The purpose of this study is to investigate the effect of TPCK trypsin on the proliferation pattern of porcine epidemic diarrhea virus in Vero cells.The TPCK trypsin and conventional tryp-sin were added for virus proliferation,and RT-qPCR technology was used to analyze the changes in virus adsorption and invasion in Vero cells.The replication ability of porcine epidemic diarrhea vi-rus in Vero cells was explored through growth curve drawing,IFA identification,and cell activity detection.The results showed that the optimal concentrations of TPCK trypsin and conventional trypsin were 1 mg/L and 6 mg/L,respectively.The virus showed a decreasing trend with the pro-longation of TPCK trypsin and conventional trypsin pretreatment time.Adding different pancreatic enzymes during the virus proliferation process did not promote the virus invasion in Vero cells.Af-ter 4 h of invasion,the virus particles of each group gradually increased.By plotting the growth curve,it was found that the virus content of the TPCK trypsin group reached its highest level at 24 h(lgTCID50=(6.30±0.14)/0.1 mL),followed by a decreasing trend at 36 h,and the fluorescence intensity produced at 24 h was higher than that of conventional trypsin.In summary,TPCK trypsin has a better promoting effect on the proliferation of porcine epidemic diarrhea virus in Vero cells.It provided theoretical basis for further research on the mechanism of TPCK trypsin affecting porcine epidemic diarrhea virus proliferation,and also provided data support for the isola-tion and purification of porcine epidemic diarrhea virus epidemic strains.

To rapidly and accurately detect Mycobacterium avium subsp.paratuberculosis(MAP),this study designed and screened primers and probes using its specific gene F57 as the detection target,established a recombinant enzyme-mediated isothermal amplification(RAA)fluorescence detection method,and applied this method to detect 116 clinical samples from cattle and sheep.The results showed that using the primer and probe combination B12F/B2R(0.4 μmol/L)+Probe B(0.12 μmol/L),MAP could be detected at a constant temperature of 42 ℃ within 20 min;this de-tection method had no cross-reaction with 11 common pathogens such as Escherichia coli,Clos-tridium,and bovine viral diarrhea in sheep and cattle;the lowest detection limit was 1.0×102 cop-ies/μL;the coefficient of variation was 3.77％—5.29％;24 clinical samples were positive,with a co-incidence rate of 88.89％with GBT27637-2011.In summary,this study established a fluorescent RAA detection method for MAP,which is simple,rapid,highly specific,sensitive,reproducible,and has a high coincidence rate with national standards,making it suitable for clinical detection and epi-demiological studies.

Swine enteric coronaviruses(SeCoV),such as porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),and porcine delta coronavirus(PDCoV),cause severe diarrhea in piglets,resulting in substantial losses in pig farming.In this study we establish a triple fluorescence reverse transcription-quantitative PCR(RT-qPCR)method for the simultaneous de-tection of PEDV,TGEV,and PDCoV.The specific primers and probes for each target virus were designed based on conserved sequences from the PEDV M gene,the TGEV ORF 1b gene,and the PDCoV ORF 1b gene respectively.Following the optimization of parameters and conditions,a triple RT-qPCR method was successfully established to simultaneously detect PEDV,TGEV,and PD-CoV.The developed assay exhibits strong specificity for these three pathogens without any cross-reaction with other common porcine viruses like CSFV,PCV2,PoRVA,PRV,and PRRSV.The de-tection limit of linear templates for pTOPO-PEDV 128,pTOPO-TGEV 116,and pTOPO-PDCoV 125 recombinant plasmids were 16.835,17.610 and 17.020 copies/μL,respectively.The intra group and inter group coefficients of variation were less than 5％,with no significant differences observed(P＞0.05).Moreover,the detection consistency rate of the developed RT-qPCR was compared with standard method and showed 100％agreement.Out of 35 small intestine tissue samples,17 tested positive for PEDV,resulting in a positive rate of 48.57％(17/35).The tests for TGEV and PDCoV yielded negative results,and no mixed infections were detected.Based on the above results,the tri-ple RT-qPCR method established is specific,sensitive,stable,and rapid,and can be used for clinical detection and differential diagnosis of PEDV,TGEV,and PDCoV simultaneously,providing a method for the detection and epidemiological investigation of porcine diarrhea coronaviruses.

Proteus mirabilis is a significant foodborne pathogen that frequently causes diseases in humans and animals.Understanding the cytokine profile alterations following gastrointestinal in-fection with P.mirabilis is crucial for developing effective treatment strategies.This study em-ployed an intragastric infection model in mice to observe clinical symptoms and pathological chan-ges.Blood samples were collected at various time points post-infection,and the concentrations of 21 cytokines in the serum of both infected and control mice were quantitatively analyzed using the Bio-Plex suspension array system.The results of the infection experiment indicated that gastroin-testinal infection with P.mirabilis in mice led to splenic hemorrhage and significant splenomegaly.A notable difference in spleen index between the infected and control groups was observed at 24 hours post-infection(P=0.001 688),with the greatest difference occurring at 96 hours post-infec-tion(P=0.000 074).Cytokine analysis revealed significant elevations in IL-2,IL-17A,KC,Eotax-in,MCP-1,RANTES,MIP-1α,MIP-1β,IFN-γ,G-CSF,and IL-10 levels in the infected group.A-mong these,the pro-inflammatory cytokines IL-17A and G-CSF exhibited the most substantial changes,positively correlating with the splenomegaly,and peaking at 96 hours post-infection,with concentrations of(104.74±3.91)and(5 184.08±280.22)ng/L,respectively,which were 26.79 and 36.25 times higher than those in the control group.These findings indicate that IL-17A and G-CSF play significant roles in the pro-inflammatory response following gastrointestinal infection with P.mirabilis.

The purpose of this study is to investigate the effect of TPCK trypsin on the proliferation pattern of porcine epidemic diarrhea virus in Vero cells.The TPCK trypsin and conventional tryp-sin were added for virus proliferation,and RT-qPCR technology was used to analyze the changes in virus adsorption and invasion in Vero cells.The replication ability of porcine epidemic diarrhea vi-rus in Vero cells was explored through growth curve drawing,IFA identification,and cell activity detection.The results showed that the optimal concentrations of TPCK trypsin and conventional trypsin were 1 mg/L and 6 mg/L,respectively.The virus showed a decreasing trend with the pro-longation of TPCK trypsin and conventional trypsin pretreatment time.Adding different pancreatic enzymes during the virus proliferation process did not promote the virus invasion in Vero cells.Af-ter 4 h of invasion,the virus particles of each group gradually increased.By plotting the growth curve,it was found that the virus content of the TPCK trypsin group reached its highest level at 24 h(lgTCID50=(6.30±0.14)/0.1 mL),followed by a decreasing trend at 36 h,and the fluorescence intensity produced at 24 h was higher than that of conventional trypsin.In summary,TPCK trypsin has a better promoting effect on the proliferation of porcine epidemic diarrhea virus in Vero cells.It provided theoretical basis for further research on the mechanism of TPCK trypsin affecting porcine epidemic diarrhea virus proliferation,and also provided data support for the isola-tion and purification of porcine epidemic diarrhea virus epidemic strains.

To rapidly and accurately detect Mycobacterium avium subsp.paratuberculosis(MAP),this study designed and screened primers and probes using its specific gene F57 as the detection target,established a recombinant enzyme-mediated isothermal amplification(RAA)fluorescence detection method,and applied this method to detect 116 clinical samples from cattle and sheep.The results showed that using the primer and probe combination B12F/B2R(0.4 μmol/L)+Probe B(0.12 μmol/L),MAP could be detected at a constant temperature of 42 ℃ within 20 min;this de-tection method had no cross-reaction with 11 common pathogens such as Escherichia coli,Clos-tridium,and bovine viral diarrhea in sheep and cattle;the lowest detection limit was 1.0×102 cop-ies/μL;the coefficient of variation was 3.77％—5.29％;24 clinical samples were positive,with a co-incidence rate of 88.89％with GBT27637-2011.In summary,this study established a fluorescent RAA detection method for MAP,which is simple,rapid,highly specific,sensitive,reproducible,and has a high coincidence rate with national standards,making it suitable for clinical detection and epi-demiological studies.

Swine enteric coronaviruses(SeCoV),such as porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),and porcine delta coronavirus(PDCoV),cause severe diarrhea in piglets,resulting in substantial losses in pig farming.In this study we establish a triple fluorescence reverse transcription-quantitative PCR(RT-qPCR)method for the simultaneous de-tection of PEDV,TGEV,and PDCoV.The specific primers and probes for each target virus were designed based on conserved sequences from the PEDV M gene,the TGEV ORF 1b gene,and the PDCoV ORF 1b gene respectively.Following the optimization of parameters and conditions,a triple RT-qPCR method was successfully established to simultaneously detect PEDV,TGEV,and PD-CoV.The developed assay exhibits strong specificity for these three pathogens without any cross-reaction with other common porcine viruses like CSFV,PCV2,PoRVA,PRV,and PRRSV.The de-tection limit of linear templates for pTOPO-PEDV 128,pTOPO-TGEV 116,and pTOPO-PDCoV 125 recombinant plasmids were 16.835,17.610 and 17.020 copies/μL,respectively.The intra group and inter group coefficients of variation were less than 5％,with no significant differences observed(P＞0.05).Moreover,the detection consistency rate of the developed RT-qPCR was compared with standard method and showed 100％agreement.Out of 35 small intestine tissue samples,17 tested positive for PEDV,resulting in a positive rate of 48.57％(17/35).The tests for TGEV and PDCoV yielded negative results,and no mixed infections were detected.Based on the above results,the tri-ple RT-qPCR method established is specific,sensitive,stable,and rapid,and can be used for clinical detection and differential diagnosis of PEDV,TGEV,and PDCoV simultaneously,providing a method for the detection and epidemiological investigation of porcine diarrhea coronaviruses.

Proteus mirabilis is a significant foodborne pathogen that frequently causes diseases in humans and animals.Understanding the cytokine profile alterations following gastrointestinal in-fection with P.mirabilis is crucial for developing effective treatment strategies.This study em-ployed an intragastric infection model in mice to observe clinical symptoms and pathological chan-ges.Blood samples were collected at various time points post-infection,and the concentrations of 21 cytokines in the serum of both infected and control mice were quantitatively analyzed using the Bio-Plex suspension array system.The results of the infection experiment indicated that gastroin-testinal infection with P.mirabilis in mice led to splenic hemorrhage and significant splenomegaly.A notable difference in spleen index between the infected and control groups was observed at 24 hours post-infection(P=0.001 688),with the greatest difference occurring at 96 hours post-infec-tion(P=0.000 074).Cytokine analysis revealed significant elevations in IL-2,IL-17A,KC,Eotax-in,MCP-1,RANTES,MIP-1α,MIP-1β,IFN-γ,G-CSF,and IL-10 levels in the infected group.A-mong these,the pro-inflammatory cytokines IL-17A and G-CSF exhibited the most substantial changes,positively correlating with the splenomegaly,and peaking at 96 hours post-infection,with concentrations of(104.74±3.91)and(5 184.08±280.22)ng/L,respectively,which were 26.79 and 36.25 times higher than those in the control group.These findings indicate that IL-17A and G-CSF play significant roles in the pro-inflammatory response following gastrointestinal infection with P.mirabilis.

Objective: To investigate the principles of diagnosis and treatment of breast cancer during pregnancy. Methods: Clinical data of patients with breast cancer during pregnancy admitted to Obstetrics and Gynecology Hospital of Fudan University between January 2012 to July 2017 were analyzed retrospectively. A total of 17 patients were diagnosed with breast cancer in pregnancy, the median age was 32 years (range from 25 to 45 years old), pathological staging revealed 2 patient with stage 0, 1 with stage Ⅱa, 7 with stage Ⅱb, 1 with stage Ⅲa, 2 with stage Ⅲc, 4 with stage Ⅳ. Results: Thirteen patients received surgical treatment in pregnancy, the gestational age at surgery was (27.7±4.6) weeks; 2 patients with ductal carcinoma in situ received mastectomy, 11 patients with breast cancer underwent modified radical mastectomy. In patients undergoing surgery during pregnancy, no prophylactic contractions were used in 4 patients who had been treated earlier, there were 2 patients with frequent contractions within 24 hours after operation in these patients. Follow-up 9 patients were given oral nifedipine to prevent contractions, no obvious contractions occurred after the operation. Seven patients received chemotherapy during pregnancy; the chemotherapy of 4 cases of triple negative breast cancer was weekly paclitaxel sequential epirubicin and cyclophosphamide, the chemotherapy of the other three patients was docetaxel sequential epirubicin and cyclophosphamide. Fifteen patients underwent cesarean section to terminate pregnancy, 2 patients underwent spontaneous labor. The gestational age of birth was (36.9 ±1.3) weeks. Less than 35 weeks of termination of pregnancy occurred in one patient, the fetus was delivered to the neonatal intensive care unit due to neonatal respiratory distress syndrome, and suffered from congenital dysaudia. The prognosis of the other 16 survived infants was good. The median follow-up time was 10 months (range from 4 to 27) months, in 13 patients of stage 0 to Ⅲc, one patient were diagnosed with bone metastasis at 12 months after surgery, the remaining 12 patients had no disease progression, the progression free survival rate was 12/13, the overall survival rate was 13/13. Among the 4 patients with stage Ⅳ, one died in 7 months after delivery, one had new liver metastasis in 8 months after delivery. The remaining 2 patients were in stable condition. Conclusions Breast cancer in pregnancy can be treated effectively, multidisciplinary cooperation and detailed assessment of maternal-fetal risks and benefits are necessary. Chemotherapy during pregnancy is safe for maternal-fetal, but it needed a large sample of clinical studies and long-term follow-up. The neonatal outcome was associated with gestational age, and therefore premature delivery was avoided as much as possible during treatment.