Engineered Staphylococcus auricularis Cas9 with high-fidelity.

CRISPR-Cas9 is a versatile gene editing tool with a broad application of basic research and clinical therapeutics. However, the potential impact caused by off-target effects remains a critical bottleneck. The small Cas9 ortholog from Staphylococcus auricularis (SauriCas9) was identified, which recognizes a 5'-NNGG-3' protospacer adjacent motif (PAM), exhibiting high activity for genome editing. Recently, we also reported enhanced-fidelity Staphylococcus aureus Cas9 (efSaCas9), which harbors a single mutation N260D. Protein sequence alignment revealed that SauriCas9 has 62.4% sequence identity with SaCas9. Because SauriCas9 is more flexible in recognizing the target sequence with PAM of 5'-NNGG-3' than SaCas9 of 5'-NNGRRT-3' PAM, we sought to test whether key mutation(N260D) or adjacent residue mutation in efSaCas9 can be appliable to SauriCas9. With this concept, two engineered SauriCas9 variants (SauriCas9-HF1, harboring the N269D mutation; SauriCas9-HF2, harboring the D270N mutation) dramatically improved targeting specificity by targeted deep sequencing and GUIDE-seq. At certain sites, reduced off-target effects (approximately 61.6- and 111.9-fold improvements) of SauriCas9-HF2 compared with wild-type SauriCas9 were observed. Overall, two identified SauriCas9 variants (SauriCas9-HF1 and SauriCas9-HF2) expand the utility of the CRISPR toolkit for research and therapeutic applications.

RPA assay coupled with CRISPR/Cas12a system for the detection of seven Eimeria species in chicken fecal samples.

Chicken coccidiosis is one of the most common and economically important diseases in the global poultry industry, and it is caused by at least one of the seven Eimeria species. A simple and reliable way to distinguish Eimeria species in infected chicken is critical for the surveillance, control, and eradication of chicken coccidiosis. In this study, a recombinase polymerase amplification (RPA) assay coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system (RPA-CRISPR/Cas12a) was developed for the detection of Eimeria species in chicken fecal samples. This assay is highly specific to the seven Eimeria species and it does not cross react between species. Assessment of analytical sensitivity revealed that a single copy of plasmid DNA could be detected. Comparative analysis revealed strong agreement between RPA-CRISPR/Cas12a assays and real-time qPCR to reliably detect all seven Eimeria species in fecal chicken samples. Importantly, the cleavage products could be visualized under a blue light instrument, making it possible for the rapid detection of Eimeria species for on-site testing. Collectively, our study demonstrated that RPA-CRISPR/Cas12a assays offer a simple and reliable diagnostic method for Eimeria species.

Prevalence and multilocus sequence typing of Clostridium perfringens isolated from different stages of a duck production chain.

Clostridium perfringens (C. perfringens) is a zoonotic microorganism and rarely reported in duck production chain. This study aimed to investigate prevalence, serotype distribution, antibiotic resistance and genetic diversity of C. perfringens at different stages of a duck production chain. In total, 319 samples were collected from a large-scale rearing and slaughter one-stop enterprise in Weifang, China, of which 42.95% of samples were positive for C. perfringens. All isolates were genotype A. Cpe and cpb2 genes were found in 2.54% and 24.87% of the isolates, respectively. Antimicrobial susceptibility testing revealed that 55.47% of the isolates resistant to at least 5 classes of commonly used antibiotics. Multilocus sequence typing (MLST) results showed that 65 representative isolates were divided into 47 sequences types (STs), 33.85% of them were included into four clonal complexes (CC). Some of isolates from breeding and slaughtering stages were distributed in the same CC or ST, indicating duck products may be contaminated by C. perfringens originated from the breeding stage. Part of duck isolates were distributed in the same CC as human isolates and systemically close with human isolates. The high contamination rates of duck products, the isolates with multi-drug antibiotic resistance or the cpe gene, and the close relationship between strains from human and ducks, indicated potential public health risks, not only control measures at slaughtering stage but also at rearing stage should be considered to reduce this risks.

An ultrasensitive, rapid and portable method for screening oseltamivir-resistant virus based on CRISPR/Cas12a combined with immunochromatographic strips.

Influenza is a significant public health challenge because of the emergence of antigenically shifted or highly virulent strains. The neuraminidase inhibitor oseltamivir is used as an antiviral drug in clinical treatment. However, its therapeutic effects can be greatly compromised by the emergence of drug-resistant mutant viruses. Thus, there is an urgent need to distinguish drug-resistant strains with a simple method. To address this, in the present study, we develop a rapid, sensitive and convenient molecular diagnosis method based on CRISPR/Cas12a technology and lateral flow detection (LFD). By targeting mutant sequences amplified by recombinase polymerase amplification (RPA) reaction, crRNA is designed to develop the CRISPR/Cas12a assay, and 2000 copies can be directly observed by the naked eye under blue light-emitting diode (LED) light. Combined with LFD, the limit of detection of RPA-CRISPR/Cas12a-LFD is about 20 copies of target sequence per reaction. Collectively, RPA-CRISPR/Cas12a-LFD method provides a novel alternative for the sensitive, specific and portable detection to diagnose oseltamivir-resistant mutant strains.

A novel fluorescence immunochromatographic assay strip for the diagnosis of schistosomiasis japonica.

BACKGROUND: Schistosomiasis japonica is a severe zoonosis. Domestic animals are the primary source of infection and play an important role in disease transmission. Surveillance and diagnosis play key roles in schistosomiasis control; however, current techniques for the surveillance and diagnosis of the disease have limitations. In this study, we developed a novel fluorescence immunochromatographic assay (FICA) strip to detect anti-Schistosoma japonicum antibodies in host serum. METHODS: A FICA strip was developed for the diagnosis of Schistosoma japonicum in domestic animals. Streptococcus protein G (SPG) and soluble egg antigen (SEA) were transferred onto a nitrocellulose (NC) membrane to form the control line (C) and the test line (T), respectively. With fluorescence activity as well as binding activity to multispecies IgG, the recombinant protein rSPG-RFP was expressed and employed as an antibody indicator in the FICA strips. RESULTS: The dual gene fusion plasmid was verified by PCR and restriction enzyme digestion. The expressed recombinant protein was 39.72 kDa in size, which was consistent with the predicted molecular weight. The western blot results showed binding activity between rSPG-RFP and IgGs from different hosts. Fluorescence microscopy also showed the fluorescence activity of the protein present. The affinity constant (Ka) values of rSPG-RFP with rabbit, donkey, mouse and goat IgG were 1.9 × 105, 4.1 × 105, 1.7 × 105 and 5.4 × 105, respectively. Moreover, based on the recombinant protein, the test strip for detecting S. japonicum in buffaloes could distinguish positive from negative serum. The lower limit of detection of the FICA strip was 1:10,000. Compared with ELISA, the FICA strips exhibited similar results in the diagnosis of infection in clinical bovine serum samples, with a kappa value of 0.9660 and P < 0.01. The cross-reactivities of the FICA strips with Haemonchus contortus and Schistosoma turkestanicum (30.15% and 91.66%, respectively) were higher than those of ELISA (26.98% and 87.5%, respectively). CONCLUSIONS: Based on the rSPG-RFP protein that we developed, strip detection can be completed within 15 min. Heightened sensitivity allows the strip to accurately identify schistosome antibodies in serum. In conclusion, this method is convenient, feasible, rapid and effective for detecting S. japonicum.

Trichinella infectivity and antibody response in experimentally infected pigs.

The objective of the present study was to investigate the infectivity and antibody response of four Trichinella species (Trichinella spiralis, Trichinella britovi, Trichinella pseudospiralis and Trichinella murrelli) in experimentally infected pigs. A total of 120 Large White pigs (30 animals per group) were inoculated with 10,000 muscle larvae (ML) of T. spiralis, T. britovi, T. pseudospiralis, and T. murrelli. The pigs were sacrificed at 12-21 days post-infection (dpi) to examine the viability and infectivity of ML. A total of 54 Large White pigs (6 animals per group) were inoculated with 25, 50, 100, 200, 400, 600, 800, 1000 and 10,000 T. spiralis ML. The pigs were sacrificed, and the average numbers of larvae per gram (lpg) from six different muscle tissues were calculated at 120 dpi. The results showed that the larvae first be detectable for T. spiralis, T. britovi, and T. pseudospiralis at 16 dpi, 17 dpi, and 16 dpi, respectively. Viable larvae and average lpg were significantly increased with time from 17 to 21 dpi. The T. spiralis ML burden was dependent of the inoculation dose with an average lpg of 0.003, 0.005, 0.007, 0.17, 0.82, 2.89, 4.90, 28.30 and 226.18, respectively. The IgG antibody response was dose-dependent to generate and increased throughout the experimental period. And the IgG1 isotype was significantly higher than IgG2a, which meant that T. spiralis infection induced the Th2 immune response. The time of detecting IgM antibodies was significantly earlier than IgG antibody detection. These results provide important information in the primary characterization of pigs infected with Trichinella.

Establishment of a fluorescence staining method for Schistosoma japonicum miracidia.

Currently the diagnosis of schistosomiasis is mainly determined by observing the presence of eggs in host stool samples. Because of the overwhelming number of impurities in the stool, eggs are rarely observed. Therefore, the stool hatching method is used to observe the miracidia in the water. However, the miracidia of Schistosoma japonicum are small and difficult to detect, and missed detection is likely to occur when the infection level is low. In this study, recombinant streptococcal protein G-enhanced green fluorescent protein (rSPG-EGFP) was expressed, purified, and used as a fluorescence staining reagent for miracidia. A preliminary miracidium fluorescence staining method based on antigen and antibody bindingwas established by combining recombinant protein staining with the stool hatching method. The stool hatching method was used to collect the miracidia of S. japonicum, and Schistosoma-positive serum and the recombinant protein were mixed to assess the feasibility of fluorescence staining of miracidia. The miracidia of S. japonicum and Schistosoma turkestanicum were incubated with S. japonicum-positive serum and S. turkestanicum-positive serum, respectively, to identify miracidia species. When the fluorescence staining method was used to observe living miracidia, the miracidiawere labelled by the recombinant protein, and their motility status was not affected.

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.

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

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.

A perspective for improving the sensitivity of detection: The application of multi-epitope recombinant antigen in serological analysis of buffalo schistosomiasis.

The sensitivity and specificity are two crucial aspects of addressing the efficacy of diagnostic antigens. Achilles' heel of low sensitivity rate exists in current diagnostic recombinant antigens for schistosomiasis detection. This study focused on the diagnosis of water buffalo schistosomiasis japonica and a perspective of improving recombinant antigens' sensitivity was assessed using archived 220 water buffalo sera (114 positive sera, 92 negative sera and 14 Paramphistomum-infected sera) and the method of enzyme-linked immunosorbent assay (ELISA). The subjects included two trivalent recombinant proteins, one bivalent antigen and two single-molecular antigens. The crude antigen SEA (soluble egg antigen) was employed as reference antigen. The highest sensitivity rate in the five recombinant antigens assigned to the trivalent multi-epitope antigen PA4 (95.61%, 109/114), no significant difference with SEA (100%, 114/114, p = .836), and showing remarkable differences with the two single-molecular antigens (p < 0.01). In term of specificity, two trivalent multi-epitope antigens PA4 (97.83%, 90/92), PA5 (100%, 92/92) and the bivalent antigen PA3 (98.91%, 91/92) had few differences with one monovalent antigens PA1 (97.83%, 90/92, p = .304/0.103/0.640), significant differences with another monovalent antigens PA2 (92.39%, 85/92, p < 0.01) and SEA (82.61%, 76/92, p < 0.01). Additional, all the recombinant antigens had low cross-reactivity (7.14%, 1/14, 0% for PA5) with serum samples of paramphistomiasis, contrast with that of SEA (50%, 7/14, p < 0.01). The results indicated that multi-epitope antigens have the possibility to improve diagnostic sensitivity and the trivalent multi-epitope antigen PA4 possesses greater likelihood to be a diagnostic antigen for water buffalo schistosomiasis.

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.

Suppression of VAMP2 Alters Morphology of the Tegument and Affects Glucose uptake, Development and Reproduction of Schistosoma japonicum.

Schistosomiasis caused by schsitosomes is a serious global public health concern. The tegument that surrounds the worm is critical to the schistosomes survival. The tegument apical membrane undergoes a continuous process of rupture and repair owing to membranous vacuoles fusing with the plasma membrane. Vesicle-associated membrane protein 2 (VAMP2), a member of soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNAREs) is required for membrane fusion. Here, we used RNA interference (RNAi) to knock down the expression of VAMP2 of Schistosoma japonicum (SjVAMP2), and both real-time PCR and western blot analysis confirmed the suppression of this molecule, as well as the suppression of the transcript levels of schistosome glucose transporters (SGTP1 and SGTP4), and insulin receptors (SjIR1 and SjIR2). SjVAMP2-suppressed worms exhibited a lower viability, and phenotypic alterations were also observed in the tegument. Moreover, the glucose consumption of SjVAMP2-suppressed worms decreased significantly in 4 and 6 days, respectively, as well as a significant reduction in egg production. We also observed a significant reduction in worm burden and hepatic eggs burden in two independent RNAi experiment in vivo, and minor pathological changes in mice treated with SjVAMP2 specific small interfering (si)RNA. These findings reveal that SjVAMP2 may play important roles in the maintenance of tegument, glucose uptake, worm development and egg production in schistosomes.

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

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.

Nested-PCR assay for detection of Schistosoma japonicum infection in domestic animals.

BACKGROUND: Schistosomiasis japonica is a common zoonosis. Domestic animals are the primary source of infection and play an important role in disease transmission. The prevalence and infectivity of this disease in domestic animals in China have significantly decreased and, for this reason, diagnostics with a higher sensitivity have become increasingly necessary. It was reported that polymerase chain reaction (PCR)-based methods could be used to detect schistosome infection in humans and animals and presented a high sensitivity and specificity. The present study aimed to develop a PCR-based method for detection of Schistosoma japonicum infection in domestic animals. METHODS: A specific nested-PCR assay was developed to detect S. japonicum infection in domestic animals via amplification of a 231-bp DNA fragment of retrotransposon SjR2. The developed assay was first used in sera and dry blood filter paper (DBFP) from goats and buffaloes at different time points of infection. Then, 78 DBFPs from 39 artificially-infected bovines at 14 and 28 days post-infection and 42 DBFPs from schistosome-negative bovines from the city of Huangshan in the Anhui province were used to evaluate the diagnostic validity. Furthermore, this assay was used to detect S. japonicum infection in domestic animals in Dongzhi and Wangjiang counties. RESULTS: The expected PCR product was detected in eggs and adult worms of S. japonicum and blood samples from S. japonicum-infected goats and water buffaloes, but not from Fasciola and Haemonchus contortus worms. The nested-PCR assay could detect the target S. japonicum DNA in DBFPs from goats and buffaloes after day 3 post-infection. The sensitivity in buffaloes at 14 and 28 days post-infection was 92.30% (36/39) and 100% (39/39), respectively. The specificity was 97.60% (41/42). The positivity rates in Dongzhi and Wangjiang counties were 6.00% and 8.00% in bovines and 22.00% and 16.67% in goats, respectively. The positivity rates in goats in both counties were higher than those in bovines with a significant difference in Dongzhi County but not in Wangjiang County (P < 0.05 and P = 0.23, respectively). CONCLUSIONS: Our results suggest that the developed nested-PCR assay may be used for the diagnosis of S. japonicum infection in domestic animals, and the control of S. japonicum infection in goats should be paid more attention.

Proteome-wide analysis of lysine acetylation in adult Schistosoma japonicum worm.

UNLABELLED: Lysine acetylation, a ubiquitous and conserved posttranslational modification, has recently been shown to participate in many diverse non-chromatin-associated biological processes in prokaryotes and eukaryotes. However, the full extent and functional significance of acetylation in Schistosoma japonicum is still unknown. To investigate the nature, extent, and biological functions of lysine acetylation in schistosomes, immunoaffinity-based acetyl-lysine peptide enrichment, integrated with mass spectrometry, was used to comprehensively characterize the lysine-acetylated proteins in this parasite. In total, 1109 acetylated proteins and 2393 acetylation sites in S. japonicum were identified, representing the largest acetylome yet reported in a parasite. In a bioinformatic analysis showed that these acetylated proteins were mainly enriched in the biological process categories of metabolism, gene expression, translation, and transport. The classification according to molecular function revealed that the largest class involved the catalytic activity of different enzymes, including oxidoreductase, transferase, and pyrophosphatase activities. Most of the acetylated proteins in the cellular component category occurred in the cytoplasm, membrane, cytoskeleton, and nucleus. These data demonstrate the generality of lysine acetylation and provide the first global survey of acetylation in schistosomes. Our findings are an exciting starting point for the further exploration of the functions of acetylation in the biology of this parasite. SIGNIFICANCE: Schistosomiasis is one of the world's most prevalent and neglected tropical parasitic zoonotic diseases, and it causes almost 200,000 deaths annually. To control and eradicate schistosomiasis, effective vaccines are urgently required, and drug targets that are essential for schistosome survival must be identified in fundamental studies of schistosome biology. Posttranslational modifications are complex, fundamental, and important mechanisms that regulate the physiological functions of organisms. Lysine acetylation, a ubiquitous and conserved posttranslational modification, has recently been shown to participate in many diverse non-chromatin-associated biological processes in prokaryotes and eukaryotes. However, the full extent and functional significance of acetylation in Schistosoma japonicum is still unknown. To investigate the nature, extent, and biological functions of lysine acetylation in S. japonicum, we employ immunoaffinity-based acetyl-lysine peptide enrichment, integrated with mass spectrometry to comprehensively characterize the lysine-acetylated proteins in this parasite. The results of our data demonstrate the generality of lysine acetylation and provide the first global survey of acetylation in schistosomes. Our findings are an exciting starting point for the further exploration of the functions of acetylation in the biology of this parasite. Meanwhile, identifying the mechanisms and proteins targeted by acetylation may also provide a promising avenue for specific drug design and the development of sophisticated therapeutic strategies.

Evaluation of recombinant multi-epitope proteins for diagnosis of goat schistosomiasis by enzyme-linked immunosorbent assay.

BACKGROUND: Schistosomiasis is a huge threat to human and animal health. Apart from bovines, goats play an important role in the transmission of schistosomiasis in some endemic areas of China. An accessible, quality-assured goat schistosomiasis diagnostic technique is needed. Recently, our laboratory identified two recombinant diagnostic antigens, SjPGM and SjRAD23 via an immuno-proteomic method. The application of these two recombinant antigens to develop a higher sensitivity and specificity technique for the sheep schistosomiasis diagnosis is urgently needed. METHODS: Epitopes of SjPGM and SjRAD23 were predicted and three polypeptides, two from SjRAD23 and one from SjPGM, were selected. Recombinant plasmids containing two to three DNA sequences encoding predicted polypeptides or large hydrophilic region of Sj23 (LHD-Sj23) were constructed and expressed. Eight recombinant schistosome antigens including four multi-epitope proteins and four recombinant single-molecule antigens as well as SEA, were assessed by ELISA in 91 sera from schistosome-infected goats, 44 sera from non-infected goats, 37 sera from Orientobilharzia-infected goats, and 12 from Haemonchus contortus-infected goats. RESULTS: ELISA tests showed that three multi-epitope proteins had higher sensitivity than the four single-molecule antigens (rSjRAD23, rSjPGM, rBSjRAD23-1, rBSj23) and the multi-epitope protein rBSjPGM-BSjRAD23-1-BSj23 had the highest sensitivity (97.8 %, 89/91) and maintained good specificity (100 %, 44/44) as well as low cross-reactivity with haemonchosis (8.33 %, 3/12) and orientobilharziasis (13.51 %, 5/37) in the diagnosis of goat schistosomiasis. In contrast, when SEA was applied as a diagnosis antigen, it had 100 % (91/91) sensitivity, 75 % (33/44) specificity, 25 and 83.78 % cross-reactivity with haemonchosis (3/12) and orientobilharziasis (31/37), respectively. CONCLUSIONS: The application of recombinant multi-epitope proteins may increase the sensitivity of diagnosis technique and retain high specificity of single-molecule antigens for schistosomiasis, and the recombinant antigen rBSjPGM-BSjRAD23-1-BSj23 has the potential to be used as a diagnosis antigen for goat schistosomiasis.

Excretory/secretory proteome of 14-day schistosomula, Schistosoma japonicum.

Schistosomiasis remains a serious public health problem, with 200 million people infected and 779 million people at risk worldwide. The schistosomulum is the early stage of the complex lifecycle of Schistosoma japonicum in their vertebrate hosts, and is the main target of vaccine-induced protective immunity. Excretory/secretory (ES) proteins play a major role in host-parasite interactions and ES protein compositions of schistosomula of S. japonicum have not been characterized to date. In the present study, the proteome of ES proteins from 14 day schistosomula of S. japonicum was analyzed by liquid chromatography/tandem mass spectrometry and 713 unique proteins were finally identified. Gene ontology and pathway analysis revealed that identified proteins were mainly involved in carbohydrate metabolism, degradation, response to stimulus, oxidation-reduction, biological regulation and binding. Flow cytometry analysis demonstrated that thioredoxin peroxidase identified in this study had the effect on inhibiting MHCII and CD86 expression on LPS-activated macrophages. The present study provides insight into the growth and development of the schistosome in the final host and valuable information for screening vaccine candidates for schistosomiasis.

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

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSION: 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.

Cloning, expression and enzymatic characterization of 3-phosphoglycerate kinase from Schistosoma japonicum.

In the present study, a full-length cDNA encoding the Schistosoma japonicum 3-phosphoglycerate kinase (SjPGK) with an open reading frame of 1251 bp was isolated from 42-day-old (42-d) schistosome cDNAs. Real-time quantitative reverse transcription PCR analysis revealed that SjPGK was expressed in all investigated developmental stages and at a higher transcript levels in 21- and 42-d worms. Moreover, the SjPGK mRNA level was significantly downregulated in 10-d schistosomula from Wistar rats (non-susceptible host). SjPGK was subcloned into pET28a(+) and expressed as both supernatant and inclusion bodies in Escherichia coli BL21 cells. The enzymatic activity of recombinant SjPGK protein (rSjPGK) was 125 U/mg. Kinetic analyses with respect to 3-phosphoglycerate (3-PGA) as substrate gave a Km of 2.69 mmol/L and a Vmax of 748 µmol/min/mg protein. rSjPGK was highly stable over a range of pH 8.0-9.0 and temperature of 30°C-40 °C under physiological conditions. Immunolocalization analysis showed that SjPGK was mainly distributed in the tegument and parenchyma of schistosomes. Western blotting showed that rSjPGK had good immunogenicity. We vaccinated BALB/c mice with rSjPGK combined with Seppic 206 adjuvant. However, there were no significant reductions in the numbers of worms of eggs in the liver, as compared to adjuvant or blank control groups in two independent vaccination tests. This study provides the basis for further investigations into the biological function of SjPGK, although it might not be suitable as a potential vaccine candidate against schistosomiasis.

The Differential Expression of Immune Genes between Water Buffalo and Yellow Cattle Determines Species-Specific Susceptibility to Schistosoma japonicum Infection.

Water buffalo are less susceptible to Schistosoma japonicum infection than yellow cattle. The factors that affect such differences in susceptibility remain unknown. A Bos taurus genome-wide gene chip was used to analyze gene expression profiles in the peripheral blood of water buffalo and yellow cattle pre- and post-infection with S. japonicum. This study showed that most of the identified differentially expressed genes (DEGs) between water buffalo and yellow cattle pre- and post-infection were involved in immune-related processes, and the expression level of immune genes was lower in water buffalo. The unique DEGs (390) in yellow cattle were mainly associated with inflammation pathways, while the unique DEGs (2,114) in water buffalo were mainly associated with immune-related factors. The 83 common DEGs may be the essential response genes during S. japonicum infection, the highest two gene ontology (GO) functions were associated with the regulation of fibrinolysis. The pathway enrichment analysis showed that the DEGs constituted similar immune-related pathways pre- and post-infection between the two hosts. This first analysis of the transcriptional profiles of natural hosts has enabled us to gain new insights into the mechanisms that govern their susceptibility or resistance to S. japonicum infections.