Efficacy of Lactobacillus rhamnosus GG in treatment of acute pediatric diarrhea: A systematic review with meta-analysis

BACKGROUND Diarrhea is a major infectious cause of childhood morbidity and mortality worldwide.In clinical trials,Lactobacillus rhamnosus GG ATCC 53013(LGG)has been used to treat diarrhea.However,recent randomized controlled trials(RCTs)found no evidence of a beneficial effect of LGG treatment.AIM To evaluate the efficacy of LGG in treating acute diarrhea in children.METHODS The EMBASE,MEDLINE,PubMed,Web of Science databases,and the Cochrane Central Register of Controlled Trials were searched up to April 2019 for metaanalyses and RCTs.The Cochrane Review Manager was used to analyze the relevant data.RESULTS Nineteen RCTs met the inclusion criteria and showed that compared with the control group,LGG administration notably reduced the diarrhea duration[mean difference(MD)-24.02 h,95%confidence interval(CI)(-36.58,-11.45)].More effective results were detected at a high dose≥1010 CFU per day[MD-22.56 h,95%CI(-36.41,-8.72)]vs a lower dose.A similar reduction was found in Asian and European patients[MD-24.42 h,95%CI(-47.01,-1.82);MD-32.02 h,95%CI(-49.26,-14.79),respectively].A reduced duration of diarrhea was confirmed in LGG participants with diarrhea for less than 3 d at enrollment[MD-15.83 h,95%CI(-20.68,-10.98)].High-dose LGG effectively reduced the duration of rotavirus-induced diarrhea[MD-31.05 h,95%CI(-50.31,-11.80)]and the stool number per day[MD-1.08,95%CI(-1.87,-0.28)].CONCLUSION High-dose LGG therapy reduces the duration of diarrhea and the stool number per day.Intervention at the early stage is recommended.Future trials are expected to verify the effectiveness of LGG treatment.

Prevalence and Genotypes of Rotavirus A and Human Adenovirus among Hospitalized Children with Acute Gastroenteritis in Fujian, China, 2009-2017

Gastroenteritis is an infectious diarrhea that has been considered as an important cause of hospitalizations and death in children aged < 5 years, particularly in developing countries. Unsanitary water, contaminated food, poor hygiene, and inadequate disposal of waste and feces are all risk factors for gastroenteritis, resulting in the higher incidence in developing countries. Gastroenteritis is generally caused by viral infections, among which rotavirus (RV) infections have been reported to be the most common, especially among young children aged < 5 years with acute gastroenteritis in Asia and Africa[1]. Other viruses associated with acute gastroenteritis include human Adenovirus (HAdV), Norovirus, Sapovirus (SaV), human Astrovirus (HAstV), and Aichi virus. Recent research has reported that adenovirus types 40 and 41, belonging to species F, cause gastroenteritis and were therefore termed as enteric adenoviruses. In addition, non-enteric HAdV species such as A, B, C, and D have been associated with diarrheal.

A blind, randomized comparison of racecadotril and loperamide for stopping acute diarrhea in adults

AIM: Racecadotril is a specific enkephalinase inhibitor that exhibits intestinal antisecretory activity without affecting intestinal transit. Loperamide is an effective anti-diarrheal agent, but it usually induces constipation. This study is to compare the efficacy, safety, and tolerability of racecadotril versus loperamide in the outpatient treatment of acute diarrhea in adults. METHODS: A two-center, randomized, parallel-group, single-blind study was carried out to compare the efficacy, tolerability, and safety of racecadotril (100 mg thrics daily) and loperamide (2.0 mg 2 twics daily) in 62 adult patients suffering from acute diarrhea. The main efficacy criterion used was the duration of diarrhea after beginning the treatment (in hours). Other signs and symptoms were also evaluated. RESULTS: The clinical success rates for these anti-diarrheal treatments were 95.7% and 92.0% for racecadotril and loperamide respectively. Patients on racecadotril had a median duration of diarrhea of 19.5 h compared with a median of 13 h for patients on loperamide. Rapid improvement in anal burn and nausea was found for each drug. However, more patients on loperamide suffered from reactive constipation (29.0% vs 12.9%). Itching, another adverse event was notably higher in the racecadotril group (28.6% vs 0%). With regard to other adverse events, the two medications showed similar occurrence rates and similar concomitant medication usage rates. CONCLUSION: Racecadotril and loperamide are rapid, equally effective treatments for acute diarrhea in adults, but loperamide treatment is associated with a higher incidence of treatment-related constipation.

Prevalence of shiga toxins(stx_1,stx_2),eaeA and hly genes of Escherichia coli O157:H7 strains among children with acute gastroenteritis in southern of Iran

Objective:To survey the prevalence severe diarrhea arising from these bacteria in children under 5 years old in Marvdasht.Methods:In this study faecal sample from 615 children aged <5years old who were hospitalized lor gastroenteritis in Fars hospitals in Iran were collected and then enriched in Escherichia coli(E.coli) broth and modified tryplone soy broth with novobiocin media,fermentation of sorbitol,lactose and β— glucoronidase activity of isolated strains was examined by CT—SMAC,VRBA and chromogenic media respectively.Then isolation of E.coli O157:H7 have been confirmed with the use of specific antisera and with multiplex PCR method presence of virulence genes including:xtx_1.stx_2,eae.A.hly has been analyzed.Results:E.coli O157:H7 was detected in 7(1.14%) stool specimens.A significanl difference was seen between detection rale of isolated bacteria from age groups 18-23 months and other age groups(P=0.004).Out of considered virulence genes.only 1 of the isolated strains(0.16%)he stx,and eaeA genes were seen and also all isolated hacleria had resistance to penicillin,ampicillin and erythromycin antibiotics.Conclusions:We found thai children < 2 years of age were at highest risk of infection with E.coli O157:H7.Regarding severity of E.coli O157:H7 pathogenesis,low infectious dose and lack of routine assay for detection ol these bacleria in clinical laboratory,further and completed studies on diagnosis and genolyping of this E.coli O157:H7 strain has been recommended.

Clinicopathogenic studies of acute diarrhea in children

AIM To demonstrate the etiologic pathogens of acute diarrhea in children and the diagnostic value of stool pH. METHODS From May 1988 to April 1992, 368 children with acute diarrhea were studied. Fresh stools were examined routinely, and stool pH were tested with pH paper, then the samples in the culture medium of the Cary Blair were sent to the lab for bacteria isolation and identification. The rotavirus was identified by the method of ELISA from the supernatant. RESULTS Thirty one pathogens and 385 bacteria strains were found in the 368 examples, with a detectable rate of 67 7%, including 37 8% of mixed infections. Among bacteria families, vibrionaceae is the most common (39 7%), and among bacteria genuses, aeromonas is the most common (26 8%). In bacterial diarrhea, the stool pH tends to be basic while in viral diarrhea to be acid. CONCLUSION There are 31 pathogens for the children′s acute diarrhea in this area. It is quite difficult to make an etiologic diagnosis only by clinial signs. However, stool pH is of some value for early diagnosis of the disease.

Resolution of acute gastroenteritis symptoms in children and adults treated with a novel polyphenol-based prebiotic

AIM: To test efficacy and durability of a polyphenol-based prebiotic treatment for acute gastroenteritis in a 300 patient double-blinded clinical study.

Efficacy of stool multiplex polymerase chain reaction assay in adult patients with acute infectious diarrhea

BACKGROUND Recently,stool multiplex polymerase chain reaction(PCR)tests have been developed for identifying diarrhea-causing bacterial pathogens.Furthermore,fecal calprotectin is a well-known effective marker for intestinal mucosal inflammation.AIM To evaluate the efficacy of stool multiplex PCR and fecal calprotectin in acute infectious diarrhea.METHODS Overall,400 patients with acute infectious diarrhea were enrolled from Kangdong Sacred Heart Hospital(January 2016 to December 2018).Multiplex PCR detected 7 enteropathogenic bacteria including Salmonella,Campylobacter,Shigella,Escherichia coli O157:H7,Aeromonas,Vibrio,and Clostridium difficile.We reviewed clinical and laboratory findings using stool multiplex PCR.RESULTS Stool multiplex PCR test detected considerably more bacterial pathogens than stool culture(49.2%vs 5.2%),with Campylobacter as the most common pathogen(54%).Patients with positive stool PCR showed elevated fecal calprotectin expression compared to patients with negative stool PCR(1124.5±816.9 mg/kg vs 609±713.2 mg/kg,P=0.001).C-reactive protein(OR=1.01,95%CI:1.001-1.027,P=0.034)and sigmoidoscopy-detected colitis(OR=4.76,95%CI:1.101-20.551,P=0.037)were independent factors in stool PCR-based detection of bacterial pathogens.Sensitivity and specificity of calprotectin were evaluated to be 70.5%and 60.9%,respectively(adjusted cut-off value=388 mg/kg).CONCLUSION Stool multiplex PCR test has increased sensitivity in detecting pathogens than conventional culture,and it is correlated with calprotectin expression.Stool multiplex PCR and calprotectin may be effective in predicting clinical severity of infectious diarrhea.

Impact of rotavirus vaccine on acute gastroenteritis emergency department visits and hospitalizations in a highly-vaccinated urban cohort

Background: Rotavirus vaccines (RVV) have significantly reduced rotavirus disease in children over the past 4 years in the United States. In this study, we describe the impact of RVV in preventing acute gastroenteritis (AGE) hospital encounters in a highly-vaccinated urban pediatric network during the 2007 and 2008 rotavirus seasons. Methods: We used 5 urban practices from a practice-based network to conduct a retrospective cohort study comparing the numbers of AGE emergency department (ED) visits and hospitalizations in RVV-immunized (exposed) and non-immunized (unexposed) children during the first 2 full seasons following RVV introduction. We determined incident rate ratios (IRR), using Poisson regression, and vaccine effectiveness for each outcome. Results: The 2007 and 2008 cohorts were analyzed separately. 62% of the 2007 cohort was vaccinated and 88% of the 2008 cohort. AGE hospitalizations were significantly reduced among RVV-immunized children from the 2007 cohort in the 2008 season with vaccine effectiveness of 67%. Sub-analysis of this cohort by age revealed that RVV was most protective against hospitalizations in the youngest age group (IRR = 0.21, 95% CI (0.06, 0.82). A trend toward protection against hospitalization was detected for both cohorts in the first season following immunization that did not reach a statistically significant level. For AGE ED visits, no significant difference was seen between RVV-immunized and non-immunized children in either cohort, although there was a trend toward protection (IRR’s: 0.67 - 0.7). Conclusions: RVV was highly effective in preventing AGE hospitalizations for a subset of our cohort in 2008. Given reports of RVV effectiveness, we hypothesize that herd immunity is responsible for the inability to detect a significant difference between RVV-immunized and non-immunized children in our highly- vaccinated cohort.

Formulation of an Early Warning Infectivity Score System for Adult Patients with Acute Bacterial Diarrhea

The aim of our study was to develop a scoring system to predict whether diarrhea is of a bacterial origin and whether the diarrheal patients constitute a potential source of infection to others. Adults with acute diarrhea （n=424） were enrolled in the study. Logistic regression and standard regression coefficients were used to formulate the Early Warning Infectivity Score System for Adults with Acute Bacterial Diarrhea （EWIS-ABD）. Four risk factors were identified by logistic regression, including body temperature （P〈0.01）, abdominal pain （P〈0.01）, leukocyte count in stool （P〈0.01）, and unclean dietary history （P〈0.01）. EWIS-ABD was thus developed, in which the value 〉5 points was set as an indicator of bacterial diarrhea. The incidence of bacterial diarrhea increased along with the elevated score. EWIS-ABD was more specific for bacterial diarrhea than for viral diarrhea. The accuracy and reliability of EWIS-ABD was high by prospective validation in 478 patients with acute diarrhea.

Frequency of E.coli pathotypes in acute diarrhea of children and its related factorsat Beassat hospital,Sanandaj

Objective:Diarrhea is a leading cause of morbidity and mortality among children in developing countries.The bacterial pathogen most commonly associated with childhood diarrhea is Escherichia coli.A one-year prospective study was carried out in Sanandaj to determine the prevalence and roles of the different E.coli pathotypes in children less than five years of age with acute diarrhea.Methods:Rectal swab were collected prospectively from children with acute diarrhea and transported to the Department of Microbiology,School of Medicine, KUMS,Sanandaj during 2008.The study was approved by the institutional ethics committee.Results:During this study period,rectal swabs were investigated from a total of 466 children 1 to 144 months of age(mean, 29.97 months±S.D) with diarrhea.Among the children,191(41%,191/466) were girls,and 275 (59%,275/466) were boys.The age-specific incidence rates of acute diarrhea among children 13-24 and 1 - 12 months of age were 37.37%(37/99) and 26.26%(26/99),respectively,during the study period.A total of 99 strains of E.coli were detected.EPEC 59(59.59%) and EIEC 22(22.22%),were the most commonly found Escherichia coli strains detected in stools from children.Disk diffusion testing showed E.coli strains resistance to tetracycline(89.89%),chloramphenicol(88.88%),Ampicillin(79.79%),Amoxicillin (75.75%) and Ceficime(75.75%).Among risk factors like age,sex,haemoglubin,fathers and mothers education,food and weight of children only mother's education was significant(P =0.018).Conclusion: In most of the clinical laboratories in Iran,E.coli does not considered as an etiologic agent responsible for diarrhea. Results in this study revealed that E.coli should be considered as an etiologic agent causing acute diarrhea among children.We therefore,recommend the routine isolation and identification of E.coli strains in all the clinical laboratories in Sanandaj.Guidelines for appropriate use of antibiotics in Sanandaj need updating.

Prolonged acute diarrhea in adults: Decades of observation from epidemiological perspective in urban Bangladesh

The study aimed to determine the epidemiological, clinical characteristics, and etiology of adults aged ≥20 years presented with prolonged acute diarrhea (≥7 days). A total of 18,210 adults aged ≥20 years were enrolled in the Diarrheal Disease Surveillance System of icddr,b between 1993-2012 and included into the analysis. Of these, 17,631 (97%) had duration of diarrhea ≤6 days, 418 (2%) diarrhea presented with a duration of 7 - 13 days and rest 161 (1%) had history of diarrhea ≥14 days. A higher proportion of adult individuals who had duration of diarrhea ≥14 days (70% vs. 56%;p male. At least 73% of all patients used oral rehydration solution;but proportion was lower among prolonged acute diarrhea groups. Use of antimicrobials was higher among those with duration 7 - 13 days (81%) and ≥14 days (81%). Diarrhea lasting ≥14 days, 47% were suffering from chronic energy deficiency and 30% had history of smoking. Individuals with diarrhea ≥7 days less frequently presented with vomiting, watery stool, frequency of stool >10 times/24 hours, drowsy or lethargy, fast breathing, some or severe dehydration, received intravenous saline for initial corrections of dehydration and stayed for longer duration in hospital (≥24 hours) but more often presented with abdominal pain. Stool microscopic examination showed less frequent presence of red blood cells (36% vs. 44%;p = 0.043) and fecal leucocytes (50% vs. 59%;p = 0.029) among individuals with diarrhea ≥14 days compared to those with ≤6 days. None was infected with Vibrio cholerae (≥14 days) (3% for 7 - 13 days);however, isolation rate of Aeromonas was higher among adults with duration for ≥14 days (11%). Only 15% with ≥14 days were positive for Shigella contrary to 19% (7 - 13 days) and 56% (≤6 days). Differences in sociodemographic, clinical presentation and etiology varied with duration of diarrhea among adults.

Clinical factors predicting rotavirus diarrhea in children:A crosssectional study from two hospitals

BACKGROUND Rotavirus is still a significant contributing morbidity and mortality in pediatric patients.AIM To look at clinical signs and symptoms and laboratory findings that can predict rotavirus gastroenteritis compared to non-rotavirus gastroenteritis.METHODS This was a cross-sectional study with medical records obtained from December 2015 to December 2019.Inclusion criteria for this study include all hospitalised pediatric patients(0-18 years old)diagnosed with suspected rotavirus diarrhea.The receiver operating curve and Hosmer-Lemeshow test would be used to assess the final prediction findings'calibration(goodness of fit)and discrimination performance.RESULTS This study included 267 participants with 187(70%)rotavirus-diarrhea cases.The patients were primarily male in both rotavirus(65.2%)and non-rotavirus(62.5%)groups.The median age is 1.33 years old(0.08-17.67 years old).Multivariate analysis shows that wet season(OR_(adj)=2.5;95%CI:1.3-4.8,Padj=0.006),length of stay(LOS)≥3 days(OR_(adj)=5.1;95%CI:1.4-4.8,Padj=0.015),presence of abdominal pain(OR_(adj)=3.0;95%CI:1.3-6.8,Padj=0.007),severe dehydration(OR_(adj)=2.9;95%CI:1.1-7.9,Padj=0.034),abnormal white blood cell counts(OR_(adj)=2.8;95%CI:1.3-6.0,Padj=0.006),abnormal random blood glucose(OR_(adj)=2.3;95%CI:1.2-4.4,Padj=0.018)and presence of fecal leukocytes(OR_(adj)=4.1,95%CI:1.7-9.5,Padj=0.001)are predictors of rotavirus diarrhea.The area under the curve for this model is 0.819(95%CI:0.746-0.878,P value<0.001),which shows that this model has good discrimination.CONCLUSION Wet season,LOS≥3 d,presence of abdominal pain,severe dehydration,abnormal white blood cell counts,abnormal random blood glucose,and presence of fecal leukocytes predict rotavirus diarrhea.

A scoring system for assessing the severity of acute diarrhea of adult patients

BACKGROUND: Diarrhea is frequently seen in developed and developing countries, and severe diarrhea is characterized by the high risk of death. Thus, it is very important to assess the severity of diarrhea early. We conducted a multi-center study to identify risk factors for the severity of diarrhea in adult patients and formulate an adult diarrhea state score(ADSS) for out-patient clinicians.METHODS: A total of 219 adult patients with acute diarrhea were divided into two groups: 132 patients with mild diarrhea and 87 with severe diarrhea. Logistic regression was used to determine risk factors for the severity of diarrhea. The risk factors were assessed and an ADSS was formulated. Receiver operating characteristic(ROC) analysis was made to evaluate the diagnostic accuracy of ADSS, and the Kappa test was used to confi rm the diagnostic reliability.RESULTS: Five risk factors for evaluating the severity of diarrhea in adults included age(P<0.05), axillary temperature(P<0.01), mean arterial pressure(P<0.01), white blood cell count(WBC; P<0.01), and WBC in stool(P<0.01). The area under the ROC curve for ADSS was 0.958 when the cut off value was 4(a sensitivity of 0.909; a specif icity of 0.874), and the Kappa value was 0.781(P<0.05).CONCLUSION: The risk factors associated with the pathogenic condition of diarrhea were identifi ed, quantifi ed and formulated into an ADSS, which has high diagnostic accuracy and reliability for the early identifi cation of patients with severe acute diarrhea.

TREATMENT OF 70 CASES OF ACUTE GASTROENTERITIS WITH ELECTROACUPUNCTURE OF MAIN ACUPOINT NEIGUAN AND GONGSUN

Objective: To observe clinical therapeutic effect of electro-acupuncture (EA) of Neiguan (PC 6), Gongsun (SP 4), etc. in the treatment of acute gastroenteritis. Methods: A total of acute gastroenteritis patients observed were randomized into two groups, i.e. 70 cases in the treatment group receiving EA of main acupoint Neiguan (PC 6) and Gongsun (SP 4) and acupuncture of supplementary acupoint Guanyuan (CV 4), Zusanli (ST 36), Pishu (BL 20) and 50 cases in the control group treated by oral administration of Norfloxacin capsules. Results: Statistically, the therapeutic results of the 2 groups were not significantly different (P>0.05), showing that EA of Neiguan (PC 6) and Gongsun (SP 4) is an effective therapeutic method for acute gastroenteritis.

黄芪建中汤合暖胃汤治疗患儿脾胃虚寒型急性肠胃炎

目的观察黄芪建中汤合暖胃汤治疗脾胃虚寒型急性肠胃炎患儿的疗效。方法选取2021年2月-2023年5月医院收治的74例脾胃虚寒型急性肠胃炎患儿,以随机数字表法分为研究组与对照组,各37例。2组均接受对症治疗,对照组在此基础上采用奥美拉唑肠溶胶囊治疗,研究组在对照组的基础上接受黄芪建中汤合暖胃汤治疗。2组均治疗14 d。对比2组中医证候评分、病症恢复时间、临床疗效、炎症反应、胃肠激素及不良反应。结果治疗后,2组主症评分、次症评分、总评分明显降低(P<0.05),且研究组降低更为明显(P<0.05)。研究组发热、腹痛、呕吐、腹泻等病症恢复时间短于对照组(P<0.05)。研究组总有效率高于对照组(P<0.05)。治疗后,2组白细胞介素-17(IL-17)、基质金属蛋白酶-3(MMP-3)、中性粒细胞与淋巴细胞比值(NLR)水平明显降低(P<0.05),且研究组降低更为明显(P<0.05)。治疗后,2组胃泌素(GSA)水平明显降低(P<0.05),且研究组降低更为明显(P<0.05)。治疗后,2组胃动力素(MTL)水平明显升高(P<0.05),且研究组升高更为明显(P<0.05)。2组不良反应总发生率比较差异无统计学意义(P>0.05)。结论黄芪建中汤合暖胃汤治疗脾胃虚寒型急性肠胃炎患儿疗效确切,可改善患儿临床症状与胃肠功能,促进患儿康复,减轻炎症反应,且安全可靠。

急性腹泻患儿粪便病原微生物检验结果及耐药情况分析

目的探讨急性腹泻患儿粪便病原微生物检验结果及耐药情况。方法选取2021年1月-2023年1月我院收治的167例急性腹泻患儿,采集所有患儿粪便标本送检,开展病原微生物培养与药敏试验。统计病原微生物检检验结果、分析急性病原微生物分布特点、志贺氏菌属、沙门氏菌属的耐药情况。结果167急性腹泻患儿共检出109株病原微生物,其中细菌51株(46.79%)、病毒48株(44.04%)、真菌10株(9.17%);51株细菌(46.79%)以志贺氏菌属(13.76%)、沙门氏菌(22.02%)、大肠埃希菌(4.59%)为主;48株病毒(44.04%),以轮状病毒(22.94%)、诺如病毒(15.60%)为主,真菌10株(9.17%);志贺氏菌属、沙门氏菌属对氨苄西林、哌拉西林、四环素的耐药性偏高,均>50%;对氨苄西林/舒巴坦、阿莫西林/克拉维酸、哌拉西林/他唑巴坦、头孢他啶、头孢吡肟、氯霉素、环丙沙星、左氧氟沙星、氨曲南的耐药性偏低,均﹤50%;对厄他培南、亚胺培南、美罗培南未产生耐药性。结论急性腹泻患儿粪便病原微生物以病毒、志贺氏菌属、沙门氏菌属、大肠埃希菌多见,临床需据此开展针对性治疗,以加快患者病情转归。

小儿肠胃康颗粒联合蒙脱石散剂辅助治疗儿童急性细菌性腹泻病的疗效

目的:探讨小儿肠胃康颗粒联合蒙脱石散剂辅助治疗儿童急性细菌性腹泻病的临床疗效。方法:选取2021年3月至2022年2月我院门诊收治的96例急性细菌性腹泻病患儿按简单随机化分组法分为对照组和联合组各48例。两组患儿均给予对症治疗(依据细菌培养及药敏试验结果选择敏感性抗菌药物、调整饮食结构、纠正电解质紊乱及菌群失调等),在此基础上,对照组给予蒙脱石散剂治疗,联合组在对照组治疗基础上给予小儿肠胃康颗粒,疗程为2周。比较两组患儿胃肠道不适症状改善时间,检测治疗前后两组患儿血清胃肠激素及免疫功能变化情况,统计不良反应发生情况。结果:治疗后,联合组治疗总有效率高于对照组(100.00%vs.91.67%),但差异无统计学意义(P>0.05),联合组疗效等级优于对照组(Z=2.691,P<0.05)。治疗后,两组患儿大便次数较同组治疗前减少(P均<0.01),联合组大便次数较对照组更少(t=5.771,P<0.05),且联合组腹泻、脱水、食欲减退、腹痛、腹胀缓解时间及大便性状改变时间均较对照组更短(P均<0.05)。治疗后,联合组胃动素(MOT)、胃泌素(GAS)水平低于对照组,生长抑素(SS)、P物质(SP)水平高于对照组,差异均有统计学意义(P均<0.05)。治疗后,联合组CD3^(+)、CD4^(+)及CD4^(+)/CD8^(+)水平高于对照组(P均<0.05)。两组患儿不良反应发生率比较差异无统计学意义(χ^(2)=0.546,P=0.460)。结论:小儿肠胃康颗粒联合蒙脱石散剂治疗儿童急性细菌性腹泻病整体价值优于单用蒙脱石散剂治疗,可缩短患儿胃肠道不适症状缓解时间,改善胃肠道动力学及免疫功能,安全性较高。

猪急性腹泻综合征冠状病毒S蛋白多克隆抗体的制备及在检测该病毒感染中的应用

为制备猪急性腹泻综合征冠状病毒(SADS-CoV)纤突蛋白(S)的多克隆抗体(PAb),本研究经PCR扩增SADS-Co V S蛋白S1亚基C端结构域(S1-CTD)基因片段(384 bp),并将其克隆至原核表达载体p GEX-6p-1中,构建重组质粒p GEX-6p-1-S1-CTD,经双酶切和测序鉴定正确后,转化大肠杆菌BL21(DE3)感受态细胞,利用IPTG诱导表达,通过western blot鉴定重组S1-CTD蛋白(rS1-CTD)的表达及反应原性。结果显示,r S1-CTD以包涵体的形式表达,在40 ku处出现特异性条带。诱导表达后的r S1-CTD经不同浓度尿素重悬并超声离心,SDS-PAGE检测后切胶纯化,得到纯化的重组蛋白。利用BCA试剂盒测得蛋白的浓度为33μg/m L。将该重组蛋白乳化后经3次免疫新西兰大白兔,并在3免一周后采血,分离血清获得S1-CTD蛋白PAb。将SADS-Co V感染Vero E6细胞24 h后,以获得的兔PAb为一抗,分别采用western blot和间接免疫荧光试验(IFA)检测该PAb的反应原性。Western blot结果显示,在约250 ku处出现特异性条带,而阴性对照组无该条带;IFA结果显示,SADS-Co V感染的细胞中出现绿色荧光,而阴性对照细胞无绿色荧光。将SADS-Co V感染仔猪的回肠组织制备病理切片,以制备的PAb为一抗,通过免疫组织化学(IHC)检测SADS-Co V的抗原。结果显示,该组织切片中出现棕色阳性信号,而阴性对照仔猪回肠组织切片则无该棕色信号。表明该PAb可与感染SADS-Co V的仔猪回肠组织中的相应抗原发生特异性免疫反应。综上所述,本实验制备的S1-CTD蛋白PAb具有良好的反应原性和免疫原性,可以用于western blot、IFA、IHC检测体内外SADS-Co V的感染,为后续SADS-Co V检测方法的建立及S蛋白生物学功能的研究奠定基础。

PEDV、TGEV与PDCoV S蛋白表位基因三联疫苗的构建及其鉴定

[目的]构建一种同时预防猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪德尔塔冠状病毒(PDCoV)的三联表位疫苗,以预防上述病原导致的猪的相关疾病。[方法]本研究运用免疫信息学在线软件对3种猪肠道冠状病毒(SeCoVs)的S蛋白B、T细胞表位进行预测分析,构建新的表位肽段,命名为PPT,通过ExPASy、VaxiJen、TMHMM、SOPMA、SOLpro和AlphaFlod2等在线软件对PPT进行生物信息学分析,并利用C-IMMSIM在线软件对其免疫反应进行模拟。通过T2A将PPT与PEDV的COE序列、TGEV的SAD序列及PDCoV的CTD序列连接并构建至真核表达载体pEGFP-N1,经PCR和双酶切鉴定正确后,将获得的重组质粒转染HEK293A细胞,经DAPI染色、CCK8、RT-PCR及Western blotting试验验证重组质粒在体外表达情况。[结果]构建的表位蛋白PPT由17条表位肽段组成,经生物信息学软件分析,该蛋白为非跨膜蛋白,结构稳定,具有抗原性和可溶性,亲水性高,无过敏性。C-IMMSIM结果显示,PPT能引起机体树突状细胞(DC)增加,B、T细胞免疫反应,刺激免疫球蛋白IgG、IgM和细胞因子γ-干扰素(IFN-γ)、白细胞介素-2(IL-2)水平升高。重组质粒经PCR和双酶切鉴定结果显示,分别在3359、2375、1064、944、764和467 bp出现特异性目的条带,与预期相符;重组质粒转染HEK293A细胞后,可见绿色荧光;RT-PCR扩增分别在3359、2375、1064、944、764和467 bp处获得与目的基因大小相符的条带;CCK-8检测结果表明,重组质粒对细胞均无明显毒性作用;Western blotting检测结果显示,分别在31.7、16.1、37.9和27.5 ku处出现与目的蛋白分子质量大小一致的条带,重组质粒成功在HEK293A细胞中表达。[结论]本研究基于计算机软件分析设计的PPT表位蛋白成功构建三联疫苗,且在体外表达,为评价PEDV-TGEV-PDCoV三联表位疫苗的免疫效果提供依据。

猪急性腹泻综合征冠状病毒S1蛋白抗体间接ELISA检测方法的建立

为建立猪急性腹泻综合征冠状病毒(SADS-CoV)S1蛋白抗体的间接ELISA检测方法,本研究通过构建重组真核分泌型表达质粒p CAGGS-S1-His并转染HEK293F悬浮细胞进行可溶性表达S1蛋白。转染重组质粒5 d后的细胞上清利用HisTrapTMExcel亲和层析柱纯化,浓缩后的蛋白经SDS-PAGE、western blot鉴定显示获得了分子量约为130 ku且大于理论大小的重组S1蛋白,表明该蛋白为分泌表达且具有良好的翻译后修饰。利用获得的重组S1蛋白作为包被抗原,通过优化反应条件,初步建立了SADS-CoV S1蛋白抗体的间接ELISA检测方法。该方法与猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪德尔塔冠状病毒(PDCoV)、猪轮状病毒(PoRV)和猪圆环病毒2型(PCV2)阳性血清均无交叉反应,仅与SADS-CoV阳性血清反应,表明该方法特异性较强;该方法检测SADS-CoV阳性血清,当稀释至1:3 200时仍为阳性,表明该方法具有较高的敏感性;对4份阳性血清的批内和批间重复性试验的变异系数均小于10%,表明该方法具有良好的重复性和稳定性;利用该方法和间接免疫荧光试验(IFA)对50份临床样品检测,结果显示该间接ELISA检测到9份阳性样品,41份阴性样品;IFA检出5份阳性样品,45份阴性样品,二者的总符合率为92%。综上表明,本研究建立的间接ELISA检测方法能够特异、灵敏地检测SADS-CoV S1蛋白抗体,可以用于SADS-CoV灭活疫苗的免疫效果评价以及该病原的血清学流行病学调查和免疫水平监测,为SADS-CoV所致疫病的鉴别诊断和防控奠定基础。