Dynamics of lingual antimicrobial peptide,lactoferrin concentrations and lactoperoxidase activity in the milk of cows treated for clinical mastitis

The aim of the present study was to examine changes in innate immune factors in the milk of mastitic dairy cows treated with antibiotics. Cows in the antibiotics group (n = 13) were infused into the mammary gland with cefazolin on the sixth day after mastitis was diagnosed (the day of the mastitis diagnosis = day ?6). The control group (n = 12) was not treated. Milk samples were collected once every 2 days from days ?6 to 12 and somatic cell count (SCC), lingual antimicrobial peptide (LAP), and lactoferrin (LF) concentrations and lactoperoxidase (LPO) activity were measured. SCC and LF concentrations in the antibiotics group markedly decreased after the antibiotic treatment. When cows in the antibiotics group were divided according to SCC on day 0, LAP concentrations and LPO activity in cows with a lower SCC on day 0 (<5 × 10⁶ cell/mL) were significantly higher and lower than those in cows with a higher SCC, respectively. These results suggest that LF concentration decreased with decrease in SCC after treatment and that LAP concentration and LPO activity differed depending on the severity of mastitis. This is the first report to reveal the dynamics of innate immune factor in milk of cows treated for clinical mastitis.     

Lingual antimicrobial peptide and lactoferrin concentrations and lactoperoxidase activity in bovine colostrum are associated with subsequent somatic cell count

The present study was undertaken to examine whether potential levels of innate immune factors (lingual antimicrobial peptide (LAP), lactoferrin (LF) and lactoperoxidase (LPO)) in colostrum are associated with subsequent milk somatic cell count (SCC) in dairy cows. Quarter milk samples were collected daily for 1 week postpartum to measure LAP and LF concentrations and LPO activity. SCC in milk was determined weekly for 2 months postpartum and its correlations to concentrations of LAP and LF and LPO activity were examined. Only small variations of all immune factors were found among four udders in each individual cow, whereas there were great differences in these factors among cows. Negative correlation was detected only between LPO activity and mean and maximum SCC, whereas its relationship was not significant. LAP and LF concentrations were significantly correlated positively to mean, maximum and minimum SCC. These results suggest that the great difference in innate immune factors among animals and high LAP and LF concentrations in colostrum may be associated with subsequent high incidence of SCC increase.     

Immunolocalization of lingual antimicrobial peptide (LAP) in the bovine mammary gland

Lingual antimicrobial peptide (LAP), a member of the β-defensin family in cows, is involved in the innate immune system and plays a crucial role in killing a large variety of microorganisms. The aim of the present study was to demonstrate the immunolocalization of LAP in the mammary glands of cows. A LAP antibody was raised in a rabbit by immunity with a synthetic 11 amino acid sequence out of a 42-amino acid sequence of the mature form of LAP. The specificity of the LAP antibody was checked using a competitive immunoassay and Western blotting. Paraffin sections of the mammary gland were immunostained with LAP antibody. In the competitive immunoassay, an increase of synthetic LAP concentration suppressed the optical density. Western blotting analysis for LAP revealed the presence of the LAP peptide in mammary alveolar tissue. When the mammary gland was immunostained with LAP antibody, epithelial cells of both infected and non-infected alveoli were immunopositive. These results indicate that LAP is localized in the epithelium of non-infected as well as infected alveolus in the mammary gland in cows.     

Regression tree analysis of the relationship between the concentrations of antimicrobial components and the microbiota of normal milk from dairy cows

The purpose of this study was to determine the concentrations of antimicrobial components (immunoglobulin A (IgA), lactoferrin (LF), lingual antimicrobial peptide (LAP), and S100A7) in normal milk and their relation to host factors (Age, somatic cell count (SCC), days in milk, richness, and alpha diversity of the milk microbiota) in dairy cows using multivariate regression tree analyses, and to clarify how the milk microbiota is related to the obtained results. Thirty normal milk samples were collected from a commercial dairy farm in June 2020. The thresholds that predicted the concentration of each antimicrobial component in milk were obtained by regression tree analysis, and the beta-diversity of the milk microbiota composition between groups divided according to each threshold was compared by an analysis of similarities test. The IgA and LF concentrations were mainly predicted by the SCC (177,500 and 70,000 cells/ml, respectively), and the LAP and S100A7 concentrations were predicted by Age (29.667 and 40.3 months, respectively). No relationship was observed between the concentration of IgA, LAP, or S100A7 and the milk microbiota composition between the groups divided by the threshold for prediction, but the milk microbiota composition was significantly different between the groups divided by the threshold for predicting the LF concentration. Our results indicated that the LF concentration in normal milk may be associated with the milk microbiota composition.     

Variation in somatic cell counts of milk samples from individual cows

Somatic cell counts of 2570 milk samples from 765 cows collected bimonthly from November 1975 to May 1976 were transformed to logarithmic values and analysed statistically. Components of variance were estimated as follows: Herds 0.033 (13 %), age groups 0.021 (8%), cows (within herds and age groups) 0.080 (31%), months 0.014 (6%), residual 0.107 (42%). Correction of actual cell counts for the influence of milk yield on the day of sampling led to only small changes in the magnitude of the various components. The coefficient of correlation between samples from the same cow was computed as 0.60 when the samples were from the same lactation, and 0.37 for samples from consecutive lactations.The proportionately small variation among herds as compared to the variation among cows of the same herd throws doubt on the efficiency of cell counting in samples of herd milk as a way of identifying cows with high cell counts.     

Potential of differential somatic cell counts as indicators of mastitis in quarter milk samples from dairy cows

Bacteriological status, somatic cell counts and proportions of lymphocytes, granulocytes and monocytes were determined in 1,659 quarter milk samples from 39 dairy cows. Discriminant analysis was performed in order to assess the ability of total and differential somatic cell counts and combinations of total somatic cell count and each of the differential cell counts, to discriminate between infected and pathogen-free quarters, as well as between quarters infected with minor pathogens and quarters infected with major pathogens. Total somatic cell count classified 82.9% of all quarters correctly with respect to bacteriological status. Differential somatic cell count was less effective than total somatic cell count in discriminating between infected and pathogen-free quarters, as well as between quarters infected with minor vs. major pathogens. Combination of total and differential somatic cell counts did not improve the rate of correctly classified quarters. Inclusion of demographic data into the discriminant function increased the number of quarters correctly classified, mainly through an increase in the proportion of correctly classified infected quarters.     

Seasonal variations in the concentration of antimicrobial components in milk of dairy cows

The incidence of bovine mastitis and the bulk milk somatic cell count (BMSCC) are influenced by season, which may be associated with innate immune functions, including antimicrobial components in mammary glands. Therefore, the present study was conducted to examine the effect of season on antimicrobial components in milk. Rectal temperature and plasma cortisol, thyroxine, and derivatives of reactive oxygen metabolites (d‐ROMs) were measured as stress parameters. Concentrations of lactoferrin (LF), lingual antimicrobial peptide (LAP), psoriasin (S100A7), and Immunoglobulin A (IgA) in milk were measured as indicators of innate immune function. LF and LAP concentrations were significantly lower in summer than in winter and spring, respectively, whereas the concentration of S100A7 was significantly lower in winter than in spring and autumn. The rectal temperature was significantly higher in summer than in other seasons, whereas plasma cortisol, thyroxine, and d‐ROMs did not exhibit any seasonal variation. In conclusion, even though stress parameters were not changed, the concentration of antimicrobial components, such as LF and LAP, decreased in summer, which may explain the frequent occurrence of mastitis during this season.     

Effects of systematic influences and intramammary infection on differential and total somatic cell counts in quarter milk samples from dairy cows

Effects of bacteriological status, stage of lactation, parity and season of sampling on differential and total somatic cell counts were estimated in quarter milk samples taken from 39 dairy cows. Log somatic cell count was affected by the bacteriological status of the quarter, as well as by the bacteriological status of adjacent quarters. Differential cell counts were affected by presence or absence of pathogens in the quarters themselves, but not by the bacteriological status of the adjacent quarters. Log somatic cell count was clearly affected by stage of lactation, due mainly to physiological variation, but possibly also accentuated by variation in infection rates throughout lactation. With the exception of early lactation, little physiological variation throughout lactation was detected for differential cell counts. Presence of infections seemed to have some indirect effect on trends throughout lactation as regards percentages of granulocytes and monocytes. Variation in somatic cell counts due to parity could be explained by variation in infection rates, rather than being physiologically determined.     

Dietary factors associated with milk somatic cell counts in dairy cows in Brittany, France

A survey (Enquête Ecopathologique Bretagne) was conducted in France for 4 years (1986–1990) in 48 commercial dairy herds (25–80 cows per herd) located in Brittany. Three groups of herd-years were made up to study peripartum dietary factors associated with milk somatic cell counts in the early lactation period (ECC). The first group included 20 herd-years with high ECC (over 400 000 cells ml⁻¹). The second group included herdyears with medium ECC (n = 20, median ECC 222 000) and the last group herd-years with low ECC (n = 20, median ECC 95 000). Herd data (components of the diet, milk yield and reproduction parameters, clinical diseases, biochemical and hematological indicators, body and dirtiness scores) were analyzed using discrimination by barycentric analysis. Two separate analysis were conducted according to study period (LG period, last 60 days of gestation; EL period, first 60 days of lactation).

The high ECC group had the following characteristics: (1) higher plasma gamma glutamyl transferase levels in the LG period; (2) higher quantities of cereal-based concentrates in the diet in the EL period; (3) shorter durations of feeding with Italian rye grass silage in the EL period. The relevance of the risk factors for high ECC are discussed with reference to dietary linolenic acid/linoleic acid ratio (through the synthesis of leukotrienes as chemotactic agents for the polymorphonuclear leukocytes), dietary energy supplies and liver fluke infestation in the herds. We speculate that nutritional factors could modulate the leukocyte activity in the milk and that supplementations with certain polyunsaturated fatty acids would be able to prevent the development of inflammation in the udder.     

Differential immunolocalization between lingual antimicrobial peptide and lactoferrin in mammary gland of dairy cows

Lingual antimicrobial peptide (LAP), one of the β-defensins in bovines, and lactoferrin (LF) are synthesized in mammary epithelium and have bactericidal and bacteriostatic functions. However, it is not known whether they have similar expression patterns. Therefore, the present study was undertaken to compare (1) immunolocalization of LAP and LF in the mammary gland and (2) changes in concentration of these two components in milk after lipopolysaccharide (LPS) challenge. Bovine mammary tissues without LPS challenge were collected and their sections were immunostained with antibodies to LAP or LF. Milk from our previous study was collected every hour up to 12h and twice daily from d 1 to 7 after LPS challenge (the day of infusion was considered as d 0). These milk samples were measured for LAP but not LF in our previous report. Therefore, concentration of LF was measured by enzyme immunoassay in the present study. Epithelial cells of some alveoli showed immunopositive reaction for LF, but negative for LAP. Conversely, some alveoli were LAP positive in their epithelial cells but LF negative. Many alveoli had immunoreactions for neither LAP nor LF. The concentration of LAP in milk was elevated significantly at 3h after LPS infusion compared with pre-infusion values and remained at a high level until 12h. However, LF concentration in milk remained low at d 0 and increased at d 2. These results suggest that LAP and LF were mostly differentially localized in the alveolar epithelium in mammary glands. The different spatial expressions between them may be associated with their different temporal expression mechanisms.     

低酸度酒精阳性乳患牛乳汁蛋白组分及氨基酸含量变化

采用聚丙烯酰胺凝胶电泳和酸水解法对低酸度酒精阳性乳患牛乳汁中蛋白组分及17种氨基酸含量进行了测定。结果显示,酒精阳性乳患牛乳汁中总蛋白、SA、lgG-H、-αLG、-βLG相对百分含量无显著变化(P0.05),tCN相对百分含量显著降低(P0.05);在酪蛋白中,-αCN相对百分含量无显著变化(P0.05),-βCN相对百分含量上升(P0.05),-κCN相对百分含量极显著下降(P0.01);17种氨基酸中,Cys含量极显著下降(P0.01),而Arg含量极显著上升(P0.01),Leu含量亦呈上升趋势(P0.05)。结果表明,酒精阳性乳患牛乳汁中主要蛋白组分及氨基酸的含量均发生了变化,乳汁中酪蛋白微球的稳定性遭到了严重破坏,乳汁胶体的稳定性降低,导致酒精试验呈阳性反应。     

Transport stress increases somatic cell counts in milk, and enhances the migration capacity of peripheral blood neutrophils of dairy cows

The present study was designed to determine the effects of physiological stress on milk-somatic cell counts (SCC) and function of bovine peripheral blood leukocytes (PBL). Nine healthy lactating cows were used in the examination. Five cows were transported 100 km for 4 hr (transported group; TG), and 4 cows were penned (non-transported group; NTG). Blood and milk samples were collected at 0, 2, and 4 hr after loading, and at 2 hr, and 1, 2, 3, and 6 days after unloading. The following activities were measured: adhesion receptor (CD 18 and L-selectin) expression of neutrophils and monocytes, migration capacity and percentage of apoptotic cells of neutrophils, serum soluble L-selectin (sL-selectin), plasma cortisol, and SCC. A significant increase in plasma cortisol and milk SCC was observed in TG. Leukocytosis, derived from neutrophils was recorded in TG, and was indicated by apoptotic measurement as an increase of young cells from the marginal pool. Increased migration and decreased surface expression of both L-selectin and CD 18 in neutrophils were observed after transportation. Elevated serum sL-selectin was also noted as a result of transportation. The present study indicated that transport stress modulates peripheral blood neutrophil function, particularly enhancing migration capacity, and causes diapedesis across the mammary epithelium. Increased milk SCC in transported cattle might be due to these phenomena, and severe physiological stress may bring about an increase in SCC in milk.     

中国鲎(Tachypleus tridentatus)基因工程抗菌肽的制备及其抗菌活性

从中国鲎血细胞RT-PCR扩增抗菌肽tachyplesins基因,经改造构建原核和真核表达载体pET-KRN、pPIC9-KRY,并分别在大肠杆菌DE3和毕赤酵母GSll5中诱导表达.用Tris-Tricine SDS-PAGE检测并进一步电洗脱纯化表达产物,经体外生物活性测定表明,表达产物KRN对大肠杆菌、金黄色葡萄球菌、枯草杆菌、伤寒沙门菌的抑菌环为25、22、25、16mm;MIC值为3.57、7.14、3.57、14.28mg/L.表达产物KRY对大肠杆菌、金黄色葡萄球菌、枯草杆菌、伤寒沙门菌和李氏杆菌的抑菌环为26、22、25、18、16mm;MIC值为1.57、3.14、1.57、6.28、6.28mg/L,对水霉的抑菌效果尤其显著,抑菌环达35mm.     

康贝防治奶牛隐性乳房炎疗效观察及对乳汁体细胞数的影响

奶牛乳房炎的研究至今已有100多年的历史,虽然取得一些令人可喜的成果,但奶牛乳房炎仍是当今奶牛场的常发病和难以防治的疾病之一.特别是奶牛隐性乳房炎,由于无明显的临床症状,隐蔽性强,发病率高,主要影响奶牛泌乳量和乳汁的质量,造成严重损失,是影响世界乳业发展的重要因素之一.奶牛隐性乳房炎在我国发病率较高,据笔者调查表明,河北省部分地区奶牛隐性乳房炎发病率高,乳区阳性率为40.1%,头阳性率为68.45%,这与其他学者报道相符[1、2、3].自1945年抗生素应用到兽医临床上以来,奶牛乳房炎的防治有了重大突破.但是由于抗生素的滥用,致使病原抗药性增强,且高药物残留等问题一直是困扰兽医工作人员的难题.因此寻求一种高效、绿色、简便易行的防制奶牛隐性乳房炎的方法一直是兽医科技人员的研究热点.康贝是一种生物活性制剂,不含有任何激素及抗生素.本试验的目的在于试验研究康贝对奶牛隐性乳房炎的防治作用,试验包括康贝防治奶牛隐性乳房炎疗效观察、康贝对奶牛血液流变学指标的影响及康贝清除氧自由基试验等内容,现将康贝防治奶牛隐性乳房炎疗效及对乳汁体细胞的影响做一初报,其他内容另文发表.     

体细胞数对牛奶品质及产量的影响

根据宁夏地区某奶牛场的DHI检测获得的体细胞数据(SCS),利用SAS软件实现单因素方差分析,探讨不同乳中SCS对产奶量和乳中主要成分的影响,并对它们之间的相关性进行分析。结果表明:乳中SCS与日产奶量呈显著负相关(P0.01);与乳糖和胎次呈正相关(P0.01);乳中SCS水平与乳脂率、乳蛋白率和尿素氮没有明显的相关性。本试验通过对SCS的研究为乳品质的测定提供一定依据和参考。     

壳聚糖对奶牛产奶性能、乳体细胞数及血清免疫球蛋白的影响

试验旨在研究壳聚糖对奶牛产奶性能、乳体细胞数及血清IgG、IgM浓度的影响。试验选择12头胎次、体重、泌乳量、泌乳阶段相近(P>0.05)的成年荷斯坦奶牛,根据产奶量、产犊日期、胎次等相近的原则,采用配对分组试验,分为对照组和壳聚糖组,每组6头。对照组不添加壳聚糖,壳聚糖组每天每头牛饲粮中添加15g的壳聚糖。试验期为75d,前15d为预饲期,后60d为正试期。结果表明:在整个试验阶段,奶牛日粮中添加0.1%的壳聚糖可显著提高产奶量(P<0.05),对于乳成分含量没有明显影响,但是平均乳脂量有明显的增加趋势;日粮中添加0.1%的壳聚糖可以显著降低乳中体细胞数(P<0.05),对血清IgG、IgM浓度无明显影响。     

Controlled clinical trial of the effect of a homoeopathic nosode on the somatic cell counts in the milk of clinically normal dairy cows

Cows in a 250-cow Holstein-Friesian herd were allocated at random to be treated with either a homoeopathic nosode or a negative control, both treatments being applied by means of an aerosol spray to the vulval mucous membranes. A total of six treatments were given over a period of three days and milk samples were taken for the determination of somatic cell counts (SCC) on days -3, 3, 7, 9, 14, 21 and 28. Individuals applying the treatments or carrying out the SCC determination were unaware of which animals were receiving which treatment. Owing to the wide natural variations in SCC, the trial had only a 71 per cent possibility of detecting a 30 per cent difference in SCC between the two groups. There were no significant differences between the SCC of the two groups on any sample day, but there were significant variations between the SCC on different days (P=0.003) in both groups.