Oral vaccination with recombinant Saccharomyces cerevisiae expressing Micropterus salmoides rhabdovirus G protein elicits protective immunity in largemouth bass.

Micropterus salmoides rhabdovirus (MSRV) is one of the main pathogens of largemouth bass, leading to serious economic losses. The G protein, as the only envelope protein present on the surface of MSRV virion, contains immune-related antigenic determinants, thereby becoming the primary target for the design of MSRV vaccines. Here, we displayed the G protein on the surface of yeast cells (named EBY100/pYD1-G) and conducted a preliminary assessment of the protective efficacy of the recombinant yeast vaccine. Upon oral vaccination, a robust immune response was observed in systemic and mucosal tissue. Remarkably, following the MSRV challenge, the relative percent survival of EBY100/pYD1-G treated largemouth bass significantly increased to 66.7 %. In addition, oral administration inhibited viral replication and alleviated the pathological symptoms of MSRV-infected largemouth bass. These results suggest that EBY100/pYD1-G could be used as a potential oral vaccine against MSRV infection.

Simultaneous detection of multiple bacterial and viral aquatic pathogens using a fluorogenic loop-mediated isothermal amplification-based dual-sample microfluidic chip.

Rapid and user-friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on-site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual-sample microfluidic chip integrated with a real-time fluorogenic loop-mediated isothermal amplification assay (dual-sample on-chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on-chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10-1  pg/µl for genomic DNA of tested bacteria and 10-4 to 10-5  pg/µl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time-to-positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on-chip LAMP assay provides an effective dual-sample and multiple pathogen analysis, and thus would be applicable to on-site detection and routine monitoring of multiple pathogens in aquaculture.

Interplay between the gut microbiota and immune responses of ayu (Plecoglossus altivelis) during Vibrio anguillarum infection.

Gut microbiota plays fundamental roles in protection against pathogen invasion. However, the mechanism and extent of responses of gut microbiota to pathogenic infection are poorly understood. This study investigated the gut bacterial communities and immune responses of ayu (Plecoglossus altivelis) upon exposure to Vibrio anguillarum. The succession of V. anguillarum infection was evidenced by increased expression of immune genes and bacterial loads in ayu tissues, which in turn altered the composition and predicted functions of gut bacterial community. The dynamics of gut bacterial diversity and evenness were temporally stable in control ayu but were reduced in infected subjects, particularly at the late stages of infection. Variations in the gut microbiota were significantly associated with the expression levels of TNF-α (P = 0.019) and IL-1 ß (P = 0.013). The profiles of certain gut bacterial taxa were indicative of V. anguillarum infection. Compared with healthy controls, the ayu infected with V. anguillarum possessed less complex, fewer connected, and lower cooperative gut bacterial interspecies interaction, coinciding with significant shifts in keystone species. These findings imply that V. anguillarum infection substantially disrupted the compositions and interspecies interaction of ayu gut bacterial community, thereby altering gut microbial-mediated functions and inducing host immune responses. This study provides an integrated overview on the interaction between the gut microbiota and host immune responses to pathogen infection from an ecological perspective.

Expression of Caenorhabditis elegans-expressed Trans-HPS, partial aminopeptidase H11 from Haemonchus contortus.

Aminopeptidase H11 present in the surface of intestine microvilli in Haemonchus contortus was identified as the most effective antigen candidate. However, its recombinant forms produced in Escherichiacoli, insect cells and yeast could not provide promising protection against H. contortus challenge, probably due to the inappropriate glycosylation and/or conformational folding. Herein, partial H11 containing the potential zinc-binding domain and two predicted glycosylation sites (nt 1 bp-1710 bp, Trans-HPS) was subcloned downstream of 5' flanking region of Caenorhabditis elegans cpr-1 gene in pPD95.77 vector, with the deletion of GFP gene. The recombinant was expressed in C. elegans and verified by blotting with anti-H11 and anti-Trans-HPS rabbit polyclonal antibodies and anti-His monoclonal antibody. Stably inherited Trans-HPS in worm descendants was achieved by integration using UV irradiation. Immunization with the crude Trans-HPS extracted from transgenic worms resulted in 37.71% reduction in faecal egg counts (FEC) (P<0.05) and 24.91% reduction in worm burden, but an upward curve with moderate rate of daily FEC in goats. These results suggested an apparent delay against H. contortus egg-laying in goats, which differed from that with bacteria-origin form of partial H11 (nt 670 bp-1710 bp, HPS) (26.04% reduction in FEC and 18.46% reduction in worm burden). These findings indicate the feasibility of sufficient C. elegans-expressed H11 for the immunological research and vaccine development.

Numerical simulation research on the overturning of gantry crane by downbursts.

Based on the simulation of the fluid-structure interaction response, the cause of an overturning of a gantry crane induced by a downburst in Shenzhen is studied in this paper. According to the results, (1) Vicroy's downburst model could establish the steady-state wind field of the downburst more reasonably when there was only low-level wind speed observation data, and its simulation results were close to the two-dimensional downburst numerical simulation results; (2) Compared with the normal exponential vertical profile of wind speed, the disturbance caused by the front girder of the double-girder gantry crane structure under the downburst wind field was more severe, which increases the probability of the gantry crane overturning. (3) The downwind displacement of the main girder of the gantry crane under the condition of downburst is far greater than that under the normal condition. At the same time, under the condition of downburst, the pressure difference on the surface of the gantry crane was greater, and the distribution of the support reaction force was more uneven, resulting in a stronger overturning tendency of the gantry crane. (4) Under the condition of downburst, the overturning moment and the shearing force borne by the foundation of gantry crane exceeded the critical value to maintain the stability of the gantry crane by the gravity, resulting in the overturning of the gantry crane.

Parallel detection of multiple zoonotic parasites using a real-time fluorogenic loop-mediated isothermal amplification-based quadruple-sample microfluidic chip.

Zoonotic parasites pose significant health risks globally. In the present study, we combined a microfluidic chip with loop-mediated isothermal amplification (on-chip LAMP) to detect five zoonotic parasites: Toxoplasma gondii, Cryptosporidium parvum, Cryptosporidium hominis, Clonorchis sinensis, and Taenia solium. This method enabled the simultaneous parallel analysis of five genetic markers from a maximum of four samples per chip. The on-chip LAMP assay was conducted in a highly automated format via the addition (by pipetting) of each sample in a single operation. The reaction was performed in volumes as low as 5 µL at a temperature of 65°C for 60 min, achieving limits of detection ranging from 10-2 to 10-3 pg./µL of recombinant plasmid DNA. All the time-to-positive values were less than 40 min, and almost all the coefficients of variation were less than 10%, even when using limit of detection concentrations for multiple pathogens, indicating robust reproducibility among replicates. The clinical sensitivity and specificity for detecting 135 field samples were 98.08 and 97.59%, respectively, compared with traditional biological methods, indicating good applicability in the detection of field samples. This on-chip LAMP assay allows for low reagent consumption, ease of operation, and multiple analyses of samples and genetic targets, and is applicable for on-site detection and the routine monitoring of multiple zoonotic parasites.

Anticoccidial activity of traditional Chinese herbal Dichroa febrifuga Lour. extract against Eimeria tenella infection in chickens.

The study was conducted on broiler birds to evaluate the anticoccidial efficacy of an extract of Chinese traditional herb Dichroa febrifuga Lour. One hundred broiler birds were assigned to five equal groups. All birds in groups 1-4 were orally infected with 1.5 × 10(4) Eimeira tenella sporulated oocysts and birds in groups 1, 2 and 3 were medicated with 20, 40 mg extract/kg feed and 2 mg diclazuril/kg feed, respectively. The bloody diarrhea, oocyst counts, intestinal lesion scores, and the body weight were recorded to evaluate the anticoccidial efficacy. The results showed that D. febrifuga extract was effective against Eimeria infection; especially 20 mg D. febrifuga extract/kg feed can significantly increase body weight gains and reduce bloody diarrhea, lesion score, and oocyst excretion in comparison to infected-unmedicated control group.

Anticoccidial effect of halofuginone hydrobromide against Eimeria tenella with associated histology.

Halofuginone (stenorol) has been used as an effective anticoccidial reagent for decades but very little is known about its mode of action. In this study, chickens were inoculated with Eimeria tenella oocysts on 14-day-old and medicated with halofuginone at days 0, 1, 2, 3, 4, 5 and 6 post inoculations (groups 0, 1, 2, 3, 4, 5 and 6, respectively). Chickens in group 7 were taken as challenge-unmedicated control and in group 8 unchallenged-unmedicated control. The survival rate, body weight gains (BWG), oocysts production, cecal scores, bloody diarrhea and histological examinations were analyzed to evaluate the anticoccidial efficacy of halofuginone and to initially elucidate its mechanisms. Results showed that halofuginone which acted as a coccidiostatic can significantly enhance the BWG, and decrease both the oocyst shedding and cecal destruction caused by E. tenella infection. The histological slide examination noted that halofuginone was effective when provided 0-2 days post inoculation but only partially effective when applied 3-7 days post infection. The second-generation schizonts treated with halofuginone appeared vacuolated and degenerated. It is concluded that halofuginone can inhibit the parasite's invasion of host cecal hypothetical cell at the early stages of life cycle and later disturb the parasite's development by vacuolation of the schizonts. The resulting abnormal schizonts could not divide into schizoites and were eventually eliminated by the host's immune response.

Adjuvant effect of ginsenoside-based nanoparticles (ginsomes) on the recombinant vaccine against Eimeria tenella in chickens.

An experiment was conducted to study the adjuvant effect of ginsomes on the recombinant profilin in coccidian-infected breeding birds. Three-day-old chickens were vaccinated with Eimeria tenella recombinant profilin antigen (10, 50, and 100 µg per chicken) with or without 50 µg ginsomes per chicken. The boost vaccination was carried out 14 days later. Two weeks after the booster, the chickens were challenged with 1.5 × 10(4) homologous sporulated oocysts. The specific antibody response, lymphocyte proliferation, and IL-1 release from lymphocyte were measured at 1-42 days after boost vaccination. Seven days post-challenge, the rate of survival, body weight gains (BWG) were examined then all chickens were sacrificed and lesion scores and oocysts per gram were monitored to evaluate the protective effects of the vaccination after challenge. Compared with the group of vaccinating with profilin only, groups of 50 and 100 µg antigen plus ginsomes significantly enhanced lymphocyte proliferation and IL-1 secretion. The profilin specific antibody level in the four vaccinated groups was significantly higher than in the control group and in groups vaccinated with profilin containing ginsomes than profilin only. In the groups vaccinated with profilin plus ginsomes, the BWG was significantly higher than that of group of profilin only, but there was no significant difference between profilin plus adjuvant ginsomes, diclazuril medicated and uninfected-unmedicated-unvaccinated control groups. The lesion scores in groups immunized with profilin plus ginsomes was significantly lower than that both of groups unimmunized-challenged-unmedicated control and group vaccinated with profilin only. Oocyst excretion in groups vaccinated with 50 or 100 µg profilin plus ginsomes was lower than that of groups vaccinated with profilin only. These results demonstrate that the adjuvant ginsomes can promote subunit vaccine to induce a strong immune response and protective effects.

Pharmacological inhibition of KDM5A for cancer treatment.

Lysine-specific demethylase 5A (KDM5A, also named RBP2 or JARID1A) is a demethylase that can remove methyl groups from histones H3K4me1/2/3. It is aberrantly expressed in many cancers, where it impedes differentiation and contributes to cancer cell proliferation, cell metastasis and invasiveness, drug resistance, and is associated with poor prognosis. Pharmacological inhibition of KDM5A has been reported to significantly attenuate tumor progression in vitro and in vivo in a range of solid tumors and acute myeloid leukemia. This review will present the structural aspects of KDM5A, its role in carcinogenesis, a comparison of currently available approaches for screening KDM5A inhibitors, a classification of KDM5A inhibitors, and its potential as a drug target in cancer therapy.

The gene structure and promoter region of the vaccine target aminopeptidase H11 from the blood-sucking nematode parasite of ruminants, Haemonchus contortus.

Aminopeptidase H11, an integral membrane glycoprotein present only in the gut of Haemonchus contortus, could provide substantial protection as shown by 90% reduction in fecal egg counts, while its recombinant version expressed in E. coli induced little. To investigate the characteristics further, we amplified mRNA of H11 gene via reverse transcriptase polymerase chain reaction, followed by isolation of its 1,517-bp 5'-flanking region and determination of its genomic organization. The H11 gene contained 25 exons separated by 24 introns and spans 14,959 bp of genomic DNA. Analysis of the 1,517 bp 5'-flanking region of the H11 gene revealed a putative "TATA-less" promoter. Partial sequences of the last exon and its 3'-UTR of H11 isoform H11-4 were also identified upstream to the H11 gene with the same transcription orientation. The 1,517-bp 5'-flanking region and part of the first exon of the H11 gene were subcloned into the vector upstream of green fluorescence protein reporter gene and microinjected into the gonads of Caenorhabditis elegans. The transformed animals exhibited fluorescence in the distal intestine in the L4 larvae stage and adult worms. This study characterized gene structure of aminopeptidase H11, demonstrated different transcriptional pattern of its promoter region between free-living and blood-sucking nematode species, and highlights the utility of C. elegans as a heterologous system to study the biology roles of H11 isoforms.

Mudskipper interleukin-34 modulates the functions of monocytes/macrophages via the colony-stimulating factor-1 receptor 1.

Interleukin-34 (IL-34) is a novel cytokine that plays an important role in innate immunity and inflammatory processes by binding to the colony-stimulating factor-1 receptor (CSF-1R). However, information on the function of IL-34 in fish remains limited. In the present study, we identified an IL-34 homolog from mudskippers ( Boleophthalmus pectinirostris). In silico analysis showed that the mudskipper IL-34 (BpIL-34) was similar to other known IL-34 variants in sequence and structure and was most closely related to an orange-spotted grouper ( Epinephelus coioides) homolog. BpIL-34 transcripts were constitutively expressed in various tissues, with the highest level of expression found in the brain. Edwardsiella tarda infection significantly up-regulated the mRNA expression of BpIL-34 in the mudskipper tissues. The recombinant mature BpIL-34 peptide (rBpIL-34) was purified and used to produce anti-rBpIL-34 IgG. Western blot analysis combined with PNGase F digestion revealed that native BpIL-34 in monocytes/macrophages (MOs/MФs) was N-glycosylated. In vitro, rBpIL-34 treatment enhanced the phagocytotic and bactericidal activity of mudskipper MOs/MФs, as well as the mRNA expression of pro-inflammatory cytokines like tumor necrosis factor α ( BpTNF-α) and BpIL-1ß in these cells. Furthermore, the knockdown of mudskipper CSF-1R1 ( BpCSF-1R1), but not mudskipper BpCSF-1R2, significantly inhibited the rBpIL-34-mediated enhanced effect on MO/MФ function. In conclusion, our results indicate that mudskipper BpIL-34 modulates the functions of MOs/MФs via BpCSF-1R1.

The host defense peptide ß-defensin confers protection against Vibrio anguillarum in ayu, Plecoglossus altivelis.

ß-defensin is a cationic host defense peptide actively participating in host innate immune response against pathogens. In teleost fish, ß-defensin exhibits a diversity in genotypes and functions. Herein, a ß-defensin homolog (PaBD) was identified from ayu, Plecoglossus altivelis, showing multiple tissues' upregulation against Vibrio anguillarum challenge. In vivo experiments revealed that intraperitoneal injection of chemically synthesized mature PaBD (mPaBD) increased the survival rate of V. anguillarum-infected ayu, accompanied by reduced bacterial load and decreased tissue mRNA levels of tumor necrosis factor α (PaTNF-α) and interleukin 1ß (PaIL-1ß). However, in vitro, mPaBD showed weak bactericidal activity against V. anguillarum. Interestingly, mPaBD enhanced phagocytosis, intracellular bacterial killing, and respiratory burst of ayu monocytes/macrophages (MO/MΦ). Moreover, it inhibited mRNA levels of PaIL-1ß and PaTNF-α in MO/MФ upon V. anguillarum infection. In conclusion, PaBD protects ayu against V. anguillarum challenge not only through its direct antibacterial ability, but also through its immunomodulation in MO/MΦ.

Profiling of differentially expressed genes in sheep T lymphocytes response to an artificial primary Haemonchus contortus infection.

BACKGROUND: Haemonchus contortus is a common bloodsucking nematode causing widespread economic loss in agriculture. Upon H. contortus infection, a series of host responses is elicited, especially those related to T lymphocyte immunity. Existing studies mainly focus on the general immune responses of sheep T lymphocyte to H. contortus, lacking investigations at the molecular level. The objective of this study was to obtain a systematic transcriptional profiling of the T lymphocytes in H. contortus primary-infected sheep. METHODS: Nematode-free sheep were orally infected once with H. contortus L3s. T lymphocyte samples were collected from the peripheral blood of 0, 3, 30 and 60 days post infection (dpi) infected sheep. Microarrays were used to compare gene transcription levels between samples. Quantitative RT-PCR was employed to validate the microarray data. Gene Ontology and KEGG pathway analysis were utilized for the annotation of differentially expressed genes. RESULTS: Our microarray data was consistent with qPCR results. From microarrays, 853, 242 and 42 differentially expressed genes were obtained in the 3d vs. 0d, 30d vs. 0d and 60d vs. 0d comparison groups, respectively. Gene Ontology and KEGG pathway analysis indicated that these genes were involved in metabolism, signaling, cell growth and immune system processes. Functional analysis of significant differentially expressed genes, such as SLC9A3R2, ABCB9, COMMD4, SUGT1, FCER1G, GSK3A, PAK4 and FCER2, revealed a crucial association with cellular homeostasis maintenance and immune response. Our data suggested that maintaining both effective immunological response and natural cellular activity are important for T lymphocytes in fighting against H. contortus infection. CONCLUSIONS: Our results provide a substantial list of candidate genes in sheep T lymphocytes response to H. contortus infection, and contribute novel insights into a general immune response upon infection.

Development and evaluation of a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP) for rapid and simultaneous detection of ten pathogenic bacteria in aquatic animals.

Rapid, low-cost, and user-friendly strategies are urgently needed for early disease diagnosis and timely treatment, particularly for on-site screening of pathogens in aquaculture. In this study, we successfully developed a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP), which was capable of simultaneously detecting 10 pathogenic bacteria in aquatic animals, i.e., Nocardia seriolae, Pseudomonas putida, Streptococcus iniae, Vibrio alginolyticus, Vibrio anguillarum, Vibrio fluvialis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio rotiferianus, and Vibrio vulnificus. The assay provided a nearly-automated approach, with only a single pipetting step per chip for sample dispensing. This technique could achieve limits of detection (LOD) ranging from 0.40 to 6.42pg per 1.414µL reaction in less than 30 min. The robust reproducibility was demonstrated by a little variation among duplications for each bacterium with the coefficient of variation (CV) for time to positive (Tp) value less than 0.10. The clinical sensitivity and specificity of this on-chip LAMP assay in detecting field samples were 96.2% and 93.8% by comparison with conventional microbiological methods. Compared with other well-known techniques, on-chip LAMP assay provides low sample and reagent consumption, ease-of-use, accelerated analysis, multiple bacteria and on-site detection, and high reproducibility, indicating that such a technique would be applicable for on-site detection and routine monitoring of multiple pathogens in aquaculture.

Ginsenoside Rg1 enhances immune response induced by recombinant Toxoplasma gondii SAG1 antigen.

Ginsenoside, the most important component isolated from Panax ginseng, exhibits a variety of biological activities. Particularly, ginsenoside Rg1 is known to have immune-modulating activities such as increase of immune activity of T helper (Th) cells. In the present study, we evaluated the immunomodulatory potentials of the Rg1 at three dose levels on the cellular and humoral immune responses of ICR mice against T. gondii recombinant surface antigen 1 (rSAG1). ICR mice were immunized subcutaneously with 50 µg Rg1 alone, 100 µg rSAG1 alone or with 100 µg rSAG1 dissolved in saline containing ginsenoside Rg1 (10 µg, 50 µg or 100 µg). After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that the groups immunized with rSAG1 and Rg1 (50 µg, 100 µg) developed a high level of specific antibody responses against T. gondii rSAG1, a strong lymphoproliferative response, and significant levels of cytokine production, compared with the other groups. After lethal challenge, the mice immunized with the rSAG1 and Rg1 (50 µg, 100 µg) showed a significantly increased survival time compared with control mice which died within 6 days of challenge. Our data demonstrate that by addition of ginsenoside Rg1, the rSAG1 triggered a stronger humoral and cellular response against T. gondii, and that Rg1 is a promising vaccine adjuvant against toxoplasmosis, worth further development.

Seroprevalence of Toxoplasma gondii infection in pigs, in Zhejiang Province, China.

Seroprevalence of Toxoplasma gondii infection in pigs in 10 regions of Zhejiang Province, China, was obtained by enzyme-linked immunosorbent assay (ELISA). Anti- T. gondii antibodies were found in 53.4% (434/813) of pigs. Results were analyzed by a chi-square (χ(2)) test. Differences were observed according to farm size, animal age, and sampling regions. Seroprevalences in pigs raised on small farms (71.4%) were significantly higher than that (42.7%) on large farms (P < 0.05), and seroprevalence increased progressively with age. The seroprevalence ranged from 28.1% to 66.0% in different regions, with Jiaxing having the lowest level (28.1%), followed by Hangzhou (36.0%) and Taizhou (42.0%). This is the first study on seroprevalence of T. gondii infection in pigs in Zhejiang Province.