[Construction and identification of a full-length cDNA library from Spirometra erinaceieuropaei].

The full-length pBluescript II SK cDNA library of adult Spirometra erinaceieuropaei was constructed by using the SMART method. Data showed that 95.5% of the library was recombinant and the titer of the library was 1.06 x 10(6). The average insert size of the library was about 1.4 kb. Forty-eight randomly selected clones were sequenced. A set of 36 effective expressed sequence tags (ESTs) with the average size of 674 bp was obtained after excluding clones shorter than 450 bp. The unigenes occupied 58.3% of the 36 ESTs. The rate of full-length cDNAs were 57.7% (15/26). The high-quality of full-length cDNA library could be used for large scale EST sequencing.

Diversity of soil nematodes in areas polluted with heavy metals and polycyclic aromatic hydrocarbons (PAHs) in Lanzhou, China.

This study investigated the soil nematode community structure along the Yellow River in the Lanzhou area of China, and analyzed the impact of heavy metals (Cd, Pb, Cr, Cu, and Zn) and polycyclic aromatic hydrocarbons (PAHs) on the nematode community. Soil samples from five locations (named A-E), which were chosen for soil analysis, showed significant differences in their heavy metal content (p < 0.01), as well as in the variety of nematodes (up to 41 genera) and families (up to 20) that were present. The different samples also differed significantly in the total PAH content (p < 0.05), as well as the six types of PAH present. Sites A-C showed the most severe contamination with heavy metals and PAHs; these sites had the lowest abundance of fungivores and omnivore/predators, but the proportion of bacteriovores was the highest (p < 0.05). Site E, in contrast, showed only minor pollution with heavy metals and PAHs, and it contained the highest abundance of plant parasites (p < 0.05). Several nematode ecological indicators were found to correlate with concentration of soil pollutants at all the sites tested: the maturity index (MI, in addition to plant parasites), plant parasite index (PPI), SigmaMI (including all the soil nematodes), Shannon-Wiener diversity index (H''), and Wasilewska index (WI). Disturbance to the soil environment was more severe when MI, SigmaMI, and H' values were lower. The results of the study show that the abundance and structure of the soil nematode communities in the sampling locations were strongly influenced by levels of heavy metals and PAHs in the soil. They also show that the diversity index H' and the maturity index can be valuable tools for assessing the impact of pollutants on nematodes.

[Dimo-thylidioctyl ammonium bromide-BCG polysaccharide nucleic acid adjuvant enhanced the immunogenicity of a Mycobacterium tuberculosis subunit vaccine].

OBJECTIVE: To investigate the activity of a novel adjuvant consisting of dimethyldioctyldecyl ammonium bromide (DDA) and BCG polysaccharide nucleic acid (BCG-PSN). METHODS: BCG-PSN was extracted by hot-phenol method, and combined with DDA and Mycobacterium tuberculosis fusion antigen AMM (Ag85B-MPT64(190-198)-Mtb8.4) to formulate the Mycobacterium tuberculosis subunit vaccine. Mice were immunized subcutaneously with a 2-week interval between the immunizations (0.2 ml/dose), and humoral and cell-mediated immunity were detected by ELISA and ELISPOT respectively. RESULTS: With the stimulation of Ag85B in vitro, the number of antigen specific IFN-gamma producing spleen lymphocytes were 222 +/- 79, 259 +/- 85, 230 +/- 64 per million respectively in the mice immunized with AMM + DDA + BCG-PSN, AMM + DDA, and BCG. Spleen lymphocytes in these 3 groups produced higher levels of IFN-gamma compared to the groups with the adjuvant of IFA or BCG-PSN alone or without adjuvant upon stimulation with Ag85B (t = 2.923-7.118, P < 0.05). Furthermore, the adjuvant consisting of DDA and BCG-PSN increased the ratio of Ig(2a)/IgG1 than DDA alone (0.125 vs. 0.025). Combined with AMM, the adjuvant DDA and the one consisting of DDA and BCG-PSN induced higher level of immunity than incomplete Freund's adjuvant (IFA), NaCl, and BCG-PSN alone. CONCLUSION: Mycobacterium tuberculosis subunit vaccine AMM + DDA + BCG-PSN induced a strong Th1-type immune response, and DDA + BCG-PSN, especially DDA promoted the immune response of the M. tuberculosis subunit vaccine in mice.

Long-term retrospective assessment of a transmission hotspot for human alveolar echinococcosis in mid-west China.

BACKGROUND: Human alveolar echinococcosis caused by infection with Echinococcus multilocularis is one of the most potentially pathogenic helminthic zoonoses. Transmission occurs involving wildlife cycles typically between fox and small mammal intermediate hosts. In the late 1980s/early 1990s a large focus of human AE was identified in poor upland agricultural communities in south Gansu Province, China. More detailed investigations in 1994-97 expanded community screening and identified key risk factors of dog ownership and landscape type around villages that could support susceptible rodent populations. A crash of the dog population (susceptible domestic definitive host) in the early 1990s appeared to stop transmission. METHODOLOGY/FINDINGS: We subsequently undertook follow-up eco-epidemiological studies based on human population screening and dog survey, in 2005/6 and in 2014/15. Our observations show a decrease in human AE prevalence, especially marked in the 11-30 year old age category. In 2015, although the dog population had recovered and in addition, forest protection and the reforestation of some areas may have favoured red fox (wild definitive host) population growth, there was no evidence of infection in owned dogs. CONCLUSIONS/SIGNIFICANCE: Those observations suggest that over decades socio-ecological changes resulted in a cascade of factors that exacerbated and then interrupted parasite emergence, with probable elimination of peri-domestic transmission of E. multilocularis in this area, despite the relative proximity of large active transmission foci on the eastern Tibetan Plateau. This study case exemplifies how anthropogenic land use and behavioural changes can modify emergence events and the transmission of endemic zoonotic parasite infections, and subsequently the importance of considering processes over the long-term in a systems approach in order to understand pathogen and disease distribution.

[Effect of combined pentoxifylline and albendazole against echinococcus multilocularis infection in mice].

OBJECTIVE: To observe the effect of pentoxifylline (PTX), albendazole (ABZ), and a combination of PTX and ABZ in mice infected with Echinococcus multilocularis (E.M). METHODS: The first part of the experiment was to observe the in vitro effect of PTX on the cultured E.M protoscolex. In the second part, mice were infected by abdominal inoculation of E.M and divided into groups given by ABZ 50 mg/kg-d, PTX 360 mg/kg-d, PTX 180mg/kgxd, and a combined regimen ABZ 50 mg/(kgxd)+PTX 180 mg/(kgxd). Another infected group and a uninfected group served as controls which received normal saline only. 100 days post-treatment, the mice were sacrificed for further observation. Indicators included wet weight of the cyst, cyst inhibition rate, level of serum cytokines TGF-beta determined by ELISA, IL-2 and IL-10 determined by radio-immunoassay (RIA). RESULTS: The inhibition rate on cysts of the combined ABZ and PTX was 88%, considerably higher than 58 % of the group ABZ. The serum TGF-beta and IL-10 decreased and IL-2 increased after treatment in comparison to the controls. CONCLUSION: The PTX and ABZ combination shows better effect on E.multilocularis infection than that of single ABZ. PTX might help increase immunity of the mice.

Serum sIL-2R, TNF-alpha and IFN-gamma in alveolar echinococcosis.

AIM: to approach the relationship between alveolar echinococcosis (AE) pathology and level of sIL-2R,TNF-alpha and IFN-gamma in sera and the significance of cytokines in development of AE. METHODS: After 23 patients with AE were confirmed by ELISA and ultrasound, their sera were collected and the concentrations of sIL-2R,TNF-alpha and IFN-gamma were detected by double antibody sandwich. Twelve healthy adults served as controls. According to the status of livers of AE patients by ultrasound scanning, they were divided into 4 groups: P(2), P(3), P(4) groups and C group (control). Average of concentrations of sIL-2R,TNF-alpha and IFN-gamma in homologous group was statistically analyzed by both ANOV and Newman-Keuls, respectively. RESULTS: The mean of sIL-2R in P(2) group was 97+/-29, P(3): 226+/-80, P(4): 194+/-23 and control group (111+/-30)X10(3) u/L (P<0.01). The mean of TNF-alpha in P(2) group was 1.12+/-0.20, P(3): 3.67+/-1.96, P(4): 1.30+/-0.25 and control group 0.40+/-0.19 mug/L (P<0.01). The mean of IFN-gamma in P(2) group was 360+/-20, P(3): 486+/-15, P(4): 259+/-19 and control group: 16+/-2 ng/L (P<0.01). Judged by ANOV and Newman-Keuls, the mean concentrations of sIL-2R, TNF-alpha and IFN-gamma had a significant difference among groups. Except for P(2) group, the mean sIL-2R between other groups of AE patients had a significant difference (P<0.05). The mean of TNF-alpha concentration in P(3) group was the highest (P<0.01). The mean of IFN-gamma concentration in all patients was higher than that in control group (P<0.01), but there was no difference between AE groups (P>0.05). CONCLUSION: Low sIL-2R level indicates an early stage of AE or stable status, per contra, a progression stage. Higher level of TNF-alpha might be related to the lesion of liver. The role of single IFN-gamma is limited in immunological defense against AE and it can not fully block pathological progression.

HLA-DRB1 allele in 35 patients with alveolar echinococcosis in Gansu Province of China.

OBJECTIVE: To investigate the association between histocompatibility leukocyte antigen (HLA)-DRB1 alleles and alveolar echinococcosis (AE). METHODS: Thirty-five patients with AE in high prevalence areas in Gansu Province of China were tested for the HLA-DRB1 gene using the polymerase chain reaction with sequence-specific primer (PCR-SSP) technique. The results were compared with those of 104 healthy individuals. RESULTS: The frequency of the HLA-DRB1 * 040x gene was 26% in the patient group, which was significantly higher than that in the control group (9.62%) with a relative risk (RR) of 4.45 (chi(2) = 13.67, P < 0.01), and an etiological fraction (EF) of 0.20. The frequency of the HLA-DRB1 * 0701 allele was significantly lower in the patient group (2.86%) as compared to the control group (13.94%; chi(2) = 6.67, P < 0.05) with a preventable fraction (PF) of 0.30. The frequencies of other DRB1 alleles were not significantly different. CONCLUSION: Susceptibility to AE is significantly associated with the HLA-DRB1 * 040x. HLA-DRB1 * 0701 gene might confer protection against AE in humans.

[Study on CD4+ cells deletion mechanism in experimental alveolar echinococcosis].

OBJECTIVE: To study the possible mechanism of CD4+ T cells deletion in mice with alveolar echinococcosis, particularly on the relationship between Echinococcus multilocularis infection and apoptosis of T lymphocyte subsets. METHODS: BALB/c mice were infected with E. multilocularis and uninfected mice were used as control group. CD4+ T cell and CD8+ T cells were separated 12 weeks and 25 weeks after infection. Purified CD4+ and CD8+ T cell subsets were cultured in complete medium and stimulated with EmAg, anti-CD3 mAb, rIL-2, mouse rTNF alpha and PWM respectively. After 16 h of incubation, cells were collected and assessed by electron microscopy. DNA fragmentation was observed by eletrophoresis, stained by TUNEL assays and PI, analyzed by flow cytometry. RESULTS: CD4+ and CD8+ T cells in 25 weeks experiment group presented chromatin condensation, lost nuclear membrane integrity, and formed exocytoplasmic vacuolization. DNA ladder was observed by agarose gel eletrophoresis, and the appearance of DNA fragments was equivalent to approximately 200 bp. None of these appearances were observed in control group in 12 weeks post infection and CD8+ T cell in mice of 25 weeks post infection group. The apoptosis level of CD4+ and CD8+ T cells in 12 weeks post infection group was not significantly different from the control group. While the apoptosis level of CD4+ and CD8+ T cells increased significantly in 25 weeks post infection group as compared with the control (P < 0.01). Higher apoptosis in CD4+ T cells was observed than that of CD8+ T cells. Apoptosis mainly appeared during S phase of cell cycle. CONCLUSION: Apoptosis is a prominent causation of activation-induced CD4+ T cell death in later period of E. multilocularis infection. Increase of the death-promoter signals and decrease of the death suppresser signals may have been responsible, in part, for the apoptosis in CD4+ T lymphocytes in the infected mice.

[Kinetic analysis of cytokines and immunoglobulin G subclass in BALB/c mice infected with Echinococcus alveolaris].

OBJECTIVE: To observe the dynamic change of immune response in mice infected with Echinococcus alveolaris (AE) at difference period of time, and to explore hostalveo's immune regulation. METHODS: The infection lasted and was followed up for 25 weeks. The spleen cells from BALB/c mice infected with AE stimulated with EmAg and ConA or PHA in vitro. IL-2R, IFN-gamma, TNF-alpha, IL-1 and specific IgG subclasses were determined by ELISA. NO was tested by chemical assay. RESULTS: NO level sharply rose in 16 weeks after BALB/c mice were infected with AE. The levels of IgG1 and IgG3 significantly increased 8 weeks after infection, and remained elevating throughout the period of observation. IgG3 showed slight increase, IgG2a and IgG2b appeared low level following infection. The production of IL-2R and TNF alpha increased significantly 8 weeks of infection, while IL-2R sharply decreased in 12 weeks of infection. During the period of 2-12 weeks of infection there was an increase in IL-1 secreting. The level of IL-1 and TNF alpha rapidly increased since 16 weeks post infection. High level of IFN-gamma was detected during the period of observation, and showed a peak at 12 weeks. CONCLUSION: Th1 is the major response in the early stage of infection, which is replaced by Th2 response in later period of infection.

Bioinformatic analysis for structure and function of TCTP from Spirometra mansoni.

OBJECTIVE: To predict structure and function of translationally controlled tumor protein (TCTP) from Spirometra mansoni by bioinformatics technology, and to provide a theoretical basis for further study. METHODS: Open reading frame (ORF) of EST sequence from Spirometra mansoni was obtained by ORF finder and was translated into amino acid residue by DNAclub. The structure domain was analyzed by Blast. By the method of online analysis tools: Protparam, InterProScan, protscale, SignalP-3.0, PSORT II, BepiPred, TMHMM, VectorNTI Suite 9 packages and Phyre2, the structure and function of the protein were predicted and analyzed. RESULTS: The results showed that the EST sequence was Sm TCTP with 173 amino acid residues, theoretical molecular weight was 19 872.0 Da. The protein has the closest evolutionary status with Clonorchis sinensis, Schistosoma mansoni, and Schistosoma japonicum. Then it had no signal peptide site and transmembrane domain. Secondary structure of TCTP contained two α -helices and eight ß -strands. CONCLUSIONS: Sm TCTP was a variety of biological functions of protein that may be used as a vaccine candidate molecule and drug target.

Community surveys and risk factor analysis of human alveolar and cystic echinococcosis in Ningxia Hui Autonomous Region, China.

OBJECTIVE: To determine the true community prevalence of human cystic (CE) and alveolar (AE) echinococcosis (hydatid disease) in a highly endemic region in Ningxia Hui, China, by detecting asymptomatic cases. METHODS: Using hospital records and "AE-risk" landscape patterns we selected study communities predicted to be at risk of human echinococcosis in Guyuan, Longde and Xiji counties. We conducted community surveys of 4773 individuals from 26 villages in 2002 and 2003 using questionnaire analysis, ultrasound examination and serology. FINDINGS: Ultrasound and serology showed a range of prevalences for AE (0-8.1%; mean 2%) and CE (0-7.4%; mean 1.6%), with the highest prevalence in Xiji (2% for CE, 2.5% for AE). There were significant differences in the prevalence of CE, AE and total echinococcosis between the three counties and villages (with multiple degrees of freedom). While hospital records showed 96% of echinococcosis cases attributable to CE, our survey showed a higher prevalence of human AE (56%) compared to CE (44%). Questionnaire analysis revealed that key risk factors for infection were age and dog ownership for both CE and AE, and Hui ethnicity and being female for AE. Drinking well-water decreased the risk for both AE and CE. CONCLUSION: Echinococcosis continues to be a severe public health problem in this part of China because of unhygienic practices/habits and poor knowledge among the communities regarding this disease.