噬菌体防治ETEC感染小鼠效果的初步研究

Preliminary study on the prevention and treatment effect of phage against ETEC infection in mice

为研究噬菌体防治产肠毒素大肠杆菌(ETEC)感染小鼠的效果,本研究通过双层平板法测定噬菌体DS2的效价,并利用双层琼脂空斑试验分别检测噬菌体DS2对致仔猪腹泻大肠杆菌K88、K99及987p的体外裂解作用。结果显示,噬菌体DS2的效价为6.9×10^(9)pfu/mL,且均能裂解上述3种ETEC。进一步经小鼠试验评估噬菌体在体内防治ETEC感染的效果:将噬菌体分别以2×10^(8)pfu/只(高剂量组)、2×10^(7)pfu/只(低剂量组)经腹腔注射接种小鼠,1 h后再经腹腔注射0.2 mL剂量均为3×10^(8)cfu/只的K88、K99及987p混合菌液。分别于感染后12 h、24 h、48 h无菌采集各组小鼠的肝脏,采用平板计数法检测各组小鼠肝脏载菌量;利用双层平板法检测各组小鼠肝脏中的噬菌体含量。感染后24 h,利用ELISA试剂盒检测各组小鼠血清中细胞因子IL-1β含量。感染后48 h,利用内毒素检测鲎试剂盒测定各组小鼠血浆内毒素的含量。小鼠肝脏载菌量和噬菌体含量检测结果显示:与接种LB肉汤的对照组相比,高、低剂量组小鼠的肝脏载菌量在感染后不同时间有升有降,但二者之间或者与对照组之间均无显著差异(P>0.05);随着感染时间的延长,高、低剂量组小鼠肝脏噬菌体的含量均在下降,且在感染后低剂量组小鼠肝脏噬菌体的含量极显著高于(感染后24 h)(P<0.01)或者高于(感染后48 h)高剂量组。IL-1β及内毒素检测结果显示,与对照组相比,高、低剂量组小鼠血清中IL-1β的含量显著下降(P<0.05)或者下降,但高、低剂量组小鼠血浆内毒素的含量均升高或显著升高(P<0.05)。将60只小鼠按照上述实验分组及处理,观察并记录感染后72 h内各组小鼠的发病率和死亡率,并统计感染后不同时间每组小鼠的平均体况得分。结果显示,对照组小鼠在感染接近24 h后体况计分降至25分以下,高、低剂量组小鼠在感染24 h后体况计分下降至70分及90分以上。高、低剂量组及对照组小鼠的存活率分别为65%、95%及15%。以上结果表明,DS2噬菌体可有效防治体内外ETEC的感染,且低剂量DS2噬菌体的疗效总体上要优于高剂量。本研究为防治仔猪大肠杆菌病提供了新思路。

To study the prevention and treatment of phage against enterotoxigenic Escherichia coli(ETEC)infection in mice,the titer of phage DS2 was determined by double agar overlay plaque assay,and the in vitro lysis effect of phage DS2 on E.coli K88,K99 and 987p causing piglet diarrhea was detected by double agar overlay plaque assay.The results showed that the phage DS2 had a titer of 6.9×10^(9)pfu/mL and could lyse the three strains of ETEC.The efficacy of phage against ETEC infection in vivo was further evaluated by mouse experiment;the mice were inoculated with 0.2mL phage per mouse by intraperitoneal injection of 2×10^(8)pfu/mouse(high-dose group)and 2×10^(7)pfu/mouse(low-dose group).After 1 hour,mice in each group were intraperitoneally injected with 0.2mL of piglet diarrhea E.coli mixture K88,K99 and 987p with a dose of 3×10^(8)cfu/mouse.The livers of mice in each group were collected aseptically at 12,24 and 48 hours after infection,and the amount of bacteria in the livers of mice in each group was examined by plate count method.The phage content in the livers of mice in each group was assesssed by double agar overlay plaque assay.Twenty-four hours after infection,serum IL-1βlevels of mice in each group were detected by ELISA kit.Forty-eight hours after infection,plasma endotoxin content of mice in each group was determined by limulus endotoxin test kit.Compared with the control group inoculated with LB broth,the bacterial load and phage content in the liver of mice in the highdose and low-dose groups increased or decreased at different time after infection,but there was no significant difference with control group or between the two groups(P>0.05).As the infection time prolonged,the content of phage in liver of mice in highdose and low-dose groups decreased,and the content of phage in liver of mice in low-dose group was significantly higher(24 hours after infection)(P<0.01)or higher(48 hours after infection)than high-dose group.The results of IL-1βand endotoxin detection showed that compared with the control group,the levels of IL-1βin serum of mice in high-dose and low-dose groups were significantly decreased(P<0.05)or decreased,but the plasma endotoxin content in high-dose and low-dose groups increased or significantly increased(P<0.05).Sixty mice were divided into groups and treated according to the above experiment.The morbidity and mortality of each group were observed and recorded within 72 hours after infection,and the average body condition score of each group at different times after infection was calculated.The results showed that the body condition score of mice in control group decreased to less than 25 points after nearly 24 hours of infection,and the body condition score of mice in high-dose and low-dose groups decreased to more than 70 and 90 points after 24 hours of infection.The survival rate of high-dose group,lowdose group and control group mice was 65%,95%and 15%,respectively.These results suggest that DS2 phage can effectively prevent and treat ETEC infection in vitro and in vivo,and the efficacy of low-dose DS2 phage is generally better than that of highdose phage.This study will potentially provide a new way for the prevention and treatment of piglet colibacillosis.