Effects of dietary supplementation of bacteriophages against enterotoxigenic Escherichia coli (ETEC) K88 on clinical symptoms of post‐weaning pigs challenged with the ETEC pathogen

The present study was performed to investigate the effects of dietary supplementation of bacteriophages (phages) against enterotoxigenic Escherichia coli (ETEC) K88 as a therapy against the ETEC infection in post‐weaning pigs. Two groups of post‐weaning pigs aged 35 days, eight animals per group, were challenged with 3.0 × 10¹⁰ colony forming units of ETEC K88, a third group given the vehicle. The unchallenged group and one challenged group were fed a basal nursery diet for 14 days while the remaining challenged group was fed the basal diet supplemented with 1.0 × 10⁷ plaque forming units of the phage per kg. Average daily gain (ADG), goblet cell density and villous height:crypt depth (VH:CD) ratio in the intestine were less in the challenged group than in the unchallenged group within the animals fed the basal diet (p < 0.05); the reverse was true for rectal temperature, faecal consistency score (FCS), E. coli adhesion score (EAS) in the intestine, serum interleukin‐8 (IL‐8) and tumour necrosis factor‐α (TNF‐α) concentrations and digesta pH in the stomach, caecum and colon. The ETEC infection symptom within the challenged animals was alleviated by the dietary phage supplementation (p < 0.05) in ADG, FCS, EAS in the jejunum, serum TNF‐α concentration, digesta pH in the colon, goblet cell density in the ileum and colon and VH:CD ratio in the ileum. Moreover, the infection symptom tended to be alleviated (p < 0.10) by the phage supplementation in rectal temperature, EAS in the ileum and caecum, and VH:CD ratio in the duodenum and jejunum. However, EAS in the colon, digesta pH in the stomach and caecum, and goblet cell density in the jejunum did not change due to the dietary phage. Overall, results indicate that the phage therapy is effective for alleviation of acute ETEC K88 infection in post‐weaning pigs.     

Early supplementation with Lactobacillus plantarum in liquid diet modulates intestinal innate immunity through toll-like receptor 4-mediated mitogen-activated protein kinase signaling pathways in young piglets challenged with Escherichia coli K88

This study was conducted to investigate the effects of early supplementation during 4 to 18 d of age with Lactobacillus plantarum (LP) in liquid diets on intestinal innate immune response in young piglets infected with enterotoxigenic Escherichia coli (ETEC) K88. Seventy-two barrow piglets at 4 d old were assigned to basal or LP-supplemented liquid diet (5 × 10¹⁰ CFU·kg⁻¹). On day 15, piglets from each group were orally challenged with either ETEC K88 (1 × 10⁸ CFU·kg⁻¹) or the same amount of phosphate-buffered saline. The intestinal mucosa, mesenteric lymph node (MLN), and spleen samples were collected on day 18. Here, we found that LP pretreatment significantly decreased the mRNA relative expression of inflammatory cytokines (interleukin [IL]-1β, IL-8, and tumor necrosis factor-α), porcine β-defensin 2 (pBD-2), and mucins (MUC1 and MUC4) in the jejunal mucosa in piglets challenged with ETEC K88 (P < 0.05). Moreover, LP significantly decreased the ileal mucosa mRNA relative expression of IL-8 and MUC4 in young piglets challenged with ETEC K88 (P < 0.05). Furthermore, the piglets of the LP + ETEC K88 group had lower protein levels of IL-8, secretory immunoglobulin A, pBD-2, and MUC4 in the jejunal mucosa than those challenged with ETEC K88 (P < 0.05). Besides, LP supplementation reduced the percentage of gamma/delta T cells receptor (γδTCR) and CD172a⁺ (SWC3⁺) cells in MLN and the percentage of γδTCR cells in the spleen of young piglets after the ETEC K88 challenge. Supplementation with LP in liquid diets prevented the upregulated protein abundance of toll-like receptor (TLR) 4, phosphorylation-p38, and phosphorylation-extracellular signal-regulated protein kinases in the jejunal mucosa induced by ETEC K88 (P < 0.05). In conclusion, LP supplementation in liquid diet possesses anti-inflammatory activity and modulates the intestinal innate immunity during the early life of young piglets challenged with ETEC K88, which might be attributed to the suppression of TLR4-mediated mitogen-activated protein kinase signaling pathways. Early supplementation with LP in liquid diets regulates the innate immune response, representing a promising immunoregulation strategy for maintaining intestinal health in weaned piglets.     

产肠毒素大肠杆菌双重PCR检测方法的建立

为了快速检测和鉴定产肠毒素大肠杆菌菌毛(K88和K99)基因,本研究设计合成了针对K88、K99的2对特异性引物,对扩增条件进行优化,建立了检测K88和K99的双重PCR方法。该方法对K88、K99基因的扩增产物大小分别为237和314 bp;最终确定dNTP终浓度0.4 mmol/L,K88、K99的引物终浓度均为25 μmol/L,退火温度为52℃。试验结果表明,该方法具有良好的灵敏性和特异性。用所建立的双重PCR方法对实验室分离的23株大肠杆菌进行检测,结果显示,K88单重PCR阳性2株,K99单重PCR阳性3株,K88和K99双重PCR阳性5株。本研究建立的双重PCR检测方法为致幼畜腹泻产肠毒素大肠杆菌的快速准确检测提供了方法。     

硒代蛋氨酸对断奶仔猪生长性能、抗氧化及免疫性能的影响

文章旨在研究日粮添加有机硒(硒代蛋氨酸)对断奶仔猪生长性能、血清抗氧化和免疫性能的影响。试验选择26 d断奶的杜×长×大仔猪1080头[平均体重(9.82±1.18)kg],随机分为6组,每组4个重复,每个重复45头。对照组饲喂基础日粮(硒含量为0.03 mg/kg),无机组饲喂在基础日粮中添加0.4 mg/kg硒(亚硒酸钠),有机硒组在基础日粮中分别添加0.2、0.4、0.6和0.8 mg/kg硒(硒代蛋氨酸),试验共进行42 d。结果显示:日粮添加0.4和0.8 mg/kg有机硒较对照组显著降低了断奶仔猪22～42 d的料重比(P <0.05)。0.4 mg/kg有机硒组较对照组和0.4 mg/kg无机硒组显著提高了血清GSH-Px和T-AOC活力(P <0.05)。与对照组相比,0.2和0.4 mg/kg有机硒组显著提高了肝脏GSH-Px活力(P <0.05),而0.2 mg/kg有机组肝脏T-AOC活力最高(P <0.05)。与对照组相比,无机硒组和有机硒组显著降低了肝脏和肌肉MDA含量(P <0.05)。与对照组相比,无机硒组和有机硒组显著降低了肝脏MDA含量(P <0.05)。0.6 mg/kg有机硒组较对照组、0.4 mg/kg无机硒组和0.2、0.4 mg/kg有机硒组显著提高了断奶仔猪21 d血清硒含量(P <0.05);与对照组相比,0.4和0.6 mg/kg有机硒组显著提高了仔猪42 d血清硒含量(P <0.05)。0.8 mg/kg有机硒组较对照组和0.4 mg/kg无机硒组显著提高了仔猪42 d血清硒含量(P <0.05)。结论 :日粮添加硒代蛋氨酸可以改善断奶仔猪的生长性能,提高其抗氧化性能和血清硒含量,断奶仔猪适宜的硒代蛋氨酸添加水平为0.2～0.4 mg/kg(以硒计)。     

猪K88、K99、987P、F41埃希氏大肠杆菌高密度发酵培养技术的初步探讨

采用高密度发酵和普通深层通气两种方法培养含K88、K99、987P、F41菌毛抗原的四株猪埃希氏大肠杆菌,对其培养液进行活菌数、OD值、pH值、效价的测定。实验结果表明,运用高密度发酵方法培养,各菌活菌数可达4.1×1010~4.9×1010CFU/mL,效价为211~213;运用普通深层通气方法培养,各菌活菌数为0.51×1010~0.59×1010CFU/mL,效价为24~25,可选择高密度发酵方法替代普通深层通气方法培养,用于制备猪埃希氏大肠杆菌K88、K99、987P、F41四价菌毛提纯苗。     

表达大肠杆菌K88ac-ST1-LTB融合蛋白工程菌株的免疫原性研究

已构建的能表达大肠杆菌K88ac-ST1-LTB融合蛋白的工程菌株BL21（DE3）（pXKST3LT5)及其表达产物经动物试验证实没有毒性反应。用从IPTG诱导的工程菌中提取的包涵体或经甲醛灭活的工程菌制成抗原，免疫小鼠，结果免疫小鼠至少能抵抗2MLD的大肠杆菌强毒株C83902（K88ac,ST^ ,L^ )的攻击，用提取的包涵体免疫家兔后，采集的血清能够中和天然ST1的毒性，这表明构建的工程菌株BL21（DE3）（pXKST3LT5)可以作为预防幼畜大肠杆菌性腹泻基因工程菌苗的候选株。     

Effects of dietary supplementation of lipid‐encapsulated zinc oxide on colibacillosis,growth and intestinal morphology in weaned piglets challenged with enterotoxigenic Escherichia coli

This study was aimed to evaluate the effect of dietary supplementation of lipid‐encapsulated (coated) zinc oxide ZnO on post‐weaning diarrhea (colibacillosis) in weaned piglets challenged with enterotoxigenic Escherichia coli (ETEC). Thirty‐two 35‐day‐old weaned piglets were orally challenged with 3 × 10¹⁰ colony forming units of ETEC K88 while eight piglets received no challenge (control). Each eight challenged piglets received a diet containing 100 ppm ZnO (low ZnO), 2500 ppm ZnO (high ZnO) or 100 ppm of lipid (10%)‐coated ZnO (coated ZnO) for 7 days; control pigs received the low ZnO diet. Daily gain, goblet cell density in the villi of the duodenum, jejunum and ileum, and villus height in the jejunum and ileum, which decreased due to the challenge, were equally greater in the coated ZnO and high ZnO groups versus low ZnO group. Fecal consistency score, serum interleukin‐8 concentration, subjective score of fecal E. coli shedding, and digesta pH in the stomach, jejunum and ileum, which increased due to the challenge, were equally low in the coated ZnO and high ZnO groups versus low ZnO. Results suggest that a low level of coated ZnO might well substitute for a pharmacological level of native ZnO in dietary supplementation to alleviate colibacillosis of weaned piglets.     

The preventive effect of Bacillus subtilus strain DB9011 against experimental infection with enterotoxcemic Escherichia coli in weaning piglets

Porcine edema disease (ED) is caused by Shiga toxin 2e‐producing Escherichia coli (STEC). Post‐weaned piglets often suffer from ED as a result of intestinal infection with STEC, which causes impaired growth performance and high mortality. Antimicrobial therapy is a curative treatment for piglets infected with STEC, but the emergence of antimicrobial‐resistant STEC has become a serious problem for Japanese pig farmers. Therefore, an alternative strategy other than antimicrobial therapy is needed for the prevention or treatment of ED. In this study, we evaluated the effect of oral administration of Bacillus subtilis DB9011 (DB9011) to prevent the experimental infection of STEC in weaning piglets. Eight 21‐day‐old piglets were divided into two groups: STEC challenge with the basal diet, and STEC challenge with DB9011 supplemented diet. The challenge was carried out when the animals were 25, 26 and 27 days old using STEC contained in capsules resistant against gastric digestion. All pigs were euthanized at 36 days of age. DB9011 improved the symptoms of ED and decreased the number of STEC in the ileal digesta and feces. Accordingly, oral administration of DB9011 in weaned piglets prevents ED through the suppression of the growth of STEC in the ileum.     

A between‐experiment analysis of relationships linking dietary protein intake and post‐weaning diarrhea in weanling pigs under conditions of experimental infection with an enterotoxigenic strain of Escherichia coli

Numerous experiments have demonstrated that feeding a lower protein diet decreases protein fermentation in the gastrointestinal tract (GIT) and reduces the incidence of post‐weaning diarrhea (PWD). However, there is a lack of holistic evidence underpinning the relationship between feeding a lower protein diet and PWD in relation to physiological responses and protein fermentation in the GIT. The scope of this article, therefore, will: (i) focus on the impact of dietary protein levels on selected indices of GIT health in weaned pigs without and with experimental infection with an enterotoxigenic strain of Escherichia coli; and (ii) attempt to conduct regression analysis to examine the relationships between dietary‐origin protein intake, nitrogen fermentation indices, fecal consistency and the incidence of PWD. We used datasets generated from a series of four intensive experiments in weaned pigs. The collective results derived from these datasets indicate that restriction of daily protein intake to less than 60 g through feeding a lower protein diet for as little as 7 days after weaning reduced the incidence of PWD commensurate with a reduction in protein fermentation indices.     

Effects of dietary supplementation of probiotic,Clostridium butyricum,on growth performance,immune response,intestinal barrier function,and digestive enzyme activity in broiler chickens challenged with Escherichia coli K88

Background:Colibacillosis caused by enterotoxigenic Escherichia coli(E.coli) results in economic losses in the poultry industry.Antibiotics are usually used to control colibacillosis,however,E.coli has varying degrees of resistance to different antibiotics.Therefore the use of probiotics is becoming accepted as an alternative to antibiotics.In this study,we evaluated the effects of Clostridium butyricum(C.butyricum) on growth performance,immune response,intestinal barrier function,and digestive enzyme activity in broiler chickens challenged with Escherichia coli(E.coif) K88.Methods:The chickens were randomly divided into four treatment groups for 28 days.Negative control treatment(NC) consisted of birds fed a basal diet without E.coli K88 challenge and positive control treatment(PC) consisted of birds fed a basal diet and challenged with E.coli K88.C.butyricum probiotic treatment(CB) consisted of birds fed a diet containing 2×10~7 cfu C butyricum/kg of diet and challenged with E.coli K88.Colistin sulfate antibiotic treatment(CS)consisted of birds fed a diet containing 20 mg colistin sulfate/kg of diet and challenged with E.coli K88.Results:The body weight(BW) and average day gain(ADG) in the broilers of CB group were higher(P 0.05) than the broilers in the PC group overall except the ADG in the 14-21 d post-challenge.The birds in CB treatment had higher(P 0.05) concentration of tumor necrosis factor-α(TNF-α) at 3 and 7 d post-challenge,and higher(P 0.05)concentration of interleukin-4(IL-4) at 14 d post-challenge than those in the PC treatment group.The concentration of serum endotoxin in CB birds was lower(P0.05) at 21 d post-challenge,and the concentrations of serum diamine oxidase in CB birds were lower(P 0.05) at 14 and 21 d post-challenge than in PC birds.Birds in CB treatment group had higher(P 0.05) jejunum villi height than those in PC,NC,or CS treatment at 7,14,and 21 d post-challenge.In comparison to PC birds,the CB birds had lower(P 0.05) jejunum crypt depth during the whole experiment.The birds in CB or CS treatment group had higher(P 0.05) activities of amylase and protease at 3,7,and 14 d post-challenge,and higher(P 0.05) activity of lipase at 3,7 d post-challenge than PC birds.Conclusions:In all,these results indicate that dietary supplementation with C.butyricum promotes immune response,improves intestinal barrier function,and digestive enzyme activities in broiler chickens challenged with E.coli K88.There is no significant difference between the C.butyricum probiotic treatment and the colistin sulfate antibiotic treatment.Therefore,the C.butyricum probiotic may be an alternative to antibiotic for broiler chickens.