乳铁蛋白在犊牛生产中的应用及其生物信息学分析

对乳铁蛋白在犊牛生长发育和免疫中的作用进行论述，同时将牛与其它8个物种的乳铁蛋白进行了多序列联配、序列同源性对比及进化树分析等生物信息学的分析。     

乳铁蛋白对鱼类的免疫增强作用

本文综述了乳铁蛋白的结构、性质,并从粒细胞呼吸爆发活性、噬菌细胞活性、过氧化物酶、细胞毒素和血清溶解酵素等方面讨论了乳铁蛋白对鱼类非特异性免疫水平的增强作用,初步讨论了乳铁蛋白在水产养殖上的研究及应用前景。     

牛乳铁蛋白多克隆抗体的制备、纯化及定量检测

本试验旨在制备高纯度和特异性的牛乳铁蛋白多克隆抗体,为鉴定并定量检测牛奶样品中及奶牛乳腺组织中合成的乳铁蛋白提供试验材料。选用4只健康新西兰大白兔,初次免疫乳铁蛋白4周后进行加强免疫,每2周加强免疫1次,待血清达到抗体效价后,对兔进行心脏采血并分离血清,利用饱和硫酸铵法和蛋白A树脂纯化抗体,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹(Western-blot)法分别用于鉴定纯化后抗体的纯度和特异性。测定纯化后抗体效价,并绘制抗体抑制曲线。最后利用所得抗体对市售液态奶、奶牛乳腺组织匀浆液、奶粉样品中的乳铁蛋白进行定量检测。结果表明,本试验制备的兔抗牛乳铁蛋白多克隆抗体纯度较高、特异性较强,抗体浓度为11.02 mg/m L,效价达到1∶128 000;采用该抗体测定的乳腺组织样品中乳铁蛋白浓度为16.13μg/g,液态奶中接近于0μg/g,奶粉中为5.28μg/g。总之,本试验采用经过饱和硫酸铵法和蛋白A树脂2步纯化后得到了纯度较高、特异性较强的兔抗牛乳铁蛋白多克隆抗体,可以用于牛奶等产品的乳铁蛋白鉴定。     

奶牛乳铁蛋白肽真核表达载体的构建及其在奶牛乳腺上皮细胞中的表达

实验用奶牛乳铁蛋白肽特异性PCR引物从奶牛乳腺组织中扩增奶牛乳铁蛋白肽基因,然后将其连入pCMV-N-eGFP载体中,构建奶牛乳铁蛋白肽的真核表达载体GFP-LfcinB,并将其转染进奶牛乳腺上皮细胞中,分别运用蛋白质免疫印记和牛津杯法等方法检测GFP-LfcinB蛋白在细胞中的表达及其对金黄色葡萄球菌的抗菌活性.结果表明,试验成功的建立了奶牛乳铁蛋白肽的真核表达载体GFP-LfcinB,并且此重组载体能在奶牛乳腺上皮细胞中正常表达具有抗金黄色葡萄球菌活性的GFP-LfcinB蛋白.     

乳铁蛋白的免疫调节作用及在动物生产中的应用

乳铁蛋白(lactoferrin,Lf)是一种非血红素铁结合性糖蛋白,在乳腺分泌物中含量丰富。近年的研究表明乳铁蛋白有着更广泛的生物学功能,文章就乳铁蛋白对机体免疫调节及其机理作一综述,随着乳铁蛋白免疫机制的进一步深入研究,其作为免疫调节剂将会越来越广泛地应用于动物生产实际中。     

乳铁蛋白的免疫调节作用及在动物生产中的应用

乳铁蛋白（1actoferrin，Lf）是一种非血红素铁结合性糖蛋白，在乳腺分泌物中含量丰富。近年的研究表明乳铁蛋白有着更广泛的生物学功能，文章就乳铁蛋白对机体免疫调节及其机理作一综述，随着乳铁蛋白免疫机制的进一步深入研究，其作为免疫调节剂将会越来越广泛地应用于动物生产实际中。     

荷兰成功培育出药用牛

荷兰科学家采用遗传工程技术，成功地培育出一种能从所产的牛乳中获得免疫药物的药用牛，并计划开用这种牛的牛乳制药的制药厂，大量生产免疫药品研究证实，乳铁蛋白是一种可以用来抑制细菌的药物，能用一治疗免疫力衰退的患者，如果利用药用牛的乳汁制取，不仅原料丰富，而且可大大降低成本。荷兰成功培育出药用牛     

乳铁蛋白及其活性肽的营养生理作用及应用前景

乳铁蛋白(LF)是一种多功能的糖蛋白,乳铁蛋白活性多肽(Lfcin)是从LF上被胃蛋白酶水解下来的25个氨基酸残基的小肽。文中阐述了乳铁蛋白及其活性多肽的结构,介绍了乳铁蛋白及其活性多肽的主要生理作用:抗菌、抑菌、抗病毒、抗氧化,调节机体的免疫和提高肠道对铁离子的吸收等作用。根据乳铁蛋白制备的研究进展,讨论了乳铁蛋白及其活性多肽在乳、食品和动物生产中作为添加剂的应用前景。     

荷兰成功培育出药用牛

荷兰科学家采用遗传工程技术 ,成功地培育出一种能从所产的牛乳中获得免疫药物的药用牛 ,并计划开办用这种牛的牛乳制药的制药厂 ,大量生产免疫药品。该药用牛是这样培育成功的 :先将人体所具有的乳铁蛋白质组成的遗传因子 ,植入牛的受精卵中 ,培育成一种名叫“海尔曼”的具有这种基因的公牛 ,然后再用“海尔曼”公牛的精液给母牛授精。母牛所产下的新一代母牛 ,牛乳中会含有此类基因的物质 ,可以用来制造免疫药物。研究证实 ,乳铁蛋白是一种可以用来抑制细菌的药物 ,能用于治疗免疫力衰退的患者 ,如果利用药用牛的乳汁制取 ,不仅原料丰富 ,…     

乳铁蛋白及其生理机能的研究进展

乳铁蛋白作为一种天然的具有免疫功能的糖蛋白,是转铁蛋白家族的一员。它存在于哺乳动物的各种外分泌物中,乳中的含量较高,因其广泛的生理功能,近年来越来越多地受到人们的关注。概括介绍乳铁蛋白的结构及其重要的生理功能以及乳铁蛋白的制备方法,并展望乳铁蛋白作为一种添加剂在畜牧业上的应用前景。     

Effect of milk fraction on concentrations of cephapirin and desacetylcephapirin in bovine milk after intramammary infusion of cephapirin sodium

Clinical mastitis in dairy cows is commonly treated with intramammary (IMM) antimicrobial agents. Pharmacokinetic data are used to design treatment regimens and determine withholding times. In some pharmacokinetic studies, investigators measure antimicrobial concentrations in foremilk, whereas in others, they use bucket milk or do not specify the milk fraction sampled. Our objective was to compare antimicrobial concentrations in foremilk, bucket milk, and strippings after IMM treatment of six healthy Holsteins. One mammary gland/cow was infused with 200 mg of cephapirin (CEPH) after each of the two milkings, using different milking frequencies and treatment intervals in a randomized crossover design. Treated glands were sampled at the first milking following each infusion. Antimicrobial concentrations in milk were measured using HPLC/MS/MS. CEPH concentration was higher in foremilk (geometric mean 44.2 μg/mL) than in bucket milk (15.7 μg/mL) or strippings (18.5 μg/mL), as it was true for desacetylcephapirin (DAC) (59.5, 23.0, and 30.2 μg/mL, respectively). This finding, which was based on milk samples collected at the first milking after IMM infusion, suggests that pharmacokinetic data based on drug concentrations in foremilk may be misleading. Strippings were more representative of bucket milk than foremilk. The relationship between milk fraction and antimicrobial concentration should be investigated for other IMM antimicrobial agents. Meanwhile, it is essential that pharmacokinetic and residue studies report the fraction of milk that was analyzed.     

Aquagrams of Raw Milk for Oestrus Detection in Dairy Cows

The purpose of this research was to develop rapid and cost‐effective method for oestrus detection in dairy cows by means of near infrared spectroscopy and aquaphotomics, using raw milk from individual cows. We found that aquaphotomics approach showed consistent specific water spectral pattern of milk at the oestrus periods of the investigated Holstein cows. Characteristic changes were detected especially in foremilk collected at morning milking. They were reflected in calculated aquagrams of milk spectra where distinctive spectral pattern of oestrus showed increased light absorbance of strongly hydrogen‐bonded water. Results showed that monitoring of raw milk near infrared spectra provides an opportunity for analysing hormone levels indirectly, through the changes of water spectral pattern caused by complex physiological changes related to fertile periods.     

Variations During Lactation in Total and Differential Leukocyte Counts,N-acetyl-ß-D-glucosaminidase,Antitrypsin and Serum Albumin in Foremilk and Residual Milk from Non-infected Quarters in the Bovine

Quarter samples of foremilk and residual milk were taken approximately every second week from 2 days post partum (pp) throughout lactation month 9, from 5 dairy cows in their second lactation period. Bacteriologically positive milk samples were excluded. The aim was to study the variation in total and differential leukocyte counts, N-acetyl-ß-D-glucosaminidase (NAGase), antitrypsin (ATR) and serum albumin (BSA) in milk during the lactation period and different stages of oestrous cycle. Also the between milkings variation was studied from lactation month 4 to 9. At 2 days pp, each fraction of milk contained significantly higher numbers of leukocytes and had a higher activity of NAGase and ATR than later in the lactation period. In foremilk the highest content of BSA was also recorded at 2 days pp. From lactation month 2 to 9, stage of lactation had, in general, a slight effect on the variation in the variables measured. The total leukocyte count in residual milk tended to increase as lactation proceeded. The proportion of monocyte-macrophages in foremilk was significantly decreased during the last 4 months. NAGase and BSA in both fractions and ATR in residual milk increased significantly towards the end of the lactation period. From lactation month 4 to 9 the highest recorded ranges of variation between milkings, within quarter and stage of lactation, in the total leukocyte count, proportions of neutrophils, lymphocytes, monocyte-macrophages, NAGase, ATR and BSA in foremilk were 215 × 10³ml, 42 %, 34 %, 54 %, 6.68 units, 0.36 units and 0.14 mg/ml respectively. The corresponding figures in residual milk were higher except for the variation in BSA which was slightly lower in residual milk than in foremilk. In residual milk there was a positive correlation between the proportion of neutrophils and the total leukocyte count, when calculated on data from all cows and the entire experimental period. During the oestrous periods, the proportion of neutrophils in residual milk was higher than during the dioestrous periods. Foremilk and residual milk differed in the total as well as the differential leukocyte counts in all the various stages of lactation, whereas the contents of NAGase, ATR and BSA were equal in both fractions. The exception was 2 days pp when the proportions of lymphocytes were equal in both fractions and BSA-significantly higher in foremilk than in residual milk.     

Composition and milk cell characteristics in quarter milk fractions of dairy cows with low cell count

Variation in milk composition and milk polymorphonuclear neutrophil leukocyte (PMN) characteristics and functions among quarter milk fractions were investigated in order to evaluate the optimal fraction for the determination of local immune response. Five fractions were classified during milking: foremilk (I), cisternal milk (II), main milk (III), strippings (IV) and residual milk (V). Somatic cell count (SCC), fat, protein, lactose, sodium, potassium, chloride, polymorphonuclear leukocyte necrosis, apoptosis and oxidative burst were analysed in each fraction. The logSCC and fat concentration were highest in residual milk (P < 0.05), whereas protein and lactose concentration were highest in the earliest fractions (I, II, III) (P < 0.05). Polymorphonuclear neutrophil leukocyte necrosis was lowest in strippings and residual milk (P < 0.05), and PMN apoptosis was lowest in residual milk (P < 0.001). The highest percentage of PMN with oxidative burst was found in residual milk (P < 0.05), and was associated with the highest mean fluorescence intensity (MFI) (P < 0.05). In conclusion, late fractions have more PMN and more active PMN. Nevertheless, it is suggested that each fraction is appropriate in the study of local immune response of the mammary gland, however the fraction used in the study has to be specified.     

The use of polyethylene intramammary device in protection of the lactating bovine udder against experimental infection with Staphylococcus aureus or Streptococcus agalactiae.

The susceptibility of lactating bovine udder quarters fitted with a polyethylene intramammary device to infection was investigated. Following experimental challenge with Streptococcus agalactiae or Staphylococcus aureus, the incidence of infection was significantly (p less than 0.05) lower in intramammary device-fitted quarters compared to control quarters. In general, total foremilk and strippings milk somatic cell counts for intramammary device-fitted and control quarters were not significantly (p less than 0.05) different. Differential foremilk and strippings milk somatic cell counts were significantly (p less than 0.05) higher in samples from intramammary device-fitted quarters compared to control quarters.     

Factors affecting skim milk progesterone assay results.

Five studies were performed to determine factors affecting progesterone concentration in skim milk. Results of the first study indicated that progesterone concentration was higher in skim milk of samples kept 16 hours in an ice bath (0 C) than of those left at room temperature (21 C). In the second study, this temperature effect was found to be reversible, with skim milk progesterone concentration increasing when whole milk samples were cooled prior to centrifugation. In the third study, [3H]-labeled progesterone was used to determine the relationship between fat content of foremilk (the first milk obtained from the teats), midmilk (milk obtained midway through milking), and strippings (milk obtained immediately after milking machines have been removed) samples and temperature (4 C and 21 C) on the percentage of progesterone in the skim milk fraction. The relationship between percentage of butterfat and percentage of progesterone in skim milk was linear when the log of these variables was used for calculations. In the fourth study, assayable progesterone in the skim milk fraction of foremilk, midmilk, and strippings was affected by temperature. In the fifth study, a multiple-regression procedure was used to determine the amount of variation in percentage of radioactive progesterone in the skim milk fraction. Independent variables (whole milk butterfat and temperature of incubation [1, 3, 13, 22, 37, and 50 C]) and the natural log of each variable, were entered into a stepwise multiple-regression analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentration patterns of antibacterial factors and immunoglobulin A antibody in foremilk fractions of healthy cows

Antibacterial factors act as innate immune components, which respond as soon as bacteria enter a living organism. To prevent and treat mastitis in cattle, understanding the concentrations of these substances inside the udder is important; however, they remain to be studied. In this investigation, the concentration of lingual antimicrobial peptide (LAP), S100 protein (S100A7), lactoferrin (LF), and immunoglobulin antibody were measured in the different fractions of foremilk. Lactating Holstein cows were examined, and 10 foremilk fractions were obtained from sequential samples up to 150 ml. The LAP concentrations in milk samples increased until 25 ml. The LF concentrations increased up to the 10 ml fraction, then stabilized at low level after the 50 ml fraction. For S100A7, some fractions had significantly higher (p < .05) concentrations than the 5 or 10 ml fractions. The IgA antibody concentration increased up to the 5 ml fraction, then after 50 ml fraction showed relatively low concentrations. This investigation determined the concentration patterns of LAP, LF, S100A7, and IgA antibody secreted in milk inside the udders of healthy lactating cows as baseline data. These distinct concentration patterns might indicate various protective responses.     

Pathogenicity of different species of staphylococci in caprine udder

Aseptic foremilk samples were collected from Finnish landrace goats. Ten different species of staphylococci, causing subclinical infections were detected. Twelve goats with persistent subclinical staphylococcal infection were followed on a monthly basis and compared with foremilk samples of nine goats suffering from clinical mastitis. Parameters of inflammation based on the activity of the California Mastitis Test (CMT). N-acetyl-beta-D-glucosaminidase (NAGase) and antitrypsin were determined from the milk. Staphylococci were further classified using the API STAPH system. On the basis of elevations of activities of CMT, NAGase and antitrypsin, Staphylococcus aureus was the most pathogenic in clinical and subclinical mastitis, S. hyicus showed only marginal pathogenicity. Subclinical infections were persistent and the infective organism was not always detected from milk by culture. The biochemical reactions of subclinical staphylococci seemed to vary within the same gland by time. Antitrypsin was most effective in differentiating between subclinical and clinical infection. A teat cistern puncture technique was found to be suitable for the goat.     

大豆黄酮对奶牛免疫功能和血清及乳中几种激素水平的影响

研究口服大豆黄酮对奶牛免疫功能和血清及乳中生长激素(GH)、催乳素(PRL)、生长抑素(SS)水平的影响。结果表明：1)大豆黄酮能明显提高血清及乳中特异性抗体水平,表明奶牛整体和乳腺器官的体液免疫功能明显增强。2)奶牛血清及乳中GH、PRL含量明显高于对照组,而血清SS含量显著低于对照组。本实验结果提示垂体GH、PRL和体内SS可能参与了大豆黄酮对奶牛免疫功能的调节过程。     

Effect of abraded intramammary device on outcome in lactating cows after challenge exposure with Streptococcus uberis

Intramammary devices (IMD) were abraded with medium-grade emery cloth or were left smooth. One IMD of each type was inserted into a mammary quarter of each of 5 lactating cows. The remaining 2 quarters served as controls. Quarter foremilk, bucket milk, and stripping milk samples were collected for 3 consecutive days at 2 weeks after IMD insertion, and milk somatic cell counts (SCC) were determined. Milk samples also were collected immediately after and 0.5, 1, 2, 4, 6, 8, and 11 hours after milking. All quarters were challenge exposed with 250 colony-forming units of Streptococcus uberis at 2 months after IMD insertion. Foremilk and stripping milk samples were collected for bacteriologic culture and SCC at the next 10 milkings. Mean foremilk, bucket milk, and stripping milk SCC (X 10(6) cells/ml) were 0.18, 0.07, and 0.91, respectively, for quarters with abraded IMD; 0.06, 0.05, and 0.43, respectively, for quarters with smooth IMD; and 0.03, 0.03, and 0.15, respectively, for control quarters. Mean SCC after milking (X 10(6) cells/ml) for the various intervals were 0.70, 1.29, 0.70, 0.97, 1.15, 1.17, 0.77, and 0.85 for quarters with abraded IMD; 0.43, 0.62, 0.61, 0.45, 0.64, 0.60, 0.31, and 0.26 for quarters with smooth IMD; and 0.15, 0.24, 0.15, 0.19, 0.15, 0.15, 0.14 and 0.06 for control quarters. After challenge exposure, 2 of 5 of the quarters with abraded IMD, 4 of 5 of the quarters with smooth IMD, and 8 of 9 control quarters became infected. Results indicated that abraded IMD increased SCC in stripping milk to concentrations that provided 60% protection against challenge exposure with S uberis.