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.     

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.     

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。     

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.     

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.     

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.     

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.     

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.     

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。     

Location of Staphylococcus aureus within the experimentally infected bovine udder and the expression of capsular polysaccharide type 5 in situ

The objective of this study was to locate Staphylococcus aureus in the bovine udder and to investigate the expression of capsular polysaccharide type 5 (CP5) in situ in both the early and chronic stages of experimental intramammary S. aureus infections. Bovine udder tissue was obtained in early and chronic stages of intramammary infection; i.e., 24 to 96 h and 122 d after experimental intramammary infection with S. aureus Newbould 305. The presence and location of S. aureus was investigated by Gram staining of tissue sections. The expression of CP5 by S. aureus in situ was investigated by immunochemical staining of tissue sections with specific antibodies against CP5. Both in the early and chronic stages of infection, S. aureus was located within the lumen of alveoli or lactiferous ducts, in association with the epithelium, and within phagocytic cells. The staphylococci were mainly observed in clusters and often in the presence of polymorphonuclear neutrophils. Expression of CP5 by S. aureus was observed both in the early and chronic stages of infection. In general, CP5-positive S. aureus were located in alveoli and in association with the mammary epithelium. In the chronic infection, CP5-positive S. aureus were also located deep in the interstitial tissue. These results indicate that--both in early and chronic stages of experimental S. aureus mastitis--colonization of the mammary epithelia and invasion into the interstitial tissue occurs and that CP5 is expressed by S. aureus Newbould 305 in situ. The invasion of S. aureus in the interstitial tissue and the expression of CP5 probably help the bacteria to withstand the host defense mechanisms.     

Functional properties of proteins: Possible relationships between structure and function in foams

Proteins via interactions with other components in food systems perform several desirable functions which are related to their structure and physico-chemical properties. In this paper the nature of food foams, the possible relationships between the molecular structure and the foaming capacity of different proteins, and factors affecting these are discussed.     

Effects of caffeic acid and bovine serum albumin in reducing the rate of development of rancidity in oil-in-water and water-in-oil emulsions

The antioxidant properties of caffeic acid and bovine serum albumin in oil-in-water and water-in-oil emulsions were studied. Caffeic acid (5 mmol/kg emulsion) showed good antioxidant properties in both 30% sunflower oil-in-water (OW) and 20% water-in-sunflower oil emulsions (WO), pH 5.4, during storage at 50 °C. Although bovine serum albumin (BSA) (0.2%) had a slight antioxidant effect, the combination of caffeic acid and BSA showed a synergistic reduction in the rate of development of rancidity, with significant reductions in concentration of total volatiles, peroxide value (PV) and p-anisidine value (PA) for both emulsion types. The synergistic increase in stability of the OW and WO emulsions containing BSA and caffeic acid was 102.9% and 50.4% respectively based on total oxidation (TOTOX) values, which are calculated as 2PV + PA, with greater synergy calculated if based on formation of headspace volatiles. The OW emulsion was more susceptible to the development of headspace volatiles by oxidation than the WO emulsion, even though the degree of oxidation assessed by the TOTOX value was similar.     

Activation of immune cells in bovine mammary gland secretions by zymosan-treated bovine serum

Mastitis, caused by bacterial infection of the mammary gland, is a major disease of dairy cattle. The greatest risks of intramammary infection occur at the end of lactation and at the initiation of the next lactation when the cow calves. Treating serum with zymosan (yeast cell wall preparation) causes the complement to cleave, allowing this serum to serve as a source of complement fragment 5a (C5a), a potent chemoattractant and activator of the immune system. Our hypothesis was that intramammary infusion of zymosan-treated serum (ZTS) would recruit polymorphonuclear neutrophils (PMN) and generate prolonged activity in lymphocytes within the mammary gland. Ultimately this could help prevent bacterial infections in cows at dry-off and at the initiation of lactation. Two ipsilateral quarters of the mammary gland of each cow were infused with ZTS (12.5 mL/quarter), and 2 contralateral quarters were infused with saline in 8 cows shortly after lactation ended. Mammary secretions were collected periodically throughout the dry period and the first 2 wk of the next lactation. Activation status of lymphocytes and PMN in those secretions was assessed based on the intracellular presence or absence of IFN-γ and IL-8 as determined by flow cytometry. The ZTS infusion greatly increased PMN numbers in mammary secretions for the first week only. The percentage of IFN-γ positive lymphocytes and PMN, and the percentage of IL-8 positive PMN, exhibited a sustained increase in secretions from ZTS-treated quarters through the first 2 wk of lactation. The ZTS can stimulate PMN and lymphocyte-mediated immune defense mechanisms in the mammary gland, which may provide a useful means of preventing new intramammary infections during the dry period as well as at the initiation of lactation.     

Interference of lipid with determination of bovine serum albumin by electroimmunodiffusion

Electroimmunodiffusion of ultra-high-temperature processed milk demonstrated an apparent increase in bovine serum albumin at some processing temperatures. The cause of the phenomenon was traced to the lipid component of the milk. Caproic acid was used in model studies to evaluate lipid effects in unheated and heated systems. Addition of caproic acid to bovine serum albumin without heat treatment reduced the apparent concentration of bovine serum albumin. Heating at 78 degrees C resulted in an initial increase in the apparent bovine serum albumin concentration, followed by a decrease with further heating. These results indicate that the reaction of specific antibody with bovine serum albumin in milk is inhibited by lipid and that this inhibition is reversed by heat treatments below that causing marked conformational changes in the protein.     

Bacterial flora associated with udder cleft dermatitis in Dutch dairy cows

Udder cleft dermatitis (UCD) or udder sores is a skin lesion, characteristically located around the anterior junction between the udder and abdomen of dairy cows. It is a worldwide problem in dairy herds with a large effect on animal welfare. The etiology and possible infectious origins of UCD are largely unknown; however, specific bacterial or parasitic causes are suggested in the literature. Therefore the aim of this study was to investigate the possible bacteriological, mycological, or parasitic involvement in clinically scored UCD lesions. Bacteriological culture was performed on subcutaneous tissue samples taken postmortem at a depth of 5 to 10 mm of 87 mild or severe UCD lesions or from the same place in healthy cows. Fungal culture was performed on a subset of 22 subcutaneous tissue samples of severe UCD postmortem. To investigate the superficial flora, swabs were taken from normal skin or skin lesions of 15 live animals equally divided over 3 groups: healthy skin or mild and severe UCD lesions. Histopathology, to describe and classify the lesions and to assess the presence of mites, fungi, or bacteria, was performed on 128 tissue samples, taken separately. In severe UCD lesions, Trueperella pyogenes and Bacteroides pyogenes were more frequently present in deep tissue layers and in superficial layers, compared with the same layers in mild UCD lesions or healthy skin. Culturing and histopathology indicated no sign of involvement of treponemes, Staphylococcus aureus, Escherichia coli, fungi, or mites in the UCD lesions. Histopathological examination showed that the majority of the lesions were characterized by chronic aspecific inflammation. Severe UCD lesions more frequently showed chronic active inflammation on histopathology, compared with mild UCD lesions. Due to the cross-sectional character of this study, it is difficult to differentiate cause and effect; however, future preventive and curative measures against UCD should take into account the chronic and anaerobic nature of this illness.     

Heritability of clinical mastitis incidence and relationships with sire transmitting abilities for somatic cell score, udder type traits, productive life, and protein yield

The objective of this study was to determine the relationships among daughter clinical mastitis during first and second lactations and sire transmitting abilities for somatic cell score, udder type traits, productive life, and protein yield. Data on clinical mastitis during first lactation were available for 1795 daughters (in six Pennsylvania herds, one Minnesota herd, and one Nebraska herd) of 283 Holstein sires. Data on clinical mastitis during second lactation were available for 1055 of these daughters. A total of 479 cows had 864 clinical episodes during first lactation, and 230 cows had 384 clinical episodes during second lactation. Clinical mastitis incidence and the total number of clinical episodes during each lactation were regressed on herd-season of calving (a classification variable), age at first calving, lactation length, and sire transmitting abilities taken one at a time. Linear effects, nonlinear effects, and odds ratios were estimated for sire transmitting abilities. Separate analyses were conducted on dependent variables that considered clinical mastitis from: all organisms, coagulase-negative staphylococci, coliform species, streptococci other than Streptococcus agalactiae, and the most common environmental organisms (coliform species and streptococci other than Streptococcus agalactiae). Heritability of clinical mastitis ranged from 0.01 to 0.42. Daughters of sires that transmit the lowest somatic cell score had the lowest incidence of clinical mastitis and the fewest clinical episodes during first and second lactations. Daughters of sires that transmit longer productive life, shallower udders, deeper udder cleft, and strongly attached fore udders had either fewer clinical episodes or lower clinical mastitis incidence during first and second lactations. The incidence of clinical mastitis and the number of clinical episodes per lactation may be reduced by selection for lower somatic cell score, longer productive life, shallower udders, deeper udder cleft, or strongly attached fore udders.