Comparative analysis of microRNA expression profiles of adult Schistosoma japonicum isolated from water buffalo and yellow cattle

Abstract Background Yellow cattle and water buffalo are important natural reservoir hosts and the main transmission sources of Schistosoma japonicum in endemic areas of China. The worms from the two hosts have marked differences in general worm morphology and ultrastructure, gene transcription and protein expression profiles. Results To investigate microRNAs (miRNAs) involved in the regulation of schistosome development and survival, we compared miRNA expression profiles of adult schistosomes derived from yellow cattle and water buffalo by using high-throughput sequencing with Illumina Hiseq Xten. Schistosoma japonicum from water buffalo and yellow cattle yielded 63.78 million and 63.21 million reads, respectively, of which nearly 50% and 49% could be mapped to selected miRNAs in miRbase. A total of 206 miRNAs were identified, namely 79 previously annotated miRNAs of S. japonicum and 127 miRNAs that matched with the S. japonicum genome and were highly similar to the annotated miRNAs from other organisms. Among the 79 miRNAs, five (sja-miR-124-3p, sja-miR-219-5p, sja-miR-2e-3p, sja-miR-7-3p and sja-miR-3490) were significantly upregulated in the schistosomes from water buffalo compared with those from yellow cattle. A total of 268 potential target genes were predicted for these five differentially expressed miRNAs. Eleven differentially expressed targets were confirmed by qRT-PCR among 15 tested targets, one of which was further validated through dual-luciferase reporter assay. Among the 127 ‘possible’ S. japonicum miRNAs, ten were significantly differentially expressed in the schistosomes from these two hosts. Conclusions These results highlight the important roles of miRNAs in regulating the development and survival of schistosomes in water buffalo and yellow cattle and facilitate understanding of the miRNA regulatory mechanisms in schistosomes derived from different susceptible hosts

Characterization of VAMP2 in Schistosoma japonicum and the Evaluation of Protective Efficacy Induced by Recombinant SjVAMP2 in Mice.

The outer-tegument membrane covering the schistosome is believed to maintain via the fusion of membranous vesicles. Fusion of biological membranes is a fundamental process in all eukaryotic cells driven by formation of trans-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes through pairing of vesicle associated v-SNAREs (VAMP) with complementary t-SNAREs on target membranes. The purpose of this study was to characterize Schistosoma japonicum vesicle-associated membrane protein 2 (SjVAMP2) and to investigate its potential as a candidate vaccine against schistosomiasis.The sequence of SjVAMP2 was analyzed, cloned, expressed and characterized. SjVAMP2 is a member of the synaptobrevin superfamily harboring the v-SNARE coiled-coil homology domain. RT-PCR analysis revealed that significantly higher SjVAMP2 levels were observed in 14-, 28- and 42-day-old worms, and SjVAMP2 expression was much higher in 42-day-old female worms than in those male worms. Additionally, the expression of SjVAMP2 was associated with membrane recovery in PZQ-treated worms. Immunostaining assay showed that SjVAMP2 was mainly distributed in the sub-tegument of the worms. Western blotting revealed that rSjVAMP2 showed strong immunogenicity. Purified rSjVAMP2 emulsified with ISA206 adjuvant induced 41.5% and 27.3% reductions in worm burden, and 36.8% and 23.3% reductions in hepatic eggs in two independent trials. Besides, significantly higher rSjVAMP2-specific IgG, IgG1, IgG2a levels were detected in rSjVAMP2-vaccinated mice.Our study indicated that SjVAMP2 is a potential vaccine candidate against S. japonicum and provided the basis for further investigations into the biological function of SjVAMP2

An ELISA based on soluble egg antigens for the serodiagnosis of animal schistosomiasis turkestanica.

BACKGROUND:The existing diagnostic techniques for detecting schistosomiasis turkestanica, such as aetiological assays, identify infection by parasitic worms via the incubation of miracidia from faeces or observing eggs under microscopy. However, they are limited in the diagnosis of low-grade and prepatent infections, which lead to a high misdetection rates. Therefore, a new method for parasite diagnosis with increased sensitivity is urgently needed. METHODS:Goats in Nimu County (Tibet, China) infected with Schistosoma turkestanicum in an epidemic area were selected according positivity for the infection by faecal examination. Adult worms were collected, eggs were extracted by the sodium hydroxide (NaOH) erosion method, and soluble worm antigen preparation (SWAP) and soluble egg antigen (SEA) were isolated. The best coating concentration of the antigens and the best degree of dilution for serum were determined by square array experiments, and the optimal blocking solution and serum diluents were selected. The specificity, sensitivity and crossover of the ELISA method were determined using 48 samples of goat sera positive for S. turkestanicum, 100 samples of goat sera negative for S. turkestanicum, and 54 samples of buffalo sera positive for S. japonicum. Serological assays were established with samples from goats naturally grazed in a rural area of Nimu County, Tibet Province, by using the indirect ELISA method for the diagnosis of schistosomiasis, and faeces were collected for miracidia hatching. The sensitivity of the two detection methods was compared. RESULTS:Eggs of S. turkestanicum were distributed in the host duodenum and small intestine. Eggs in the host intestinal wall were extracted by the NaOH erosion method, which provided intact eggs with reduced impurities. The testing results obtained by isolating SEA were more stable than those obtained by using SWAP and less affected by the coating concentration and serum dilution. Additionally, the value of positive serum/negative (P/N) serum for SEA was much higher than that for SWAP. The optimal coating concentration of SEA was 0.5 μg/ml, and the optimal serum dilution was 1:100. The specificity and sensitivity of the indirect ELISA based on SEA (S. turkestanicum) were both 100%, and no cross-reactivity was found with schistosomiasis japonica. An epidemiological survey of goats in naturally infected areas showed that the prevalence rate of schistosomiasis turkestanica was 93%, and the infection rate increased with the ages of the goats. CONCLUSION:We aimed to develop a sensitive method to utilize in the mass field screening of livestock. As a diagnostic antigen, SEA (S. turkestanicum) was more suitable for serological testing than SWAP (S. turkestanicum). The indirect ELISA using SEA (S. turkestanicum) exhibited good sensitivity, specificity and no cross-reactivity with schistosomiasis japonica. The degree of infectivity and prevalence of S. turkestanicum infection in endemic areas are serious and should be a focus of concern among local departments

Chitosan/Alginate Nanoparticles with Sustained Release of Esculentoside A for Burn Wound Healing

Burn injury remains one of the most devastating burdens on global public health. The inflammation, caused by burn injury and transplantation of dermal scaffolds, often leads to delayed burn wound healing. Esculentoside A (EsA), with a strong anti-inflammatory capacity, is an available agent that might contribute to the treatment of burn wounds. However, the poor stability and toxicity of EsA limit its clinical application. In the present study, we constructed chitosan/alginate nanoparticles (EsA-CS/ALG-NPs) to improve sustainability and reduce toxicity followed by impregnation of the prepared EsA-CS/ALG-NPs into a collagen/chitosan scaffold (EsA-CS/ALG-NPs@CCS). The particle size, structural morphology, thermal properties, and chemical interaction of repaired nanoparticles were evaluated using the Nanometrics instrument, differential scanning calorimetry, transmission electron microscopy, and Fourier transform infrared spectroscopy, respectively. The hybrid EsA-CS/ALG-NPs@CCS was evaluated for physical characteristics, in vitro drug release, biocompatibility, and anti-inflammation capacity with RAW 264.7 cells and in vivo burn wound healing studies with SD rats. The results showed that we successfully constructed CS/ALG-NPs and optimized the preparation process to achieve the highest encapsulation efficiency. The hybrid EsA-CS/ALG-NPs@CCS, with reduced cytotoxicity and sustained release of EsA, could alleviate inflammation, decrease the ratio of M1 macrophages, and increase the proportion of M2 macrophages in vitro. It was demonstrated that 5 μg EsA CS/ALG-NPs@CCS not only reduces inflammatory cytokines secretion and inhibits M1 macrophages but also promotes the release of anti-inflammatory cytokines and activates M2 macrophages, thereby achieving accelerated and high-quality healing of burn wounds ultimately. In summary, our work suggests that the synergistic combination of EsA, nanoparticles, and scaffolds provided a promising strategy for treating burn injuries

iTRAQ-Based Comparative Proteomic Analysis of Adult Schistosoma japonicum from Water Buffalo and Yellow Cattle

Schistosomiasis japonicum is one of the most severe zoonotic diseases in China. Water buffalo and yellow cattle are important reservoir hosts and the main transmission sources of Schistosoma japonicum in endemic areas. The susceptibility of these two hosts to schistosome infection is different, as water buffaloes are less susceptible to S. japonicum than yellow cattle. In this study, iTRAQ-coupled LC-MS/MS was applied to compare the protein expression profiles of adult schistosomes recovered from water buffalo with those of yellow cattle. A total of 131 differentially expressed proteins (DEPs) were identified, including 46 upregulated proteins and 85 downregulated proteins. The iTRAQ results were confirmed by Western blotting and quantitative real-time PCR. Further analysis indicated that these DEPs were primarily involved in protein synthesis, transcriptional regulation, protein proteolysis, cytoskeletal structure and oxidative stress response processes. The results revealed that some of the differential expression molecules may affect the development and survival of schistosomes in these two natural hosts. Of note, this study provides useful information for understanding the interplay between schistosomes and their final hosts

A novel colloidal gold immunochromatography assay strip for the diagnosis of schistosomiasis japonica in domestic animals

Abstract Background Schistosomiasis remains a major public health concern in China and an epidemiological survey has revealed that schistosome-infected bovines and goats are the main transmission sources for the disease. Therefore, development of a sensitive technique for the diagnosis of schistosomiasis in domestic animals is necessary. Method A novel colloidal gold immunochromatography assay (GICA) strip was developed for detecting Schistosoma japonicum in domestic animals. The colloidal gold was conjugated with recombinant streptococcal protein G (rSPG). As the test and control lines, the schistosome soluble egg antigen and rSPG, respectively, were blotted on nitrocellulose membrane. Results The lowest detectable serum dilution was 1∶640 for schistosome-infected buffaloes. The cross-reaction rate of GICA was 14.29% with Paramphistomum sp. in buffaloes, 16.67% with Haemonchus sp. in goats, and 33.33% with Orientobilharzia sp. in goats. These results were slightly lower and similar to those obtained through ELISA. Moreover, the strips for detecting S. japonicum in mice, rabbits, buffaloes, and goats showed high sensitivity (100.00%, 100.00%, 100.00%, and 100.00%, respectively) and specificity (100.00%, 100.00%, 94.23%, and 88.64%, respectively). And the sensitivity or specificity of the GICA strips did not present any significant differences after storage for 12 months at room temperature. When compared with ELISA, the GICA strips exhibited similar sensitivity and specificity in the diagnosis of schistosomiasis in mice, rabbits, buffaloes, and goats. Besides, only 5 μl of serum are required for the test and the detection can be completed within 5 min. Conclusion This study is the first to develop a GICA strip using gold–rSPG conjugate for the diagnosing of schistosomiasis in domestic animals, and preliminary results showed that the developed strip may be suitable for large-scale screening of schistosomiasis in endemic areas

Effect of PZQ on SjVAMP2 transcription.

<p>(A) Mice were treated with a single dose of 40 mg/kg of PZQ (incomplete treatment dose) and sacrificed at various times post-treatment. (B) Mice were treated with single dose of 200 mg/kg of PZQ (treatment dose) and sacrificed at various times post-treatment. Statistically significant differences compared with the control group are denoted by *** (P<0.001).</p

Comparison of the number of adult worms and EPG between the rSjVAMP2-immunized group and the control groups.

<p>Comparison of the number of adult worms and EPG between the rSjVAMP2-immunized group and the control groups.</p

Immunolocalization of SjVAMP2 in <i>S</i>. <i>japonicum</i>.

<p>A, B, section of 28-day-old worm probed with anti-rSjVAMP2 mouse serum; C, D, section of 28-day-old worm probed with native mouse serum (negative control). The brown color indicates binding of the anti-rSjVAMP2 mouse serum, and the arrows show the location of the SjVAMP2 protein. The slides were analyzed by brightfield microscopy (A, C, magnification, ×100; B, D, magnification, ×400).</p