奶牛乳房炎乳中结合珠蛋白含量的夹心ELISA检测方法研究

为准确检测奶牛乳房炎乳中结合珠蛋白(Hp)含量,本研究采用牛血红蛋白(Hb)包被酶标板捕获乳清中Hp,利用特异性抗血清和酶标抗体建立夹心ELISA方法,并对建立的方法进行优化,用于检测牛奶中Hp含量.结果表明,ELISA方法检测下限为0.08μg/mL,与其他几种常见乳房炎乳中急性期蛋白质无交叉反应,批内试验和批间试验变异系数分别为3.27%～4.98%和5.46%～8.31%.应用本实验建立的夹心ELISA方法和商品化试剂盒分别检测50份已知患乳房炎牛乳清中Hp含量,同时对检测结果做相关性分析.结果表明,夹心ELSIA方法与商品化试剂盒具有很好的相关性(R2=0.996 2),同时验证了乳清中体细胞数与Hp含量呈正相关.夹心ELISA方法可准确测定牛奶中Hp含量,并可为奶牛乳房炎的诊断提供一种简易有效的辅助手段.     

隐性乳房炎患牛乳汁pH值变化与体细胞数关系的研究

采集奶牛乳样604份, 测定乳汁pH值与体细胞的含量, 确定奶牛隐性乳房炎与牛乳pH值的变化关系。结果表明, 牛乳pH值的变化与体细胞的含量呈正相关。正常乳汁的pH值在6 4~6 6之间; 体细胞在20万~50万/mL区间时, 奶牛乳汁pH值在6 6~6 8之间; 体细胞在50~150万/mL区间时, 奶牛乳汁pH值在6 8~7 0之间; 体细胞在150万~500万/mL区间时, 奶牛乳汁pH值在7 0~7 2之间; 体细胞大于500万/mL时, 奶牛乳汁pH值在7 2以上。     

"乳源康"治疗奶牛乳房炎药效学研究

"乳源康"是新型中草药注射剂产品,是由提取、分离并经鉴定的中草药经科学组方而成.体外抑菌试验结果表明,"乳源康"对奶牛乳房炎常见的8种病原菌均有一定的抑菌作用,对G 菌效果更好,总体抑菌效果与环丙沙星相当,优于青、链霉素.应用"乳源康"采用乳房给药在兰州各大奶牛场试用3年,共收治各类型临床乳房炎病牛1 638头次,2 203乳区,平均治愈率为93.3%,平均疗程4.5 d,与临床常用的青、链霉素相比差异非常显著(P＜0.01).     

奶牛临床型乳房炎Nisin抗菌肽治疗后日产奶量及主要乳成分的变化

选取临床型乳房炎自然发病病例9头,乳房内灌注乳酸链球菌素(Nisin)进行治疗,通过检测日产奶量以及停药后2,7,14,21 d的主要乳成分指标,确定Nisin乳房灌注剂对临床型乳房炎病例泌乳性能的影响情况。结果显示,乳房内灌注Nisin后患病乳区乳腺组织得到一定程度的修复,日产奶量逐渐恢复,停药后8 d的日产奶量与发病前6 d相比,平均降幅2.5 kg。治愈后患病乳区牛奶乳脂肪、乳蛋白、乳糖和非脂乳固体含量都呈现上升趋势,但与同期相比,略低于非用药乳区混合牛奶;其中,乳糖含量增加幅度较快,除停药后2 d的治疗乳区外,乳糖含量均在4.7%以上,提示奶牛乳房炎病例经Nisin乳房灌注治疗后,受损乳腺组织修复较快,乳腺上皮细胞合成乳成分的能力大幅度提高。由此可见,Nisin对奶牛临床型乳房炎具有良好的治疗作用。     

Neutrophil recruitment in endotoxin-induced murine mastitis is strictly dependent on mammary alveolar macrophages

Mastitis, inflammation of the mammary tissue, is a common disease in dairy animals and mammary pathogenic Escherichia coli (MPEC) is a leading cause of the disease. Lipopolysaccharide (LPS) is an important virulence factor of MPEC and inoculation of the mammary glands with bacterial LPS is sufficient to induce an inflammatory response. We previously showed using adoptive transfer of normal macrophages into the mammary gland of TLR4-deficient C3H/HeJ mice that LPS/TLR4 signaling on mammary alveolar macrophages is sufficient to elicit neutrophil recruitment into the alveolar space. Here we show that TLR4-normal C3H/HeN mice, depleted of alveolar macrophages, were completely refractory to LPS intramammary challenge. These results indicate that alveolar macrophages are both sufficient and essential for neutrophil recruitment elicited by LPS/TLR4 signaling in the mammary gland. Using TNFα gene-knockout mice and adoptive transfer of wild-type macrophages, we show here that TNFα produced by mammary alveolar macrophages in response to LPS/TLR4 signaling is an essential mediator eliciting blood neutrophil recruitment into the milk spaces. Furthermore, using the IL8 receptor or IL1 receptor gene-knockout mice we observed abrogated recruitment of neutrophils into the mammary gland and their entrapment on the basal side of the alveolar epithelium in response to intramammary LPS challenge. Adoptive transfer of wild-type neutrophils to IL1 receptor knockout mice, just before LPS challenge, restored normal neutrophil recruitment into the milk spaces. We conclude that neutrophil recruitment to the milk spaces is: (i) mediated through TNFα, which is produced by alveolar macrophages in response to LPS/TLR4 signaling and (ii) is dependent on IL8 and IL1β signaling and regulated by iNOS-derived NO.     

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.     

Plasma haptoglobin and immunoglobulins as diagnostic indicators of deoxynivalenol intoxication

This study aimed to discover potential biomarkers for dioxynivalenol (DON) intoxication. B6C3F1 male mice were orally exposed to 0.83, 2.5 and 7.5 mg/kg body weight (bw) DON for 8 days and the differential protein expressions in their blood plasma were determined by SELDI - Time-of-Flight/Mass Spectrometry (TOF/MS) and the immunoglobulins (Igs) G, A, M and E in the serum were investigated. 11.7 kDa protein was significantly highly expressed according to DON administration and this protein was purified by employing a methyl ceramic HyperD F column with using optimization buffer for adsorption and desorption. The purified protein was identified as a haptoglobin precursor by peptide mapping with using LC/Q-TOF/MS and MALDI-TOF/MS and this was confirmed by western blotting and ELISA. IgG and IgM in serum were decreased in a dose-dependent manner and IgA was decreased at 7.5 mg/kg bw DON administration, but the IgE level was not changed. To compare the expressions of haptoglobin and the Igs patterns between aflatoxin B1 (AFB1), zearalenone (ZEA) and DON intoxications, rats were orally administered with AFB1 1.0, ZEA 240 and DON 7.5 mg/kg bw for 8 days. Haptoglobin was increased only at DON 7.5 mg/kg bw, while it was slightly decreased at ZEA 240 mg/kg bw and it was not detected at all at AFB1 1.0 mg/kg bw. IgG and IgA were decreased by DON, but IgG, IgA, IgM and IgE were all increased by AFB1. No changes were observed by ZEA administration. These results show that plasma haptoglobin could be a diagnostic biomarker for DON intoxication when this is combined with examining the serum Igs.     

头孢噻呋治疗隐性乳房炎对乳汁中主要致病菌区系的影响

采用细菌分离鉴定和高通量测序技术,比较分析了头孢噻呋治疗隐性乳房炎对乳汁中主要致病菌区系的影响。结果:经5～10 d的乳头灌注头孢噻呋治疗,有效率为68. 75%(11/16),治愈率仅为18. 75%(3/16)。无乳链球菌、酵母菌和金黄色葡萄球菌是治疗前乳汁中的主要致病菌,分别占分离菌株总数的33. 33%(6/18)、27. 78 (5/18)%和16. 67%(3/18);治疗后乳汁中主要致病菌是酵母菌,比例为58. 33%(7/12),其次是大肠杆菌(16. 67%)和其他链球菌(16. 67%)。治疗前后乳汁中主要真菌组成差异不显著,且假丝酵母属(Candida,63. 01%)、Meyerozyma属(25. 15%)和德巴利酵母属(Debaryomyces,5. 73%)是主要的3种真菌属。因此,在该奶牛场使用头孢噻呋治疗慢性隐性乳房炎效果并不理想,原因可能是抗菌治疗后真菌特别是酵母菌的大量繁殖。     

Test-day somatic cell score, fat-to-protein ratio and milk yield as indicator traits for sub-clinical mastitis in dairy cattle

Test-day (TD) records of milk, fat-to-protein ratio (F:P) and somatic cell score (SCS) of first-lactation Canadian Holstein cows were analysed by a three-trait finite mixture random regression model, with the purpose of revealing hidden structures in the data owing to putative, sub-clinical mastitis. Different distributions of the data were allowed in 30 intervals of days in milk (DIM), covering the lactation from 5 to 305 days. Bayesian analysis with Gibbs sampling was used for model inferences. Estimated proportion of TD records originated from cows infected with mastitis was 0.66 in DIM from 5 to 15 and averaged 0.2 in the remaining part of lactation. Data from healthy and mastitic cows exhibited markedly different distributions, with respect to both average value and the variance, across all parts of lactation. Heterogeneity of distributions for infected cows was also apparent in different DIM intervals. Cows with mastitis were characterized by smaller milk yield (down to -5 kg) and larger F:P (up to 0.13) and SCS (up to 1.3) compared with healthy contemporaries. Differences in averages between healthy and infected cows for F:P were the most profound at the beginning of lactation, when a dairy cow suffers the strongest energy deficit and is therefore more prone to mammary infection. Residual variances for data from infected cows were substantially larger than for the other mixture components. Fat-to-protein ratio had a significant genetic component, with estimates of heritability that were larger or comparable with milk yield, and was not strongly correlated with milk and SCS on both genetic and environmental scales. Daily milk, F:P and SCS are easily available from milk-recording data for most breeding schemes in dairy cattle. Fat-to-protein ratio can potentially be a valuable addition to SCS and milk yield as an indicator trait for selection against mastitis.     

Reliability in somatic cell count measurement of clinical mastitis milk using DeLaval cell counter

Somatic cell counts (SCC) measurements are typically performed using quantitative methods, such as the Breed method (Breed) and the Fossomatic method (FSCC). The DeLaval cell counter (DCC) developed recently is a quantitative somatic cell counter with a low initial cost and superior portability. However, since the DCC was specifically developed for measuring SCC of ≤ 4 × 10⁶ cells/mL milk from bulk tanks or individual cows, its reliability for estimating SCC that exceed this concentration has not yet been clarified. This study therefore examined whether it is possible to accurately measure SCC by diluting milk samples with initial SCC of 4 × 10⁶ cells/mL, as seen in clinical mastitis milk. We collected milk samples from 99 quarters of 99 Holstein cows with clinical mastitis. These milk samples were diluted 10‐fold with saline and thoroughly mixed before performing SCC measurement with the DCC. The correlation coefficients of SCC measured by the FSCC, Breed and DCC methods indicated strong correlations between each pair of methods. The findings showed that DCC can be used to identify bovine clinical mastitis milk and is useful as a quantitative SCC measurement device on farm sites.     

21种中药对奶牛乳房炎3种病原菌的体外抑菌试验

采用琼脂平板扩散法 (打孔法 ) ,选择连翘、金银花、黄芩、黄柏、鱼腥草等 2 1种中草药对从奶牛乳房炎分离出的 3种主要致病菌进行了体外抑菌试验。结果发现 ,诃子、黄芩、石榴皮等的综合效果好 ,从而为中草药防治奶牛乳房炎的科学配方筛选提供了依据     

Influence of mastitis metritis agalactia (MMA) on bone and fat metabolism

Mastitis metritis agalactia (MMA) is a common disease in post-partum sows and has a negative effect on sows’ longevity as well as on sows’ and piglets’ health. MMA leads to an inflammatory state. The aim was to investigate the impact of MMA on bone and fat metabolism. The hypothesis was that it is possible to predict MMA by measuring ketone bodies and bone markers. Blood samples from 175 sows were taken within 72 hr after farrowing. Serum was analysed for 25-hydroxyvitamin D (25-(OH)-D), serum crosslaps (CTX-I), osteocalcin (OC), alkaline phosphatase (ALP), calcium (Ca), magnesium (Mg), phosphorus (P), parathyroid hormone (PTH), triglycerides (TG), beta-hydroxybutyric acid (BHB), tumour necrosis factor-alpha (TNF-α) and haptoglobin. Spontaneous urine was collected, and pH value was measured in addition to Ca and P. A proximate analysis of the sows’ diets was performed. Age, litter size, body condition score (BCS) and clinical signs of MMA were recorded for each sow. A multivariable logistic regression was undertaken with disease status (MMA or healthy) as the dependent variable. Significance was accepted at p < .05. MMA sows had a poorer BCS (p < .001) in relation to healthy sows. Age and number of piglets did not differ. MMA sows showed increased serum CTX-I (p = .004) and decreased serum OC (p < .001). Concentrations of P (p = .007), activity of ALP (p = .002) and BHB (p = .019) as well as TNF-α (p < .001) and haptoglobin (p = .048) concentrations were increased in MMA sows. No difference in urinary pH value between MMA and healthy sows was found. Our results are in accordance with the known fact that sows are in an extreme catabolic state peripartum. Bone metabolism in MMA sows is much more negatively affected than in healthy sows post-partum, due to inflammatory processes shown by higher concentrations of pro-inflammatory cytokines.     

An improved method for the study of equine haptoglobin heterogeneity

Equine serum haptoglobin was separated by polyacrylamide gel isoelectric focusing and visualized by protein staining or Western blotting. Conventional protein staining revealed up to three bands in the pI range 4.17 to 4.44. The blotting technique, however, showed an anodal group of 8 to 10 bands with a pI range of 4.11 to 4.52 and a cathodal group of 4 to 6 bands with a range of 4.55 to 5.14. The blotting method revealed that equine haptoglobin migrates outside the prealbumin area, in contrast to previous reports.     

Subclinical mastitis causes alterations in nitric oxide, total oxidant and antioxidant capacity in cow milk

The aim of this study was to investigate total antioxidant (TAC), and oxidant capacity (TOC) and nitric oxide (NO) levels in milk of cows with subclinical mastitis. Brown Swiss and Holstein breed cows were screened with California Mastitis Test (CMT) to determine mammary glands with subclinical mastitis. Moreover, somatic cell counts (SCC) were determined electronically in all milk samples. Mammary quarters were classified as healthy (n = 25) or subclinical mastitis (n = 35) based on CMT scores and somatic cell count (SCC: ?200,000/ml or >200,000/ml) in milk. Nitric oxide, TOC and SCC levels were significantly higher (p < 0.001, p < 0.005 and p < 0.001, respectively) in milk from mammary quarters with subclinical mastitis compared to those from healthy mammary quarters. In conclusion, subclinical mastitis results in higher NO concentrations, TOC and SCC, and NO and TOC were positively correlated with SCC. Moreover, alterations in NO levels and TOC in milk could be used as an alternative diagnostic tool to screen for subclinical mastitis.     

原料乳和临床乳房炎金黄色葡萄球菌毒力基因检测及药敏分析

对临床乳房炎（57株）和原料乳（44株）金黄色葡萄球菌菌株,用PCR方法检测mecA基因、PVL基因、ETs基因、SEs基因和TSST-1基因;采用CLSI指导说明执行琼脂稀释法药敏性试验。结果显示原料乳菌株中,84.09%携带有毒素基因,其中PVL的检出率为84.09%,肠毒素的检出率为52.27%,主要流行的肠毒素基因为sea（56.82%）,均未检测到携带mecA、ETs、TSST-1、sei和sej基因的菌株;同时得到10种毒素基因型,其主要流行的毒素基因型为PVL＋sea（29.55%）和PVL（27.27%）。临床菌株中,78.95%携带有毒素基因,其中PVL的检出率为28.07%,肠毒素的检出率为77.19%,主要流行的肠毒素基因为sea（47.37%）,没有检测到携带ETs、TSST-1和seh基因菌株;同时得到25种毒素基因型,其主要流行的毒素基因型为sea（19.30%）,其次是seb（7.02%）,sea＋sed＋sej（3.51%）和PVL＋sea＋seb＋sec＋seg＋sei（3.51%）。6株（10.53%）携带有mecA基因菌株均含有较多毒素基因。原料乳分离株对甲氧苄啶和头孢西丁的耐药率较高,分别为100%和86.36%,其次对氯霉素、红霉素、苯唑西林、头孢哌酮和庆大霉素的耐药率分别为11.36%、4.55%、2.27%、2.27%和6.82%,所有原料乳菌株均对环丙沙星敏感,同时得到8种耐药谱,多重耐药率达22%;临床乳房炎菌株对红霉素和甲氧苄啶的耐药率较高,分别为100%和71.93%,其次对氯霉素、庆大霉素、环丙沙星、头孢西丁和苯唑西林的耐药率分别为28.07%、26.07%、24.56%、19.30%和7.02%,临床乳房炎菌株对头孢哌酮和四环素的敏感率为100%,同时得到13种耐药谱,多重耐药率达77.19%。所有原料乳和临床乳房炎菌株均对万古霉素和阿米卡星敏感。临床乳房炎菌株携带的毒素基因和多重耐药率比原料奶菌株高,同时在临床乳房炎乳中检测到MRSA菌株,提示我们应加强乳及其乳制品的管理,并对奶牛乳房炎加以重视。     

Concentrations of haptoglobin in bovine plasma determined by ELISA or a colorimetric method based on peroxidase activity

The objective was to compare different procedures for determination of haptoglobin in bovine plasma. Nine Angus steers were vaccinated against Mannheimia haemolytica to stimulate an acute‐phase response. Blood samples were collected immediately prior to vaccination (day 0), and on days 1, 3, 5, 7 and 10. Plasma samples were frozen in duplicates at ?80 °C. One set of the duplicates was analysed for haptoglobin concentrations using a commercial ELISA kit. A day effect was detected (p < 0.01) because haptoglobin peaked on day 3 and returned to baseline on day 7 relative to vaccination. The second duplicate was analysed using a procedure that measures haptoglobin–haemoglobin complexing by estimating differences in peroxidase activity (CPPA) with results expressed as optical density. Further, based on the ELISA results, the plasma sample with the greatest haptoglobin concentration was also serially diluted into a plasma sample with negligible haptoglobin concentration from the same steer (1:1 through 1:1024 dilution). These dilutions were used within the CPPA method to generate a standard curve and estimate plasma haptoglobin concentrations (CPPA + STD). A linear standard curve was generated (r² = 0.99). A day effect similar to the ELISA method was detected for the CPPA and CPPA + STD methods (p < 0.01). Results obtained from CPPA and ELISA methods were positively correlated (r = 0.97; p < 0.01). The values generated by the CPPA + STD procedure were similar (p = 0.38) compared to the values generated by the ELISA method. In conclusion, assessing concentrations of haptoglobin in bovine plasma using the CPPA and CPPA + STD methods generate highly correlated or similar results, respectively, compared to ELISA. Therefore, the CPPA + STD and CPPA methods can be used as a less expensive alternative to ELISA to determine concentrations or monitor changes in plasma haptoglobin in bovine samples.     

Gelatinase activities and haptoglobin concentrations in healthy and in degenerative articular cartilage of pigs

The objectives of this study were to investigate the activity of the matrix metalloproteinase (MMP)‐2 and MMP‐9 in healthy and in degenerative cartilage and to characterize the relation with the acute phase protein haptoglobin (HP) in articular cartilages of pigs. Joint surfaces of the proximal and distal humerus and femur of fattening pigs were histopathologically classified. In addition, cartilage homogenates and synovia were obtained. The tissue homogenates were analysed for gelatinase activity by zymography and by activity assay. The concentrations of HP in cartilage homogenates, in synovia and in serum were analysed by ELISA. High enzymatic activity of the MMP‐2 latent form was observed in zymography in all samples. Zymographic activities of MMP‐2 active form and MMP‐9 (active and latent form) were detected at low levels in some samples. Comparison of the zymographic activities of gelatinases in unaltered vs. altered cartilages yielded no differences. In contrast to zymography, cartilage homogenates were negative for MMP‐2 and MMP‐9 in the activity assays. The concentrations of HP in cartilage homogenates and in synovia from samples without alteration and from samples with massive alterations were not different. When classified according to their HP concentration, cartilage homogenates with increased HP concentrations had higher (p < 0.05) zymographic activities of the MMP‐2 active form. For the two MMPs investigated, there was no detectable relationship with degenerative processes in the cartilage.     

Exfoliation rate of mammary epithelial cells in milk on bovine mastitis caused by Staphylococcus aureus is associated with bacterial load

The exfoliation rate of mammary epithelial cells (MECs) in milk is affected by physiological, breeding and environmental factors. Little is known about the relationship between the MEC exfoliation into milk and mammary‐infected Staphylococcus aureus (S. aureus) load on bovine mastitis caused by S. aureus. The aim of this study was to investigate the relationship between S. aureus load and the proportion of MEC exfoliation in milk using five substantial bovine mastitis models. In 64 randomly extracted milk samples from udders at 3–21 days after S. aureus infusion, there were various samples with different numbers of S. aureus counts and somatic cell counts. No significant correlations were found between the S. aureus counts and somatic cell count (r = 0.338). In contrast, a significant correlation was noted between S. aureus counts and the proportion of cytokeratin‐positive cells in the milk from the infused udders (r = 0.734, P < 0.01). In conclusion, the increasing MEC exfoliation rate in milk from mastitis udders caused by S. aureus may contribute to reduced milk yield.