Airway necrosis and granulation tissue formation caused by Rhizopus oryzae leading to severe upper airway obstruction: a case report.

Pulmonary Mucormycosis is a fatal infectious disease with high mortality rate. The occurrence of Mucormycosis is commonly related to the fungal virulence and the host's immunological defenses against pathogens. Mucormycosis infection and granulation tissue formation occurred in the upper airway was rarely reported. This patient was a 60-year-old male with diabetes mellitus, who was admitted to hospital due to progressive cough, sputum and dyspnea. High-resolution computed tomography (HRCT) and bronchoscopy revealed extensive tracheal mucosal necrosis, granulation tissue proliferation, and severe airway stenosis. The mucosal necrotic tissue was induced by the infection of Rhizopus Oryzae, confirmed by metagenomic next-generation sequencing (mNGS) in tissue biopsy. This patient was treated with the placement of a covered stent and local instillation of amphotericin B via bronchoscope. The tracheal mucosal necrosis was markedly alleviated, the symptoms of cough, shortness of breath, as well as exercise tolerance were significantly improved. The placement of airway stent and transbronchial microtube drip of amphotericin B could conduce to rapidly relieve the severe airway obstruction due to Mucormycosis infection.

Multiple Lung Cavity Lesions, Thoracic Wall Abscess and Vertebral Destruction Caused by Streptococcus constellatus Infection: A Case Report.

Disseminated infection caused by Streptococcus constellatus was seldom occurred. We reported a case of Streptococcus constellatus infection, presenting as multiple pulmonary cavities, thoracic wall abscess and vertebral destruction. The 37-year-old male had recurrent fever, chest wall swelling and pain, and lower limb numbness, he had weak physical condition and previously suffered from poorly controlled diabetes and severe periodontal disease for 3 years. Definite diagnosis of Streptococcus constellatus infection was made by metagenomic next­generation sequencing (mNGS) in abscess drainage fluid. Systemic antibiotics and thoracic wall drainage were given, and the pulmonary cavity and the thoracic intermuscular abscess were significantly decreased. Few to no study reported the disseminated infection (pulmonary cavities, thoracic wall abscess and vertebral destruction) caused by Streptococcus constellatus. This case report highlighted the importance of mNGS for accurate diagnosis, as well as the timely drainage and antibiotics for effective treatment of Streptococcus constellatus infection.

Oral Vaccination With Recombinant Pichia pastoris Expressing Iridovirus Major Capsid Protein Elicits Protective Immunity in Largemouth Bass (Micropterus salmoides).

Largemouth bass iridovirus (LMBV) can cause high mortality and lead to heavy economic loss in the cultivation of largemouth bass, but there was no effective treatment. Here, the present study constructed a recombinant Pichia pastoris expressing LMBV major capsid protein (MCPD). The recombinant GS115-pW317-MCPD was then used to immunize largemouth bass via oral administration, and mucosal immune response mediated by immunoglobulins (Igs) was measured after oral immunization. Serum antibody levels were measured by ELISA, neutralizing antibody titers were determined by serum neutralization test (SNT), antigen presentation-related gene expressions were detected by RT-PCR, and the histopathological characteristics of immunized fish were assessed after challenging with 0.1 ml 107.19 TCID50/ml LMBV. The relative percentage survival (RPS) was also determined. Our results showed that the serum antibody titers of immunized fish were significantly higher than that of control groups (P < 0.05). IgT and IgM expressions in gut were increased significantly after vaccination with GS115-pW317-MCPD; however, much stronger response in gut was observed as compared with gill. The expression levels of major histocompatibility complex (MHC) II, CD8, and T-cell receptor (TCR) were significantly elevated in GS115-pW317-MCPD group (P < 0.05), while CD4 and MHC I transcription levels remained unchanged after oral immunization (P > 0.05). The RPS of fish orally immunized with 1.0 × 108 CFU/g GS115-pW317-MCPD was reached up to 41.6% after challenge with 0.1 ml 109.46 TCID50/ml LMBV. Moreover, orally immunizing with GS115-pW317-MCPD can relieve the pathological damage caused by LMBV. Therefore, GS115-pW317-MCPD showed a promising potential against LMBV.

Hepatopancreas-Specific Lectin Participates in the Antibacterial Immune Response by Regulating the Expression of Antibacterial Proteins.

The hepatopancreas is an important digestive and immune organ in crustacean. There were low but stable numbers of microbes living in the hemolymph of crustacean, whereas the organs (including hepatopancreas) of crustacean were immersed in the hemolymph. It is very important to study the immune mechanism of the hepatopancreas against bacteria. In this study, a novel CTL (HepCL) with two CRDs, which was mainly expressed in the hepatopancreas, was identified in red swamp crayfish (Procambarus clarkii). HepCL binds to bacteria in vitro and could enhance bacterial clearance in vivo. Compared with the C-terminal CRD of HepCL (HepCL-C), the N-terminal CRD (HepCL-N) showed weaker bacterial binding ability in vitro and stronger bacterial clearance activity in vivo. The expression of some antimicrobial proteins, such as FLP, ALF1 and ALF5, was downregulated under knockdown of HepCL or blocked with Anti-HepCL after challenge with Vibrio in crayfish. These results demonstrated that HepCL might be involved in the antibacterial immune response by regulating the expression of antimicrobial proteins.

A crayfish ALF inhibits the proliferation of microbiota by binding to RPS4 and MscL of E. coli.

Antimicrobial peptides (AMPs), most of which are small proteins, are necessary for innate immunity against pathogens. Anti-lipopolysaccharide factor (ALF) with a conserved lipopolysaccharide binding domain (LBD) can bind to lipopolysaccharide (LPS) and neutralize LPS activity. The antibacterial mechanism of ALF, especially its role in bacteria, needs to be further investigated. In this study, the antibacterial role of an anti-lipopolysaccharide factor (PcALF5) derived from Procambarus clarkii was analyzed. PcALF5 could inhibit the replication of the microbiota in vitro and enhance the bacterial clearance ability in crayfish in vivo. Far-western blot assay results indicated that PcALF5 bound to two proteins of E. coli (approximately 25 kDa and 15 kDa). Mass spectrometry (MS), far-western blot assay, and pull-down results showed that 30S ribosomal protein S4 (RPS4, 25 kD) interacted with PcALF5. Further studies revealed that another E. coli protein binding to PcALF5 could be the large mechanosensitive channel (MscL), which is reported to participate in the transport of peptides and antibiotics. Additional assays showed that PcALF5 inhibited protein synthesis and promoted the transcription of ribosomal component genes in E. coli. Overall, these results indicate that PcALF5 could transfer into E. coli by binding to MscL and inhibit protein synthesis by interacting with RPS4. This study reveals the mechanism underlying ALF involvement in the antibacterial immune response and provides a new reference for the research on antibacterial drugs.

Facile Synthesis of Urchin-Like and Bouquet-Like Silver Nanoparticles Using Gas Assisted Wet Chemistry Method.

A facile synthesis approach of urchin-like and bouquet-like silver nanoparticles (AgNPs) using gas assisted wet chemistry method with silver nitride as source materials, ascorbic acid as reducing agent, polyvinylpyrrolidone (PVP) as passivator and NO2/O2 as ventilation mixture is proposed It was demonstrated that the urchin-like and bouquet-like AgNPs evolved from spherical nanoparti cles and/or clusters of Ag as a result of strong adsorption and passivation of newly-formed Ag {100} facets by PVP, which effectively boost preferential growth. The NO2/O2 as the ventilation mixture provides an equilibrium of aggregation and detachment of Ag atoms on the surface, thus confining the shapes of AgNPs generated. This study provides an alternative approach for synthesis AgNPs in specific shapes and facilitates their applications.

Influences of nitrite on paracetamol degradation in dielectric barrier discharge reactor.

The frequent detection of paracetamol in natural water increased environmental concerns. The dielectric barrier discharge (DBD) technology is an effective paracetamol removing method, however, this research showed that the removal of paracetamol using DBD technology at 30 min dropped from 100% to 53.3% as the initial paracetamol concentration increased from 10 mg/L to 100 mg/L, due to the formation of more competitive intermediate products at higher paracetamol concentration. The removal of TOC was found to be much slower than that of paracetamol, as paracetamol was removed completely after 5 min treatment, the removal rate of TOC was 46.3% after 20 min treatment under 500 W discharge power and 50 mL/min air flow rate. The orthogonal experiment showed that the removal of TOC was significantly influenced by the treatment time, discharge power and recirculating flow rate, while less influenced by the discharge frequency. In the removal process of paracetamol, nitrite ion that generated during DBD treatment reacted with paracetamol to form an intermediate product of 3-nitro-4-acetamidophenol. The presence of nitrite ion retarded the removal of 3-nitro-4-acetamidophenol and thus the TOC, however, the nitrate ion did not. The degradation of paracetamol followed a sequence of 3-nitro-4-acetamidophenol, nitrosophenol/acetamide, N-methylacetamide, acetamide and small molecule organic acids in the DBD reactor, and these intermediates were finally oxidized to CO2, H2O and NO3-.

Proteomic analysis of differentially expressed proteins in the two developmental stages of Ichthyophthirius multifiliis.

Ichthyophthirius is a severe disease of farmed freshwater fish caused by the parasitic ciliate Ichthyophthirius multifiliis (Ich). This disease can lead to considerable economic loss, but the protein profiles in different developmental stages of the parasite remain unknown. In the present study, proteins from trophonts and theronts of Ich were identified by isobaric tags for relative and absolute quantitation (iTRAQ). A total of 2300 proteins were identified in the two developmental stages, of which 1520 proteins were differentially expressed. Among them, 84 proteins were uniquely expressed in the theronts stage, while 656 proteins were expressed only in trophonts. The differentially expressed proteins were catalogued (assorted) to various functions of Ich life cycle, including biological process, cellular component, and molecular function that occur at distinct stages. Using a 1.5-fold change in expression as a physiologically significant benchmark, a lot of differentially expressed proteins were reliably quantified by iTRAQ analysis. Two hundred forty upregulated and 57 downregulated proteins in the trophonts stage were identified as compared with theronts. The identified proteins were involved in various functions of the I. multifiliis life cycle, including binding, catalytic activity, structural molecule activity, and transporter activity. Further investigation of the transcriptional levels of periplasmic immunogenic protein, transketolase, zinc finger, isocitrate dehydrogenase, etc., from the different protein profiles using quantitative RT-PCR showed identical results to the iTRAQ analysis. This work provides an effective resource to further our understanding of Ich biology, and lays the groundwork for the identification of potential drug targets and vaccines candidates for the control of this devastating fish pathogen.

Live recombinant Lactococcus lactis vaccine expressing immobilization antigen (i-Ag) for protection against Ichthyophthirius multifiliis in goldfish.

The parasite Ichthyophthirius multifiliis (Ich) has been reported in various freshwater fishes worldwide and results in severe losses to both food and aquarium fish production. Lactobacillus strains have a number of properties that make them attractive candidates as delivery vehicles for the presentation to the mucosa of compounds with pharmaceutical interest, in particular vaccines. Here, the present study was conducted to evaluate a live recombinant Lactococcus lactis vaccine expressing immobilization antigen (IAG-52X) in protection against I. multifiliis. A 1266 bp gene fragment containing a potential antigenic epitope of the 48 kDa immobilization antigen of I. multifiliis was assembled from six synthetic ohgonucleotides and cloned into pSIP409 and electrotransformed into Lactobacillus plantarum NC8. The recombinant vaccine candidate was then orally fed into goldfish. The expression of immune-related genes: complement component 3 (C3), MHC I, IgM gene in blood from goldfish at different time points after immunization were evaluated. Immunized fish were than challenged with a lethal dose of infectious I. multifiliis. The cumulative mortality and relative percentage survival (RPS) were also determined. Our results showed that the antibody level in the blood and skin of the immunized fish was statistically significant (P < 0.05) in relation to the control groups. Goldfish orally immunized with NC8-pSIP409- IAG-52X had high serum antibody titers that ranged from 32 to 256 after 28d post immunization, while fish fed with NC8-pSIP409 or PBS had no detectable immobilizing antibody response. Expression of IgM, C3, MHC I genes in the group immunized with IAG-52X were significantly (P < 0.05) up regulated as compared with control group, indicating that different immune cells were actively involved in cellular immune response. The results showed that the average survival rate of fish orally immunized with 108 and 106NC8-pSIP409-IAG-52X was 60% and 50% respectively. Therefore, NC8-pSIP409-IAG-52X could become a promising oral vaccine candidate against I. multifiliis.

Evaluation of nystatin isolated from Streptomyces griseus SDX-4 against the ciliate, Ichthyophthirius multifiliis.

The present study was conducted to evaluate the in vitro and in vivo antiparasitic efficacy of active compounds from the bacterial extracellular products of Streptomyces griseus SDX-4 against Ichthyophthirius multifiliis. Bioassay-guided fractionation and isolation of compounds with antiparasitic activity were performed on n-butanol extract of S. griseus yielding a pure bioactive compound, nystatin (Nys), identified by comparing spectral data (EI-MS, (1)H NMR, and (13)C NMR) with literature values. Results from in vitro antiparasitic assays revealed that Nys could be 100% effective against I. multifiliis theronts and encysted tomonts at the concentration of 6.0 mg L(-1), with the median effective concentration (EC50) values of 3.1 and 2.8 mg L(-1) for theronts and encysted tomonts (4 h), respectively. Results of in vivo test demonstrated that the number of I. multifiliis trophonts on the gold fish treated with Nys was markedly lower than the control group at 10 days after exposed to theronts (p < 0.05). In the control group, 85.7% mortality was observed owing to heavy I. multifiliis infection at 10 days after the exposure. On the other hand, only 23.8% mortality owing to parasite infection was recorded in the groups treated with the Nys (4.0 and 6.0 mg L(-1)). In addition, our results showed that the survival and reproduction of I. multifiliis tomont exited from the fish were significantly reduced after treated with the 6.0 mg L(-1) Nys. The median lethal dose (LD50) of Nys for goldfish was 16.8 mg L(-1). This study firstly demonstrated that Nys has potent antiparasitic efficacy against I. multifiliis, and it can be a good candidate drug for chemotherapy and control of I. multifiliis infections.

Antiparasitic efficacy of dihydrosanguinarine and dihydrochelerythrine from Macleaya microcarpa against Ichthyophthirius multifiliis in richadsin (Squaliobarbus curriculus).

Ichthyophthirius multifiliis is a holotrichous protozoan that invades the gills and skin surfaces of fish and can cause morbidity and high mortality in most species of freshwater fish worldwide. The present study was undertaken to investigate the antiparasitic activity of crude extracts and pure compounds from the leaves of Macleaya microcarpa. The chloroform extract showed a promising antiparasitic activity against I. multifiliis. Based on these finding, the chloroform extract was fractionated on silica gel column chromatography in a bioactivity-guided isolation affording two compounds showing potent activity. The structures of the two compounds were elucidated as dihydrosanguinarine and dihydrochelerythrine by hydrogen and carbon-13 nuclear magnetic resonance spectrum and electron ionization mass spectrometry. The in vivo tests revealed that dihydrosanguinarine and dihydrochelerythrine were effective against I. multifiliis with median effective concentration (EC(50)) values of 5.18 and 9.43 mg/l, respectively. The acute toxicities (LC(50)) of dihydrosanguinarine and dihydrochelerythrine for richadsin were 13.3 and 18.2mg/l, respectively. The overall results provided important information for the potential application of dihydrosanguinarine and dihydrochelerythrine in the therapy of serious infection caused by I. multifiliis.

In vivo anthelmintic activity of chelidonine from Chelidonium majus L. against Dactylogyrus intermedius in Carassius auratus.

Dactylogyrus intermedius is one of the most common and serious cause of parasitic diseases of freshwater fish in aquaculture, and can cause morbidity and high mortality in most species of freshwater fish worldwide. To attempt controlling this parasite and explore novel potential antiparasitic agents, the present study was designed to ascertain the anthelmintic activity of Chelidonium majus L. whole plant and to isolate and characterize the active constituents against D. intermedius. The ethanol extract from C. majus whole plant showed significant anthelmintic activity against D. intermedius [EC(50) (median effective concentration) value = 71.5 mg L(-1)] and therefore subjected to further isolation and purification using various chromatographic techniques. A quaternary benzo[c]phenanthridine alkaloid exhibited significant activity against D. intermedius was obtained and identified as chelidonine. In vivo anthelmintic efficacy tests exhibited that chelidonine was 100% effective against D. intermedius at a concentration of 0.9 mg L(-1), with EC(50) value of 0.48 mg L(-1) after 48 h of exposure, which is more effective than the positive control, mebendazole (EC(50) value = 1.3 mg L(-1)). In addition, the 48-h median lethal concentration (LC(50)) for chelidonine against the host (Carassius auratus) was 4.54 mg L(-1). The resulting therapeutic index for chelidonine was 9.46. These results provided evidence that chelidonine might be potential sources of new antiparasitic drugs for the control of Dactylogyrus.

Effect of sanguinarine from the leaves of Macleaya cordata against Ichthyophthirius multifiliis in grass carp (Ctenopharyngodon idella).

The ciliate Ichthyophthirius multifiliis is one of the most pathogenic parasites of fish maintained in captivity. In this study, effects of crude extracts, fractions, and compounds from the leaves of Macleaya cordata against I. multifiliis were investigated under in vitro conditions by bioactivity-guided isolation method. The dried ethanol extract of M. cordata was extracted successively in a separating funnel with petroleum ether, ethyl acetate, chloroform and n-butanol. Among them, only the chloroform extract showed promising activity and therefore, was subjected to further separation and purification using various chromatographic techniques. Four compounds were isolated from chloroform extract, but only one compound showed potent activity. The structure of the active compound was elucidated as sanguinarine by hydrogen, carbon-13 nuclear magnetic resonance spectrum and electron ionization mass spectrometry. In vitro antiparasitic efficacy tests exhibited that sanguinarine was 100% effective against I. multifiliis at a concentration of 0.7 mg l(-1), with LC(50) and LC(90) values of 0.437 and 0.853 mg l(-1) after 4 h of exposure. In vivo antiparasitic efficacy tests showed that the number of I. multifiliis on the gills in the treatment group (in 0.9 mg l(-1) sanguinarine) was reduced by 96.8%, in comparison to untreated group at 25°C for 48 h. Mortality of fish did not occur in the treatment group during the trail, although 40% of untreated fish died. Our results indicate that the studied plant extracts, as well as sanguinarine might be potential sources of new antiparasitic drug for the control of I. multifiliis.

[Preparation and characterization of the mAb against Vibrio harveyi].

AIM: To prepare the mAb against Vibrio harveyi and analyse their biological properties. METHODS: 8-week-old female BALB/c mice were immunized with pathogenic V.harveyi GYC1108-1, which was isolated from the diseased great yellow croaker in Zhejiang China in 2003. Spleen cells collected from the immunized mice after the fifth immunization were fused with Sp2/0-Ag-14 myeloma cells. ELISA was used to screen hybridoma cells and identify the specificity and sensitivity of mAbs. RESULTS: Limited dilution method was used to subclone the positive clones. After three cycles of subcloning, 5 mAbs against GYC1108-1 were obtained and were proved to have high titer of ELISA. The test of specificity suggested that 1B7 reacted on V.harveyi, V.alginolyticus and V.parahaemolyticus but did not react on V.mimicus, V.anguillarum, Aeromonas hydrophila and the other isolates among the 14 bacterium species tested. 1B7 was used to detect V.harveyi at the concentration of 10(4) CFU/mL by indirect ELISA. CONCLUSION: The obtained mAbs could be used to diagnosis and prevent of V.harveyi in aquaculture rapidly.