奶牛多形核白细胞凋亡的分子调控与乳腺免疫

多形核白细胞(polymorphonuclear neutrophil leukocytes,PMN)在奶牛乳腺防御病原菌感染中充当首要防线,而PMN的凋亡在解除奶牛乳腺的炎性反应中发挥关键作用。作者综述了PMN凋亡的分子调控及其在奶牛乳腺免疫中的重要作用和意义。     

奶牛乳房炎的预防和治疗方法

正奶牛乳房炎是危害奶牛生产最主要的疾病之一。笔者介绍几种无药残治疗奶牛乳房炎的方法。1预防加强饲养管理,提高奶牛抵抗力,是最关键有效的预防措施。首先应注重提高犊牛的先天性免疫力,增强奶牛的第一道防线,防止病原体入侵机体而发病。其次严把饲料关,保证饲料和饮水的清洁卫生,防止饲料霉变。     

不同牛奶体细胞数下牛奶体细胞分型及奶牛泌乳性能变化

本试验旨在通过研究不同牛奶体细胞数(SCC)下牛奶体细胞分型特点,进而评估奶牛泌乳性能。选择102头荷斯坦奶牛进行牛奶样品采集,检测SCC、产奶量和乳成分。根据SCC检测结果将这102份牛奶样品分为3组,低SCC组(SCC100 000个/mL)、中等SCC组(SCC为100 000~400 000个/mL)和高SCC组(SCC400 000个/mL)。通过流式细胞仪对牛奶样品中的体细胞进行分选,检测淋巴细胞、巨噬细胞和多形核嗜中性白细胞(PMN)的比例。采用标准平板计菌法对菌落总数进行计数,并通过细菌学检查对牛奶样品中的细菌进行分类鉴定。结果表明:1)在SCC100 000个/mL范围内,SCC在13 000~76 000个/mL时,淋巴细胞比例为89.43%,是健康乳腺中的主要细胞群体,当SCC增加到76 000~100 000个/mL时,PMN比例极显著增加(P0.01),达到79.68%,成为乳腺内主要细胞群体,表明早期炎症已经开始发生。SCC200 000个/mL时,PMN比例与SCC呈正相关;淋巴细胞比例与SCC呈负相关;巨噬细胞比例与SCC无明显相关性。2)鉴别出的主要病原体有金黄色葡萄球菌、凝固酶阴性葡萄球菌(CNS)、无乳链球菌等;次要病原体有绿脓杆菌、芽孢杆菌、乳酸乳球菌等。主要病原体组的牛奶样品中PMN比例(48.93%)极显著高于无病原体组(17.23%)(P0.01),显著高于次要病原体组(P0.05)。3)细菌培养阳性的牛奶样品中,感染传染型病原体的奶牛,产奶量显著下降(P0.05),酪蛋白/乳蛋白及乳糖含量显著减少(P0.05);在细菌培养阴性牛奶样品中,中等SCC(10 000~40 000个/mL)和高SCC(SCC40 000个/mL)牛奶样品中观察到酪蛋白与和乳糖含量显著降低(P0.05),高SCC牛奶样品中观察到乳蛋白含量显著降低(P0.05)。由此可见,体细胞分型可用于对奶牛乳腺健康状况做出更详细分析,特别是在低SCC的牛奶中更早地识别炎症起始发生。乳腺早期炎症反应的发生明显低于目前公认的健康乳腺牛奶SCC阈值(100 000个/mL),并且CNS可能是诱发早期乳腺炎症的潜在病原体。细菌培养阴性样品中,炎症导致高SCC牛奶中酪蛋白/乳蛋白和乳糖含量的显著降低。     

动物免疫的常用方法及注意事项

免疫是人和动物都存在的一种用来抵御内外病原体，保护自身安全的生理功能。人和动物的免疫从广义上讲有三道防线。一是由皮肤、皮肤黏膜、与外界相通的器官分泌的杀菌体液组成的壁垒系统。执行着隔离与清除的免疫功能。二是由体液中的杀菌物质和吞噬细胞组成的非特异性免疫系统。执行着识别、吞噬、清除各种病原体的功能。这两道防线是人和动物在进化过程中逐渐建立起来的天然防御功能，特点是每个个体生来就有，不针对某一种特定的病原体，对多种病原体都有防御作用，因此叫做非特异性免疫（又称先天性免疫）多数情况下，这两道防线可以防止病原体对机体的侵袭。     

奶牛乳房炎的预防和治疗方法

1预防方法加强饲养管理,提高奶牛抵抗力,是最关键有效的预防措施。优秀先进的饲养管理可以让奶牛活动的环境更干净,避免奶牛置于众多病原体的环境中;提高奶牛的先天性免疫力,增强奶牛的第一道防线。在生产过程中,不可避免的一些因素     

家禽肠道健康的检测方法研究进展

肠道是家禽抵御外界病原体入侵的第一道防线,研究家禽肠道健康对家禽生产具有重要意义。文章综述了家禽肠道消化吸收功能、机械屏障功能、微生物屏障功能以及免疫屏障功能四个方面检测肠道健康的相关方法,为后续进行更深入的肠道健康研究提供参考。     

奶牛血和乳中性粒细胞的形态与功能研究概述

中性粒细胞(PMN)是感染性奶牛乳腺炎的重要防御屏障。文章着重介绍了奶牛PMN在光学显微镜、透射电子显微镜和扫描电子显微镜下的形态特点,并叙述了PMN动力学和各类PMN的生长期、PMN生存期调节与发生奶牛乳腺炎之间的关系,最后提出奶牛PMN的研究方向。     

鸡抗马立克氏病毒免疫应答机制研究

自100多年前鸡马立克病(MD)首次报道以来,人们一直致力于病原体及其防控策略的研究。近几年来,由于马立克氏病毒(MDV)的不断进化,引起诸多地区屡次暴发MD,如何改进预防措施显得刻不容缓,因此,必须了解病毒与宿主之间的相互作用。宿主的免疫系统与病毒的入侵与扩散息息相关,主要通过两种机制应对病毒入侵,作为机体第一道免疫防线的天然防御机制包括可溶性因子的分泌以及巨噬细胞和自然杀伤细胞等免疫细胞的参与,天然免疫反应促使免疫系统形成特异的抵抗MDV的抗体和细胞免疫应答。除免疫系统外,遗传和表现遗传机制有助于鸡应答MDV感染。本文综述了目前对MDV诱导的免疫应答的理解以及免疫应答的遗传因子。     

禽异嗜性粒细胞与机体免疫研究进展

禽异嗜性粒细胞是存在于禽类血液中的一种主要的颗粒性白细胞,类似于哺乳动物的中性粒细胞,是禽类抵御外界微生物病原体入侵的一道重要防线,也是机体非特异性免疫系统中的重要成员和主要调节者。近年来,对于该细胞在机体急性炎症反应和吞噬杀伤作用中的研究已经深入到了基因和蛋白水平,为从禽类异嗜性粒细胞方面入手治疗禽类疾病,提供了理论依据和研究方向。论文从异嗜性粒细胞与中性粒细胞在形态结构,急性炎症反应和吞噬杀伤病原体作用中的区别,以及近年来该细胞的其他研究热点等方面综述了禽类异嗜性粒细胞的研究进展。     

奶牛激素相关的乳腺MicroRNA研究进展

MicroRNA(miRNA)是一种非编码RNA,在调节细胞增殖、分化和凋亡的过程中起重要作用.研究表明,激素通过单独或相互作用完成对奶牛乳腺发育和泌乳进程的调控.本文从激素对奶牛乳腺miRNA表达谱影响及miRNA通过激素受体及其下游通路调控乳腺功能2个方面,综述了激素与相关miRNA的研究现状,以期助力于乳腺生物学...     

The bovine neutrophil: Structure and function in blood and milk

Migration of polymorphonuclear neutrophil leukocytes (PMN) into the mammary gland provide the first line of defense against invading mastitis pathogens. Bacteria release potent toxins that activate white blood cells and epithelial cells in the mammary gland to secrete cytokines that recruit PMN that function as phagocytes at the site of infection. While freshly migrated PMN are active phagocytes, continued exposure of PMN to inhibitory factors in milk such as fat globules and casein, leads to altered PMN morphology and reduced phagocytosis. In the course of phagocytosing and destroying invading pathogens, PMN release chemicals that not only kill the pathogens but that also cause injury to the delicate lining of the mammary gland. This will result in permanent scarring and reduced numbers of milk secretory cells. The life span of PMN is limited by the onset of apoptosis. To minimize damage to mammary tissue, PMN undergo a specialized process of programmed cell death known as apoptosis. Macrophages quickly engulf and phagocytose apoptotic PMN, thereby minimizing the release of PMN granular contents that are damaging to tissue. The PMN possess an array of cell surface receptors that allow them to adhere and migrate through endothelium and to recognize and phagocytose bacteria. One receptor found on phagocytes that is receiving considerable attention in the control of infections by Gram-negative bacteria is CD14. Binding of lipopolysaccharide (LPS) to membrane bound CD14 causes release of tumor necrosis factor-alpha and sepsis. Binding of LPS to soluble CD14 shed from CD14-bearing cells results in neutralization of LPS and rapid recruitment of PMN to the site of infection. Recent advances in the fields of genomics and proteomics should greatly enhance our understanding of the PMN role in controlling intramammary infections in ruminants. Further, manipulation of PMN, through either recombinant proteins such as soluble CD14 that enhance PMN response or agents that mediate PMN apoptosis, may serve as novel therapeutics for the treatment of mastitis.     

Bactericidal activity of macrophages against Streptococcus uberis is different in mammary gland secretions of lactating and drying off cows

The aim of this study was to compare the ability of milk macrophages and macrophages from the mammary gland secretions during the mid-dry period for their interaction with the mastitis-causing Streptococcus uberis. We also aimed to determine if S. uberis induced the release of the cytokine tumour necrosis alpha (TNF-alpha) and the bactericidal moiety nitric oxide (NO) from milk macrophages of lactating cows and macrophages from the mammary gland secretions at the mid-dry period. Macrophages were isolated from the mammary gland secretions of cows during the mid-lactation or mid-dry period, and compared with blood monocytes for their interaction with the important mastitis-causing pathogen S. uberis. When infected in vitro with S. uberis, milk macrophages from lactating cows with S. uberis released modest amounts of the cytokine tumour necrosis factor alpha (TNF-alpha) (139 pg/ml) and the bactericidal moiety nitric oxide (NO) (3-4 microM of nitrite). Blood monocytes from lactating cows released significantly higher amounts of TNF-alpha (345 +/- 143 pg/ml) and NO (7 +/- 2 microM of nitrite) after interaction with S. uberis, compared to milk macrophages (P < 0.01 for both TNF-alpha and NO). Stimulation of blood monocytes with the cytokine interferon-gamma (IFN-gamma) enhanced significantly the release of NO and TNF-alpha, but IFN-gamma did not significantly enhance the production of NO and TNF-alpha by milk macrophages from lactating cows. Milk macrophages from all lactating cows failed to kill S. uberis efficiently, and this lack of killing was unaffected by prior treatment with gamma interferon (IFN-gamma) (P > 0.05). Rather, S. uberis multiplied significantly inside infected milk macrophages from lactating cows, with a two-fold increase in bacterial numbers at 2 h post-infection. Milk macrophages from lactating cows were able however, to kill a significant proportion (50-60%, P < 0.01) of phagocytosed Staphylococcus aureus. Blood monocytes from all cows were found to exert significant bactericidal activity against S. uberis. There were no significant differences in the bactericidal activity of milk macrophages obtained from lactating cows with low somatic cell counts (SCC; < 10(5) ml(-1)) compared with those with a mildly elevated SCC (> 10(5) ml(-1)) (P > 0.05). In contrast, mammary gland secretion macrophages isolated from the same cows in the mid-dry period killed a significant proportion of phagocytosed S. uberis (50-65% of ingested S. uberis killed, P < 0.01) although cytokine production in response to in vitro bacterial infection was low. We conclude that the bactericidal activity of mammary gland secretion macrophages against a virulent strain of S. uberis is low during the lactation period. In addition, our data indicate that S. uberis is not a strong inducer of NO and TNF-alpha in macrophages from the milk or mammary gland secretions of cows during the drying off period. Finally, IFN-gamma does not activate milk macrophages or macrophages from cows during the lactating period or mammary gland secretions during the drying off period.     

Proteomic analysis of mammary tissues from healthy cows and clinical mastitic cows for identification of disease-related proteins

To investigate the disease-related proteins and understand molecular mechanism of mastitis at the protein level, this project presents the protein changes in the mammary gland between healthy cows and clinical mastitic cows using two-dimensional gel electrophoresis (2-DE), after stained with colloidal Coomassie Bright Blue, six spots of differentially expressed protein were detected by PDQuest software and subjected to ion trap mass spectrometer equipped with a HPLC system, and five proteins were identified. Hemoglobin beta, kappa-casein and tryptophanyl-tRNA-synthetase (TrpRS) in healthy dairy cows, while hemoglobin beta, cytochrome C oxidase and annexin V in clinical mastitic cows were identified, they were involved in binding, transport and catalytic activity. The results may provide valuable information for the investigating of the host mammary immune system response to defense against pathogens at the protein level and potential protein targets for treatment.     

自噬对奶牛乳腺发育和功能影响的研究进展

自噬是细胞降解自身受损细胞器和长寿命蛋白的过程,有助于在饥饿、应激等情况下保护细胞存活。奶牛乳腺组织在青春期和妊娠期经历生长发育,在泌乳后期和干奶期进行退化再生,自噬在这2个过程中均发挥重要作用,既可以保护乳腺上皮细胞存活,又可以促使细胞发生程序性死亡。自噬也与机体中许多生理、病理过程相互作用,并受体内外多种因素的影响。本文就自噬的过程、自噬在奶牛乳腺组织生长发育和退化再生过程中发挥的作用及其影响因素进行综述,以期为通过调控自噬改善奶牛生产性能提供理论依据。     

Severity of E. coli mastitis is mainly determined by cow factors

Intramammary infections of dairy cows with Gram-positive bacteria such as Staphylococcus aureus (major cause of mastitis) have received a lot of attention because of their major economic impact on the dairy farm through production losses induced by an increase in somatic cell count. Management strategies, including greater awareness for efficient milking and hygienic measures, have limited the spread of Gram-positive bacteria and resulted in a significant decrease of proportion of S. aureus isolates and subclinical mastitis worldwide. Other organisms such as coliform subspecies and Streptococcus uberis, both environmental bacteria that cause clinical mastitis, have received less attention. Escherichia coli causes inflammation of the mammary gland in dairy cows around parturition and during early lactation with striking local and sometimes severe systemic clinical symptoms. This disease affects many high producing cows in dairy herds and may cause several cases of death per year in the most severe cases. It is well known that bacterial, cow and environmental factors are interdependent and influence mastitis susceptibility. Many studies, executed during the last decade, indicate that the severity of E. coli mastitis is mainly determined by cow factors rather than by E. coli pathogenicity. During E. coli mastitis, the host defense status is a cardinal factor determining the outcome of the disease. Today, we know that the neutrophil is a key factor in the cows' defense against intramammary infection with E. coli. Effective elimination of the pathogen by neutrophils is important for the resolution of infection and the outcome of E. coli mastitis. This review is a compilation of some major findings over the last 15 years concerning mainly host factors that modulate and influence neutrophil function and the mammary inflammatory reaction. The individual chapters address: virulence factors of E. coli strains, how neutrophils kill E. coli, connection between endotoxins, tumor necrosis factor-alpha and nitric oxide, severity classification of E. coli mastitis, lifespan of neutrophils, host factors that influence severity, tissue damage and production loss.     

Apoptosis of polymorphonuclear leukocytes of the juvenile bovine mammary gland during induced influx

The dynamics of apoptosis of polymorphonuclear leukocytes (PMN) during induced influx of PMN into the cavity system of the juvenile bovine mammary gland in order to investigate the role of apoptosis of PMN in the resolution of mastitis was studied. The instillation of a synthetic analogue of muramyl dipeptide into teat sinus of the sixteen mammary glands was followed by a massive influx of PMN culminating after 24 h and resolving after 96 h. Every 24 h following the influx, apoptotic PMN were microscopically detected, based on morphological characteristics. Twenty four hours after the stimulation, apoptotic PMN were already observed, and peak counts of apoptotic PMN were reached 48 h after the stimulation. The lowest differential count of apoptotic PMN, corresponding to the pre-stimulation value, was found 96 h after the stimulation. The presence of macrophages (MAC) containing phagocytized apoptotic PMN was observed by histochemical staining for myeloperoxidase (MPO) and electron microscopy. The percentage of MPO-positive macrophages increased during the resolution phase to reach peak values 48 h after the stimulation. Apoptosis of PMN and phagocytosis by macrophages may represent a removal mechanism that is important in the resolution of the induced influx of PMN in the cavity system of juvenile bovine mammary gland.     

CD44 is highly expressed on milk neutrophils in bovine mastitis and plays a role in their adhesion to matrix and mammary epithelium

Mastitis, inflammation of the mammary gland, is a common and economically important disease in dairy animals. Mammary pathogenic organisms, such as Escherichia coli, invade the teat canal,milk ducts, and mammary alveolar space, replicate in mammary secretions, and elicit a local inflammatory response characterized by massive recruitment of blood polymorphonuclear neutrophil leukocytes (PMN) into the alveoli and milk ducts. CD44 is a trans-membrane glycoprotein previously shown to play a role in mediation and control of blood PMN recruitment in response to inflammatory signals. Here we show, for the first time, increased expression of CD44 on recruited milk PMN in bovine mastitis and the expression of a CD44 variant, CD44v10, on these PMN. Furthermore, we demonstrate that CD44 mediates specific adhesion of bovine blood PMN to hyaluronic acid and mammary epithelial cells. Our results suggest that in mastitis CD44 plays a role in recruiting blood PMN into the mammary glands, the exact nature of this role needs to be elucidated.     

Comparison between stearoyl‐CoA desaturase expression in milk somatic cells and in mammary tissue of lactating dairy cows

Stearoyl‐CoA desaturase (SCD) is an important enzyme in the bovine mammary gland, where it inserts a cis‐double bond at the Δ9 position in a wide range of fatty acids. Investigating SCD expression in the bovine mammary gland generally requires invasive biopsy to obtain mammary tissue. The aim of this study was to evaluate the use of milk somatic cells as a non‐invasive alternative to biopsy for measuring mammary SCD expression in dairy cows. Both milk somatic cells and mammary tissue were collected from 14 Holstein‐Friesian cows and used for analysis of SCD expression by real‐time PCR. The SCD5 mRNA levels in mammary tissue compared with SCD1 were low, and for several milk somatic cell samples, SCD5 expression was even below the limit of detection. A significant relationship was found between SCD1 expression in milk somatic cells and in mammary tissue. In addition, SCD1 expression in milk somatic cells was significantly related to Δ9‐desaturase indices in milk, which are commonly used as an indicator of SCD1 activity within the mammary gland. Our study showed that milk somatic cells can be used as a source of mRNA to study SCD1 expression in dairy cows, offering a non‐invasive alternative to mammary tissue samples obtained by biopsy.