背景:新生儿和断奶后腹泻是由产肠毒性大肠杆菌菌毛F4(F4+ETEC)在猪场引起的关注疾病。腹泻爆发通常是严重和昂贵的,因为疾病在同一群体中的高流行和传播。自体疫苗是保护猪免受F4+ETEC感染的战略解决方案之一。在目前的研究中,我们在妊娠母猪和保育猪中进行了两项使用粗制F4菌毛提取物疫苗接种的试验。
方法:在实验1中(20头母猪;未接种疫苗的对照,n=10),我们在分娩前4周和2周接种了两次妊娠母猪(n=10),并评估了疫苗接种对母体免疫的影响。接种前收集母猪血清和初乳,接种疫苗后2周和4周,分娩后6小时,分别,和来自两组的仔猪血清(2只仔猪/母猪,每组10只仔猪)也在3日龄时收集以测量F4特异性IgG,F4特异性IgA使用内部ELISA试剂盒。在实验2中,优化仔猪候选疫苗的剂量和剂量,将18头仔猪(3头仔猪/组)分为5个免疫组和1个对照组(未免疫组),我们在4周龄和6周龄以不同剂量免疫仔猪两次(即,0、50、100、150、200µg),剂量为150微克,我们用1ml和2ml两种剂量免疫。用3ml剂量的3×109CFU/ml产肠毒素大肠杆菌(F4ETEC)细菌培养物攻击仔猪,以评估疫苗的功效。挑战之后,每天观察仔猪的临床症状,每天进行直肠拭子检查,使用PCR技术研究大肠杆菌(F4ETEC)的粪便脱落。之前收集血清,接种疫苗后2周和4周以及攻击后1周测量F4特异性IgG,使用内部ELISA试剂盒和细胞因子水平的F4特异性IgA(即,IL-1β,IL-6,IL-8和TNFα)在攻击前和攻击后1周使用商业ELISA试剂盒。
结果:抗体水平结果显示,实验1中,接种母猪血清和初乳中F4特异性IgG和F4特异性IgA的抗体水平在接种疫苗后均显著升高。免疫母猪的仔猪血清中F4特异性IgG和F4特异性IgA的抗体水平均明显高于未免疫母猪的仔猪(p<0.01)。在实验2中,无论不同的剂量和剂量,免疫组之间F4特异性IgG和F4特异性IgA水平无差异。然而,与未接种疫苗组(>5天)相比,所有接种疫苗的仔猪在攻击后表现出更高的F4特异性IgG和F4特异性IgA水平,并且在粪便中消除大肠杆菌(F4+ETEC)更快(<3天)。对于细胞因子水平,更高水平的IL-1β,与未接种组的水平相比,发现接种组的攻击后1周的IL-6、IL-8和TNFα。
结论:我们的结果表明,粗制F4菌毛提取物自体疫苗是保护仔猪免受产肠毒性大肠杆菌(F4ETEC)引起的腹泻病的候选疫苗,并且在分娩前两次对孕母猪进行疫苗接种是为新生仔猪提供母源抗体以抵抗早期产肠毒性大肠杆菌(F4ETEC)的策略之一。
BACKGROUND: Neonatal and post-weaning diarrhea is a concern disease caused by enterotoxigenic Escherichia coli fimbriae F4 (F4+ETEC) in pig farms. Diarrhea outbreaks are often severe and costly due to the high prevalence and spread of the disease within the same herd. Vaccine is one of strategic solution in protecting pig against F4+ETEC infection in particular pig farm. In present study, we conducted two trials of vaccination with crude F4 fimbriae extract vaccine in pregnant sow and nursery pigs.
METHODS: In experiment 1 (20 sows; non-vaccinated control, n=10), we vaccinated pregnant sows (n=10) twice at 4 wk and 2 wk before farrowing and evaluated impact of vaccination on maternal immunity. The sow serum and colostrum were collected before vaccination, 2 and 4 weeks after vaccination, 6 hours after farrowing, respectively, and the piglet\'s serum from both groups (2 piglet/sow, 10 piglets from each group) were also collected on 3 days old to measure F4 specific IgG, F4 specific IgA using in house ELISA kit. In experiment 2, to optimize doses and dosage of candidate vaccine in piglets, 18 piglets (3 piglets/group) were allocated into five immunized groups and one control group (unimmunized group), we immunized piglets twice at 4 and 6 weeks old with difference doses (i.e., 0, 50, 100, 150, 200 µg), and for a dose 150 µg, we immunized with two dosages at 1 ml and 2 ml. Piglets were challenged with a 3 ml dose of 3 × 109 CFU/ml bacterial culture of enterotoxigenic Escherichia coli (F4+ETEC) in order to evaluate the efficacy of vaccine. After challenging, the clinical sign of the piglets was daily observed and the rectal swab was performed every day for investigation of the fecal shedding of Escherichia coli (F4+ETEC) by using PCR technique. Serum were collected before, 2 and 4 weeks after vaccination and 1 week after challenge to measure F4 specific IgG, F4 specific IgA using in house ELISA kit and cytokines levels (i.e., IL-1 beta, IL-6, IL-8 and TNF alpha) before and 1 week after challenge using commercial ELISA kit.
RESULTS: The levels of antibody results showed that in experiment 1, the anti-F4 antibody levels both F4 specific IgG and F4 specific IgA in serum and colostrum of vaccinated sow increased significantly after vaccination. The piglets of immunized sows have antibody level both F4 specific IgG and F4 specific IgA in their serum higher than those piglets of unimmunized sows significantly (p < 0.01). In experiment 2, irrespective of different doses and dosage, there is no difference in term of F4 specific IgG and F4 specific IgA levels among immunized groups. However, all of vaccinated piglets showed F4 specific IgG and F4 specific IgA levels higher and the elimination of Escherichia coli (F4+ETEC) in feces post challenge faster (< 3 days) than unvaccinated group (> 5 days). For cytokines levels, a higher level of IL-1 beta, IL-6, IL-8 and TNF alpha at 1 week after challenge in vaccinated groups was found when compared with the levels in non-vaccinated group.
CONCLUSIONS: Our results suggest that crude F4 fimbriae extract autogenous vaccine is a candidate vaccine for protecting piglets against diarrhea disease caused by enterotoxigenic Escherichia coli (F4+ETEC) and vaccination the pregnant sow twice before farrowing is one of strategies to provide maternal derived antibody to the newborn piglets for against enterotoxigenic Escherichia coli (F4+ETEC) during early life.