Preparation and application of biocontrol formulation of nematode-trapping fungus-Duddingtonia flagrans.

The use of nematophagous fungi as a biological control strategy for parasitic gastrointestinal nematodes (GINs) in livestock holds promise as an innovative alternative approach. This study aimed to evaluate the clinical efficacy of a lyophilized Duddingtonia flagrans preparation, utilized in association with the anthelmintics ivermectin or albendazole, to control GINs in Tibetan sheep on a farm based in Qinghai Province. The experimental design included five groups: D. flagrans lyophilized preparation group; D. flagrans+ ivermectin combination tablets treatment group (0.6 tablets for each 10 kg b.w. containing 106 chlamydospores of D. flagrans); D. flagrans+ albendazole combination capsules treatment group (5 capsules for each 10 kg b.w. containing 106 chlamydospores of D. flagrans); ivermectin group (0.2 mg/kg); albendazole group (15 mg/kg), and a control group; The effect of these strategies was evaluated through the analysis of feces collected directly from the animals in each group at 24 h, 48 h, 72 h,96 h and 120 h after administration, by estimating the counts of fecal egg count reduction percentage (FECR) and larval development reduction percentage (LDR). The combination of D. flagrans lyophilized preparation with either ivermectin or albendazole yielded fecal egg and larval reduction rates of up to 100% within 72 h after oral administration, outperforming the groups treated with a single anthelmintic. Moreover, the application of the lyophilized preparation of D. flagrans chlamydospores in isolation demonstrated an 89.8% larval reduction rate. The formulation containing D. flagrans showed high predatory capacity after passage through the gastrointestinal tract of sheep and was effective for controlling gastrointestinal nematodes, which greatly reduced the pollution of the grassland, and avoid reinfection.

Quantitative label-free proteomic analysis of excretory-secretory proteins in different developmental stages of Trichinella spiralis.

Trichinella spiralis (T. spiralis) is a zoonotic parasitic nematode with a unique life cycle, as all developmental stages are contained within a single host. Excretory-secretory (ES) proteins are the main targets of the interactions between T. spiralis and the host at different stages of development and are essential for parasite survival. However, the ES protein profiles of T. spiralis at different developmental stages have not been characterized. The proteomes of ES proteins from different developmental stages, namely, muscle larvae (ML), intestinal infective larvae (IIL), preadult (PA) 6 h, PA 30 h, adult (Ad) 3 days post-infection (dpi) and Ad 6 dpi, were characterized via label-free mass spectrometry analysis in combination with bioinformatics. A total of 1217 proteins were identified from 9341 unique peptides in all developmental stages, 590 of which were quantified and differentially expressed. GO classification and KEGG pathway analysis revealed that these proteins were important for the growth of the larvae and involved in energy metabolism. Moreover, the heat shock cognate 71 kDa protein was the centre of protein interactions at different developmental stages. The results of this study provide comprehensive proteomic data on ES proteins and reveal that these ES proteins were differentially expressed at different developmental stages. Differential proteins are associated with parasite survival and the host immune response and may be potential early diagnostic antigen or antiparasitic vaccine candidates.

Immunosuppressive Ability of Trichinella spiralis Adults Can Ameliorate Type 2 Inflammation in a Murine Allergy Model.

BACKGROUND: There is an increase in the global incidence of allergies. The hygiene hypothesis and the old friend hypothesis reveal that helminths are associated with the prevalence of allergic diseases. The therapeutic potential of Trichinella spiralis is recognized; however, the stage at which it exerts its immunomodulatory effect is unclear. METHODS: We evaluated the differentiation of bone marrow-derived macrophages stimulated with T spiralis excretory-secretory products. Based on an ovalbumin-induced murine model, T spiralis was introduced during 3 allergy phases. Cytokine levels and immune cell subsets in the lung, spleen, and peritoneal cavity were assessed. RESULTS: We found that T spiralis infection reduced lung inflammation, increased anti-inflammatory cytokines, and decreased Th2 cytokines and alarms. Recruitment of eosinophils, CD11b+ dendritic cells, and interstitial macrophages to the lung was significantly suppressed, whereas Treg cells and alternatively activated macrophages increased in T spiralis infection groups vs the ovalbumin group. Notably, when T spiralis was infected prior to ovalbumin challenge, intestinal adults promoted proportions of CD103+ dendritic cells and alveolar macrophages. CONCLUSIONS: T spiralis strongly suppressed type 2 inflammation, and adults maintained lung immune homeostasis.

Identification of antigens in the Trichinella spiralis extracellular vesicles for serological detection of early stage infection in swine.

BACKGROUND: Several studies have reported the roles of Trichinella spiralis extracellular vesicles in immune regulation and pathogen diagnosis. Currently, the T. spiralis muscle larvae excretory/secretory product (Ts-ML-ES) is the antigen recommended by the International Commission on Trichinellosis (ICT) for serological diagnosis of trichinellosis. However, it can only be used to detect middle and late stages of infections, and cross-reactions with other parasite detections occur. Therefore, there is a need to identify antigens for specific detection of early stage trichinellosis. METHODS: Extracellular vesicles of T. spiralis muscle larvae (Ts-ML-EVs) were isolated by ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, flow cytometry and western blot. Ts-ML-EVs protein profiles were analyzed by LC-MS/MS proteomics for identification of potential antigens (Ts-TTPA). Ts-TTPA were cloned into pMAL-c5X vector and expressed as recombinant proteins for evaluation of potential as detected antigens by western blot and ELISA. RESULTS: Isolated Ts-ML-EVs were round or elliptic (with diameters between 110.1 and 307.6 nm), showing a bilayer membrane structure. The specific surface markers on the Ts-ML-EVs were CD81, CD63, enolase and the 14-3-3 protein. A total of 53 proteins were identified by LC-MS/MS, including a variety of molecules that have been reported as potential detection and vaccine candidates. The cDNA of Ts-TTPA selected in this study has a total length of 1152 bp, encoding 384 amino acids with a molecular weight of 44.19 kDa. It contains a trypsin domain and can be recognized by anti-His antibody. It reacted with swine sera infected with 10,000 T. spiralis at 15, 25, 35 and 60 days post-infection (dpi). At 10 µg/ml, this antigen could detect T. spiralis antibodies from the swine sera at 13 dpi. There were no cross-reactions with the swine sera infected with other parasites including Clonorchis sinensis, Toxoplasma gondii, Taenia suis, Ascaris suis and Trichuris suis. CONCLUSIONS: This study identifies potential early stage detection antigens and more thoroughly characterizes a serine protease domain-containing protein. Extracellular vesicle proteins may be explored as effective antigens for the early stage detection of trichinellosis.

Trichinella spiralis excretory/secretory products from adult worms inhibit NETosis and regulate the production of cytokines from neutrophils.

Upon encountering exogenous pathogens, polymorphonucleocytes (PMNs) engage in various processes to destroy them, including releasing neutrophil extracellular traps (NETs) that trap pathogens and induce phagocytosis and cytokine production. Parasites have unique strategies with which to evade the host's immune response. However, the strategy employed by Trichinella spiralis in response to the reaction of PMNs has yet to be elucidated. This study explored the effect of excretory/secretory products (ESP) on three major functions: NETs, phagocytosis, and cytokine production. Specifically, PMNs were pre-treated with the ESP of 3-day-old adults and then stimulated with phorbol 12-myristate 13-acetate (PMA). We found that in PMNs pretreated with ESP, PMA-induced NET generation was suppressed by ESP. ROS production is a hallmark of PMA-induced NETosis. The LDH assay results showed that ESP inhibits NETs by suppressing ROS rather than promoting PMN death. Furthermore, ESP enhanced Escherichia coli engulfment by PMNs, improving overall phagocytic function. Finally, cytokine analysis revealed an increase in pro-inflammatory cytokine IL-1ß, and other cytokines (IL-10, TNF-α), while IL-4 displayed a significant reduction. In conclusion, this study has unraveled T. spiralis' evasion and regulation mechanisms against innate immune cells, providing insights into parasite strategies to manipulate host immunity, potentially informing new treatments for NET-related autoimmune diseases.

Functional Analysis of a Glutamine Biosynthesis Protein from a Psychrotrophic Bacterium, Cryobacterium soli GCJ02.

A putative glutamine synthetase (GS) was detected in a psychrophilic bacterium, Cryobacterium soli GCJ02. For gaining greater insight into its functioning, the gene was cloned and expressed in a heterologous host, Escherichia coli. The monomer enzyme with a molecular weight of 53.03 kDa was expressed primarily in cytosolic compartment. The enzyme activity was detected using glutamate and ATP. The optimum conditions of its biosynthesis were observed to be 60 °C and pH value 7.5. Its thermostability was relatively high with a half-life of 50 min at 40 °C. GS activity was enhanced in the presence of metal ions such as Mg2+ and Mn2+, whereas Fe2+, Cu2+ and Ca2+ proved inhibitory. The consensus pattern [EXE]-D-KP-[XGXGXH] in the GS lies between residues 132 and 272. The catalytic active sites consisting of EAE and NGSGMH were verified by site-directed mutagenesis. Based on the analysis of the consensus pattern, the GS/glutamate synthase cycle of C. soli GCJ02 is expected to contribute to the GS synthesic activity.

Green Synthesis of the Flavor Esters with a Marine Candida parapsilosis Esterase Expressed in Saccharomyces cerevisiae.

The green synthesis of the flavor esters, n-propyl acetate, isobutyl acetate and isoamyl acetate had the advantages over the chemical synthesis. The esterase from Candida parapsilosis could transform n-propanol, isobutanol and isoamyl alcohol into n-propyl acetate, isobutyl acetate and isoamyl acetate, respectively. The esterase was expressed in Saccharomyces cerevisiae. At 30 °C for 1 d, the concentration of n-propyl acetate, isobutyl acetate and isoamyl acetate synthesized by the esterase expressed in Saccharomyces cerevisiae was 24.6 mg/100 mL, 8.3 mg/100 mL, 5.6 mg/100 mL, respectively. Expression of the esterase has a practical significance for flavor ester synthesis by green biochemical process.

Tandem integration of aerobic fungal cellulase production, lignocellulose substrate saccharification and anaerobic ethanol fermentation by a modified gas lift bioreactor.

Cellulase production, lignocellulose saccharification and bioethanol fermentation were integrated to efficiently produce bioethanol. A modified gas lift bioreactor was developed for bioethanol production by the integrated process. Cellulase production was achieved using Aspergillus niger mycelia immobilized within the reactor in wire meshes, and Saccharomyces cerevisiae cells were immobilized in resin beads. During four repeated batches fermentation, cellulase activities were more than 6.28 U/mL and bioethanol production was over 45.9 g/L for 48 h. The factual bioethanol conversion efficiency was 86.8%. By the modification of the modified gas lift bioreactor, immobilization of Aspergillus niger mycelia and Saccharomyces cerevisiae cells, aerobic cellulase production, substrate saccharification and anaerobic bioethanol fermentation were successfully integrated in tandem. The integrated processes is of great significance in bioethanol production.