Targeted Gene Insertion and Replacement in the Basidiomycete Ganoderma lucidum by Inactivation of Nonhomologous End Joining Using CRISPR/Cas9.

Targeted gene insertion or replacement is a promising genome-editing tool for molecular breeding and gene engineering. Although CRISPR/Cas9 works well for gene disruption and deletion in Ganoderma lucidum, targeted gene insertion and replacement remain a serious challenge due to the low efficiency of homologous recombination (HR) in this species. In this work, we demonstrate that the DNA double-strand breaks induced by Cas9 were mainly repaired via the nonhomologous end joining (NHEJ) pathway, at a frequency of 96.7%. To establish an efficient target gene insertion and replacement tool in Ganoderma, we first inactivated the NHEJ pathway via disruption of the Ku70 gene (ku70) using a dual single guide RNA (sgRNA)-directed gene deletion method. Disruption of the ku70 gene significantly decreased NHEJ activity in G. lucidum. Moreover, ku70 disruption strains exhibited 96.3% and 93.1% frequencies of targeted gene insertion and replacement, respectively, when target DNA with the orotidine 5'-monophosphate decarboxylase (ura3) gene and 1.5-kb homologous 5'- and 3'-flanking sequences was used as a donor template, compared to 3.3% and 0%, respectively, at these targeted sites for a control strain (Cas9 strain). Our results indicated that ku70 disruption strains were efficient recipients for targeted gene insertion and replacement. This tool will advance our understanding of functional genomics in G. lucidum. IMPORTANCE Functional genomic studies in Ganoderma have been hindered by the absence of adequate genome-engineering tools. Although CRISPR/Cas9 works well for gene disruption and deletion in G. lucidum, targeted gene insertion and replacement have remained a serious challenge due to the low efficiency of HR in these species, although such precise genome modifications, including site mutations, site-specific integrations, and allele or promoter replacements, would be incredibly valuable. In this work, we inactivated the NHEJ repair mechanism in G. lucidum by disrupting the ku70 gene using the CRISPR/Cas9 system. Moreover, we established a target gene insertion and replacement method in ku70-disrupted G. lucidum that possessed high-efficiency gene targeting. This technology will advance our understanding of the functional genomics of G. lucidum.

[Effect of Toxoplasma gondii Infection in Female Mice on Dopamine Level in the Brain of the Male Offspring].

OBJECTIVE: To investigate the effect of Toxoplasma gondii infection in female mice on dopamine level in the brain of male offspring. METHODS: Thirty-six ICR female mice were randomly divided into control group and infection group, 18 mice in each group. Each mouse in infection group was orally infected with 10 cysts of T. gondii Prugniaud strain. On the 90th day after infection, the infected female mice were mated with normal male ICR mice at 1:1 ratio. On the 20th day of pregnancy, 2 mice in each group were delivered for fetal mice by cesarean section, and the brain of male fetal mice (n = 6) in each group were collected. On the 14th and 63rd day after birth, 6 male offspring mice in each group were sacrificed, and the brain were collected. Dopamine levels in the cortex, cerebellum, hippocampus, and striatum were analyzed by high-performance liquid chromatography-electrochemical detection (HPLC-ECD). RESULTS: Three mice in infection group died during the experiment, and 6 out of 15 female mice mated successfully. The number of fetal mice and F1 generation mice in infection group was 12 (male: 7) and 21 (male: 15), respectively. All the mice in control group mated successfully. The number of fetal mice and F1 generation mice was 23 (male: 12) and 179 (male: 92), respectively. The dopamine level in the cerebellum of fetal mice of infection group and control group was (413.25 ± 21.78) ng/g and (346.30 ± 51.83) ng/g, respectively (P < 0.01). No significant difference was found in dopamine content in the cortex between the two groups (P > 0.05). Compared with the control group, on the 14th day and 63rd day after birth, the dopamine content in cortical areas [(462.50 ± 24.80) ng/g and (1215.77 ± 113.64) ng/g], cerebellum area [(271.55 ± 26.19) ng/g and (1328.82 ± 39.62) ng/g], hippocampus area [(225.78 ± 24.17) ng/g and (1322.70 ± 58.34) ng/g], and striatum area [(455.23 ± 61.53) ng/g and (991.32 ± 54.31) ng/g] of the male offspring in infection group were significantly higher than that of the control (P < 0.05, P < 0.01). CONCLUSION: T. gondii infection in female mice causes an increase of dopamine level in the brain of F1 generation male mice.

Stem cell therapy for the treatment of parasitic infections: is it far away?

Stem cell therapy is an interventional treatment that introduces new cells into damaged tissues, which help in treating many diseases and injuries. It has been proved that stem cell therapy is effective for the treatment of cancers, diabetes mellitus, Parkinson's disease, Huntington's disease, cardiovascular diseases, neurological disorders, and many other diseases. Recently, stem cell therapy has been introduced to treat parasitic infections. The culture supernatant of mesenchymal stem cells (MSCs) is found to inhibit activation and proliferation of macrophages induced by the soluble egg antigen of Schistosoma japonicum, and MSC treatment relieves S. japonicum-induced liver injury and fibrosis in mouse models. In addition, transplantation of MSCs into naïve mice is able to confer host resistance against malaria, and MSCs are reported to play an important role in host protective immune responses against malaria by modulating regulatory T cells. In mouse models of Chagas disease, bone marrow mononuclear cell has been shown effective in reducing inflammation and fibrosis in mice infected with Trypanosoma cruzi, and transplantation of the bone marrow mononuclear cells prevents and reverses the right ventricular dilatation induced by T. cruzi infection in mice. Preliminary clinical trials demonstrate that transplantation of bone marrow derived-cells may become an important therapeutic modality in the management of end-stage heart diseases associated with Chagas disease. Based on these exciting results, it is considered by stating that it is firmly believed that, within the next few years, we will be able to find the best animal models and the appropriate stem cell type, stem cell number, injection route, and disease state that will result in possible benefits for the patients with parasitic infections, and stem cell therapy, although at an initial stage currently, will become a real therapeutic option for parasitic diseases.

[Dynamic changes of sciatic nerve conduction velocity in rats infected with Toxoplasma gondii].

OBJECTIVE: To observe the dynamic changes of sciatic nerve conduction velocity of Toxoplasma gondii-infected rats at different time points. METHODS: Twenty SD rats were randomly divided into control group and Toxoplasma gondii infection group. Rats in T. gondii infection group were intraperitoneal injected with 4x10(7) T. gondii tachyzoites, while those in control group were given equivalent normal saline. Motor and sensory nerve conduction velocities (MNCV, SNCV) in sciatic nerve were measured by Medtronic Keypoint4 Workstation electromyography at pre-infection, and 2, 4, 8, 12 months post-infection. RESULTS: Within two months after infection, there was no difference in SNCV and MNCV between control group and infection group (P>0.05). From 4 months after T. gondii injection, infected rats began to show the slowness of SNCV and MNCV, which progressed with the course of infection. At 4, 8, and 12 months after infection, SNCV and MNCV of infection group were (35.26±3.02) and (25.94±3.20) m/s, (33.57±2.27) and (22.75±2.31) m/s, and (32.38±2.38) and (22.03±2.08) m/s, respectively. Compared with control group, SNCV and MNCV of infection group reduced by (7.47±2.11)% and (12.57±1.89)%, (8.92±2.64)% and (13.72±2.65)%, and (12.18±1.94)% and (15.46±2.37)%, respectively (P<0.05). CONCLUSION: From 4 months after infection, Toxoplasma gondii-infected rats show a slowness of motor and sensory nerve conduction velocities in sciatic nerve.

[Proteomic study of the hippocampus tissue from rats with chronic Toxoplasma gondii infection].

OBJECTIVE: To observe the proteome changes in the hippocampus tissue of rats with chronic Toxoplasma gondii infection. METHODS: Six male SD rats were randomly divided into control group and infection group. Each rat in infection group was intraperitoneally injected with 4 x 10(7) purified T. gondii tachyzoites. Rats in the control group received equivalent volumes of sterile normal saline. At the fifth day post-infection, blood samples were taken from the lateral tail vein and Ciemsa staining of blood cells was performed to find Toxoplasma gondii. Rats were dissected at the 10th week post-infection, total protein in the hippocampus was separated by using two-dimensional gel electrophoresis (2-DE). After Coomassie blue staining, the Image Analysis software was used to select and separate proteins on the gel. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used for peptide mass fingerprint PMF). Proteins were identified by using Mascot software to search the MSDB and SwissProt databases. RESULTS: Microscopy examination of blood smears confirmed that the rats in infection group were all infected by 11 gondii. The number of protein spots of rats from infection group and control group was 311 +/- 19 and 327 +/- 13 respectively. Compared with the control group, 5 protein spots disappeared, 4 protein spots were up-regulated and 7 were down-regulated in the infection group. The 9 differentially expressed protein spots were identified by MALDL-TOF-MS: phosphoglycerate kinase 1, similar to alpha-enolase, glutamine synthetase, creatine kinase, creatine kinase B-type, ATP synthase, aconitase 2, mitochondrial precursor, actin and an unnamed protein. The first three proteins were up-regulated and the other five proteins were down-regulated in infection group. CONCLUSION: Nine differential expression proteins are found from the hippocampus tissue in rats chronically infected with T. gondii and normal SD rats.

Increased production and anti-senescence activity of exopolysaccharides in Ganoderma lingzhi by co-overexpression of ß-1,3-glucan synthase and UDP-glucose pyrophosphorylase.

A ß-1,3-glucan synthase gene (gls) was cloned and overexpressed in Ganoderma lingzhi. The content of intracellular polysaccharides (IPS) in G. lingzhi overexpressing gls was 22.36 mg/100 mg dry weight (DW), 19 % higher than those in the wild-type (WT) strain. Overexpression of gls did not affect the expression of the phosphoglucomutase gene and the UDP-glucose pyrophosphorylase gene (ugp) in the polysaccharide biosynthesis. The gls and ugp were then simultaneously overexpressed in G. lingzhi for the first time. The combined overexpression of these two genes increased the IPS content and exopolysaccharides (EPS) production to a greater extent than the overexpression of gls independently. The maximum IPS content of the overexpressed strain was 24.61 mg/100 mg, and the maximum EPS production was 1.55 g/L, 1.31- and 1.50-fold higher than that in the WT strain, respectively. Moreover, the major EPS fractions from the overexpression strain contained more glucose (86.7 % and 72.5 %) than those from the WT strain (78.2 % and 62.9 %). Furthermore, the major fraction G+U-0.1 from the overexpression strain exhibited stronger antioxidant and anti-senescence activities than the WT-0.1 fraction from the WT strain. These findings will aid in the hyperproduction and application of Ganoderma polysaccharides and facilitate our understanding of mushroom polysaccharide biosynthesis.

Study of the Microstructure of Coal at Different Temperatures and Quantitative Fractal Characterization.

In order to understand the influence of underground coal fires on coal fractures and pores, mercury intrusion porosimetry (MIP) and scanning electron microscopy (SEM) are combined to study the development of coal pore and fracture under high-temperature treatment and calculate the fractal dimension to analyze the relationship between the development of coal pore and fracture and the fractal dimension. The results show that the volume of pores and fractures of the coal sample (C200) treated at 200 °C (0.1715 mL/g) is greater than that of the coal sample (C400) treated at 400 °C (0.1209 mL/g), and both are greater than the original coal sample (RC) (0.1135 mL/g). The volume increase is mainly due to mesopores and macropores, and the proportions of mesopores and macropores in C200 were 70.15 and 59.97% in C400. The MIP fractal dimension shows a decreasing trend with the increase of temperature, and the connectivity of coal samples improved with the increase of temperature. The changes in volume and three-dimensional fractal dimension of C200 and C400 showed the opposite trend and are related to the different stress of coal matrix at different temperatures. The experimental SEM images confirm that the connectivity of coal fractures and pores improves with the increase of temperature. Based on the SEM experiment, the larger the fractal dimension, the more complex the surface is. The SEM surface fractal dimensions indicate that the surface fractal dimension of C200 is the smallest and that of C400 is the largest, which is consistent with the observations made by SEM. The combination of the two fractal dimensions is used to characterize the self-similarity of coal using the fractal dimension difference. When the temperature increased to 200 °C, the unordered expansion of the coal sample resulted in the largest fractal dimension difference and the lowest self-similarity. When heated to 400 °C, the fractal dimension difference of the coal sample is the smallest, and the microstructure of coal shows a regular groove-like development.

Overexpression of phosphomannomutase increases the production and bioactivities of Ganoderma exopolysaccharides.

In this study, we explored a novel approach to enhancing the production and bioactivities of Ganoderma exopolysaccharides. The homologous phosphomannomutase gene (PMM1) was cloned and overexpressed in Ganoderma for the first time. As a result, the maximum production of exopolysaccharides by the PMM1 transformant was 1.53 g/L, which was 1.41-fold higher than of a wild-type (WT) strain in a 5-L bioreactor. The transcription levels of PMM1 and PMM2 increased 40.5- and 2.4-fold, respectively, whereas the value of the GDP-D-mannose pyrophosphorylase gene did not change significantly in this transgenic strain. Furthermore, the major exopolysaccharide fractions from PMM1 transformants contained higher amounts of mannose (56.5 % and 21.1 %) than those from a WT strain (26.7 % and 9.3 %). Moreover, the major fractions from PMM1 transformants exhibited stronger regulation effects on macrophage. In conclusion, this study is helpful for the efficient production and application of Ganoderma exopolysaccharides and facilitates an understanding of polysaccharide biosynthesis regulation.

Kinetics of circulating antigen 14-3-3 in sera of rabbits firstly infected with Schistosoma japonicum and treated with/without praziquantel.

A sandwich ELISA was developed for the detection of circulating antigen 14-3-3 in the sera of rabbits. Rabbits that were infected with 500 cercariae of Schistosoma japonicum were grouped and the kinetics of 14-3-3 was observed. For the treated group, the 14-3-3 protein could be detected as early as 2-4 weeks postinfection and then its levels rose rapidly and reached a peak at around 6 weeks. The 14-3-3 levels in the sera significantly decreased after the infected rabbits were treated with praziquantel at 6 weeks postinfection and declined to the initial level about 8 weeks posttreatment. While in the untreated group, 14-3-3 levels reached a peak in 8 weeks postinfection and then remained at plateau level for about 6 weeks. Our findings showed that detection of S. japonicum 14-3-3 has an important value for diagnosis of acute infection of S. japonicum and evaluation of chemotherapy.

Effect of praziquantel prolonged administration on granuloma formation around Schistosoma japonicum eggs in lung of sensitized mice.

Schistosomiasis remains a major public health problem and it is an immune disease. The schistosome egg is the primary parasite factor responsible for the overt disease. The eggs release the soluble antigen, which induces intensive tissue reaction, a granulomatous reaction to the eggs. If granuloma formation could be suppressed, overt disease might not develop. Praziquantel is an effective antischistosomal drug especially for adult worms. However, whether praziquantel has a suppressing effect on granuloma formation around schistosome eggs directly remains unclear. The purpose of the present study was to investigate the effect of praziquantel, especially administered persistently, on granuloma formation around Schistosoma japonicum eggs in the lung of sensitized mice. Thirty-six mice were divided into three groups averagely. Group A was a control group. First, the mice were injected with schistosomal eggs hypodermically in abdomen, and 10 days later injected with schistosomal eggs intravenously via a tail vein. Group B was a praziquantel short administration group. In addition to the injections of schistosomal eggs as the same of Group A, the mice were administered with praziquantel in a daily dose of 300 mg/kg for 3 days, from 1 day before the intravenous injection of the eggs. Group C was a praziquantel prolonged administration group. In addition to the injections of schistosomal eggs as the same of Group A, the mice were administered with praziquantel in a daily dose of 150 mg/kg for 5 days weekly until the mice were sacrificed. Three mice of each group were sacrificed on days 7, 14, 28, and 56, respectively after the intravenous injection of the eggs, and the lung tissues were fixed with formalin and the slices were HE stained. The granulomas containing eggs in their centers were selected, and 25-30 granulomas from the animals of each group were measured at each time period. The mean areas of egg granulomas of each group were calculated, and the neutrophilic granulocytes, eosinocytes, lymphocytes, fibroblasts, and macrophages within the egg granulomas were counted. The mean numbers of them of each group were calculated. All the data of each group were analyzed and compared statistically. On day 56 after the intravenous injection of the eggs, the mean area of schistosomal egg granulomas in group B was (227.4 ± 728.0) × 10(3) µm(2), less than that of [(297.9 ± 153.3) × 10(3) µm(2)] in group A, and the suppression rate was 23.7% (P < 0.05). On days 7, 14, 28, and 56, the mean areas of schistosomal egg granulomas in group C were (575.8 ± 155.6) × 10(3) µm(2), (310.5 ± 854.0) × 10(3) µm(2), (267.7 ± 513.3) × 10(3) µm(2), and (214.9 ± 446.4) × 10(3) µm(2), respectively, significantly less than those of [(692.7 ± 232.6) × 10(3) µm(2), (439.4 ± 165.0) × 10(3) µm(2), (385.7 ± 129.3) × 10(3) µm(2), and (297.9 ± 153.3) × 10(3) µm(2)] in group A. The suppression rates were 16.9%, 29.3%, 30.6%, and 27.9%, respectively (P values <0.05). On day 56, the mean numbers of neutrophilic granulocytes were 11.4 ± 5.0 in group A and 5.2 ± 3.1 in group C, respectively, with the suppression rate of 54.4% in group C (P < 0.05). On day 56, the mean numbers of eosinocytes within the egg granulomas were 2.3 ± 2.0, 0.1 ± 0.3, and 0.3 ± 0.6 in groups A, B, and C, respectively, with the suppression rate of 95.7% in group B and 87.0% in group C (P values <0.05). On day 56, the mean numbers of macrophages within egg granulomas were 14.3 ± 6.9 in group C, compared with 18.6 ± 8.2 in group A, the suppression rate was 23.1% (P < 0.05). On day 56, the mean numbers of fibroblasts within the egg granulomas were 6.6 ± 4.4 and 5.8 ± 2.6 in groups B and C, respectively, and compared with 14.3 ± 7.8 in group A, the increasing extents decreased by 53.8% and 59.4%, respectively (P values <0.05). Therefore, the administration of praziquantel, especially the prolonged administration, can suppress the formation of schistosomal egg granulomas, including reduction in the areas of granulomas and suppression of the inflammatory cells and the hyperplasia of fibroblasts within granulomas.

[Impairment of learning and memory ability in mice with latent infection of Toxoplasma gondii].

OBJECTIVE: To detect the learning and memory ability in mice model of latent Toxoplasma gondii infection with object recognition test and Morris water maze test. METHODS: Thirty-six Kunming mice were divided into control group, infection group with 6 cysts each mouse (low infection group), and infection group with 12 cysts each mouse (high infection group) averaged. Mice in the two infection groups were orally infected with T. gondii Prugniaud (PRU) low virulence strain. Object recognition test was conducted at the 63rd day after infection. After the first day of adaptation and the second day of familiarization in the test, the time expended on exploring new and familiar objects was recorded on the third day and the discrimination index (DI) was calculated. Morris water maze test was conducted at the 66th day. The ability of spatial learning, spatial memory retention and working memory capacity was evaluated by place navigation test, spatial probe test, and working memory test, respectively. The mice were sacrificed at the 74th day after infection. The left cerebral hemisphere of mice was fixed, sliced, and stained with eosin-hematoxylin for pathological examination. The right hemisphere was used to detect the activity of superoxide dismutase (SOD) and malondialdehyde (MDA) content. RESULTS: The results of object recognition test showed that the discrimination index of high infection group and low infection group was (14.3 +/- 5.2)% and (17.5 +/- 5.6)%, respectively, significantly lower than the control [(28.9 +/- 7.1)%] (P < 0.01). In the place navigation test, the latency to find the platform in the two infection groups was longer than the control, with significant difference on the second and third day (P<0.05). In the spatial probe test, the percentage of the distance across the platform quadrant in the total swimming distance of high infection group and low infection group were (19.9 +/- 5.0)% and (23.9 +/- 6.8)%, respectively, significantly lower than the control [(27.4 +/- 3.6)%] (P < 0.05). In the working memory test, at the fourth day of test the latency of high infection group and low infection group [(365 +/- 14.2) s and (35.3 +/- 13.7) s] was significantly longer than the control [(30.4 +/- 12.5) s] (P<0.05). In all the tests, there was no statistical significance between low infection group and high infection group (P > 0.05). The brain sections of two infection groups showed cysts of T. gondii, proliferation of glial cells, widened gap around small blood vessels, and a phenomenon of "vascular cuff". The activity of SOD in the mice brains of two infection groups was significantly lower than the control, while MDA level was significantly higher (P < 0.05). SOD and MDA showed no significant difference between two infection groups (P > 0.05). CONCLUSION: Latent infection of T. gondii may lead to learning and memory impairment in mice.

Developing a rat model of dilated cardiomyopathy with improved survival.

To compare the continuous infusion and intermittent bolus injection administration protocols of doxorubicin (Dox) under the same cumulative dose (12 mg/kg), and establish a rat dilated cardiomyopathy model with improved survival, a total of 150 Sprague-Dawley (SD) rats were divided into three groups: a control group, administered with normal saline; a Dox 1 group, administration twice a week at 1 mg/kg; a Dox 2, administration once a week at 2 mg/kg. Mortality rates in the Dox 1 and Dox 2 groups were 22% and 48%, respectively (P<0.05). As shown by echocardiography, both Dox groups exhibited significant chamber dilatation and reduced cardiac function (all P<0.05 vs. control). Plasma brain natriuretic peptide and C-reactive protein concentrations were significantly increased (P<0.05) with both Dox regimens. The concentrations of Caspase-3 in myocardial tissues of rats significantly increased in both doxorubicin regimens. Myocardial metabolism imaging by histology and 18F-fluoro-deoxyglucose-positron emission tomography (18FDG-PET) both revealed decreased myocardial viability and necrosis, and even interstitial fibrosis, in left ventricles (LVs) in both Dox groups. Serum creatinine and aspartate aminotransferase concentrations were significantly higher in the Dox 2 model than in the Dox 1 model. Doxorubicin given at both regimens induced dilated cardiomyopathy, while its administration at lower doses with more frequent infusions reduced the mortality rate.

[Fusion expression and antigenicity analysis of miracidial antigen from eggs of Schistosoma japonicum].

OBJECTIVES: To express the miracidial antigen from eggs of Schistosoma japonicum (Chinese mainland strain) (SjMP10), and investigate the role of the miracidial antigen during the hepatic granuloma formation of schistosomiasis. METHODS: A pair of specific primers was designed and synthesized according to the nucleotide sequence of the open reading frame of the miracidial antigen gene. The open reading frame of the miracidial antigen gene was amplified, digested by restrictive enzyme (BamHI, SolI), and cloned directly into the expression plasmid pGEX-4T-3 to construct the recombinant plasmid. The recombinant plasmids were transformed into E. coli BL21(DE3), and induced by IPTG to express the fusion protein of GST-SjMP10. The expressed fusion protein was purified by electric elution method, and its antigenicity was examined by Western blotting and lymphocyte proliferation test. RESULTS: The gene of miracidial antigen was cloned into the expression plasmid pGEX-4T-3. After induced by IPTG, the recombinant expressed a fusion protein of GST-SjMP10, with a molecular weight of 39 000 approximately. The purified fusion protein showed proper antigenicity that could be recognized by the sera of rabbits heavily infected by Schistosoma japonicum and could stimulate the proliferation of splenic lymphocytes of infected BALB/c mice. CONCLUSION: The miracidial antigen from eggs of Schistosoma japonicum was expressed successfully.

[Enhancement of the protective effect of SjC23 DNA vaccine against Schistosoma japonicum infection by immunostimulatory sequence].

OBJECTIVE: To investigate the effect of immunostimulatory sequence on SjC23 DNA vaccine against Schistosoma japonicum infection. METHODS: SjC23 gene fragment was inserted into pcDNA3. 1-CpG to construct pcDNA3.1-SjC23/CpG. BALB/c mice in 4 groups were immunized intramuscularly 3 times at 2 week intervals, with 100 microg plasmid DNA per injection. Four weeks after the 3rd immunization, all mice were challenged with 45 +/- 1 cercariae of S. japonicum by abdominal skin penetration. After 45 days post-challenge, mice were perfused and the number of recovered worms and of eggs in liver was counted. Blood samples were collected from the tail vein of all mice 2 days before the 1st immunization and before challenge respectively. IgG, IgG1 and IgG2a in sera were detected. Three weeks after the 3rd inoculation, the spleen cells of 2 mice from each group were cultured and stimulated with ConA and recombinant peptide. The supernatant was collected to detect IL-2, IL-4 and IFN-gamma. Simultaneously, the cytotoxic activity was detected with 51Cr release assay in vitro. RESULTS: The worm reduction rate in SjC23 group and SjC23/CpG group was 28.1% and 35.1%, the hepatic egg reduction rate was 21.6% and 26.5%, respectively, compared with the control group. The level of protection in SjC23/CpG group was higher than that in SjC23 group (P<0.05). ELISA results indicated that mice immunized with pcDNA3.1-SjC23 and SjC23/CpG produced specific IgG to rSjC23, while mice immunized with pcDNA3.1 and pcDNA3.1-CpG did not. Mice in SjC23 group and SjC23/CpG group also produced IgG1 and IgG2a antibody isotypes, with the ratio of IgG2a/IgG1 10.1 and 12.2, respectively. In comparison with the control, the level of IL-2 and IFN-gamma in mice immunized with pcDNA3.1-SjC23 and pcDNA3.1-SjC23/CpG was augmented. The cytotoxic activity of spleen cells from mice in SjC23/CpG group was augmented from 9.7% to 40.0% compared with that in SjC23 group. CONCLUSION: The study indicates that immunostimulatory sequence appears to increase the level of protection induced by immunization with pcDNA3.1-SjC23 vaccine.

[Establishment and diagnostic performance of biotin-avidin complex enzyme linked immunosorbent assay of detecting specific IgG4 of clonorchiasis].

OBJECTIVE: To explore the performance of the biotin-avidin complex enzyme linked immunosorbent assay of detecting specific IgG4 for the diagnosis of clonorchiasis. METHODS: The avidin-biotin complex enzyme linked immunosorbent assay of detecting specific IgG4 (IgG4-ABC-ELISA)against Clonorchis sinensis was established, and used to detect the serum samples of patients with clonorchiosis sinensis, schistosomiasis japonica, paragonimiasis, toxoplasmosis, echinococcosis, cysticercosis and sparganosis mansoni. At the same time, these sera were analyzed by the ELISA of detecting IgG4 (IgG4-ELISA) and ELISA of detecting the total IgG (IgG-ELISA) as controls. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and the respective diagnostic performance of the three methods were compared. RESULTS: The IgG4-ABC-ELISA for diagnosis of clonorchiasis was established successfully. The sensitivity and specificity of the IgG4-ABC-ELISA for detecting clonorchiasis were 90.0% and 98.2% respectively, and PPV and NPV were 93.8% and 97.0% respectively. Its diagnostic performance was 96.3%. The sensitivity and specificity of the IgG4-ELISA for detecting clonorchiasis were 86.0% and 98.2% respectively, and PPV and NPV were 93.5% and 95.9% respectively. Its diagnostic performance was 95.4%. The sensitivity and specificity of the IgG-ELISA for detecting clonorchiasis were 94.0% and 88.1% respectively, and PPV and NPV were 70.1% and 98.0% respectively. Its diagnostic performance was 89.4%. The sensitivity of IgG4-ABC-ELISA was higher than that of IgG4-ELISA (P < 0.05), and the specificity of IgG4-ABC-ELISA was higher than that of IgG-ELISA (P < 0.05). CONCLUSIONS: IgG4-ABC-ELISA of detecting specific antibody IgG4 against Clonorchis sinensis has high sensitivity and specificity. Therefore, it has a good application value in the diagnosis of clonorchiasis.

[Study on immunologic function of thioredoxin glutathione reductase from Schistosoma japonicum].

OBJECTIVE: To study the immunogenicity and the immuno-protection of thioredoxin glutathione reductase from Schistosomajaponicum (SjTGR) against schistosome infection in mice. METHODS: Seventy-five mice were randomly divided into 5 groups, namely, blank group, PBS group, CpG2 immunized group, TGR immunized group and TGR + CpG2 co-immunized group. Each mouse was immunized for 3 times. The mice were tail bled before the first immunization and 2 weeks after the third immunization. The serum antibody levels of total IgG, IgG1 and IgG2a against SjTGR were assayed by ELISA. Two weeks after the third immunization, each mouse was infected with 40 ± 2 S. japonicum cercariae by abdominal skin penetration. Forty-two days later, all the mice were sacrificed to collect schistosome adult worms and liver eggs. The worm and egg reduction rates were calculated respectively. The single splenocyte of mouse was collected 2 weeks after the third immunization, and the expressions of CD44high, CD4+CD44high or CD8+CD44high on splenocytes of mice were examined by flow cytometry. After 72 h incubation with recombinant SjTGR, the levels of IL-2, IL-4, IL-10, and IFN-γ in the single-cell supernatant were determined by using ELISA kit. RESULTS: Two weeks after the third immunization, the titers of serum IgG against SjTGR in mice immunized with SjTGR and co-immunized with SjTGR and CpG2 were higher than 1:200 000. The IgG2a: IgG1 ratio (IgG2a/IgG1) increased slowly with time in both TGR immunized group and TGR + CpG2 co-immunized group. There were obviously higher levels of IFN-γ and IL-2 in the cell supernatant in the TGR immunized group and TGR + CpG2 co-immunized group compared to the blank, PBS and CpG2 groups (P < 0.05). The increased subpopulations of CD44high, CD8+CD44high and CD4+ CD44high cells in the splenocytes from mice immunized by SjTGR and co-immunized by SjTGR and CpG2 were found comparing to the blank, PBS and CpG2 groups (P < 0.05). The TGR immunization and TGR + CpG2 co- immunization caused 9.4% and 10.5% reductions in the number of adult worms and 9.2% and 32.8% reductions in the number of eggs, respectively. CONCLUSIONS: SjTGR displays strong immunogenicity inducing Th1 type immune response in mice. However, it could not produce protective efficacy against S. japonicum infection. CpG2 ODN may be a broadly effective Th1 adjuvant.

[Protective immunity induced by the nucleic acid vaccine of SjC 21.7 in mice].

OBJECTIVE: To observe the protective immunity induced by the nucleic acid vaccine of 21.7 kDa membrane protein molecule of Schistosoma japonicum Chinese mainland strain (SjC 21.7) in BALB/c mice. METHODS: A pair of primers (P1 and P2) was synthesized according to the DNA sequence of the SjC21.7. The ORF sequence of SjC21.7 was amplified by PCR, and the Kozark sequence was added to the position of initiator. The gene fragment was inserted into the eukaryotic expression plasmid pcDNA3.1 to form the recombinant plasmid SjC21.7-pcDNA3.1. Forty-eight BALB/c mice were divided into three groups: control, test and boost. Each mouse was injected in quadriceps femoris with plasmid pcDNA3.1 (control) or recombinant plasmid SjC21.7-pcDNA3.1 (test, boost); for the boost group, with additional P35-pcDNA3.1 and P40-pcDNA3.1. All mice were immunized three times with an interval of 2 weeks, challenged each with 45 cercariae of S. japonicum at the 30th day after final immunization. At day 45 after challenge, all mice were sacrificed, the numbers of worms and hepatic eggs were counted. Antibody level in the sera of mice before and two weeks after immunization was determined with ELISA. The expression of the target gene in quadriceps femoris was observed with immunohistochemistry. RESULTS: The immunohistochemistry analysis showed that there were specific antigens expressed in the local tissue of the test group mice. There was specific IgG in the serum of partial mice in test and boost groups. Compared with the control group, the worm reduction rate was 29.9% and its egg reduction rate 13.8% in the test group; 31.9% and 28.0% respectively in the boost group. The egg reduction rate in the boost group was higher than that of the test group (P < 0.05). CONCLUSION: The SjC21.7 nucleic acid vaccine could induce partial protective immunity against Schistosoma japonicum in BALB/c mice.

[Cloning and function analysis of high mobility group box 1 (HMGB1) protein of Schistosoma japonicum (Mainland strain)].

OBJECTIVE: To clone and express a high mobility group box 1(HMGB1) protein of Schistosomajaponicum (Mainland strain) and analyze its function. METHODS: The DNA fragment of open reading frame encoding Sj HMGB 1 protein was amplified by RT-PCR from the mRNA of S. japonicum worms, then it was subcloned into the expression vector pET28a(+) to form the recombinant expression plasmid SjHMGB1-pET28a. The recombinant expression plasmid was transformed into the component E. coli BL21(DE3), and the tranformant containing recombinant expression plasmid was induced with IPTG to express the recombinant protein SjHMGB1. The recombinant SjHMGB1 protein was purified by affinity chromatography with nickel chelating affinity chromatography agarose gel. The Gel retard experiment and animal immunization were performed to analyze the DNA binding capacity and the immunologic property of recombinant SjHMGB1. The expression levels of HMGB1 in different life cycle stages of S. japonicum were analyzed by Western bloting and RT-PCR. Female ICR mice were immunized with the recombinant SjHMGB1 protein and infected with 45 +/- 2 cercariae of S. japonicum after three immunizations. Forty-two days post-infection, the worms and eggs of S. japonicum were recovered from the portal vein and liver tissue, respectively. The worm and egg reduction rates were calculated respectively. RESULTS: A 530 bp of specific DNA fragment was amplified from mRNA of S. japonicum by RT-PCR, which was the open reading frame (ORF) encoding SjHMGBlprotein confirmed by DNA sequencing analysis. The recombinant expression plasmid SjHMGB1-pET28a was constructed by cloning the ORF of SjHMGB1 into a expression vector pET28a(+). The bacterium transformants containing the recombinant plasmid expressed a soluble recombinant protein about 28 kDa after induced by IPTG, and the recombinant SjHMGB1 protein was purified by nickel chelating affinity chromatography. The gel retard experiment showed that the recombinant SjHMGB1 protein could bind to both supercoiled DNA and linear DNA, and the recombinant protein immunized mice produced high titers of antiserum IgG. Western bloting indicated that the recombinant SjHMGB1 protein was recognized specifically by the S. japonicum-infected mice serum. Above results showed that the recombinant SjHMGB1 protein possessed both functional activity and immunogenicity as the natural protein. RT-PCR and Western blot results showed that SjHMGB1 was abundantly expressed in the adult and egg stages whereas barely detectable in the cercaria stage. The immune protection experiment showed that the recombinant SjHMGB1 induced mice to produce high titers of specific antibody IgG but failed to conduct an effective immune protection against S. japonicum. CONCLUSION: The gene encoding HMGB1 from S. japonicum and the soluble recombinant SjHMGB1 protein with natural functional activity are obtained, and the recombinant SjHMGB1 has a high immunogenicity but is not able to induce an effective immune protection against S. japonicum.

Identification of proteins inducing short-lived antibody responses from excreted/secretory products of Schistosoma japonicum adult worms by immunoproteomic analysis.

The excretory/secretory antigens of Schistosoma japonicum (Sj ESAgs) play important roles in host-parasite immune interactions. In this study, the antibody response patterns to Sj ESAgs in sera of individual rabbits at the healthy stage, 2-6 weeks post-infection and 4-16 weeks after treatment were examined. Antigens inducing short-lived antibody responses were selected by comparing differences in immune recognition of proteins in sera across the different stages by Western blotting and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS). The diagnostic value of these short-lived antibody responses for schistosomiasis was investigated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as a major antigen inducing a short-lived antibody response in Sj ESAgs. The antibody response against Sj GAPDH decreased at week 4 and disappeared between weeks 8-12 after effective chemical treatment of rabbits, and this response declined to negative levels in schistosomiasis patients one year after treatment. The intensity of the antibody response against Sj GAPDH was dependent on parasite load in mice. The sensitivity and specificity of IgG antibodies against recombinant Sj GAPDH for schistosomiasis diagnosis were 82.5% and 91.3%. Our findings suggest that Sj GAPDH induces short-lived antibody responses in the host, and detecting IgG against this antigen provides the basis for developing a potential method for diagnosis and evaluating treatment effects for schistosomiasis japonicum. BIOLOGICAL SIGNIFICANCE: Schistosomiasis is one of the world's major public health problems. Developing effective diagnostic methods for detecting schistosome-specific antibodies to effectively identify active infections is part of a critical strategy for blocking transmission of the parasite and eradicating schistosomiasis. The excretory/secretory antigens of S. japonicum (Sj ESAgs) play important roles in host-parasite immune interactions. In our study, we examine the antibody response patterns to Sj ESAgs within individual rabbits at the healthy, schistosome infection and post-treatment stages by Western blotting. Proteins among the Sj ESAgs inducing short-lived antibody responses were identified by Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS), and their potential as immune markers for diagnosis and evaluating therapeutic effects in schistosomiasis was evaluated. Our findings suggest that S. japonicum glyceraldehyde-3-phosphate dehydrogenase (GAPDH) induces short-lived antibody responses in the host, and detecting IgG against this antigen provides the basis for developing a potential method for diagnosis and evaluating treatment effects for schistosomiasis japonicum.

The South-to-North Water Diversion Project: effect of the water diversion pattern on transmission of Oncomelania hupensis, the intermediate host of Schistosoma japonicum in China.

BACKGROUND: The South-to-North Water Diversion Project (SNWDP) is the largest national water conservancy project in China. However, the Eastern Route Project (ERP) of SNWDP will refer to the habitats of Oncomelania hupensis, the intermediate host of Schistosoma japonicum. The present study was aimed at investigating the effects of some factors relating to the water diversion pattern on the spread north of O. hupensis and transmission of S. japonicum. METHODS: Marked snails were attached to the floating debris, and then placed on the water surface, the passage of snails through water pumps was observed. Some marked living adult snails were placed under water in the 5 spots, 15, 30, 60, 90 and 120 days later, their survival and transfer under water were investigated. 2, 4, 8, 16, 32, 64 and 128 juvenile snails, with a male: female ratio of about 1, were caged, 1 year later, their reproductions were calculated. RESULTS: The snails attached on the floating debris at 100-, 50- and 20-cm-distance from the inlet pipe of the big pump (with a diameter of 80 cm), could be absorbed into the pumps, with passing rates of 2.45%, 3.93% and 43.46%, respectively, compared with 72.07% and 91.00% for the snails at 20 cm and 10 cm-distance from the inlet pipe of the small pump (with a diameter of 20 cm). A total of 36,600 marked living snails were put into 5 ponds and ditches, with the water depths of 1-1.6 m, 15-120 days later, no marked ones were found along the ponds and ditches or in the straw packages. The juvenile snails did not reproduce until their density reached up to 8 snails (ratio of male: female of 1)/0.16 m2. CONCLUSIONS: During the construction of ERP of SNWDP, the risk of northward spread of schistosomiasis japonica will be decreased or eliminated as long as long-term reliable interventions for snail control are implemented.