Abstract:[Background] Previous studies in our laboratory have demonstrated that Mycoplasma hyopneumoniae Mhp367 protein is a humoral immunodominant proteinic antigen. However, the reactivity of different regions of Mhp367 to the convalescent sera remains unclear. [Objective] The purpose of this study is to identify the reactivity of different regions of Mhp367 to the convalescent sera. [Methods] Nine DNA fragments of mhp367 were amplified with different primer combinations. The amplified products were respectively ligated into pGEX-6P-1, pGEX-4T-3, or pGEX-5X-3, and the ligation products were transformed into Escherichia coliDH5α competent cells. The constructed plasmids were extracted from the cells and confirmed by digestion with BamHⅠ and XhoⅠ and nucleotide sequencing. The identified plasmids were then transformed into E.coliBL21(DE3) to express target proteins (different fragments of Mhp367) under the induction of IPTG. After ultrasonic disruption of the recombinant bacteria, the protein was purified with glutathione-conjugated agarose beads. The expression of proteins was visualized by SDS-PAGE. ELISA was employed to identify the reactivity between different fragments of Mhp367 protein and convalescent sera. [Results] Nine recombinant strains that could express different fragments of Mhp367 in soluble form were successfully constructed. All of the nine fragments were humoral immunodominant. The region aa 394–524 in Mhp367 had the strongest reactivity with convalescent sera and was a good antigen fragment for vaccine candidates. [Conclusion] This study provides a candidate of genetic engineering subunit vaccines against M.hyopneumoniae.

Abstract:[Background] The type VI secretion system of bacteria acts as a weapon to kill eukaryotic predators or prokaryotic competitors by releasing toxins, known as effectors. Although some effectors have been identified, the functions of most effectors remain unknown. [Objective] To study the role of the effector Rhs encoded by rhs gene in Salmonella typhimurium. [Methods] The rhs-deleted mutants of Salmonella typhimuriumCVCC541 were constructed via Red recombination, including single gene deletion strains CVCC541Δrhs1 and CVCC541Δrhs2 and the double genes deletion strain CVCC541Δrhs1-2. Meanwhile, the corresponding gene complemented strains C-Δrhs1, C-Δrhs2 and C-Δrhs1-2 were constructed. The biochemical characteristics, biofilm formation, antibiotic resistance, competition between bacteria, bactericidal ability of complement in antiserum, dynamic distribution in vivo and the amounts of IL-18 and IL-1β released by host after infection were compared among the wild-type strain, gene-deleted strains and gene-complemented strains. [Results] Rhs did not affect the metabolism, biofilm formation, antibiotic resistance or complement bactericidal ability of Salmonella typhimurium. The competition index (CI) of CVCC541Δrhs1, CVCC541Δrhs2 and CVCC541Δrhs1-2 was 0.85, 0.77 and 0.87, respectively, which indicated slightly weakened virulence. Compared with the wild-type strain, CVCC541Δrhs1, CVCC541Δrhs2 and CVCC541Δrhs1-2 showed decreased number in the liver and spleen of mice (P<0.05). The host secretion of IL-1β and IL-18 was correlated with the Rhs of Salmonella typhimurium(P<0.05). [Conclusion] The Rhs effectors of Salmonella typhimuriumplay a role in the competition between bacteria, the colonization of bacteria in host organs, and the regulation of the secretion of inflammatory cytokines IL-1β and IL-18 in the host.

Abstract:[Background] A severe infectious disease characterized with gout has attacked the goslings in Shandong, Anhui, Jiangsu, and Guangdong provinces in China since 2016. [Objective] To study the genetic characteristics of goose astrovirus (GAstV) causing this disease. [Methods] A strain of GAstV, named JSSQ, was isolated from the liver sample of a gosling suspected to be undergoing gout from a goose farm in Jiangsu province. The virus genes were sequenced for the genetic characterization. [Results] GAstV JSSQ strain stably propagated in LMH cells without obvious cytopathic changes. The strain had a full-length genome of 7 175 nt and shared the similarity of 57.3%–99.1% with other reported reference strains of GAstV. The phylogenetic analysis showed that JSSQ was close related to the reference strain AHAU3 and they were in the same branch. Two recombination events occurred at the sites 807 and 2 818 in GAstV JSSQ genome, originating from GD AHAU2 (major parent) and AHAU4 (minor parent). The antigenic epitope analysis showed that there were several amino acid mutations, such as P64S, A224T, A228S, and Q229P, in ORF2 region, which resulted in changes in antigenic epitope. [Conclusion] The biological characteristics may vary between the GAstVs in different areas, and thus attention should be paid to the prevention and control of such viruses.

Abstract:[Background] Protopine, sanguinarine, and chelerythrine are main components of the new veterinary drugs Boluohui powder and Bopuzongjian powder in China, which have multiple pharmacological effects. [Objective] To preliminarily reveal the influence and mechanisms of the alkaloids from Macleaya cordata on the physiological activities of extraintestinal pathogenic Escherichia coli (ExPEC), we evaluated the effects of sub-inhibitory concentrations of sanguinarine, chelerythrine, and protopine on the expression of outer membrane protein (OMP), their regulatory genes, and type II toxin-antitoxin (T-A) systems of ExPEC. [Methods] We measured the minimum inhibitory concentrations (MICs) of protopine, sanguinarine, chelerythrine, and antibiotics on the ExPEC mutants with the deletion of yafON, hicAB, and prlF-yhaV. We then compared the expression of OMP genes (ompC,ompX,tolC, and ompF), their regulatory genes (acrR,rob,marR,rpoS, andsoxS), and type II T-A systems (yafON,hicAB, and prlF-yhaV) between WT and the tolC-deleted strain (ΔtolC) exposed to 1/2 MIC of protopine, sanguinarine, and chelerythrine. [Results] The sensitivity ofhicAB-and prlF-yhaV-deleted strains to chloramphenicol increased by 2–4 times, and that of hicAB-deleted strain to sanguinarine also increased by 2 times. Chelerythrine at 1/2 MIC significantly up-regulated the expression of ompX and tolC in WT and ΔtolC, while sanguinarine inhibited the expression of tolC in WT. Protopine, sanguinarine, and chelerythrine at 1/2 MIC up-regulated the expression of ompX and ompC while down-regulated that ofompF in ΔtolC. Sanguinarine and chelerythrine at 1/2 MIC significantly promoted the marR expression in WT and ΔtolC. Moreover, sanguinarine improved the expression of rpoS. Protopine at 1/2 MIC significantly lowered the expression of yafN in WT while significantly increased that in ΔtolC. Protopine, sanguinarine, and chelerythrine at 1/2 MIC down-regulated the expression of hicA and hicB in WT while up-regulated that in ΔtolC. They improved the expression of prlF in WT and ΔtolC, while sanguinarine significantly inhibited the expression of yhaV. [Conclusion] Type II T-A systems and OMPs participate in the responses of ExPEC to alkaloids. The integrity of OMPs (such as TolC) affects the role of T-A systems in bacterial responses to stress, the mechanisms of which remain unclear.

Abstract:[Background] The infection of Nocardia seriolae, a typical opportunistic pathogen, has been documented in many kinds of fishes such as Channa argus and Micropterus salmoides, particularly wounded fishes or fishes with decreased immunity. The resulted nocardiosis lasts a long time, causing great loss to the aquaculture. [Objective] This paper aims to clarify the pathogenicity of Nocardia seriolae isolated from hybrid snakehead (C. maculata♀×C. argus♂), as well as the whole genome and virulence factors information of the pathogen, which is expected to lay a basis for the future research on etiology ofNocardia seriolae, prevention and control of this pathogen, and vaccine development. [Methods] Through challenge test and pathological analysis of target snakehead organs, the virulence and pathogenic characteristics of Nocardia seriolae NK201610020 were elucidated. In addition, based on whole-genome sequencing and comparative genomics analysis, the genomic features and virulence genes information of the strain were clarified. [Results] The result of challenge test showed that the death rates in the five infection groups were up to over 90% except the group of 1.5×10³, and the LD₅₀is1.079×10³ CFU/mL, which indicating the strong virulence of the strain. Histopathological results revealed serious pathological damages in liver, spleen, and kidney, as well as formation of granulomas. The whole genome of NK201610020 was 8 294 329 bp with GC content of 68.10% and 7 812 coding genes. The genomes of Nocardia seriolae from different hosts and different regions showed little difference (>99.9% similarity). According to the alignment with virulence gene database, 171 coding genes in the genome of NK201610020 might be virulence genes, which were involved in cell wall synthesis, nutrient metabolism, and persistent bacterial infection. [Conclusion] Nocardia seriolae has strong virulence, and conserved genome sequence containing diverse virulence genes. The results lay a basis for further analysis of Nocardia seriolae pathogenic mechanism.

Abstract:[Background] Free gossypol in cottonseed meal restricts the application of the meal as a dietary protein resource, and thus the degradation of gossypol has become a research hotspot. We previously found that Bacillus subtilis M-4 can degrade gossypol and it has been used in the industrial detoxification of cottonseed meal. [Objective] To improve the ability of M-4 to degrade gossypol in cottonseed meal and expand the application of cottonseed meal in breeding industry. [Methods] We employed atmospheric and room temperature plasma (ARTP) for mutagenesis of M-4, and preliminarily screened the forward mutants with the index of gossypol degradation rate in liquid medium. Furthermore, according to the degradation rate of gossypol in solid fermentation medium, the free gossypol-degrading ability of the forward mutants was evaluated. In addition, based on gossypol residue and degradation rate of gossypol, the solid fermentation conditions for cottonseed meal were optimized by single-factor experiment and the optimal fermentation parameters were obtained. [Results] A total of 19 forward mutants were screened out and MY-4-17 was identified to be most efficient. In solid medium, the gossypol degradation rate of MY-4-17 reached 97.15%, 2.55% higher than that of M-4. MY-4-17 showed genetic stability after 5 passages. The optimal fermentation conditions for cottonseed meal with MY-4-17 are as follows: 4% corn flour as sugar source, water content in raw material of 45%–50%, MY-4-17 dose at 1.0×10¹³ CFU/t, and fermentation duration of 13–15 days. Under such conditions, the content of gossypol in cottonseed meal was reduced from 980 mg/kg to 22 mg/kg and the degradation rate was over 97.75%. [Conclusion] The mutant screened by ARTP can effectively degrade the gossypol in cottonseed meal. The result can serve as a reference for the development and production of fermentation agent for cottonseed meal.

Abstract:[Background] Lovastatin is a secondary metabolite of Monascus and an important clinically used lipid-lowering drug. Under liquid fermentation conditions, the production of lovastatin from Monascus is low, which is difficult to meet the requirements of industrial production. [Objective] In this study, a Monascus strain with high lovastatin yield was screened, and the production of lovastatin was improved by optimizing liquid fermentation conditions. [Methods] A high lovastatin yielding Monascus strain was selected from red yeast rice, which was identified based on morphological characteristics, physiological and biochemical characteristics, and 18S rRNA gene sequence analysis, response surface methodology (RSM) was applied for the optimization of the liquid fermentation conditions for lovastatin production. [Results] A lovastatin-producing fungus (Monascus purpureus M4) was obtained. With glycerol at 57.80 g/L, yeast extract powder at 5.52 g/L, and inoculation size at 6.90%, the lovastatin output (173.60 mg/L) was 4.8 times higher than that before optimization. [Conclusion] The establishment of the optimal liquid fermentation conditions for lovastatin production by strain M4 provided technical support for the large-scale production of lovastatin and the industrial application of the strain.

Abstract:[Background] Beauveria bassiana, a major entomopathogenic fungus, infects silkworm larvae, producing the white muscardine silkworm Bombyx Batryticatus. The bioactive substances in Bombyx Batryticatus are widely used in medical, healthcare, and cosmetic industries. [Objective] In order to provide high-quality strain resources for the preparation of Bombyx Batryticatus, a highly pathogenic B.bassiana strain was screened from 7 B. bassiana strains from different samples of Bombyx Batryticatus, and the fermentation conditions of this strain were optimized. [Methods] We isolated 7 B. bassiana strains (SDJC-1, SDJC-2, SDJC-3, GXHC-1, SDND-BB, AT-3006 and SQJC-1) from different samples of Bombyx Batryticatus. Based on the results of biological characteristics, protease and chitinase activities, and the lethality in silkworm, a strain with high pathogenicity and excellent traits was screened out. Moreover, the fermentation conditions of this strain were optimized. [Results] The strain SDJC-3 had excellent biological characteristics, high sporulation, and the lethal rate of 75% to silkworm. The Bombyx Batryticatus prepared with SDJC-3 was large and plump and had clear ring of silk gland in the cross-section, exhibiting good quality. The optimum fermentation conditions of SDJC-3 were 27 ℃ of the temperature, 180 r/min of the rotational speed, and 1/5 of the broth volume ratio (250 mL culture flask). [Conclusion] The findings of this study provide a theoretical basis for the use of B.bassiana to prepare and improve the yield and quality of Bombyx Batryticatus.

Abstract:[Background] In recent years, tellurium nanoparticles (TeNPs) have been widely used in optoelectronics, energy, medicine, and other fields. Featuring mild synthesis conditions and low toxicity, biosynthetic TeNPs have attracted wide attention. However, there are few studies on the synthesis of TeNPs by fungi. [Objective] To investigate the ability of Mariannaea sp. HJ to synthesize TeNPs and the antibacterial and antioxidant properties of the TeNPs. [Methods] We used HJ to synthesize TeNPs and optimize the synthesis conditions. XRD, SEM, and DLS were employed to characterize the yielded TeNPs. In addition, we tested the antibacterial and antioxidant properties of TeNPs through experiments. [Results] The optimal synthesis conditions are as follows: 1.5 g (wet weight) of HJ, and TeO₃²⁻ at 5 mmol/L. The yielded TeNPs were mainly spherical with a hexagonal crystal system (XRD). FTIR showed that functional groups such as hydroxyl, carboxyl, and amino were involved in the synthesis of TeNPs. The TeNPs inhibited Staphylococcus aureus and DPPH (inhibition rate=80%at 500 mg/L). [Conclusion] This study provides a TeNPs-synthesizing fungal strain, which lays a theoretical basis for the biosynthesis and application of TeNPs.