Dana Huang

Epidemiological study on Clonorchis sinensis infection in Shenzhen area of Zhujiang delta in China.

To study the transmission route and epidemiological features of Clonorchis sinensis infection in Shenzhen area, which is the biggest immigration city in the south of China, we examined 1,473 individuals (710 males and 763 females) to assess the current status of C. sinensis infection among the people in a village of Shenzhen in Zhujiang delta of Guangdong province, China. Freshwater snails, 630, of different species known as the first intermediate host of C. sinensis were collected and examined for cercaria infection, and 430 freshwater fishes of different species as the second intermediate host were examined for metacercaria infection. Among 1,473 people examined, 70 (4.75%) were found infected with C. sinensis. By counting eggs per gram feces (EPG), it was found that the intensity of infection in males was stronger than that of females, and the average EPG was 41.87 in all population. Snails, 1.15%, were infected with cercariae of C. sinensis. The average infection rate of freshwater fishes of 15 species with metacercariae of C. sinensis was 16.97%, and the carps reached the highest infection rate (40.74%). A questionnaire was designed with 12 questions covering socioeconomic conditions and human behavior, contamination of the environment, and fishponds. Of 1,473 interviewees, 54% did not know about fluke disease or its transmission route, 12% of those who knew about the fluke believed that the infection causes no harm or only slight harm to their health. Of the interviewees, 27%, ate raw fish at least one to two times per month. Of families, 5% used the same utensils for both raw fish and cooked food. Of the fishpond owners, 40% fed their fishes with feces of domestic animals and humans. All these factors of unhealthy behaviors, poor knowledge, inappropriate farming/fishery practices, and eating raw fish have made the prevalence of clonorchiasis increase in humans in the Shenzhen area. It is urgent to perform a control program, including health education, environmental modification, reform of traditional farming/fishery practice, mass screening, and chemotherapy for humans, and the management of domestic animals to decrease C. sinensis infection in the human population in Shenzhen.

FOXO3a is involved in the apoptosis of naked oocytes and oocytes of primordial follicles from neonatal rat ovaries

Inhibition of the forkhead transcription factor, FOXO3a, can promote the transition from primordial to primary follicle and subsequent follicle development in mammalian ovaries. Stem cell factor (SCF) initiates anti-apoptotic signaling from its membrane receptor, c-kit, to Bcl-2 family members through PI3K/AKT in oocytes of primordial follicles. However, whether FOXO3a mediates the apoptosis of naked oocytes and oocytes of primordial follicles remains unknown. In the present study, oocytes from nests and primordial follicles from neonatal rat ovaries were cultured, and oocyte apoptosis was examined using the TUNEL technique. The pro-apoptotic action of FOXO3a and the potential signal transduction pathways were investigated using RT-PCR, Western blot, and immunocytochemistry. Culturing oocytes in the presence of SCF did not affect the level of total FOXO3a protein, but rapidly elevated the level of phosphorylated FOXO3a (indicating functional suppression). As phosphorylated FOXO3a increased, oocyte apoptosis was inhibited. The specific PI3K/Akt inhibitor, LY 294002, abolished the phosphorylation of FOXO3a and the anti-apoptotic action of SCF. SCF down-regulated the expression of p27KIP1 and pro-apoptotic factors such as Bim, Bad, and Bax, and this activity was reversed by LY 294002. SCF up-regulated the expression of MnSOD, which was also inhibited by LY 294002. However, SCF had no effect on Bcl-2 protein. These results suggest that FOXO3a is involved in oocyte apoptosis in the neonatal rat ovary, and the SCF-PI3K/Akt-FOXO3a signaling pathway mediates oocyte apoptosis and primordial follicle formation.

Enzootic angiostrongyliasis in Shenzhen, China.

To the Editor: Angiostrongylus cantonensis is a zoonotic parasite that causes eosinophilic meningitis in humans after they ingest infective larvae in freshwater and terrestrial snails and slugs, paratenic hosts (such as freshwater fish, shrimps, frogs, and crabs), or contaminated vegetables. With the increase of income and living standards, and the pursuit of exotic and delicate foods, populations around the world have seen angiostrongyliasis become an important foodborne parasitic zoonosis (1–9). Shenzhen municipality is situated in the most southern part of mainland People’s Republic of China between the northern latitudes of 22°27′ to 22°52′ and eastern longitudes of 113°46′ to 114°37′; it shares a border with the Hong Kong Special Administrative Region, China, in the south. The climate is subtropical, with an average annual temperature of 23.7°C. The city is 1,952.84 km2 and has a population of 10 million. Since 2006, thirty-two sporadic cases of human eosinophilic meningitis caused by consumption of undercooked aquacultured snails have been documented in Shenzhen (Shenzhen Center for Disease Control and Prevention, unpub. data). To identify the source of these infections and assess the risk for an outbreak of eosinophilic meningitis, we conducted a survey to investigate whether A. cantonensis occurs in wild rats and snails in Shenzhen. To examine A. cantonensis infection in intermediate host snails, 302 terrestrial snails (Achatina fulica) were collected from 10 investigation sites across Shenzhen, and 314 freshwater snails (Pomacea canaliculata)were sampled from 6 investigation sites. We examined the snails for A. cantonensis larvae by using pepsin digestion standardized procedures (3). To survey the prevalence of adult A. cantonensis in definitive host rats, we collected 187 Rattus norvegicus rats and 121 R. flavipectus rats collected from 4 sites where positive snails positive for A. cantonensis were found. These rats were examined for the presence of adult A. cantonensis in their cardiopulmonary systems. A. cantonensis larvae were found in 96 (15.6%) of 616 examined snails. Of these, P. canaliculata had an average infection rate of 20.7% (65/314), significantly higher (p<0.01) than that of A. fulica (10.3%, 31/302), an indication that P. canaliculata may be the principal intermediate host for A. cantonensis in Shenzhen. A. cantonensis adults were recovered from the cardiopulmonary systems of 37 (12%) of 308 examined rats. Infection rate for R. norvegicus rats was 16.6% (31/187), significantly higher (p<0.01) than that for R. flavipectus (4.9%, 6/121), an indication that R. norvegicus may be the principal definitive host for A. cantonensis in Shenzhen, possibly due to the rat’s preference for eating snails. Infection rates were higher for female rats (25.6% for R. norvegicus and 7.8% for R. flavipectus) than for male rats (8.9% for R. norvegicus, 2.9% for R. flavipectus), possibly because female rats eat more snails to supply proteins for reproduction. This report of enzootic A. cantonensis infection in wild rats and snails in Shenzhen demonstrates the existence of natural origins of infection with A. cantonensis for humans in this city. Persons in Shenzhen eat raw or undercooked freshwater and terrestrial snails and slugs. This practice provides opportunities for infection with A. cantonensis, particularly given that P. canaliculata has been aquacultured intensively for human consumption. The prevalence of A. cantonensis in wild rats and snails in Shenzhen poses substantial risk for future outbreaks of human eosinophilic meningitis. Moreover, public health officials, epidemiologists, researchers, clinical technicians, medical practitioners, parasitologists, and veterinarians, as well as the general public, should be aware of such risks, and integrated strategies should be taken to reduce or eliminate such risks.

Differentially expressed genes between female and male adult Anopheles anthropophagus

The aim of the present study was to identify sex-specific genes in adult Anopheles anthropophagus. As the major malaria vector and Brugia malayi vector in the Asian continent, female Anopheles mosquitoes take blood meals and transmit pathogens through this pathway, while males are nectar feeders. This complex behavior is controlled at several levels, but is probably initiated by the genetic background difference between these two groups. In our study, a subtractive cDNA library for female A. anthropophagus was constructed using the suppression subtractive hybridization (SSH) technique and then 3,074 clones from the female SSH library were analyzed using a microarray-based survey. Genes that were expressed differentially according to sex in A. anthropophagus were screened using real-time polymerase chain reaction and reverse transcription polymerase chain reaction. In our results, we report a series of genes which may be involved in female-specific mosquito behavior, including an inorganic phosphate transporter, a serine protease, the salivary protein GP35-2, and the D7 cluster salivary protein. These findings will provide clues to the nature of insect vectors and open up unprecedented opportunities to develop novel strategies for the control of mosquito-borne diseases.

Monoclonal antibodies against excretory/secretory antigens of Angiostrongylus cantonensis.

In the present study, four murine monoclonal antibodies (MAbs) were generated against the excretory/secretory (ES) products of Angiostrongylus cantonensis adult worms; two represented IgG1 and two represented IgM MAbs, and they were designated 12D5, 15F8, 21B7 and 14G10, respectively. Immunoblotting revealed that all of the MAbs predominantly recognized a 98 kDa antigen in the ES products of A. cantonensis adult worms, and no cross reactions were found with the whole worm antigens of some other common parasites, namely, Schistosoma japonicum, Clonorchis sinensis, Paragonimus westermani, Ascaris lumbricoides, Trichinella spiralis, Anisakis sp., Echinococcus granulosus, Taenia solium, and Spirometra erinacei. Immunolocalization showed that all of the four MAbs reacted with the cuticle of the adult parasite, the external surface of its intestinal canal and reproductive organs, and its egg and first-stage larvae in the lungs of rats experimentally infected with A. cantonensis. The generation and characterization of four specific MAbs against A. cantonensis ES antigens provide foundation for the development of specific immunological diagnostic techniques for human infections with A. cantonensis.

Identification of differentially expressed genes in female Culex pipiens pallens

Culex pipiens pallens is the mosquito vector of a number of human pathogens such as Wuchereria bancrofti, Brugia malayi, and epidemic encephalitis B virus. Female C. pipiens pallens play an important role in transmitting pathogens by sucking blood, which is essential for reproduction. In the present study, a subtractive cDNA library for female C. pipiens pallens was constructed by the suppression subtractive hybridization (SSH) technique and then 100 clones from the female SSH library were sequenced and analyzed. Female-differentially expressed genes in C. pipiens pallens were screened using semiquantitative RT-PCR. The full-length cDNA of an EST sequence (fs68) that was specifically expressed in female C. pipiens pallens was characterized by 3′ and 5′ rapid amplification of cDNA ends (RACE). The characteristics of the female-specific gene were further analyzed using bioinformatics and Northern blot. It was shown that the female-specific gene was a previously uncharacterized gene and may encode a salivary peptide. This putative salivary peptide could be a very important molecule in the blood feeding of female C. pipiens pallens.

Development of Lateral Flow Immunoassay for Antigen Detection in Human Angiostrongylus cantonensis Infection

Angiostrongyliasis is difficult to be diagnosed for the reason that no ideal method can be used. Serologic tests require specific equipment and are not always available in poverty-stricken zone and are time-consuming. A lateral flow immunoassay (LFIA) may be useful for angiostrongyliasis control. We established a LFIA for the diagnosis of angiostrongyliasis based on 2 monoclonal antibodies (mAbs) against antigens of Angiostrongylus cantonensis adults. The sensitivity and specificity were 91.1% and 100% in LFIA, while those of commercial ELISA kit was 97.8% and 86.3%, respectively. Youden index was 0.91 in LFIA and 0.84 in commercial ELISA kit. LFIA showed detection limit of 1 ng/ml of A. cantonensis ES antigens. This LFIA was simple, rapid, highly sensitive and specific, which opened an alternative approach for the diagnosis of human angiostrongyliasis.

Seroprevalence of Angiostrongylus cantonensis Infection in Humans in China

Abstract A seroepidemiological survey was carried out in China during 2009–2010 to determine the extent of circulating antigens (CAg) for Angiostrongylus cantonensis in the Chinese population using the gold immunochromatographic assay, with the objective of elucidating the nationwide prevalence of angiostrongyliasis in China. A total of 1,730 blood samples was collected and assayed from the general adult population (the “general group”), and those involved in aquaculture or processing of snails Achatina fulica and Pomacea canaliculat (the “occupational group”) from 5 provinces (Fujian, Hunan, Guangdong, Guangxi, and Zhejiang) and 1 municipal city (Beijing). The overall seroprevalence for the “occupational group” was 7.4% (40/540), which was significantly higher (P < 0.001) than that of the “general group” (0.8%, 9/1,190). The seroprevalence in males (9.5%) was significantly higher than in females (4.2%) (P < 0.05). These results demonstrate that angiostrongyliasis represents a significant zoonotic disease in China, requiring the strengthening of food safety for control of this food-borne disease.

Highly Sensitive Naked-Eye Assay for Enterovirus 71 Detection Based on Catalytic Nanoparticle Aggregation and Immunomagnetic Amplification

Development of sensitive, convenient, and cost-effective virus detection product is of great significance to meet the growing demand of clinical diagnosis at the early stage of virus infection. Herein, a naked-eye readout of immunoassay by means of virion bridged catalase-mediated in situ reduction of gold ions and growth of nanoparticles, has been successfully proposed for rapid visual detection of Enterovirus 71 (EV71). Through tailoring the morphologies of the produced gold nanoparticles (GNPs) varying between dispersion and aggregation, a distinguishing color changing was ready for observation. This colorimetric detection assay, by further orchestrating the efficient magnetic enrichment and the high catalytic activity of enzyme, is managed to realize highly sensitive detection of EV71 virions with the limit of detection (LOD) down to 0.65 ng/mL. Our proposed method showed a much lower LOD value than the commercial ELISA for EV71 virion detection. Comparing to the current clinical gold standard polymerase chain reaction (PCR) method, our strategy provided the same diagnostic outcomes after testing real clinical samples. Besides, this strategy has no need of complicated sample pretreatment or expensive instruments. Our presented naked-eye immunoassay method holds a promising prospect for the early detection of virus-infectious disease especially in resource-constrained settings.

Anti-Idiotypic Antibodies Specific to prM Monoantibody Prevent Antibody Dependent Enhancement of Dengue Virus Infection

Dengue virus (DENV) co-circulates as four serotypes (DENV1-4). Primary infection only leads to self-limited dengue fever. But secondary infection with another serotype carries a higher risk of increased disease severity, causing life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Serotype cross-reactive antibodies facilitate DENV infection in Fc-receptor-bearing cells by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody dependent enhancement (ADE). Most studies suggested that enhancing antibodies were mainly specific to the structural premembrane protein (prM) of DENV. However, there is still no effective drugs or vaccines to prevent ADE. In this study, we firstly confirmed that both DENV-2 infected human sera (anti-DENV-2) and DENV-2 prM monoclonal antibody (prM mAb) could significantly enhance DENV-1 infection in K562 cells. Then we developed anti-idiotypic antibodies (prM-AIDs) specific to prM mAb by immunizing of Balb/c mice. Results showed that these polyclonal antibodies can dramatically reduce ADE phenomenon of DENV-1 infection in K562 cells. To further confirm the anti-ADE effect of prM-AIDs in vivo, interferon-α and γ receptor-deficient mice (AG6) were used as the mouse model for DENV infection. We found that administration of DENV-2 prM mAb indeed caused a higher DENV-1 titer as well as interleukin-10 (IL-10) and alaninea minotransferase (ALT) in mice infected with DENV-1, similar to clinical ADE symptoms. But when we supplemented prM-AIDs to DENV-1 challenged AG6 mice, the viral titer, IL-10 and ALT were obviously decreased to the negative control level. Of note, the number of platelets in peripheral blood of prM-AIDs group were significantly increased at day 3 post infection with DENV-1 compared that of prM-mAb group. These results confirmed that our prM-AIDs could prevent ADE not only in vitro but also in vivo, suggested that anti-idiotypic antibodies might be a new choice to be considered to treat DENV infection.