This study aims to establish an efficient protoplast-mediated genetic transformation sys-tem for Pochonia chlamydosporia(P.chlamydosporia)to facilitate gene transformation,genetic modification,and subsequent biological function research.Through orthogonal testing,the study systematically optimized key factors influencing electroporation,including voltage,osmotic stabi-lizer,pulse time,nucleic acid concentration,and protoplast concentration.The results showed that the optimal electroporation conditions were:voltage of 250 V,osmotic stabilizer as 0.6 mol/L su-crose solution,pulse time of 10 ms,plasmid concentration of 1％,and protoplast concentration of 1×107 protoplasts/mL,yielding a transformation efficiency of 0.4 × 103 CFU/pg.The interaction analysis revealed that the five factors affected transformation efficiency in the following order:plasmid concentration＞protoplast concentration＞pulse time＞osmotic stabilizer type＞voltage.Based on the positive transformants,the study further evaluated their colony morphology,growth rate,conidial yield,mycelial dry weight,and the ability to infect the eggs of three types of animal gastrointestinal nematodes.The results indicated that the electroporation process did not signifi-cantly affect the biological functions of P.chlamydosporia.The transformants successfully ex-pressed enhanced green fluorescent protein(EGFP),and PCR analysis confirmed the successful in-tegration of the thiostrepton resistance gene.In conclusion,this study successfully established a protoplast-based electroporation-mediated genetic transformation system for P.chlamydosporia,providing a valuable foundation for further research into the mechanism of egg parasitism and the underlying genes involved in its biocontrol activity.

This study aims to establish an efficient protoplast-mediated genetic transformation sys-tem for Pochonia chlamydosporia(P.chlamydosporia)to facilitate gene transformation,genetic modification,and subsequent biological function research.Through orthogonal testing,the study systematically optimized key factors influencing electroporation,including voltage,osmotic stabi-lizer,pulse time,nucleic acid concentration,and protoplast concentration.The results showed that the optimal electroporation conditions were:voltage of 250 V,osmotic stabilizer as 0.6 mol/L su-crose solution,pulse time of 10 ms,plasmid concentration of 1％,and protoplast concentration of 1×107 protoplasts/mL,yielding a transformation efficiency of 0.4 × 103 CFU/pg.The interaction analysis revealed that the five factors affected transformation efficiency in the following order:plasmid concentration＞protoplast concentration＞pulse time＞osmotic stabilizer type＞voltage.Based on the positive transformants,the study further evaluated their colony morphology,growth rate,conidial yield,mycelial dry weight,and the ability to infect the eggs of three types of animal gastrointestinal nematodes.The results indicated that the electroporation process did not signifi-cantly affect the biological functions of P.chlamydosporia.The transformants successfully ex-pressed enhanced green fluorescent protein(EGFP),and PCR analysis confirmed the successful in-tegration of the thiostrepton resistance gene.In conclusion,this study successfully established a protoplast-based electroporation-mediated genetic transformation system for P.chlamydosporia,providing a valuable foundation for further research into the mechanism of egg parasitism and the underlying genes involved in its biocontrol activity.

Objective To study the effect of IL-22 on the colonic barrier and its relationship with Nrf2 pathway in liver fibrosis mice.Methods The mice were divided into four groups:the control group(CON group),the model group(MOD group),the interleukin-22 group(IL-22 group),and the IL-22+ML385 group(ML385,an inhibitor of Nrf2),with 10 mice in each group,and the modeling cycle was 8 weeks.Liquid feed containing alcohol and carbon tetrachloride olive oil were given intraperitoneally in all groups except the CON group;IL-22 was given on top of this in the IL-22 group;and ML385 was injected intraperitoneally in the IL-22+ML385 group one hour before IL-22 treatment.At the end of modeling,the livers were stained with HE and Masson staining to clarify whether fibrosis occurred in the mice;the feces were collected to detect the cocci to bacillus ratio and observe the growth of intestinal flora;the colons were stained with HE staining,immunofluorescence and immunohistochemistry,and analyzed for the expression of tight junction proteins ZO-1,Occludin,and the Nrf2 pathway proteins(Nrf2,HO-1,and NQO1).The expression of these proteins was analyzed by immunohistochemistry.Results Compared with the CON group,mice in the MOD group showed significant fibrosis in the liver tissue,inflammatory cell infiltration in the colon tissue,and decreased expression of tight junction proteins(P<0.05).No overgrowth of various pathogenic bacteria was seen in fecal media.And there was no significant difference in the bulb-to-bar ratio.Compared with the MOD group,both liver and colon histopathologic damage were reduced in the IL-22 group,and tight junction protein expression was elevated,in addition,the expression levels of Nrf2,NQO1,and HO-1 were also elevated(P<0.05),whereas there was no significant change in the IL-22+ML385 group.Conclusion IL-22 improved the colonic barrier function in liver fibrosis mice,and the mechanism was related to the activation of Nrf2 anti-oxidative stress pathway.

Objective:To discuss the challenges and problems of the prevention and control of emerging infectious diseases in Shanghai as a megacities.Methods:An imported case of cutaneous anthrax occurred in Shanghai on May 30, 2019. Shanghai Municipal Center for Disease Control & Prevention conducted an epidemiological investigation and treatment of the cases; after this case, the data of patients with cutaneous anthrax were collected, and an epidemiological study was conducted. Meanwhile, the wound and blood samples of the patient were collected for laboratory testing.Results:Of the seven wound samples of the patient, 6 were positive for the Bacillus anthracis nucleic acid test, and the double serological test results showed a 4-fold increase in the titer of anthrax antibodies. Shanghai CDC conducted an epidemiological investigation of the confirmed cases and observed its contacts. After treatment, the patients recovered, and no other issues appeared among the 19 contacts. Conclusions:Shanghai must strengthen the training of clinicians on emerging infectious diseases to achieve early detection, diagnosis, and treatment of imported infectious diseases and reduce the incidence, spread, and death of the diseases. At the same time, multi-department joint prevention and control are needed to prevent and control secondary cases.

Objective: To investigate the molecular characterization of adult diarrhea cases caused by enterotoxic Escherichia coli (ETEC) and explore the practical model of epidemiology for laboratory technique and data needs based on the surveillance network. Methods: Epidemiological design and sampling targeted adult cases ETEC caused diarrhea in epidemic season. The enterotoxin type, serogroup, resistance, colonization factor and molecular type of ETEC were identified. Multiple dynamic phenotypic characteristics of ETEC were indicated by multidimensional and multivariable data. Results: From 2016 to 2018, 84 eligible ETEC strains were detected. The dominant serums/toxins were O∶6 (STh), O∶25 (LT), O∶159 (STh), O∶153 (STh). O∶6 (STh+CS21), which replaced O∶25 and O∶159 as the popular clones in 2018. Six cases of O∶153 (STh+CFA/I+CS8+PT34) in outbreak in 2017 were imported ones. The resistance rates of ETEC strains detected in adults to sulfasoxazole, naproxinic acid, ampicillin and azithromycin were more than 30%, multidrug resistance (MDR) reached 58.3%. Serum/toxin types suggested that attenuated strains were more likely to become MDR. Molecular typing confirmed that the genetic similarity of the dominant clone of O∶6 serogroup (PT20-24) was higher than O∶25 and O∶159. There was a high correlation between the minimal inhibitory concentration (MIC) of azithromycin and the resistant gene mphA (87.5%, 28/32). O∶6 (STh+CS21+mphA) resistant clone was first detected in 2016. Conclusion A new epidemic clone in adult ETEC diarrhea cases in Shanghai was O∶6 (STh+CS21+mphA). For the first time the association between azithromycin resistance gene mphA and a serum group of ETEC was observed. Multidimensional and multivariate analysis techniques based on epidemiology can help reveal the potential transmission pattern of ETEC for the accurate surveillance and early warning of outbreaks.

Biochemical indexes and blood composition in early lactation health,ketosis and hypocalcemia dairy cows were analyzed to make sure the milk composition characteristics with related diseases,the correlation analysis between early lactating dairy milk composition and blood biochemical were also make in order to provide support for the cattle health assessment.According to theblood index,72 Holstein cows 7-21 d postpartum,were divided into group subclinical hypocalcemia,ketosis test group and control group,24 heads each group.The blood and milk of cows were collected and used to analyze the correlation between blood biochemical indexes and milk composition.The results showed that ketosis and hypocalcemia induced the level of milk protein and non fat milk solids decreased,while the content of citric acid in milk increased.The correlation equation between citric acid in milk and serum NEFA,BHBA and GLU was y=3.192x-0.802,(R2 =0.363),y=4.594x-0.793,(R2 =0.320),y=1.228x+0.775,(R2 =0.261),in which x was the content of citric acid in milk.The results showed that the content of citric acid in milk had positively related to blood NEFA,which could be used as an early marker for the diagnosis of negative energy balance.The levels of BUN and ALB in blood can be used to evaluate the levels of milk protein and urea.