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。     

牛奶过敏原β-乳球蛋白抗体的制备及免疫检测方法的建立

本文将牛奶过敏原β-乳球蛋白抗原分别免疫新西兰大白兔和BALB/c小鼠制备其多克隆抗体和单克隆抗体,并对所获得的抗体进行了效价等特性测定;采用蛋白A亲和层析法将多克隆抗体进行了纯化,采用辛酸-硫酸铵法对单抗细胞株3A7进行了纯化;实验建立了双抗体夹心的ELISA检测方法,并对一些蛋白样品进行了初步的检测。实验结果表明,所制备的抗β-乳球蛋白兔多克隆抗体效价达到了1:6.56×106;Western-blotting鉴定结果显示所制备的多克隆抗体能够与β-乳球蛋白特异性反应;3株单抗的效价均在106以上,纯化后多抗仅与酪蛋白有一定的交叉反应,而纯化后单抗与乳中主要蛋白及其它过敏原蛋白基本没有交叉反应,特异性很好;利用所建立的方法对一些蛋白样品进行检测,准确率100%。     

苹果浓缩汁中嗜酸耐热菌多克隆抗体的制备及纯化

从苹果浓缩汁中分离得到嗜酸耐热菌,首次以耐热菌繁殖体及芽胞体分别作为免疫抗原,采用耳静脉注射及肌肉注射2种途径,对8只日本大耳白兔进行免疫,分别获得多克隆抗体。使用试管凝集法分别检测得到的抗体效价及其特异性,并采用饱和硫酸铵沉淀法及DEAE离子交换法纯化多克隆抗体,最终经SDS-PAGE电泳进行鉴定。以耳静脉注射和肌肉注射为免疫途径,制备得到的抗体效价分别达到1∶2560和1∶640。耳静脉注射得到的抗体效价高且免疫周期短,因此,耳静脉注射途径为最佳免疫途径;抗体特异性及纯化效果好。首次建立了苹果浓缩汁中耐热菌的家兔免疫程序,获得了多克隆抗体。     

一株肠出血性大肠杆菌O157H7免疫奶牛实验即抗EHEC O157H7免疫乳的试制

用1株O157H7菌株和另外11株人类肠道主要病原细菌配制O157H7单菌疫苗和多菌联合多价疫苗,对荷斯坦牛进行免疫实验。对免疫常乳中抗EHEC O157H7特异性抗体凝集价进行了长期监测,并对受试奶牛的免疫反应、应激反应及疫苗对奶牛可能产生的毒性作用进行了观察。结果证明,疫苗免疫效果很好,所有奶牛在整个泌乳期一直保持较高的特异性抗体效价(滴度),均在27以上,下个泌乳期开始阶段也未见明显下降的迹象。本次实验意外发现约有30%的未进行过免疫注射的对照组乳样有较高的O157H7特异性抗体滴度,个别乳样凝集价高达27,这可能说明这些牛群中有较高的O157H7感染率或携带率。O157H7是人类近二十余年来才开始认识到的出血性结肠炎(HC)的主要致病菌,能造成出血性腹泻和其它严重并发症。是值得研究和重点防范的病原细菌。     

德氏乳杆菌保加利亚亚种atpA抗原的表达及抗体制备

利用原核表达德氏乳杆菌保加利亚亚种atpA抗原并进行抗体制备,以便用于Western blot分析.首先利用PCR的方法扩增出atpA蛋白的基因片段,利用大肠杆菌pQE 40原核表达系统表达重组atpA抗原,镍柱亲和层析纯化表达蛋白后,常规免疫两只新西兰长耳白兔.利用ELISA检测兔血清的效价来制备多克隆抗体,然后用Protein A纯化抗体.最后得到的atpA抗体进行Western blot检测验证抗体的特异性.结果表明,两只新西兰长耳白兔的血清效价分别为1∶20000和1∶80000;Western blot检测表明,制备的抗体能够与H+-AT-Pase蛋白进行特异性的结合.本研究成功的在大肠杆菌中表达了德氏乳杆菌保加利亚亚种的atpA蛋白,并利用其制备了效价较高的多克隆抗体,该抗体可作为一抗用于Western blot分析.     

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。     

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。     

抗副溶血弧菌IgY的制备及活性检测

副溶血性弧菌(Vibrio parahaemololyticus,Vp)是我国微生物食源性疾病的主要病因,且是海水养殖业中的常见病原菌,造成巨大的经济损失。本研究制备副溶血弧菌免疫原,并以5×107、5×108CFU/ml两种剂量分别免疫母鸡,获得特异性的抗副溶血弧菌IgY;并建立ELISA方法检测免疫鸡蛋中的抗体效价,高剂量免疫组的抗体效价可达1:57600,低剂量免疫组的抗体效价达1:28800;低剂量组抗体效价可维持250d,高剂量组抗体效价可维持300d以上。     

副溶血弧菌多克隆抗体的制备及其特性分析

确定了副溶血弧菌多克隆抗体的制备方法:用体积分数0.05%甲醛于30℃灭活副溶血弧菌4 h制备抗原,并将此抗原分多次,以不同剂量免疫新西兰大白兔获得特异性多克隆抗体,以山羊抗兔IgG-HRP作为酶标二抗,通过间接ELISA法测定其多克隆抗体的效价、敏感性和特异性。结果表明:在免疫的第5周产生大量高效价抗体,效价最高可达1.6×105;此多克隆抗体对副溶血弧菌检测灵敏度为1×105CFU/mL;交叉反应和阻断试验结果显示,此多克隆抗体具有很强的特异性。     

抗变链球菌鸡卵黄免疫球蛋白(IgY)的制备及活性检测

以远缘链球菌(S.sorbrinus 6715血清型g)免疫产卵母鸡,应用酶联免疫吸附测定法(ELISA)测定鸡卵黄中免疫球蛋白(Immunoglobulin of Yolk,IgY)的活性,研究鸡免疫应答并制备出高免抗-S.sorbrinus IgY.结果显示以109 cfu/只剂量免疫的母鸡,第一次免疫后的第七天卵黄中出现特异性IgY,加强免设后抗体效价上升更快,经ELISA法检测,卵黄中的抗体效价到1384.而冷冻干燥后的IgY,其效价要超过12000.     

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。     

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。     

Modulation of the humoral immune response in rats by antibodies from the maternal milk

The influence of specific antibodies of mother's milk on the parameters of humoral immune response and the degree of sensitization to ovalbumin in the progeny was studied in experiments on non-suckling rats. On the second postnatal day lactating rats were immunized subcutaneously with ovalbumin in a dose of 200 micrograms with aluminium hydroxide (10 mg) as adjuvant. On day 22 after birth the young rats which were fed by immunized (test) or intact (control) mothers, received intraperitoneal injections of 100 micrograms ovalbumin. After 8 days the humoral immune response in the young rats was estimated by the number of antibody-forming cells in the spleen, by the count of rosette-forming cells and by the humoral antibody titer. The sensitization degree was evaluated by the titers of reagin antibodies in the passive skin anaphylactic reaction. The number of antibody-forming cells in the spleen of young rats in the test group was 20 times lower as compared to those in the control group. In the test group the humoral antibody titers were suppressed and the sensitization degree was decreased. It has been concluded that the mother's milk antibodies induce immunosuppressive effect on the parameters of the humoral immune response of the progeny and on the degree of their sensitization.     

Efficacy of immunization with ferric citrate receptor FecA from Escherichia coli on induced coliform mastitis

The effects of immunization with the ferric citrate receptor FecA on antibody responses and on experimentally induced mastitis following intramammary challenge were investigated. Twenty-one cows were assigned to seven blocks of three cows based on expected parturition. Cows within block were randomly assigned to one of three treatments: 1) FecA immunization, 2) Escherichia coli J5 immunization, and 3) unimmunized controls. Challenge was by infusion of approximately 60 cfu of E. coli 727 into one uninfected mammary gland between 13 and 31 d after parturition. Cows within block were challenged on the same day. Cows immunized with FecA had higher immunoglobulin (Ig)G titers against FecA in serum and in mammary secretions at calving, immediately before challenge, and 7 d after challenge than did cows immunized with E. coli J5 or control cows. Immunization with FecA also increased IgG titers against whole-cell E. coli 727 in serum and in mammary secretions at calving. Serum IgM titers against FecA were higher in FecA immunized cows than in other treatment groups immediately before challenge. Bacterial counts in milk, duration of bacterial isolation in milk, rectal temperature, and milk somatic cell counts following intramammary challenge were similar among treatments. Milk production and dry matter intake did not differ among treatments. The ferric citrate receptor FecA was immunogenic in cows, but immunization had minimal effect on the clinical severity of experimentally induced E. coli mastitis.     

Anti-idiotypic responses of lactating cows immunized with monoclonal antibodies against bovine somatotropin.

The objective of this study was to produce anti-idiotypic antibodies with bovine somatotropin (bST)-like activity by active immunization of lactating cows and to determine their effects on milk yield. Several monoclonal antibodies against bST were evaluated for their interaction with bST in a rat growth bioassay. Two bST-agonist monoclonal antibodies (1 and 2), and two bST-antagonist monoclonal antibodies (3 and 4) were selected. Cows were immunized with immunoglobulin G as a control (n = 12) or with one of the four anti-bST monoclonal antibodies (1, 2, 3, 4; n = 12) on d 3, 24, 45, 66, 87, 108, 129, and 150 of lactation. From wk 3 of lactation, all cows immunized with each of the four anti-bST monoclonal antibodies developed anti-idiotypes until wk 30 of lactation. Total lactation yields were not different among monoclonal antibodies 2, 3, and 4 and control cows (9299, 9321, 9733, and 9415 kg, respectively). However, cows immunized with anti-bST monoclonal antibody 1 had reduced lactation yield compared with cows on other treatments (8136 kg). Daily milk yield of cows immunized with monoclonal antibody 1 was decreased from wk 9 of lactation [36.2 vs. 40.9 kg/d (control)] until the end of lactation, concomitantly with decreased bST concentration from wk 9 of lactation. Cows immunized with anti-bST monoclonal antibody 4 had increased milk yield compared with that of controls during wk 3 to 6 and wk 18 to 21 of lactation. Therefore, anti-idiotypes directed against anti-bST 1 had bST-antagonistic effects on lactation performance; anti-idiotypes against anti-bST 4 transiently increased milk yield.     

Effect of enterotoxigenic Escherichia coli vaccine on innate immune function of bovine mammary gland infused with lipopolysaccharide

The effects of using an enterotoxigenic Escherichia coli vaccine on innate immune responses following intramammary infusion of lipopolysaccharide (LPS) were investigated in midlactation Holstein-Friesian cows. Seven out of 14 cows were inoculated with E. coli vaccine. Three weeks later, 100μg of LPS dissolved in 10mL of saline was infused into 1 quarter of all cows. Milk was collected every hour from infusion to 12h after infusion, and twice daily (at 0900 and 1600h) for 4d. Blood samples were collected 0, 4, 8, 24, 48, 72, and 96h after infusion. Rectal temperatures and milk yields were measured. The somatic cell count (SCC), lingual antimicrobial peptide concentration, lactoperoxidase (LPO) activity, and lactoferrin (LF) concentration in milk, and haptoglobin concentration in serum were determined. The mean rectal temperature in vaccinated cows was higher than in control cows at 10h. The mean milk yield was decreased significantly in the infused quarter of control cows at 24h compared with pretreatment, but not in vaccinated cows. The mean SCC in milk from vaccinated cows at 12 and 55h was significantly lower than that of control cows. The lingual antimicrobial peptide and LF concentrations were significantly lower at 8h and 55h, respectively, in vaccinated cows than in control cows. The mean antibody titer in the serum against the vaccine at the time of LPS infusion into vaccinated cows was significantly higher than in control cows. These antibody titers were positively correlated with the peak concentrations of LPO and LF in milk following challenge; therefore, cows with a high antibody titer were accompanied by high LPO activity and LF concentration in milk. These results suggest that vaccination suppresses the innate immune reaction after intramammary LPS infusion; however, the elevated antibody titer was unlikely to be responsible for the modification of the innate immune reaction.     

Nonspecific antibacterial factors in milk from cows immunized with human oral bacterial pathogens.

Both the immunoglobulins and non-specific antibacterial factors in milk from cows immunized with pathogenic oral bacteria have the potential to influence the oral microflora during passive immunization studies. The first six milks after calving were collected from 2 cows immunized with adjuvant and from 14 cows immunized with adjuvant and heat-killed strains of periodontopathic Actinomyces, Porphyromonas, Prevotella, and Fusobacterium. Analysis of the products from the first to the sixth milks revealed that the protein and lysozyme content decreased approximately 66 and 72%, respectively; the mean specific activity of the enzyme remained relatively constant. In contrast, the mean lactoperoxidase activity increased 2.3-fold in the second milking and increased further in the fourth and sixth milkings. The mean iron-binding activity increased 1.2-fold from the first to the second milkings and then decreased 3.6-fold through the sixth milking. Cows immunized with adjuvant alone showed similar responses. Per unit volume, the milk contained approximately 150 times less lysozyme than whole human saliva obtained from six subjects but higher concentrations of lactoperoxidase and iron-binding components. Purified bovine nonspecific factors prevented the growth of the bacteria used for immunization when bacteria were tested at concentrations similar to those found in saliva and milk. Because bovine nonspecific antibacterial factors could influence both the pathogenic target bacteria and the indigenous microflora in oral passive immunization studies with bovine immunoglobulins, the presence of these proteins should be considered.     

The relationship between milk production and antibody response to ovalbumin during the peripartum period

Suboptimal innate and immune mechanisms of host resistance during the peripartum period may contribute to increased incidence of mastitis. To evaluate associations between antibody response to ovalbumin and milk production during the peripartum period, 136 Holstein cows and heifers from three herds with known antibody response profiles, were evaluated for projected 305-d milk, protein, and fat yield. Using a previously described index (Wagter et al., 2000), cows were quantitatively classified based on their profile of antibody response to ovalbumin into high, average, or low antibody response groups. The single-effect antibody response group contributed significantly to variation in fat and protein yield, but not milk yield. The interaction between antibody response and parity significantly contributed to the variation in milk, fat, and protein yields; therefore the effects of group were reported on a within-parity basis. Among first-parity cows, low responders had a higher fat and protein yield than high or average antibody responder animals. Among older cows (parity 3 or greater) milk yield was significantly higher for those in the high antibody response group compared with average and low response groups. However, no significant differences in fat or protein yields were observed between high and low antibody response groups. These results suggest the possibility to select cows for enhanced immune response with no adverse effects on yield. That first-parity cows with low antibody response produce more fat and protein may be offset by the fact that mastitis occurrence was highest in this group in two out of three herds investigated. Selection for high immune response may prove beneficial to herd life by maintaining optimal yield, yet minimizing occurrence of disease.     

Changes occurring in immune responsiveness of single- and twin-bearing Comisana ewes during the transition period

Changes induced by twin and single lambing in the immune response of 16 periparturient Comisana ewes were studied. Cell-mediated immune responses were evaluated by means of skin tests performed from 3 wk before and up to d 35 after parturition. At d 21 and 7 before lambing, the sheep received an intramuscular injection of the antigen keyhole limpet hemocyanin (KLH), to which the animals had not been previously exposed, to determine their humoral immune response. Starting 3 wk before lambing and up to d 35 postlambing, the ewes were sampled to determine the plasma concentrations of anti-KLH antibody (IgG), IL-6, and IL-1 β. From parturition through d 35 postpartum, individual milk samples were collected for determination of anti-KLH IgG titers and IL-6 and IL-1β concentrations by means of a capture ELISA. The number of lambs born affected IL-6 concentrations in ewe plasma; IL-6 secretion always was higher in ewes birthing twins than in single-lambing ewes. Apart from the number of lambs born, the concentrations of plasma IL-6 in ewes were higher at lambing than at d 21 antepartum and at d 35 postpartum. An interaction of number of lambs born × time of sampling was observed for plasma antibody titers to KLH. The IgG concentrations were significantly higher in single-bearing ewes than in twin-bearing ewes before parturition and were very similar across groups after parturition. A time effect was found for the cell-mediated immune response and for anti-KLH IgG concentrations in milk, such that at parturition, cellular responses were lowest, and the anti-KLH IgG concentration was highest. A significant correlation was found for IgG titers to KLH in plasma and milk. Results indicate that IL-6 concentrations in blood can be considered a reliable indicator of stress connected to lambing and that the mammary gland is a microenvironment unrelated to blood stream with respect to interleukins expression. In contrast, a relationship was found for the IgG secretions in milk and blood, which suggests that the assessment of humoral immune status may be combined with milking routine in dairy animals.