Blastocystis sp. is a widespread unicellular protozoan mainly parasitizing the intestines of humans and animals. This parasite has rich genetic diversity, with 42 subtypes (ST1–ST17, ST21, ST23–ST46) reported, of which ST1, ST2, and ST3 are prevalent. However, the pathogenicity levels and the pathogenicity differences among different subtypes have been controversial. [Objective] To determine the pathogenicity of ST1, ST2, and ST3 and explore whether there are differences in the pathogenicity between the three subtypes of Blastocystis sp. [Methods] Blastocystis sp. strains were isolated from fresh human and macaque faeces and identified by morphological observation, 18S rRNA gene amplification and sequencing, and cluster analysis. After BALB/c mice were infected with strains of ST1, ST2, and ST3, the distribution characteristics of the parasites in vivo were observed. In addition, the clinical signs, weight gain, feed conversion rate, and mortality of the infected mice were recorded to compare the pathogenicity of the three subtypes. Finally, the pathological changes in the intestines of mice were observed. [Results] Three subtypes of Blastocystis sp., ST1, ST2 and ST3, were successfully isolated and identified. Animal challenge results showed that the parasites predominantly colonized the intestine and negatively affected the health of mice. A high dose of ST3 resulted in death in mice, while ST1, ST2, and low doses of ST3 did not affect the survival of mice. Blastocystis sp. damaged the intestinal tissue of challenged mice. [Conclusion] Blastocystis sp. causes harm to the host by damaging the intestine. The pathogenicity varies among different subtypes and ST3 has stronger pathogenicity. This study lays a foundation for further studying the pathogenicity mechanism and provides data for assessing the threat of Blastocystis sp. to public health.

[Objective] Bovine bones contain a large amount of beneficial nutrients such as proteins, mineral salts, and vitamins for human health. With the calcium-to-phosphorus ratio meeting the optimal absorption ratio for the human body, bovine bones serve as a valuable nutrient source. [Methods] Lactic acid bacteria with high yields of L-lactic acid were screened by the calcium-dissolving ring method and the performance of strains in L-lactic acid production was then determined via fermentation of bovine bone meal. Metabolomics was employed to explore the differential metabolites and main pathways during the fermentation. [Results] One strain LP15, identified as Lactobacillus plantarum, was isolated from the liquid sample in the production of sour bamboo shoots for its strong ability to produce L-lactic acid. After fermentation of bone meal for 96 h, the strain produced 3.89 g/L L-lactic acid and 69.82 mg/100 mL free calcium, which represented a 32.6-fold increase compared with that in the control group. The metabolomic analysis revealed ABC transporters, metabolic pathway, and biosynthesis of secondary metabolites as the most important differential metabolic pathways during the fermentation of bovine bone meal. Meanwhile, a huge number of physiological active substances, such as cyclocreatine and 2-hydroxy-2-methylbutyric acid, were detected in the fermented bone meal. [Conclusion] Fermentation of bovine bone meal by L. plantarum LP15 is an effective strategy to promote calcium dissolution and produce beneficial active substances. This study lays a practical foundation for developing health food products by microbial fermentation of bovine bone meal.

In recent years, antimicrobial peptides (AMPs) are considered alternatives to antibiotics and have received increasing attention. AMPs have a broad antibacterial spectrum and extensive sources and are not prone to drug resistance. At present, AMPs are mainly produced with three methods: extraction from natural sources, chemical synthesis, and microbial expression via genetic engineering. The application of the former two methods is limited due to their complicated processes, high costs, and low yields. Microbial expression via genetic engineering is more economical, scientific, and effective than the above two methods. This article introduces and compares the various expression systems and summarizes the strategies for increasing the heterologous expression levels, with a view to providing theoretical support for large-scale production of AMPs with low costs.

Pseudorabies virus (PRV) is a member of the genus Varicellovirus in the Herpesviridae family. It primarily causes pseudorabies characterized by reproductive failure in sows, and neurological and respiratory symptoms in piglets, posing a significant threat to pig production. Vaccination is the most important measure to prevent PRV in pigs. However, due to variations of the virus and its latent infection characteristics, the effectiveness of traditional vaccines is compromised. Consequently, there is an urgent need for new drug preparations to assist vaccine immunization. It has been found that natural plant polysaccharides and small molecules such as flavonoids, phenols, and acids can inhibit PRV infection either by directly blocking the viral infection process or by regulating the immune response. In addition, host antiviral protein type Ⅰ interferon and its downstream interferon-stimulated genes have significant inhibitory effects on PRV infection. Host defense peptides, including antimicrobial peptides and defensins, also show good inhibitory effects on PRV infection. Interestingly, researchers have recently found that extracts and metabolites from bacteria and fungi also exhibit anti-PRV effects, and it is expected that these bacteria and fungi and their products could be applied for the prevention and treatment of viral diseases in the future. This study focused on the recent research progress of natural bioactive molecules against PRV infection, aiming to provide important references for the research and development of anti-PRV infection drugs.

The pneumococcal vaccines play an indispensable role in defending against various invasive and non-invasive diseases caused by Streptococcus pneumoniae. The capsular polysaccharide is a key antigen, while the impurity residues in the capsular polysaccharide cause side effects of pneumococcal polysaccharide vaccines and conjugate vaccines. As the global demand for high-valent pneumococcal vaccines covering a wide range of serotypes keeps increasing, it is urgent and crucial to develop efficient and scalable production technologies for high-purity capsular polysaccharide antigens. The safety and quality attributes of capsular polysaccharide are influenced by multiple factors encompassing the selection of pathogen strains, formulation of culture media, control of fermentation processes, and purification technologies. Following the concept of Quality by Design (QbD) and the process development path, this article systematically summarizes the knowledge and experience throughout the entire process ranging from upstream strain screening and optimization, refined fermentation process design, to downstream purification, aiming to provide comprehensive and in-depth theoretical guidance and practical strategies for building and improving the production system of capsular polysaccharide.

[Objective] To explore the effects of different concentrations of chlortetracycline on the characteristics of microbial communities involved in inorganic phosphorus (Pi) dissolution and organic phosphorus (Po) mineralization in the soil applied with organic fertilizer, focusing on soil P transformation and availability. [Methods] The purple soil collected from Tongnan District of Chongqing was used for a pot experiment with the addition of chicken manure as the organic fertilizer. Three chlortetracycline treatments (No-CTC, Low-CTC, and High-CTC) were designed with the addition levels of 0.0, 0.1, and 4.0 mg/kg, respectively. The soil samples were collected on days 7 (D7) and 30 (D30) after pepper ('Xinxiang 8') was planted. Real-time qPCR and Illumina MiSeq high-throughput sequencing were employed to analyze the community characteristics of the bacteria carrying the key genes (pqqC and phoD) of Pi dissolution and Po mineralization, respectively. Furthermore, the sequencing results and biologically based P (BBP) fractionation were employed to examine the effects of CTC addition on soil P transformation. [Results] High-CTC increased the content of Citrate-P and Enzyme-P by 8.2% and 44.0%, respectively, compared with No-CTC on D7. Low-CTC and High-CTC increased the content of Enzyme-P by 44.0% and 65.6%, respectively, compared with No-CTC on D30. The addition of CTC suppressed alkaline phosphatase (ALP) activity and affected the community structures of pqqC and phoD-harboring bacteria in the soil. The Mantel test results showed that Citrate-P was significantly associated with the dominant pqqC-carrying taxa Pseudomonas, Geodermatophilus, and Saccharothrix on D7. The dominant phoD-carrying taxa Bradyrhizobium, Ensifer, and Skermanella exhibited notable correlations with Enzyme-P on D7, and such correlations weakened over time. The average degree of the community network of the bacteria carrying pqqC increased in the Low-CTC treatment and decreased in the High-CTC treatment on D7. The average degree of this network decreased in High-CTC and Low-CTC treatments on D30. The average degree of the community network of the bacteria carrying phoD decreased with the increase in CTC addition on D7, while this trend was opposite on D30. [Conclusion] The addition of CTC significantly affected soil Enzyme-P by regulating the community structure of pqqC- and phoD-carrying bacteria as well as acid phosphatase (ACP) and ALP activities, thereby affecting the P forms and availability in the soil. This study contributes to a deeper understanding of alterations in microbial communities associated with P cycling in the soil-plant system contaminated by CTC. Moreover, it lays a scientific foundation for enhancing nutrient utilization efficiency in the soil applied with antibiotics.

[Objective] Human norovirus (HuNoV) is one of the most common pathogens causing acute gastroenteritis in humans worldwide. Currently, there are no approved vaccines to prevent this disease. This study aimed to prepare virus-like particles (VLPs) of HuNoV in the flashBAC baculovirus expression system, laying a foundation for the development of vaccines against HuNoV. [Methods] After codon optimization, the full-length gene sequence of the VP1 protein of HuNoV GⅡ.4 was synthesized and cloned into the baculovirus pBacPAK9 transfer vector to obtain the recombinant plasmid pBacPAK9-8his-VP1. After enzyme digestion and sequencing, the recombinant plasmid was co-transfected with the linear baculovirus plasmid (Bacmid) into SF9 cells to obtain a recombinant baculovirus carrying the VP1 gene. Hi-Five (HF) cells were infected by the recombinant baculovirus for protein expression, and the expression of VP1 was analyzed by SDS-PAGE and Western blotting. VP1 was purified by Ni-NTA affinity chromatography and identified by SDS-PAGE and Western blotting. The purity of VP1 was examined by high performance liquid chromatography (HPLC). The VLPs were observed by transmission electron microscopy. [Results] A transfer plasmid pBacPAK9-8his-VP1 was constructed, and the VP1 protein, with a molecular weight of approximately 58 kDa, was mainly expressed in the cytoplasm of HF cells. The HPLC results showed that the purity of VP1 was over 99%. The VLPs with a regular shape, uniform sizes, and diameters of 30–40 nm were observed by transmission electron microscopy. [Conclusion] The VLPs of HuNoV GⅡ.4 were prepared with a baculovirus expression system, laying a foundation for the development of HuNoV vaccines.

The aquaculture industry has rapidly expanded in recent years in China, whereas it faces the challenge brought by bacterial diseases. Antibacterial agents have been the primary tools to combat these diseases. However, prolonged and haphazard usage of antibacterial agents in aquaculture has exacerbated antimicrobial resistance and led to severe antimicrobial residues. Considering these challenges, scholars worldwide have been exploring natural alternatives, such as Chinese herbal medicines. Among them, Pithecellobium clypearia stands out due to its antibacterial, antiviral, and anti-inflammatory properties, coupled with its safety and lack of antimicrobial resistance. Nonetheless, the potential of P. clypearia in the prevention and control of aquatic diseases remains underexplored. [Objective] This study evaluated the in vitro inhibitory activity of P. clypearia aqueous extract against aquatic pathogenic bacteria, including an artificially induced antimicrobial-resistant strain. Additionally, we investigated changes in bacterial cell membrane permeability and observed cellular alterations by transmission electron microscopy to elucidate the mechanism of the extract. Our findings are expected to pave the way for developing P. clypearia as an environmentally friendly antibacterial agent, reducing antibacterial agent dependency, and mitigating pathogen resistance in aquaculture. [Methods] We employed the microbroth method to assess the antimicrobial resistance of 107 pathogen strains attacking aquatic animals and analyzed the inhibitory activity of P. clypearia aqueous extract against aquatic pathogenic bacteria, including an artificially induced antimicrobial-resistant bacterial strain. Furthermore, we determined the extracellular K⁺ content and ultrastructural changes in Streptococcus agalactiae and Vibrio parahaemolyticus after treatment with the extract. [Results] The resistance rate of 107 pathogen strains to sulfonamides was as high as 67.29%, and 46.73% of the strains showed multidrug resistance, among which Aeromonas sp. showed the most severe resistance. The aqueous extract (12.50 mg/mL) of P. clypearia exerted inhibitory effects on all the pathogenic bacteria, especially on Aeromonas sp. with the minimum inhibitory concentration (MIC) as low as 0.39 mg/mL. The MICs of the extract were similar for the strains belonging to the same genus but with different antimicrobial resistance characteristics. The aqueous extract of P. clypearia showed stronger inhibitory effect on the artificially induced enrofloxacin-resistant strain of A. schubertii than on the original strain, with the MICs of 0.78 mg/mL on the original strain and 0.20 mg/mL on the resistant strain. In addition, the treatment with P. clypearia aqueous extract significantly increased the extracellular K⁺ concentration, leading to damage to the bacterial cell membrane structure, leakage of intracellular contents, and vacuolation of cytoplasm, which suggested that P. clypearia exerted the antibacterial effect by destroying the bacterial membrane structure. [Conclusion] P. clypearia exerts in vitro inhibitory effects on aquatic pathogenic bacteria and demonstrates great potential for further research and development in the prevention and treatment of bacterial diseases in aquatic animals. The antibacterial mechanism of P. clypearia appears to involve disrupting bacterial cell membranes. The application of P. clypearia in aquaculture promises to reduce antibiotic dependency and pathogen resistance, paving the way for a healthy and sustainable aquaculture industry.

[Objective] We isolated the aerobic bacteria capable of effectively degrading polylactic acid (PLA) and characterized the bacterial growth and degradation, aiming to lay a theoretical foundation for the bioremediation of PLA contaminated environment. [Methods] The degrading bacterium was identified by 16S rRNA gene sequencing. Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) were employed to analyze the morphological and chemical changes of PLA films before and after degradation. [Results] A strain of Bacillus sp. JA-4 was screened from activated sludge, and it caused the PLA weight loss of 10.6% after 30 days. The weight loss of PLA reached 5.6% after incubation with the strain at an inoculation amount of 20%, pH 8.0, and 30 ℃ for 7 days. Gelatin significantly enhanced the biodegradation of PLA. In the presence of 3% gelatin, the weight loss of PLA reached 23.1% after 10 days of degradation, and the degradation rate was greatly increased. FTIR results indicated that Bacillus sp. JA-4 degraded PLA by hydrolyzing the ester bonds. [Conclusion] This study enriched the microbial resources for the biodegradation of PLA and provided technical support for the effective degradation of PLA waste in the environment.

[Objective] To screen microbial strains with high production of exopolysaccharides (EPS) and provide strain resources for the development of soil improvement agents. [Methods] The string test with a LB-aniline blue plate was employed to qualitatively screen the strains of plant growth-promoting rhizobacteria (PGPR) with EPS production. After fermentation with each strain in four media, the EPS content in the fermentation liquid was determined by low temperature alcohol precipitation and the sulfate-anthranone colorimetric method, on the basis of which the PGPR strain with high EPS production was screened out. The fermentation conditions of the strain screened out were optimized by orthogonal test with EPS content in fermentation liquid as the indicator. The influence of the fermentation liquid of strain F1 on the content of macro-aggregates in sandy loam soil was analyzed by the petri dish culture experiment. [Results] Eight EPS-producing PGPR strains were primarily screened out, among which strain F1 had the highest EPS production. PDA was the best medium for F1 to produce EPS, with the EPS content of 867.54 μg/mL. Based on morphological, physiological and biochemical characteristics, phylogenetic analysis based on 16S rRNA gene sequence, and also average nucleotide identity analysis, F1 was identified as a strain of Bacillus megaterium. The optimal culture conditions for F1 to produce EPS were 28 ℃ and 180 r/min for 24 h, under which the EPS yield reached 1 123.39 μg/mL. After F1 was incubated in sandy loam soil for 40 days, the content of water-stable macro-aggregates with the grain diameter > 0.25 mm in the soil increased by 4.44 times compared with that of the control. [Conclusion] Strain F1 with high EPS production can promote the formation of water-stable macro-aggregates in sandy loam soil. The optimal conditions for F1 to produce of EPS was incubation in PDA at 28 ℃ and 180 r/min for 24 h.