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.     

抗菌肽重组逆转录病毒载体的构建、重组病毒的包装及其在牛奶中的稳定表达

将含有双抗菌肽基因的表达盒插入到逆转录病毒载体pLN-bcp-LK(R)中,获得了含有双抗菌肽基因的重组逆转录病毒载体pLN-bcp-LfcinB-CE-MA(R),利用脂质体转染PA317包装细胞,G418进行筛选得到了阳性细胞克隆,克隆扩大培养后,用病毒上清感染NIH3T3细胞进行滴度测定,最高滴度为1×104CFU/mL,用病毒上清转染妊娠中后期奶牛乳腺组织,产犊后采集的奶样经琼脂板溶圈法和Tricine-SDS-PAGE检测,结果表明乳汁中表达的抗菌肽对大肠杆菌和金黄色葡萄球菌均具有明显地抑菌活性,并能实现相对稳定的表达,表达可持续30~45 d。     

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.     

Gram-negative bacterial infections of the mammary gland in cows

Naturally acquired gram-negative bacterial intramammary infections (n = 160) were studied in 99 cows over a 2-year period. Escherichia coli, Klebsiella spp, Serratia spp, Enterobacter spp, and unidentified gram-negative bacteria were isolated from 28.8, 39.4, 9.4, 5.0, and 11.2%, respectively, of infected mammary glands. A majority (61%) of intramammary infections were first detected during the nonlactating period. Gram-negative bacteria isolated during the first half of the nonlactating period were predominantly Klebsiella spp, Serratia spp, and Enterobacter spp. Onset of E coli intramammary infections was more prevalent during the second half of the nonlactating period and during the first 7 days of lactation. The majority (59%) of infections were less than 28 days in duration, but Klebsiella spp and Serratia spp infections were of significantly (P less than 0.05) greater duration than infections with E coli. The greatest percentage (47%) of gram-negative bacterial intramammary infections were first detected during the summer.     

Effects of colostrum whey on immune function in the digestive tract of goats

The objective of the present study is to examine whether colostrum whey can have an effect on immune function in goats digestive tract. Two milliliters of colostrum whey (colostrum group) or water (control group) were administrated orally to goats every day for 3 weeks. Blood was collected twice a week for 3 weeks to measure immunoglobulin A (IgA), interleukin 8 (IL‐8), and IL‐10. At the end of the experimental period, the parotid glands, oral mucosa, lingua, esophagus, jejunum, and ileum were collected for immunohistochemical detection of IgA, cathelicidin‐7, and S100A8. The ratio of the length of IgA‐positive mucosal surface in the esophagus to the total esophageal length was significantly greater in the colostrum group than in the control group. The number of IgA‐positive cells in the labial gland and ileum in the colostrum group was significantly higher than that in the control group. There were no significant differences between the colostrum and control groups in the number of cathelicidin‐7‐positive cells in the jejunum and ileum and in the number of S100A8‐positive cells in the lingua, jejunum, and ileum. These results suggest that colostrum stimulates the recruitment of plasma cells into the labial gland, which then secrete more IgA into the saliva.     

The innate immune response of the bovine mammary gland to bacterial infection

Intra-mammary (IM) bacterial infection in cattle can result in clinical outcomes that range from being acute and life-threatening to those that are chronic and sub-clinical. The typical bacteria involved in IM bacterial infections activate the mammary immune system in different ways which can influence the severity of the outcome. A clear understanding of the mechanisms that activate and regulate this response is central to the development of effective preventative and treatment regimes. This review focuses on the different immune responses of the bovine mammary gland to common mastitis-causing pathogens. There is special emphasis on comparing the responses to Escherichia coli and Staphylococcus aureus infections, as these are typically associated, respectively, with acute/severe and chronic/sub-clinical forms of the disease.     

LPS诱导牛乳房炎相关因子及蛋白变化研究进展

乳房感染导致的炎性反应,引起乳汁中的体细胞数量增加,并激活了乳汁中的细菌抑制酶和蛋白.在脂多糖(LPS)刺激时,肿瘤坏死因子α(TNF-α)、干扰素-1β(IFN-1β)和环加氧酶-2的表达增加.在LPS刺激后的3 h达到最高.乳铁转运蛋白、溶霉素、诱导一氧化氮合成酶、L-选择蛋白、NFκB和凋亡因子caspase-3、caspase-7及Fas(FS-7相关表面抗原)升高,在刺激后6 h达到峰值.而乳汁中的α-乳白蛋白和κ-CN表达量下降,同时导致乳产量和适合制作奶酪的产量下降.     

Low expression of the antibacterial factor L‐amino acid oxidase in bovine mammary gland

In the mouse, L‐amino acid oxidase (LAO) produces hydrogen peroxide by utilizing free amino acids and is a proven antibacterial factor in mammary glands. Mastitis, a bacterial infection of the mammary gland, is the most frequent disease in dairy cattle. Here, we investigate whether LAO is expressed in the mammary gland of dairy cattle and is antibacterial. In dairy cattle, the expression level of LAO mRNA in the mammary gland was considerably lower than that in mice, and LAO activity was not observed in cattle milk that produced hydrogen peroxide. The expression of LAO mRNA was also low in Japanese Black cattle, the same as in Holstein cattle. A higher LAO mRNA expression was observed in the mastitis glands than in the lactating glands. Furthermore, spleen and lymph nodes expressed high levels of LAO mRNA in dairy cattle. We conclude that mammary glands in dairy cattle have lower ability to express the LAO gene compared to that in mice, which may result in a high incidence of mastitis.     

Effect of milk stasis on Brucella abortus infection of the mammary gland in goats

To compare the effects of milk stasis and milk flow on Brucella abortus infection of the mammary gland under the same systemic conditions, primiparous goats (n = 5) were inoculated IV with B abortus on the day of parturition, and suckling by their neonates was restricted to one mammary gland. Goats were euthanatized and necropsied at 3 weeks after inoculation, and milk, mammary glands, and supramammary lymph nodes were evaluated by bacteriologic, histologic, and immunoenzymatic staining techniques. Nonnursed mammary glands had high titers of brucellae in milk, moderate interstitial mastitis, and brucellar antigen in macrophages located primarily in alveolar and ductal lumina. Brucellae often filled the macrophage cytoplasm. In contrast, nursed mammary glands had fewer brucellae in milk, minimal inflammatory changes, and no detectable brucellar antigen in histologic sections. Hyperplastic changes were only seen in supramammary lymph nodes draining nonnursed mammary glands; these contained more brucellae than lymph nodes draining nursed mammary glands. These studies show that milk stasis may be the sole cause of increased susceptibility of nonnursed mammary glands to B abortus infection.     

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.     

甲硝唑对地塞米松预处理鸡免疫功能的影响

对地塞米松预处理鸡投以10mg／kg剂量的甲硝唑，观察后者对前者作用的影响。结果表明：地塞米松明显抑制由伴刀豆球蛋白A(ConA)引起的鸡外周血淋巴细胞(PBL)产生IL-2活性，而甲硝唑对这种抑制作用有显著的拮抗作用；地塞米松能抑制鸡对绵羊红细胞(SRBC)悬液和牛血清白蛋白(BSA)两种胸腺依赖性抗原的免疫应答和对布鲁氏菌非胸腺依赖性抗原的抗体应答，但甲硝唑会逆转这种抑制作用；甲硝唑能封闭地塞米松对补体产生的抑制作用。对比实验结果同时表明，在拮抗地塞米松对鸡免疫功能负面影响方面，左旋咪唑与甲硝唑作用非常类似。     

Factors influencing the degree of in vitro bacterial adhesion to ovine mammary gland epithelial cells

Bacterial adhesion to mammary gland epithelial cells (EC) may play a role in the pathogenesis of mastitis. In vitro adherence systems have been developed to study mastitis in cattle but little has been done in sheep. In this work, a method is described for obtaining mammary gland cell preparations containing greater than or equal to 65% EC from live or dead ewes, using a Ficoll-Hypaque flotation method (cell viability = 70-90%). An in vitro adhesion assay procedure was also developed to study the interaction between EC and ovine mastitis bacterial strains. It was observed that, under the test conditions, adherence increased as the incubation time was prolonged from 30 to 120 min (P less than 0.05). Adhesion was greater at incubation temperature of 37 degrees C than at 22 degrees C (P less than 0.001). An acidic pH (5.9) was associated with an increase in adhesion, when compared with a higher pH (7.2; P less than 0.05). Tween 20, Tween 80 and bovine serum albumin helped to eliminate a background of unbound bacteria from the test slides, but they also inhibited adhesion to some strains. Strain differences in adhesion and in ability to form a background were also observed. Some of these findings may have in vivo implications.     

抗菌肽NZ2114对来源于奶牛乳房炎的无乳链球菌及其生物膜的消杀作用

为探究抗菌肽NZ2114对无乳链球菌的体外抑制效果和相关机制,本试验以无乳链球菌ATCC 13813为研究对象,通过最小抑菌浓度（minimum inhibitory concentration,MIC）、杀菌动力学、抗生素后效应和电镜学试验对抗菌肽NZ2114杀菌特性和杀菌机制进行评价,并进一步分析其对无乳链球菌生物膜和持留菌的抑制和消除作用。结果显示,NZ2114对无乳链球菌ATCC 13813的MIC为0.23 μmol/L,对3株奶牛乳腺炎临床分离菌株CAU-FRI 1、2和3的MIC均为0.11 μmol/L,万古霉素对以上4株受试菌株的MIC值均为0.67 μmol/L,因此NZ2114的抗菌活性是万古霉素的2.91和6.09倍。同时,2×MIC、4×MIC浓度的NZ2114可在0.5 h内杀灭99.9%细菌,且持续药效作用可达270 min。扫描电镜结果显示,NZ2114处理后,细菌表面出现皱缩甚至破裂,细菌产生的生物膜消除。NZ2114在2×MIC下,24 h对早期生物膜抑制率为99.9%;在32×MIC下,24 h对成熟生物膜消除率达99.9%。此外,NZ2114在16×MIC时,对生物膜内的菌体具有99.9%以上的杀灭效果;万古霉素无法清除的持留菌经0.5×MIC的NZ2114处理后,也可以达到99%以上的消除率。激光共聚焦结果显示,NZ2114处理后的生物膜厚度从28.48 μm减少到10.32 μm,证明了其强效杀菌和抑制生物膜的作用。上述结果表明,NZ2114对无乳链球菌ATCC 13813杀菌活性高、速度快、抑制/去除生物膜能力强,并对膜内菌及持留菌具有高效杀菌作用,且作用显著高于万古霉素。因此,NZ2114具有开发成为治疗由无乳链球菌引起的奶牛乳房炎新型制剂的潜力。     

The influence of anti-inflammatory therapy on bacterial clearance following intramammary Escherichia coli challenge in goats

Coliform mastitis in dairy cattle frequently results in systemic disease with occasional deaths in association with endotoxic shock. Systemic anti-inflammatory therapy has been used to alter the course of endotoxic shock in severe cases. Use of anti-inflammatory therapy has been questioned on the basis that such treatment may compromise immune function and decrease clearance of bacteria from infected mammary glands. The purpose of this investigation was to determine whether anti-inflammatory therapy influenced bacterial clearance following intramammary challenge of lactating goats with Escherichia coli.Standardized quantities of a pathogenic coliform culture were infused through the teat canal into one half of the mammary gland in 18 goat does. The does were then randomly assigned to receive one of three intravenous treatments: saline (controls), one dose of steroid (dexamethasone), or two doses of a non-steroidal anti-inflammatory agent (flunixin meglumine). The clinical signs, milk production, complete blood counts, serum clinical chemistry values, milk bacterial cultures and milk somatic cell concentrations were monitored sequentially.Goats treated with anti-inflammatory agents exhibited some improvement in clinical response to challenge with E. coli (e.g. rectal temperature, degree of appetite suppression) as compared to saline controls. There were no significant differences between treatments in the degree of inflammation present in the mammary glands or supramammary lymph nodes examined at necropsy. The most important finding was that anti-inflammatory therapy did not adversely influence the clearance of E. coli from challenged glands.     

抗菌肽天蚕素B基因的克隆及其在山羊乳腺中的暂态表达

根据已发表的天蚕素B的cDNA序列化学合成4务引物,以重叠延伸PCR的方法合成了真核细胞偏爱密码子编码的抗菌肽天蚕素B基因,将其克隆于pMD18-T栽体,DNA序列分析证实合成的抗菌肽基因的碱基序列与设计的序列完全一致.利用山羊-β酪蛋白基因5'侧翼调控序列和真核基因表达栽体plRES1-neo,构建抗菌肽天蚕素B的乳腺组织特异性表达载体pbCP-cpin,将该载体溶于生理盐水,注入处于泌乳期的成年母山羊乳腺组织进行暂时表达.通过对大肠杆菌、金黄色葡萄球菌的抑菌试验确定了抗菌肽天蚕素B基因在山羊乳腺中的表达和抗菌活性.本试验建立了抗菌肽天蚕素B的乳腺瞬时表达系统,为验证各调控元件以及抗菌肽基因在转基因动物乳汁中分泌表达的有效性和可行性提供了一个快速检测系统.     

灌注葡萄糖对奶山羊乳腺营养摄取的影响

本试验研究了阴外动脉灌注葡萄糖对乳腺营养物质吸收和乳成分的影响。选用体重和产奶量相近的4只泌乳中期关中奶山羊,做阴外动脉和腹部皮下静脉手术安装血插管,采用4×4拉丁方设计,每天分别灌注葡萄糖0、12、24g和36g。结果表明:乳脂率随葡萄糖灌注量的增加而显著降低(P0.01)。灌注36g/d处理组乳脂产量显著低于其他处理组(P0.01);葡萄糖灌注提高了奶山羊的产奶量,24g/d处理组的产奶量显著高于对照组(P0.01);36g/d处理组乳腺对葡萄糖的摄取量和乳糖的产量都显著高于对照组(P0.01)。甘油三酯和非脂固形物的含量各组间差异不显著(P0.05),但葡萄糖灌注提高了乳蛋白产量,其中24g/d处理组乳蛋白产量最高,并显著高于对照组(P0.05)。葡萄糖的灌注显著降低了奶山羊干物质的采食量(P0.01)。葡萄糖的灌注对乳腺氨基酸的摄取影响不显著。葡萄糖的灌注使氨基酸合成乳蛋白的产出率提高1%～9%,葡萄糖合成乳糖的产出率提高0%～5%,有效改善了乳成分。     

Effect of steroid hormones on the innate immune response induced by Staphylococcus aureus in the goat mammary gland

The objective of this study was to compare the dynamics of innate immune components after intramammary infusion of Staphylococcus aureus (SA) under conditions of high oestrogen and high progesterone in goats. In one group (“E‐group”), controlled internal drug release (CIDR) devices were inserted intravaginally from days ?11 to ?4. Prostaglandin F2α was administered immediately after removal of the CIDR device at day ?3, and then oestradiol benzoate (E) was injected intramuscularly once a day from days ?2 to 3. Heat‐inactivated SA was then administered via intramammary infusion to the left udder at day 0, whilst only saline was infused to the right udder as a control. In a second group (“P‐group”), CIDR devices were inserted intravaginally from days ?3 to 7 and SA was infused at day 0 in the same way as in the E‐group. The milk yield and the concentration of innate immune components (somatic cell count (SCC), lactoferrin (LF), S100A7 and goat ß‐defensin 1 (GBD‐1)) in the milk were measured. Milk yield decreased drastically in both SA and control udders in the E‐group, whereas the P‐group exhibited increased milk yield in both SA and control udders. SCC increased after SA infusion in both E‐ and P‐groups, although it was higher in the E‐group than in the P‐group. There was no significant change in LF concentration in the E‐group, but a decrease was observed in the P‐group. Concentrations of S100A and GBD‐1 were significantly increased after SA infusion in the E‐group but not in the P‐group. These results suggest that E enhances the innate immune response induced by SA in the goat mammary gland. This effect may be due to the reduction in milk yield and upregulation of innate immune components.     

CpG-ODN enhances mammary gland defense during mastitis induced by Escherichia coli infection in goats

Seven healthy native goats in early lactation, weighing 30–40 kg, were used in this study. The right mammary gland of the seven does were infused with CpG-ODN at a dosage of 100 μg kg⁻¹ body weight on the day 5 postpartum (PP). The left glands were used as controls and infused with sterile phosphate-buffered saline (PBS). On day 8 PP, the same dosage of CpG-ODN or PBS was again infused. On day 9 PP, the mammary glands (both right and left) of the seven does were infused with 6 × 10⁶ colony-forming units (CFU) Escherichia coli and, at 0, 8, 16, 24, 48 and 72 h postinfection (PI), milk samples were collected from all glands. Goats were euthanized at 72 h PI and the mammary tissue harvested. Infusion with 6 × 10⁶ CFU ml⁻¹ E. coli induced acute mastitis. Histopathological evaluations showed that polymorphonuclear neutrophils (PMNs) were still present in alveoli at 72 h PI, but PMNs in the CpG-ODN-treated glands has disappeared. Bacteria counts in milk peaked at 16 h PI and CpG-ODN induced a significant decrease in viable bacteria from 16 h PI until the end of the experiment. This study showed that CpG-ODN promoted the expression of its specific receptor (TLR-9 mRNA) in mammary tissue, stimulated IL-6 production, reduced bacteria counts in milk, attenuated the impact of inflammation mediators on cells and significantly shortened the inflammation course. These results suggest that the CpG-ODN improved mammary gland defense and, thereby, had a beneficial effects against mastitis caused by E. coli infection in goats.     

Goat cathelicidin‐2 is secreted by blood leukocytes regardless of lipopolysaccharide stimulation

It has been reported that goat cathelicidin‐2, an antimicrobial peptide, localizes in leukocytes and is present in milk. Here, we examined whether cathelicidin‐2 is secreted by leukocytes. Different concentrations (10⁵–10⁸ cells/mL) of blood leukocytes were cultured for 0–48 h with or without lipopolysaccharide (LPS). After culture, the concentrations of cathelicidin‐2 in the conditioned media were measured. Blood was collected from male goats 0–24 h after the intravenous injection of Escherichia coli O111:B4 LPS. The plasma cathelicidin‐2 concentrations were determined and the blood leukocytes immunostained with anti‐cathelicidin‐2 antibody to calculate the proportion of cathelicidin‐2‐positive cells in the total leukocytes. When higher concentrations of leukocytes were cultured, the cathelicidin‐2 concentrations in the media increased significantly, whereas the addition of LPS to the media caused no further increase. The plasma cathelicidin‐2 concentrations did not increase with time after LPS infusion. The proportion of cathelicidin‐2‐positive cells in the total leukocytes was significantly reduced 1 h after LPS injection compared with that at 0 h, but increased again at 6 h and thereafter. These results suggest that cathlicidin‐2 is secreted by leukocytes even without LPS stimulation, whereas LPS may be required for cathelicidin‐2‐containing leukocytes to be recruited from the blood to tissues showing inflammation.     

不同程度乳腺炎血清中相关细胞因子的试验

本试验主要探讨患有不同程度乳腺炎的奶牛血清中细胞因子含量的变化与乳腺炎的关系,确定相关指标的变化是否由乳腺炎引起,为今后诊断乳腺炎提供可能参考依据.对患有不同程度乳腺炎的奶牛颈静脉采血,分离血清,采用放射免疫法测定其血清中IL-1β、IL-6、IL-8、TNF-α含量及变化趋势,对结果使用SPSS分析.结果显示奶牛发生乳腺炎后血清中IL-1β、IL-6、IL-8、TNF-α含量随炎症程度的加剧逐渐升高;但只有IL-1β在奶牛临床症状明显时血清中的含量又开始出现下降趋势.