from the experimental data of the circulating pipeline shows that when the water content is below the phase inversion point, the apparent viscosity increases and when the water content is close to the phase inversion point, the apparent viscosity increases nearly three times. Only when the water content is greater than the phase inversion point, the apparent viscosity shows a downward trend. Also, then, various viscosity prediction models with different independent variables, which mainly included temperature, water content, and dilution ratio, are selected and verified. Based on experimental data of six crude oils, a prediction model of the phase inversion point is established. Simultaneously, a method for determining the boundary conditions of heavy oils using the combined methods of heating, water blending, and dilution is proposed, while a set of simple decision diagrams of boundary conditions for heavy oil is also described. Finally, verified by the heavy oil pipeline of the Bohai LvDa oilfield, the gathering and transportation limits determined by this method are consistent with the operating parameters of the oilfield.

Distribution, genetic characteristics and public health implications of Triatoma rubrofasciata, the vector of Chagas disease in Guangxi, China.

BACKGROUND: Triatomines are natural vectors of Chagas disease and are mainly prevalent in the Americas. In China, previous data from decades ago showed that there were two species of triatomine bugs, Triatoma rubrofasciata and T. sinica. However, the distribution, genetic characteristics and public health implications of triatomines in China are still relatively unknown. In order to gain knowledge on the distribution, genetic characteristics and public health implications of the triatomines in Guangxi, China, an entomological-epidemiological study and genetic research was conducted. METHODS: Different methods were used to elucidate the distribution of triatomines in Guangxi including consultations with county-level Center for Disease Prevention and Control staff and village doctors, the distribution of educational material on triatomines though the internet and social media apps such as Wechat and QQ, and conducting manual inspections and light trapping to collect triatomines. The morphological characteristics of the collected triatomines were identified under light microscopy. The mitochondrial 16S rRNA, cytochrome b (cytb) genes and nuclear 28S rRNA gene were amplified, sequenced and used in phylogenetic analyses. RESULTS: A total of 305 triatomines were captured from 54 different sites in 13 cities in Guangxi. All collected bugs were identified as T. rubrofasciata based on morphology. Most triatomine collection sites were around or inside houses. Four triatomines bite cases were observed during the investigation indicating that triatomine bites are common, the bites can cause serious anaphylaxis and skin papules and urticaria, suggesting a systemic skin response. The 16S rRNA, 28S rRNA and cytb sequence analyses of T. rubrofasciata from Guangxi and other countries showed that T. rubrofasciata sequences from different regions exhibit a high similarity, with no geographical differences. The phylogenetic tree based on the 16S rRNA and cytb genes showed that T. rubrofasciata sequences from different regions and continents were in the same cluster, indicating no differentiation among different geographical populations. CONCLUSIONS: Our study showed that T. rubrofasciata is widely distributed in Guangxi and that people are commonly bitten by this insect in some regions. This highlights the need to enhance surveillance for and control of T. rubrofasciata and to strengthen the monitoring of imported Trypanosoma cruzi in China. The 16S rRNA, 28S rRNA and cytb sequence analyses of T. rubrofasciata from different regions and continents suggested that T. rubrofasciata populations exhibit high similarity, and the clustering in the phylogenetic analyses indicates that T. rubrofasciata has a close ancestor originating in the Americas.

Distribution of Triatomines, the Vector of Chagas Disease - Southern China, 2016-2018.

What is already known on this topic? Triatomines, also known as kissing bugs, are widespread vectors for Chagas disease which affects 6-8 million people worldwide. Two species of triatomines have been previously reported in China. What is added by this report? This study showed data from the first investigation of triatomine distribution in China. Triatoma rubrofasciata and a novel species of triatomine in 170 habitats in 30 cities in southern China were recorded in this investigation. What are the implications for public health practice? Considering the worldwide spread of Chagas disease and new species of trypanosomiasis, strengthening the monitoring of triatomines and their associated diseases in southern China is vital to prevent and control these diseases.

K13-propeller gene polymorphisms of Plasmodium falciparum and the therapeutic effect of artesunate among migrant workers returning to Guangxi, China (2014-2017).

BACKGROUND: The resistance of Plasmodium falciparum to artemisinin has been identified in Asia and some parts of Africa. The drug resistance of P. falciparum will be an obstacle to the successful elimination of malaria by 2025. Whole-genome sequencing of the artemisinin-resistant parasite line revealed mutations on the k13 gene associated with drug resistance in P. falciparum. To understand the artemisinin resistance of the imported P. falciparum cases from Africa, the mutations in the k13 gene in parasites from imported malaria cases in Guangxi Province were detected and the treatment efficiency of artesunate monotherapy was observed. METHODS: DNA was extracted from 319 blood samples from migrant workers with P. falciparum infection who returned to their hometown in Guangxi Province from Africa between 2014 and 2017. The k13-propeller gene was amplified by nested PCR, and sequencing, gene mutation frequency and geographic difference of imported P. falciparum cases were analysed by comparison with the wild-type strain. Of 319 patients, 158 were P. falciparum-infected and were treated with intravenous injection of artesunate and were observed, including the time of asexual stage clearance and the dose of artesunate used. RESULTS: Of the 319 P. falciparum samples, 12 samples had the k13-propeller mutation, and 11 point mutations were detected; 5 were non-synonymous mutations (T474I, A481T, A578S, V603E, G665S) and were not associated with artemisinin resistance. The clinical treatment observation showed that the median (IQR) dose of artesunate for peripheral blood parasite asexual stage clearance was 407.55 (360-510) mg, and the D3 parasite clearance rate was 70.25%, including the five k13-propeller mutations of P. falciparum. After 7 days of treatment, 98.73% of cases were cleared. Two cases were treated with artemisinin for 8 days with a 960-mg dose to completely clear the asexual parasite, but they did not have a mutation in the k13 gene. CONCLUSIONS: Five mutations of the k13-propeller gene in 319 P. falciparum samples from patients returning from Africa were identified. The frequency of the k13-propeller mutants was low, and the mutations were not strongly associated with artemisinin resistance. The median (IQR) dose of artesunate monotherapy in actual clinical treatment to remove asexual parasite stages was 407.55 (360-510) mg, equivalent to D3-D4. Some P. falciparum cases without a k13-propeller mutation showed obvious delayed clearance of the parasite from peripheral blood. Trial registration The diagnosis of malaria and the treatment of malaria-infected patients are the routine work of Centres for Disease Control and Prevention. Information on the patients was conveyed with the patient's approval, and the research aim, methods, risks and benefits of the study were explained in detail to the patients.

Correction to: First description of Echinococcus ortleppi infection in China.

Following publication of the original article [1], the authors reported an error in Table 2.

First description of Echinococcus ortleppi infection in China.

BACKGROUND: Echinococcosis has led to considerable social and economic losses in China, particularly in the endemic communities of the eastern Tibetan Plateau. In China, human cases of Echinococcus granulosus (sensu stricto), E. canadensis and E. multilocularis infections have been described, but no E. ortleppi (G5) infections in humans or animals have been reported. RESULTS: A case of E. ortleppi infection in a human from Guangxi, which is a non-endemic echinococcosis area in China, is described. A 17 × 12 × 20 cm (diameter) cyst was observed in the liver of the patient, and Echinococcus larvae were collected from the cyst. A morphological examination indicated that the larvae were E. ortleppi, and amplification and analysis of the nicotinamide adenine dinucleotide hydrogenase dehydrogenase subunit 1 (nad1) and cytochrome c oxidase subunit 1 (cox1) genes showed that the larvae had 99-100% homology with the corresponding E. ortleppi sequences on GenBank. CONCLUSIONS: To our knowledge, this report describes the first identification of a human E. ortleppi infection in China. Our data broaden the geographical distribution of this rarely reported species of Echinococcus.

Long Noncoding RNA Profiling from Fasciola Gigantica Excretory/Secretory Product-Induced M2 to M1 Macrophage Polarization.

BACKGROUND/AIMS: Long noncoding RNAs (lncRNAs) are well known regulators of gene expression that play essential roles in macrophage activation and polarization. However, the role of lncRNA in Fasciola gigantica excretory/secretory products (ESP)-induced M2 polarization into M1 macrophages is unclear. Herein, we performed lncRNA profiling of lncRNAs and mRNAs during the ESP-induced macrophage polarization process. METHODS: F. gigantica ESP was used to induce peritoneal cavity M2 macrophages in BALB/c mice (5-6 weeks old) in vivo, and these cells were subsequently isolated and stimulated with IFN-γ + LPS to induce M1 cells in vitro. LncRNA and mRNA profiling was performed via microarray at the end of both polarization stages. RESULTS: In total, 2,844 lncRNAs (1,579 upregulated and 1,265 downregulated) and 1,782 mRNAs (789 upregulated and 993 downregulated) were differentially expressed in M2 macrophages compared to M1 macrophages, and six lncRNAs were identified during polarization. We selected 34 differentially expressed lncRNAs and mRNAs to validate the results of microarray analysis using quantitative real-time PCR (qPCR). Pathway and Gene Ontology (GO) analyses demonstrated that these altered transcripts were involved in multiple biological processes, particularly peptidase activity and carbohydrate metabolism. Furthermore, coding and non-coding gene (CNC) and mRNA-related ceRNA network analyses were conducted to predict lncRNA expression trends and the potential target genes of these lncRNAs and mRNAs. Moreover, we determined that four lncRNAs and four mRNAs might participate in F. gigantica ESP-induced M2 polarization into M1 macrophages. CONCLUSIONS: This study illustrates the basic profiling of lncRNAs and mRNAs during F. gigantica ESP-induced M2 polarization into M1 macrophages and deepens our understanding of the mechanism underlying this process.

Corrigendum: Pathogenicity of Human ST23 Streptococcus agalactiae to Fish and Genomic Comparison of Pathogenic and Non-pathogenic Isolates.

[This corrects the article on p. 1933 in vol. 8, PMID: 29056932.].

Pathogenicity of Human ST23 Streptococcus agalactiae to Fish and Genomic Comparison of Pathogenic and Non-pathogenic Isolates.

Streptococcus agalactiae, or Group B Streptococcus (GBS), is a major pathogen causing neonatal sepsis and meningitis, bovine mastitis, and fish meningoencephalitis. CC23, including its namesake ST23, is not only the predominant GBS strain derived from human and cattle, but also can infect a variety of homeothermic and poikilothermic species. However, it has never been characterized in fish. This study aimed to determine the pathogenicity of ST23 GBS to fish and explore the mechanisms causing the difference in the pathogenicity of ST23 GBS based on the genome analysis. Infection of tilapia with 10 human-derived ST23 GBS isolates caused tissue damage and the distribution of pathogens within tissues. The mortality rate of infection was ranged from 76 to 100%, and it was shown that the mortality rate caused by only three human isolates had statistically significant difference compared with fish-derived ST7 strain (P < 0.05), whereas the mortality caused by other seven human isolates did not show significant difference compared with fish-derived ST7 strain. The genome comparison and prophage analysis showed that the major genome difference between virulent and non-virulent ST23 GBS was attributed to the different prophage sequences. The prophage in the P1 region contained about 43% GC and encoded 28-39 proteins, which can mediate the acquisition of YafQ/DinJ structure for GBS by phage recombination. YafQ/DinJ belongs to one of the bacterial toxin-antitoxin (TA) systems and allows cells to cope with stress. The ST23 GBS strains carrying this prophage were not pathogenic to tilapia, but the strains without the prophage or carrying the pophage that had gene mutation or deletion, especially the deletion of YafQ/DinJ structure, were highly pathogenic to tilapia. In conclusion, human ST23 GBS is highly pathogenic to fish, which may be related to the phage recombination.

Clonorchis sinensis infection and co-infection with the hepatitis B virus are important factors associated with cholangiocarcinoma and hepatocellular carcinoma.

To evaluate the contributions of Clonorchis sinensis and hepatitis B virus to the development of cholangiocarcinoma (ICC) and hepatocellular carcinoma (HCC), C. sinensis and hepatitis B virus infections in 20 clinical liver cancer cases from a C. sinensis- and hepatitis B virus-epidemic region were detected. Eight cases of ICC, 11 cases of HCC and one mixed ICC and HCC case were verified by CT, pathological section and (or) observations during surgery. The C. sinensis infection was detected by stool microscopy and ELISA, and the worms and eggs found during surgery and in pathological sections also allowed for diagnoses. Hepatitis B virus infections were detected by ELISA. In the 20 cases, 18 patients were diagnosed with C. sinensis infections. Eight of the 20 patients were infected with the hepatitis B virus, and seven were co-infected with C. sinensis. In the eight ICC patients, seven were diagnosed with C. sinensis infection, and two had mixed infections with the hepatitis B virus. In the 11 HCC patients, 10 were diagnosed with C. sinensis, four had mixed infections with the hepatitis B virus, and only one HCC patient presented a single infection by the hepatitis B virus. These clinical observations revealed that C. sinensis infection and C. sinensis co-infection with the hepatitis B virus are important factors in ICC and HCC.

TLR2 signal influences the iNOS/NO responses and worm development in C57BL/6J mice infected with Clonorchis sinensis.

BACKGROUND: Although the responses of inducible nitric oxide synthase (iNOS) and associated cytokine after Clonorchis sinensis infection have been studied recently, their mechanisms remain incompletely understood. In this study, we investigated the effects of toll-like receptor 2 (TLR2) signals on iNOS/nitric oxide (NO) responses after C. sinensis infection. We also evaluated the correlations between iNOS responses and worm development, which are possibly regulated by TLR2 signal. METHODS: TLR2 wild-type and mutant C57BL/6 J mice were infected with 60 C. sinensis metacercariae, and the samples were collected at 30, 60, 90 and 120 days post-infection (dpi). The total serum NO levels were detected using Griess reagent after nitrate was reduced to nitrite. Hepatic tissue samples from the infected mice were sliced and stained with hematoxylin and eosin (HE) to observe worm development in the intrahepatic bile ducts. The iNOS mRNA transcripts in the splenocytes were examined by real time reverse transcriptase polymerase chain reaction (qRT-PCR), and iNOS expression was detected by immunohistochemistry. RESULTS: Developing C. sinensis juvenile worms were more abundant in the intrahepatic bile ducts of TLR2 mutant mice than those of TLR2 wild-type mice. However, no eggs were found in the faeces of both mice samples. The serum levels of total NO significantly increased in TLR2 mutant mice infected with C. sinensis at 30 (t (5) = 2.595, P = 0.049), 60 (t (5) = 7.838, P = 0.001) and 90 dpi (t (5) = 3.032, P = 0.029). Meanwhile, no changes occurred in TLR2 wild-type mice compared with uninfected controls during the experiment. The iNOS expression in splenocytes showed unexpected higher background levels in TLR2 mutant mice than those in TLR2 wild-type mice. Furthermore, the iNOS mRNA transcripts in splenocytes were significantly increased in the TLR2 wild-type mice infected with C. sinensis at 30 (t (5) = 5.139, P = 0.004), 60 (t (5) = 6.138, P = 0.002) and 90 dpi (t (5) = 6.332, P = 0.001). However, the rising of iNOS transcripts dropped under the uninfected control level in the TLR2 mutant mice at 120 dpi (t (5) = -9.082, P < 0.0001). Both total NO and iNOS transcripts were significantly higher in the TLR2 mutant mice than those in the TLR2 wild-type mice at 30 (t (5) = 3.091/2.933, P = 0.027/0.033) and 60 dpi (t (5) = 2.667/6.331, P = 0.044/0.001), respectively. In addition, the remarkable increase of iNOS expressions was immunohistochemically detected in the splenic serial sections of TLR2 wild-type mice at 30 and 60 dpi. However, the expressions of iNOS were remarkably decreased in the splenocytes of both TLR2 wild-type and mutant mice at 120 dpi. CONCLUSIONS: These results demonstrate that TLR2 signal plays an important role in the regulation of iNOS expression after C. sinensis infection. TLR2 signal is also beneficial to limiting worm growth and development and contributing to the susceptibility to C. sinensis in which the iNOS/NO reactions possibly participate.

[Establishment of Duplex PCR for Identifying Metagonimus yokogawai and Haplorchis taichui].

OBJECTIVE: To develop a duplex PCR method for identifying Metagonimus yokogawai and Haplorchis taichui. METHODS: ITS1 sequences of M. yokogawai and H. taichui, as well as those of their homologous species were obtained from GenBank, and two sets of specific primer pairs for M. yokogawai and H. taichui were designed accordingly using Primer Premier 5.0 software. PCR reaction system and conditions were optimized. The established duplex PCR method was applied in a pool of M. yokogawai, H. taichui, and 17 related species to examine its specificity. Sensitivity was evaluated through serial dilutions of plasmids containing their specific sequences. Finally, the duplex PCR was applied to identify M. yokogawai and H. taichui among trematodes collected from the viscera of 47 cats and 40 dogs to test its practicality. RESULTS: The duplex PCR method amplified target sequences of M. yokogawai and H. taichui, generating 648 bp and 279 bp products, respectively. No cross reaction was found with the following 17 related species: Haplorchis pumilio, Clonorchis sinensis, Pharyngostomum cordatum, the metacercaria of Metorchis sp. and Exorchis sp., Echinochasmus liliputanus, Echinochasmus perfoliatus, Echinostoma friedi, Hypoderaeum conoideum, Holostephanus sp., Diplodiscus sp., Anisakis sp., Metorchis orientais, Paragonimus westermani, Watsonius watsoni, Notocotylus sp. and Hysterothylacium sp, indicating a high specificity of this method. The detection limits for DNAs of M. yokogawai and H. taichui were 1.49 x 10(-1) pg and 1.14 x 10(-1) pg, suggesting a good sensitivity for this method. Further, the duplex PCR successfully identified M. yokogawai and H. taichui from cat and dog viscera, with no cross amplification of other trematodes. CONCLUSION: The duplex PCR is effective in identifying Metagonimus yokogawai and Haplorchis taichui.

Occurrence and Characterization of Cronobacter spp. in Dehydrated Rice Powder from Chinese Supermarket.

Cronobacter spp. are emerging food-borne pathogens and have been identified as causative agents of meningitis and necrotizing enterocolitis in infants. Dehydrated rice is popular with a wide range of people and it is frequently used as a substitute for infant milk powder to baby older than four months. The occurrence of Cronobacter spp. was investigated in 1,012 samples of dehydrated rice powder collected from 14 manufacturers in China during 2010 to 2012. The isolates were identified using fusA allele sequencing and subtyped using pulsed-field gel electrophoresis. Seventy-six samples (7.5%) contained Cronobacter spp. The prevalence among manufacturers ranged from 0-28.8%. The 76 isolates included 4 species [Cronobacter sakazakii (52 isolates) Cronobacter malonaticus (14 isolates), Cronobacter dublinensis (7 isolates), and Cronobacter muytjensii (3 isolates)]. Twenty-three unique fusA alleles and sixty-six PFGE-patterns were detected. All isolated strains were observed to be sensitive or to show intermediate susceptibility to eight tested antimicrobial agents. The study revealed serious contamination of dehydrated rice powder by Cronobacter spp., with prevalence varying among manufacturers in China. Identified Cronobacter species, fusA alleles, and subtypes were diverse.