免疫初乳中乳抗体对大肠杆菌和沙门氏菌所致小鼠腹泻的被动免疫保护

以24株人肠道病原菌(包括病原性大肠杆菌12株、沙门氏菌8株、志贺氏菌3株、小肠结肠炎耶尔森氏菌1株)作为抗原，对乳牛进行系统免疫，免疫乳与非免疫乳乳中IgG含量无显著差异。系统免疫并不增加乳中IgG的含量，但IgG的抗体特异性大大增强，所得的免疫初乳中乳抗体对24种不同病原菌的凝集价为2^8-2^12，为普通初乳中乳抗体凝集价的32—256倍。采用硫酸胺盐析从免疫初乳中分离乳抗体，免疫初乳中的特异性IgG乳抗体较普通初乳中的IgG可显著抑制大肠杆菌和沙门氏菌的生长。动物试验表明，免疫乳中特异性的IgG对大肠杆菌和沙门氏菌所致小鼠腹泻具有很好的保护作用，而普通乳中非特异性的IgG则无此作用，这是由于IgG的特异性所决定。     

含24种乳抗体免疫乳的制备

以24株人肠道病原菌（包括病原性大肠杆菌12株、沙门氏菌8株、志贺氏菌3株、小肠结肠炎耶尔森氏菌1株）作为抗原，对乳牛进行系统免疫，免疫乳与非免疫乳乳中IgG含量无显著差异。系统免疫并不增加乳中IgG的含量，但IgG的抗体特异性大大增强，所得的免疫初乳中乳抗体对24种不同病原菌的凝集价为2^8-2^12，为普通初乳中乳抗体凝集价的32－256倍；免疫常乳中乳抗体对24种不同病原菌的凝集价为2^5-2^8，为普通乳中乳抗体凝集价的8－128倍。     

重放异彩的免疫乳

早在1892年,“免疫之父”德国学者伊莱恩首先提出免疫乳理论,并同时使用了“免疫乳”这个名词。所谓免疫乳,是给牛等哺乳动物定期接种一些能引起人类疾病的病原体,或者其他一些外来抗原,以此来刺激动物机体发牛免疫应答,而使其分泌出的乳汁含有特异性抗体（即针对某一细菌或病毒的抗体）,这种含有特异性抗体的乳汁即为免疫乳。     

免疫乳对肠道菌群的调节作用

以24种人肠道病原茵作为抗原对乳牛进行免疫，制得的免疫乳中乳抗体的凝集价为非免疫乳的64倍。以免疫乳作为原料制备的免疫乳粉具有调节肠道茵群的作用。结果表明免疫乳粉较普通乳粉可显著增加小鼠肠道中双歧杆茵和乳杆茵的数量以及肠道中乳酸和已酸的含量，同时可显著降低小鼠肠道中肠球茵、大肠杆茵及产气荚膜梭茵的数量。     

免疫婴儿乳粉的安全毒理学评定

以24株人肠道病原菌(包括病原性大肠杆菌12株、沙门氏菌8株、志贺氏菌3株、小肠结肠炎耶尔森氏菌1株)作为抗原,对乳牛进行系统免疫,免疫乳与非免疫乳乳中IgG含量无显著差异。系统免疫并不增加乳中IgG的含量,但IgG的抗体特异性大大增强,所得的免疫初乳中乳抗体对24种不同病原菌的凝集价为28～212,为普通初乳中乳抗体凝集价的32～256倍。以免疫初乳作为原料制备免疫初乳粉并添加到婴儿乳粉中制成免疫婴儿乳粉。婴儿免疫乳粉LD50>10g/kg;Ames试验及小鼠骨髓微核试验和小鼠精子畸变试验表明婴儿免疫乳粉无致畸变作用。大鼠30d喂养试验表明,婴儿免疫乳粉对大鼠生长无不良影响,大鼠血液指标正常,病理学检查未见任何病变。     

免疫接种次数对免疫乳中特异性抗体效价的影响

研究了免疫接种次数对免疫乳中特异性抗体效价的影响。以9株人肠道病原菌制备的混合抗原对孕牛进行系统免疫,其中对照组免疫接种4次,实验组免疫接种6次,分娩后收集乳样,检测其中特异性抗体的效价。结果表明,实验组和对照组初乳中抗体效价无统计学意义,均达到210～212;而常乳中特异性抗体效价有统计学意义:对照组效价平均为25～26,实验组效价平均为26～27,并且随着时间的推移差异越来越显著。结论:选择好免疫接种的间隔时间,并进行多次接种,能使免疫乳中特异性抗体效价在长时间内保持较高水平。     

关于免疫乳与初乳

本文综述了免疫乳和初乳的发展、制备和作用,分析了免疫乳和初乳存在的问题,提出免疫乳与牛初乳只能作为增强肠道局部免疫的保健食品,但对预防肠道传染病的时效性提出质疑,并指出长期服用可能会产生抗抗体的论点。     

抗人轮状病毒和大肠杆菌免疫乳持续性的研究

以引起婴儿腹泻的轮状病毒（Ｒｏｔａｖｉｒｕｓ,Ｒ．Ｖ）和大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ,Ｅ．ｃｏｌｉ）给妊娠后期的奶牛免疫,使之产生抗这两种病原的抗体,定期采乳,分别用试管凝集反应和反向间接血凝抑制试验检验大肠杆菌和轮状病毒抗体,并摸索其消长规律。试验结果表明：乳中轮状病毒和大肠杆菌高免抗体可持续近两个月,强化免疫,又可得以高免抗体,并能持续近一个月。     

乳矿物盐复合粉免疫调节作用的研究

研究乳矿物盐复合粉对小鼠免疫功能的调节作用。分别以0.375、0.75、2.25 g/kg·bw剂量的乳矿物盐复合粉对小鼠连续灌胃30 d后,测定各项免疫指标。乳矿物盐复合粉能促进小鼠淋巴细胞增殖转化作用,提高小鼠的抗体生成细胞及血清溶血素水平,促进小鼠的单核-巨噬细胞的吞噬功能。乳矿物盐复合粉具有增强免疫力功能作用。     

牛乳铁蛋白的酶联免疫吸附分析(ELISA)方法的建立

用牛乳铁蛋白(Bovine Laetoferrin，BLf)、纯品免疫新西兰白兔制得兔抗BLf的多克隆抗体，琼扩效价最高为l：64。经ELlSA方法检测，该抗体与牛乳中的主要蛋白质：α—乳清蛋白、β—乳球蛋白、血清白蛋白以及酪蛋白之间不存在免疫交叉性，说明抗体特异性良好。以BLf纯品为包被抗原，自制抗体为一抗，辣根过氧化物酶(HRP)标记的羊抗兔IgG为二抗，邻苯二胺(OPD)为底物，建立了BLf的竞争抑制性ELISA方法。包被抗原及抗体的浓度均由方阵稀释实验确定。所建立的ELlSA方法孔间误差1．6％，板间误差4．3％，灵敏度25μg／L，检测范围25～800μg／L。标缝曲线线性方程（Logit）Y=-1.488IgC 3.8116，相关系数R=0.990。     

免疫乳的研究与开发利用

免疫乳是一种新概念天然保健食品。本文对免疫乳和与其相关的初乳的概念、主要生理活性成份、生理作用以及国内外研究开发历史和现状进行了综述。93年起，我国一些科研工作者开始对免疫乳及其制品进行了较为深入的研究和中试开发。目前国内已有普通初乳产品，但尚无真正意义上的免疫乳制品生产。认为免疫乳及其制品具有很大的发展前景。     

Relationship Between Oligosaccharides and Glycoconjugates Content in Human Milk and the Development of the Gut Barrier

The intestinal immune barrier is considered to be the gatekeeper of the human body and rapidly develops directly after birth. Many pre‐ and postnatal factors influence the development of the gut‐barrier, which is composed of the microbiota, the mucus, the epithelial layer and the mucosal immune system. Even minor disturbances during barrier development can have consequences for health far into adulthood. Here we critically discuss the current knowledge on which pre‐ and postnatal factors influence development, maturation, and maintenance of the gut immune barrier. Human milk has a unique composition and is the gold standard for adequate development of the intestinal immune barrier. Not only the influence of human milk oligosaccharides (HMOs) but also that of glycoproteins (HMGPs) is reviewed. We discuss the influence of maternal genetic factors, such as the secretor and Lewis phenotypes on breast milk fucosylation and sialylation of HMOs and HMGPs. This diversity in HMOs and HMPGs influences microbiota composition and also the development of the immune barrier. Cow milk‐derived infant formula is often being used as an alternative for human breast milk. The consequences of this for proper development of the intestinal immune barrier and, in particular, the differences in the type of oligosaccharides and glycosylation patterns (sialic and fucose composition) between cow and human milk are critically discussed. Current and prospective strategies to promote proper gut‐immune maturation are proposed. These might include more personalized infant formulas when breast milk is not an option.     

Metataxonomic and immunological analysis of milk from ewes with or without a history of mastitis

Mastitis is a highly prevalent condition that has a great impact on milk production and animal welfare, and often requires substantial management efforts. For this reason, it is generally considered an important threat to the dairy industry. Many microbial, host, and environmental factors can protect against, predispose to, or influence the development of mastitis. The objective of this work was to characterize the milk microbiota of Manchega ewes, and to compare samples from animals with and without a history of mastitis. We analyzed milk samples from 36 ewes belonging to 2 different farms (18 ewes from each farm) using culture-dependent and culture-independent techniques. We also analyzed several immune compounds to investigate associations of mastitis with 3 main variables: farm; history of mastitis or no mastitis; and parity number. Both culture-dependent and culture-independent techniques showed that ewe milk harbored a site-specific complex microbiota and microbiome. Staphylococcus epidermidis was the main species driving the difference between farm A (where it was the dominant species) and B (where it was not). In contrast, samples from farm B were characterized by the presence of a wide spectrum of other coagulase-negative staphylococci. Some of these species have already been associated with subclinical intramammary infections in ruminants. Of the 10 immune compounds assayed in this study, 3 were related to a history of mastitis [IL-8, IFN-γ, and IFN-gamma-induced protein 10 (IP-10)]. Increases in IL-8 concentrations in milk seemed to be a feature of subclinical mastitis in sheep, and in this study, this immune factor was detected only in samples from ewes with some episodes of mastitis and from the group with the highest somatic cell count. We also observed a positive correlation between the samples with the highest somatic cell count and IFN-γ and IP-10 levels. Our results suggest that these 3 compounds could be used as biomarkers for the negative selection of mastitis-prone animals, particularly when somatic cell count is very high.     

Milk immunoglobulins for health promotion

The biological function of bovine colostral immunoglobulins is to provide the newborn calf with adequate passive immune protection against microbial infections. Immunoglobulin preparations designed for farm animals are commercially available, and some colostrum-based products are marketed also for humans as dietary supplements. The concentration of specific antibodies against a certain pathogenic microorganism can be raised in colostrum and milk by immunizing cows with this pathogen or its antigen. Advances in bioseparation and chromatographic techniques have made it possible to fractionate and enrich these antibodies and formulate so-called hyperimmune colostral or milk preparations. Their efficacy in prevention and treatment of various microbial infections has been evaluated in numerous studies. Immune milk preparations have proven effective in prophylaxis against infections caused by a variety of gastrointestinal pathogens. Their therapeutic efficacy, however, seems more limited. A few commercial immune milk products are already on market and more applications can be expected in the coming years. This article reviews the recent progress made in isolation techniques of bovine immunoglobulins and the application of colostral and immune milk preparations in fighting various microbial infectious diseases in humans.     

免疫耐受在牛乳过敏中的研究进展

食物过敏是机体对过敏原产生的一种不良免疫反应,而免疫耐受是指机体对摄入的食物抗原不产生此类免疫应答。近年来,牛乳过敏的发生率越来越高,大部分牛乳过敏的儿童3岁之后出现了免疫耐受。基于此,介绍了牛乳过敏及引发免疫耐受的相关免疫细胞包括树突状细胞、调节性B细胞、调节性T细胞在免疫耐受中的作用及3种诱导免疫耐受的方法。     

Immune modulation in suckling rat pups by a growth factor extract derived from milk whey

Oral tolerance to foreign enteral antigens is not fully developed in early neonatal life. Epidemiological evidence supports a role for maternal milk in the development of immune responses, including oral tolerance. Formula fed infants have an increased susceptibility to food allergy and the later development of autoimmune disease. This may relate to the lack in infant formula of growth factors found in maternal milk. Bovine milk contains proteins, growth factors and cytokines. Various studies have outlined the immune modulating potential of bovine milk-derived products. Fractionated whey extracts have therapeutic potential in disease states where there is an excessive inflammatory reaction, and disease preventive potential for infants who are not breast-fed. We have shown that daily oral administration of a growth factor-enriched fraction from milk whey to naturally suckling rat pups between days 4-9 postnatal can down-regulate immune activation to a specific orally administered food antigen, ovalbumin, assessed by lymphocyte proliferation. In addition, non-specific down-regulation in the intestine was observed as assessed by the expression of MHC I. Treatment of rat pups with whey extract at the time of oral sensitisation to ovalbumin also resulted in an increased secretion of TGF-beta into the culture supernatant of spleen cells incubated with specific antigen. TGF-beta is an immuno-down-regulatory cytokine involved in tolerance induction. Immune modulation by extracts derived from milk whey could be of potential benefit for formula-fed and pre-term infants in reducing susceptibility to inappropriate activation to food antigens.     

Invited review: effects of milk ration on solid feed intake, weaning, and performance in dairy heifers

A feeding regimen that allows a smooth transition from milk to solid feed is vital for successful heifer-rearing programs. In the past, research efforts have focused on the development of feeding methods that allow early weaning, perhaps because the risk of disease is highest during the milk feeding stage. To encourage early intake of calf starter, conventional feeding programs have limited the supply of milk (often to 10% of BW at birth). However, dairy calves provided free access to milk will typically consume more than twice this amount. We critically review the available literature examining the relationship between milk feeding method, solid feed consumption, and rumen development in young dairy calves and identify areas where new work is required. We conclude that milk-fed dairy calves can safely ingest milk at approximately 20% of body weight (BW)/d, and greater milk consumption supports greater BW gain, improved feed efficiency, reduced incidence of disease, and greater opportunity to express natural behaviors, which in combination suggest improved welfare. Method of weaning greatly influences feed consumption, rumen development, and growth check in calves provided higher amounts of milk. Gradual weaning encourages starter intake during the preweaning period, and both weaning age and duration of weaning influence this consumption. Increased solid feed consumption during the weaning process contributes to rumen development, permitting higher starter intake and BW gain after weaning. Growth factors in milk may also enhance the growth and maturation of the gastrointestinal tract, but more research is required to understand the role of these factors. Greater nutrient supply through increased amount of milk appears to improve immune function and long-term performance of heifer calves; for example, reducing the age at first breeding and increasing first-lactation milk yield, but more research is needed to confirm these effects.     

母乳对婴儿肠道菌群及免疫系统影响的研究进展

近年来的研究发现肠道菌群与疾病有很大关联,婴幼儿的免疫系统尚未成熟,肠道菌群的建立还未完善,极易受到疾病的感染。母乳因其含有丰富的营养元素和免疫元素作为婴幼儿饮食首选。本文综述了母乳中微生物、营养物质、免疫细胞和免疫分子对婴儿肠道菌群的建立及免疫系统的影响,并简单介绍了婴儿肠道菌群与过敏症的关系,为降低婴儿感染疾病的风险以及预防疾病提供科学依据,同时为如何改善婴幼儿奶粉配方提供可靠的理论参考。     

Exploration of long noncoding RNA in bovine milk exosomes and their stability during digestion in vitro

Previous studies have demonstrated that bovine milk contains mRNA and microRNA that are largely encapsulated in milk-derived exosomes. However, little information is available about long noncoding RNAs (lncRNA) in bovine milk. Increasing evidence suggests that lncRNA are of particular interest given their key role in gene expression and development. We performed a comprehensive analysis of lncRNA in bovine milk exosomes by RNA sequencing. We used a validated human in vitro digestion model to investigate the stability of lncRNA encapsulated in bovine milk exosomes during the digestion process. We identified 3,475 novel lncRNA and 6 annotated lncRNA. The lncRNA shared characteristics with those of other mammals in terms of length, exon number, and open reading frames. However, lncRNA showed higher expression than mRNAs. We selected 12 lncRNA of high-expression abundance and identified them by PCR. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that lncRNA regulate immune function, osteoblastogenesis, neurodevelopment, reproduction, cell proliferation, and cell–cell communication. We also investigated the 12 lncRNA using quantitative real-time PCR to reveal their expression profiles in milk exosomes during different stages of lactation (colostrum 2 d, 30 d, 150 d, and 270 d); their resulting expression levels in milk exosomes showed variations across the stages. A digestion experiment showed that bovine milk exosome lncRNA was resistant to in vitro digestion with different digestive juices, including saliva, gastric juice, pancreatic juice, and bile juice. Taken together, these results show for the first time that cow milk contains lncRNA, and that their abundance varied at different stages of lactation. As expected, bovine milk exosomal lncRNA were stable during in vitro digestion. These findings provide a basis for further understanding of the physiological role of milk lncRNA.     

不同泌乳期羊乳和牛乳的高通量定量乳清蛋白质组学

利用高分辨质谱技术获取牛初乳、牛常乳、羊初乳和羊常乳的乳清蛋白组轮廓,基于其非标记定量强度建立偏最小二乘判别分析模型。结果发现牛初乳和牛常乳在蛋白质组成方面较羊初乳和羊常乳更相似,同时筛选出羊乳中丰度较高的9 种蛋白用作标志物区分这4 组乳样。生物信息学分析发现羊乳中的高丰度蛋白大部分与免疫应答和代谢过程有关,说明羊乳更有助于新生儿建立抗微生物感染的免疫系统。该研究可加深对羊乳蛋白的认识,对牛乳及其制品的营养改良和母乳替代品的生产具有重要意义。