Pathology of Staphylococcus aureus mastitis during lactogenesis: relationships with bovine mammary structure and function

Pathological alterations of mammary parenchymal tissue from 5 dairy cows with Staphylococcus aureus mastitis were studied. Tissue from infected quarters exhibited less synthetic and secretory ability during lactogenesis, as indicated by lower percentages of luminal area, but higher percentages of stromal area compared with control tissue. Ultrastructural analysis of alveolar epithelium demonstrated decreased numbers of organelles associated with milk synthesis and secretion. Mammary secretion from 12 additional cows confirmed the loss of secretory potential in infected quarters as concentrations of alpha-lactalbumin were lower during the first 14 d of lactation compared with concentrations from controls. Higher concentrations of serum alpha-lactalbumin from cows with Staphylococcus aureus mastitis suggest leakage of mammary secretion through gaps left by damaged or sloughed alveolar epithelium. Macrophages and lymphocytes were observed frequently in large gaps between basal plasma membrane of secretory cells and the basal lamina which may contribute to epithelial damage. Ability of neutrophils and macrophages to phagocytize staphylococci may have been compromised in the prelactating gland due to the indiscriminate ingestion of accumulated fat and casein.     

Cell function in the bovine mammary gland: a preliminary study on interdependence of healthy and infected udder quarters

Udder defence mechanisms are not completely explained by current mastitis research. The anatomical construction of the udder implies that infection of one udder quarter does not influence the immune status of neighbouring quarters. To test this hypothesis, we compared the immune reactions of individual udder quarters in response to microbial attacks. In the course of immune reactions, polymorphonuclear leucocytes (PMN) release oxygen radicals, which can be determined by chemiluminescence (CL). Milk from 140 udder quarters of 36 cows was analysed for somatic cell count (SCC), differential cell count, viability and CL activity. Quarters with an SCC < 100,000 cells/ml and free of pathogens were defined as uninfected, all other quarters were categorized as infected. Three groups of cows were classified cytologically: group A (healthy, 11 animals, SCC limit < 100,000 cells/ml); group B (moderate mastitis, 8 cows, SCC > or = 100,000 and < 400,000 cells/ml in at least one quarter); and group C (severe mastitis, 17 cows, SCC > or = 400,000 cells/ml in at least one quarter). Infected and uninfected quarters in groups B and C were analysed separately. Viability of PMN leucocytes was significantly (P=0.0012) lower in group A (72.6%) than in healthy quarters of group C (84.0%). Lowering the SCC limit of healthy quarters to <50,000 cells/ml (group A: all quarters within the udder) revealed striking differences between samples of groups B and C: in addition to varying differential cell counts and viabilities, CL activity of group B<50 (2929 CL units/million PMN) was markedly lower than that of the other groups (5616 in group A<50 and 6445 CL units/million PMN in group C<50). These results allow the conclusion that the infection of one udder quarter influences the cell activity of neighbouring quarters. When the SCC threshold for healthy quarters was reduced to 50,000 cells/ml, greater differences in cell activities were detected between healthy udders and healthy quarters of infected udders.     

Invited review: Microbiota of the bovine udder: Contributing factors and potential implications for udder health and mastitis susceptibility

Various body sites of vertebrates provide stable and nutrient-rich ecosystems for a diverse range of commensal, opportunistic, and pathogenic microorganisms to thrive. The collective genomes of these microbial symbionts (the microbiome) provide host animals with several advantages, including metabolism of indigestible carbohydrates, biosynthesis of vitamins, and modulation of innate and adaptive immune systems. In the context of the bovine udder, however, the relationship between cow and microbes has been traditionally viewed strictly from the perspective of host-pathogen interactions, with intramammary infections by mastitis pathogens triggering inflammatory responses (i.e., mastitis) that are often detrimental to mammary tissues and cow physiology. This traditional view has been challenged by recent metagenomic studies indicating that mammary secretions of clinically healthy quarters can harbor genomic markers of diverse bacterial groups, the vast majority of which have not been associated with mastitis. These observations have given rise to the concept of “commensal mammary microbiota,” the ecological properties of which can have important implications for understanding the pathogenesis of mastitis and offer opportunities for development of novel prophylactic or therapeutic products (or both) as alternatives to antimicrobials. Studies conducted to date have suggested that an optimum diversity of mammary microbiota is associated with immune homeostasis, whereas the microbiota of mastitic quarters, or those with a history of mastitis, are considerably less diverse. Whether disruption of the diversity of udder microbiota (dysbiosis) has a role in determining mastitis susceptibility remains unknown. Moreover, little is known about contributions of various biotic and abiotic factors in shaping overall diversity of udder microbiota. This review summarizes current understanding of the microbiota within various niches of the udder and highlights the need to view the microbiota of the teat apex, teat canal, and mammary secretions as interconnected niches of a highly dynamic microbial ecosystem. In addition, host-associated factors, including physiological and anatomical parameters, as well as genetic traits that may affect the udder microbiota are briefly discussed. Finally, current understanding of the effect of antimicrobials on the composition of intramammary microbiota is discussed, highlighting the resilience of udder microbiota to exogenous perturbants.     

Endoscopic examination and tissue sampling of the bovine teat and udder cistern

The aim of this study was to evaluate the application of an endoscopic technique to investigate the teat and udder cisterns of the bovine mammary gland, and to biopsy tissues within the cisterns. An anesthetic protocol for application in standing animals was designed, using a combination of general and local anesthesia. Individual quarter milk production (QMP), quarter somatic cell count (SCC), and occurrence of new intramammary infection were assessed after application of the technique, and possible applications for biopsies collected were investigated. Bovine teat and gland cistern lining could be visualized and small biopsy samples could be collected. The collected biopsy samples were successfully used in histological-histopathological examination and PCR analysis. To study the impact of endoscopy on QMP, milk SCC, and bacteriology, endoscopic examination of 12 low SCC (<200,000 cells/ mL) quarters was performed in 8 different first- and second-lactation cows. Immediately following endoscopy, 8 quarters received antibiotic treatment, whereas 4 quarters remained untreated. During a 15-d follow-up, no new intramammary infection could be observed in the endoscopically treated quarters. For QMP, no significant interaction between time and treatment could be observed throughout the 15-d follow-up period. Quarter SCC did not differ among treatments (control, endoscopy with antibiotics, and endoscopy without antibiotics). In conclusion, the endoscopic technique is suitable for examination and tissue biopsy collection of the bovine mammary gland cisterns without major interference with QMP and quarter SCC.     

Short communication: Association between udder health status and blood serum proteins in dairy cows

The aim of this study was to investigate the association between udder health (UH) status and blood serum proteins (i.e., total protein, albumin, globulin, and albumin-to-globulin ratio) in dairy cows. Blood and milk samples were collected from 1,508 cows of 6 different breeds (Holstein Friesian, Brown Swiss, Jersey, Simmental, Rendena, and Alpine Grey) that were housed in 41 multibreed herds. Bacteriological analysis was performed on milk samples with somatic cell count (SCC) >100,000 cells/mL and bacteria identification was confirmed by multiplex-PCR assays. Milk samples were grouped into 7 clusters of UH status: healthy (cows with milk SCC <100,000 cells/mL and not cultured); culture-negative samples with low, medium, or high SCC; and culture-positive samples with contagious, environmental, and opportunistic intramammary infections. Data of blood serum proteins were analyzed using a linear mixed model that included the fixed effects of stage of lactation, parity, breed, herd productivity (high or low production) and UH status, and the random effect of herd-date within herd productivity. Culture-negative samples with high milk SCC, which were most likely undergoing a strong inflammatory response and whose pathogens could not be isolated because they were engulfed by macrophages or because they had already cleared, and milk samples infected by contagious and environmental bacteria were associated with greater globulin concentrations (and lower albumin-to-globulin ratio) in blood. Variation in blood serum proteins seems to be associated with inflammatory status rather than infection, as serum globulin significantly increased in UH status groups with the highest milk SCC and no differences were observed among intramammary infections pathogens. Blood serum proteins can be a mammary gland inflammation indicator, but cannot be used to differentiate among different UH status groups.     

Compensatory milk production within the bovine udder: effects of short-term non-milking of single quarters

Daily quarter-milk yields of eight high-yielding cows (24-28 kg/d) and eight heifers (14-24 kg/d) were measured to examined to examined compensatory changes in milk production between quarters within an udder. Either one, two or three quarters per cow were left unmilked for 12 d, in early or late lactation, and then all quarters of all cows were milked normally for a further period of 12 d. Concentrate feeding levels were constant throughout the experiment. The mean daily yield per cow fell to 26, 59, and 75% during the period of milking either one, two or three quarters respectively. Twelve days after resumption of normal milking the total daily yield per cow was the same for cows continuously milked in two, three or all four quarters. Daily yield in cows with only one quarter milked continuously recovered to only 78% of the level of the pretreatment period. During the treatment period, the mean daily yield of the continuously milked quarters increased by almost 14% for cows milked in one quarter only, by more than 10% for two quarters milked, and by 4% per quarter if three quarters were milked. Milk yields of these quarters remained above their pretreatment levels when milking was resumed in the adjacent quarters. There was no difference in the compensatory effect between cows and heifers in early lactation, but the compensatory effects were lower in the lower in late lactation heifers.     

放射免疫法快速检测牛血清中四环素类药物

目的采用Charm Ⅱ放射免疫分析方法检测牛血清中四环素类药物残留。方法采集6个牛血清样品进行添加检测,设定控制点,样品结果与控制点比较。评价免疫反应体系的特异性和灵敏度。测得控制点后与样品结果比较,大于控制点则为阴性,小于控制点则为阳性。结果采用此方法短时间可出检测结果,四环素类检测限为20μg/kg。该方法特异性强,操作方法简单便捷。结论此方法作为初筛方法,测得阳性结果时则需采用其他检测方式进一步确证。该方法保持了家畜鲜活状态,减少了商家的损失,既能快速得到检测结果,又能达到检测牛血清中四环素类药物残留的要求。     

Folic acid complexes with human and bovine serum albumins

The interaction of folic acid with human serum (HSA) and bovine serum albumins (BSA) at physiological conditions, using constant protein concentration and various folic acid contents was investigated. FTIR, UV–visible and fluorescence spectroscopic methods as well as molecular modelling were used to analyse folic acid binding sites, the binding constant and the effect on HSA and BSA stability and conformations. Structural analysis showed that folic acid binds HSA and BSA via both hydrophilic and hydrophobic contacts with overall binding constants of K_{folic acid–HSA} = 8.1 (±0.5) × 10⁴ M⁻¹ and K_{folic acid–BSA} = 1.0 (±0.3) × 10⁵ M⁻¹. The number of bound acid molecules per protein was 1.7 (±0.4) for HSA and 1.5 (±0.3) for BSA complexes. Molecular modelling showed participation of several amino acids in folic acid–protein complexes stabilised by hydrogen bonding network. Folic acid complexation altered protein secondary structure by major reduction of α-helix from 59% (free HSA) to 35% (acid-complex) and 62% (free BSA) to 25% (acid-complex) with an increase in random coil, turn and β-sheet structures indicating protein unfolding. The results suggest that serum albumins might act as carrier proteins for folic acid in delivering it to target molecules.     

牛初乳的营养保健功能及其生物活性成分的开发利用

天然的牛初乳含有丰富的营养及生物活性成分,有很强的营养保健功能。介绍了天然牛初乳的营养成分和生物活性物质,列举了牛初乳的一些加工特性及目前国内外牛初乳产品的开发现状,阐述了在开发功能性牛初乳制品中存在的问题,展望了牛初乳功能性保健制品的未来发展前景。     

Relationship between somatic cell count,individual leukocyte populations and milk components in bovine udder quarter milk

The somatic cell count (SCC) in bovine milk is an indicator of udder health and milk quality. Individual cell populations, i.e., macrophages, lymphocytes and polymorphonuclear neutrophils, were identified and their relationship to milk components such as acute-phase proteins (serum amyloid A (SAA) and α₁-acid glycoprotein), lactic acid and lactoferrin (LF), as well as protein, fat and lactose, were studied in foremilk from separate udder quarters. The whey proteins and peptides were studied using two-dimensional gel electrophoresis, which was shown in the present study to be a useful method of assessing udder health and milk quality. The growth of a starter culture was evaluated using a conductance method as a simulation of fermentation processes relevant for cheese production. The investigation showed that LF, SAA, l-lactate and lactose are reliable indicators of milk quality on udder quarter level. The correlations between SCC, or the individual cell populations, and the potential indicators were highly significant.     

Ultrasonographic measurement of the bovine teat: breed differences, and the significance of the measurements for udder health

The objective was to measure teat canal length and diameter, teat diameter and teat wall thickness by ultrasonographic scanning in order to determine the differences in bovine breeds, and to study the influence of teat canal length and diameter on the occurrence of mastitis. A total of 269 lactating dairy cows of four different breeds (Brown Swiss, Simmental, Simmental crossbred with Red Pied, and Holstein-Friesians) from seven Upper Austrian dairy farms were examined. Average teat canal length of Brown Swiss animals was shortest (15.7 mm) followed by Holstein-Friesians (17.2 mm) and Simmental (18.3 mm). These differences in teat canal length were highly significant (P < or = 0.001). There was no significant difference in teat canal length between pure-bred and crossbred Simmentals. Differences of teat canal diameter between breeds were significant (P < or = 0.05). Brown Swiss animals had the largest diameters (2.0 mm) and Holstein-Friesians the smallest (1.7 mm). Differences in teat diameter between Brown Swiss, Holstein-Friesian and Simmental were also significant. No differences were found between the pure-bred and crossbred Simmental cows. The narrowest teats were in Holstein-Friesians and the widest in Simmental. Holstein-Friesians also exhibited the thinnest teat walls while the Simmental had the thickest ones. Teat canal length and diameter were correlated with udder health. Teat canals of healthy udders tended to be longer (17.4 mm) and narrower (1.8 mm) than teat canals of infected udders (15.8 mm, 2.1 mm; P < or = 0.001). A logistic regression model showed significant effects of teat canal length, teat canal diameter and lactation number on udder health.     

Genetics statistics and relationships of teat and udder traits, somatic cell counts, and milk production

Teat lengths and diameters, udder clearance, cleft depth, and distances between teats were measured from 1967 to 1982 on Holstein cows. Numbers of observations per trait ranged from 5934 to 11,449. Cows were from six herds owned by the North Carolina Department of Agriculture and two research herds of North Carolina State University. Repeatabilities between adjacent lactations for physical traits of udder were mostly between .60 and .80. Repeatabilities between first and second lactations for average somatic cell count and mean log somatic cell count were .11 +/- .04 and .21 +/- .04. Heritabilities in first lactation were .63 for front teat length, .44 for teat diameter, .56 for rear udder clearance, .10 for cleft depth, .32 for mature equivalent milk, and .18 for somatic cell counts. Heritabilities of distances between teats ranged from .33 to .48. Cows with udder clearance higher from the ground, deeper clefts, smaller distances between teats, and teats of smaller diameter had lower somatic cell counts. Multiple regression analysis showed that dams' somatic cell counts and rear udder clearances were not significant predictors of daughters' somatic cell counts. Lactation average somatic cell counts of daughters were predicted with greater accuracy from averages of somatic cell counts of multiple lactations of dams than from single lactations of dams. Milk production and somatic cell counts seemed positively genetically correlated, more so in first than later lactations.     

怀山药多糖及怀牛膝皂甙与蛋白质作用研究

利用紫外可见分光光度法对怀牛膝皂甙和怀山药多糖与牛血清白蛋白（BSA）的相互作用进行研究.结果表明,牛血清白蛋白的吸光强度随怀牛膝皂甙和怀山药多糖加入量的增大而增大,并且最大吸收峰位置发生红移.     

Change in udder health and overmilking

Removing milker units as soon as milk flow stopped was compared to milking for a fixed time of 12 min in an 8-wk trial with 60 cows. Teats were dipped in broth culture of approximately 10(9) colony forming units per ml of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis for 4 wk. More new infections of quarters occurred in the 12-min group. In trials 2 and 3, 20 additional cows were milked with automatic milking machine detachers and 20 cows were milked with a flow control unit for 12 min each trial. New infection rates were highest in the two groups milked for 12 min. Age for lactation and cows were associated with most of the variation in the new infection rate. Cows with pointed teat ends had the highest rate of new infection. Effects of fixed-time milking on increased mammary infection and clinical mastitis were small.     

食用色素苋菜红与牛血清白蛋白的相互作用研究

在模拟动物体生理条件和不同温度下,采用荧光、紫外光谱法来研究苋菜红(AMR)与牛血清白蛋白(BSA)结合反应。用Stern-Volmer和Lineweaver-Burk方程分别处理试验数据,发现BSA与AMR发生反应,生成新的复合物,属于静态荧光猝灭。实验求出反应时复合物的形成常数KLB、热力学参数与结合位点数,证明苋菜红与牛血清白蛋白的结合主要为静电作用力。位点竞争实验结果显示AMR与BSA的作用位置主要在BSA的Site I(sub-domain IIA)位。通过同步荧光光谱和三维荧光光谱法,探讨AMR对BSA构象的影响,实验结果表明,AMR使BSA酪氨酸残基和色氨酸残基所处微环境的极性减弱,疏水性增强。本文为探讨AMR的染色机理、毒理效应和生物学效应提供了重要信息。     

Investigating the cow skin and teat canal microbiomes of the bovine udder using different sampling and sequencing approaches

There is a need for standardized, efficient, and practical sampling methods to support large population-based studies of the internal and external epithelial microbiomes of the bovine udder. The primary objective of this study was to evaluate different sampling devices for the isolation of microbial DNA originating from the internal and external teat epithelium. Secondary objectives were to survey and compare the microbial diversity of external and teat canal epithelial microbiomes using amplicon and shotgun metagenomic sequencing approaches. To address these objectives, we enrolled a convenience sample of 24 Holstein dairy cows and collected samples from the external epithelium at the base of udder, the external teat barrel epithelium, the external teat apex epithelium, and the teat canal epithelium. Extracted DNA was quantified and subjected to PCR amplification of the V4 hypervariable region of the 16S rRNA gene and sequenced on the Illumina MiSeq platform (Illumina Inc., San Diego, CA). A subset of samples was subjected to a shallow shotgun metagenomic assay on the Illumina HiSeq platform. For samples collected from the external teat epithelium, we found that gauze squares consistently yielded more DNA than swabs, and Simpson's reciprocal index of diversity was higher for gauze than for swabs. The teat canal epithelial samples exhibited significantly lower diversity than the external sampling locations, but there were no significant differences in diversity between teat apex, teat barrel, and base of the udder samples. There were, however, differences in the microbial distribution and abundances of specific bacteria across external epithelial surfaces. The proportion of shotgun sequence reads classified as Bos taurus was highly variable between sampling locations, ranging from 0.33% in teat apex samples to 99.91% in teat canal samples. These results indicate that gauze squares should be considered for studying the microbiome of the external epithelium of the bovine udder, particularly if DNA yield must be maximized. Further, the relative proportion of host to non-host DNA present in samples collected from the internal and external teat epithelium should be considered when designing studies that utilize shotgun metagenomic sequencing.