Identification, isolation, and partial characterization of a novel Streptococcus uberis adhesion molecule (SUAM)

The ability to attach to the host cell surface has been considered an important virulence strategy in many bovine mammary gland pathogens, including Streptococcus uberis. Research conducted in our laboratory lead to the identification of an S. uberis adhesion molecule (SUAM) with affinity for bovine lactoferrin (LF) and delineation of its role in adherence of S. uberis to bovine mammary epithelial cells. Using a selected bacterial surface protein extraction protocol and affinity chromatography, a 112-kDa protein that had a similar molecular mass and the LF affinity as one of the identified S. uberis LBP described by Fang and Oliver in 1999 was found. To further characterize SUAM, the N-terminal amino acid sequence of this protein was elucidated. A protein query versus translated database TBLASTN search of the National Center for Biotechnology (NCBI), non-redundant database, nr, with the LBP N-terminal amino acid sequence showed no significant similarity with previous entries. Antibodies directed against SUAM and a 17 amino acid long N-terminal sequence (pep-SUAM) inhibited adherence to and internalization of S. uberis UT888 into bovine mammary epithelial cells. Data presented suggests that we have discovered a novel bacterial protein involved in the pathogenesis of this economically important mastitis pathogen.     

Vaccination of dairy cows with recombinant Streptococcus uberis adhesion molecule induces antibodies that reduce adherence to and internalization of S. uberis into bovine mammary epithelial cells

Streptococcus uberis is an important environmental mastitis pathogen that causes subclinical and clinical mastitis in lactating and nonlactating cows and heifers throughout the world. Previous work from our laboratory suggests that S. uberis adhesion molecule (SUAM) is involved in S. uberis pathogenesis and may be an excellent target for vaccine development. The objective of this study was to evaluate the antibody response of cattle vaccinated with recombinant SUAM (rSUAM). Uninfected primiparous dairy cows (n=30) in late lactation were divided randomly into three groups of 10 cows each: control, 200 μg rSUAM, and 400 μg rSUAM. Cows in groups vaccinated with 200 μg and 400 μg rSUAM received an emulsion containing adjuvant, phosphate-buffered saline (PBS) and affinity purified rSUAM. Cows in the control group received an emulsion containing adjuvant and PBS. Cows were vaccinated subcutaneously in the neck region at drying off (D-0), 28 d after drying off (D+28) and within 7 d after calving. Serum was collected at D-0, D+28, at calving (C-0), calving vaccination (CV), and during early lactation (CV+14). Serum antibody responses were measured by an ELISA against rSUAM. Following the first vaccination a significant increase in anti-rSUAM antibodies was detected at D+28 in cows from groups vaccinated with 200 μg and 400 μg rSUAM when compared to the control group. This increase in anti-rSUAM antibodies continued following the second immunization at D+28; reaching the highest levels in the post-parturient sampling period (C0), after which antibodies appeared to plateau. S. uberis UT888 pretreated with several dilutions of heat-inactivated serum from cows vaccinated with rSUAM, affinity purified antibodies against rSUAM, and to a 17 amino acid long peptide from the N terminus of SUAM (pep-SUAM) were co-cultured with bovine mammary epithelial cells and adherence to and internalization of S. uberis into epithelial cells was measured. Compared to untreated controls, opsonization of two strains of S. uberis with sera from cows vaccinated with rSUAM, with affinity purified rSUAM antibodies, or with affinity purified pep-SUAM antibodies significantly reduced adherence to and internalization of this pathogen into bovine mammary epithelial cells. In conclusion, subcutaneous vaccination of dairy cows with rSUAM during physiological transitions of the mammary gland either from or to a state of active milk synthesis induced antibodies in serum and milk and these antibodies reduced adherence to and internalization of S. uberis into mammary epithelial cells under in vitro conditions. SUAM appears to be an excellent candidate for vaccine development.     

Binding of host glycosaminoglycans and milk proteins: possible role in the pathogenesis of Streptococcus uberis mastitis

The role of indirect binding of host proteins through glycosaminoglycans (GAGs) on adherence and internalization of Streptococcus uberis to bovine mammary epithelial cells was evaluated. Preincubation of S. uberis with GAGs followed by incubation with fetal bovine serum (FBS), bovine milk or milk proteins resulted in greater adherence to and internalization of S. uberis into mammary epithelial cells than observed in untreated controls. Highest values were detected, when final incubation was done with milk. Greater adherence to and internalization into mammary epithelial cells were observed when heparin sulfate (HEP) and milk were used compared with any other GAG and FBS. When individual milk proteins were used, greatest adherence and internalization were observed when S. uberis strains were pretreated with HEP followed by treatment with beta-casein. The findings of this study illustrate a pathogenic strategy of S. uberis that may occur during the very early stages of infection.     

Role of collagen in adherence of Streptococcus uberis to bovine mammary epithelial cells

We reported previously that pre-incubation of Streptococcus uberis with collagen induced expression of S. uberis surface proteins. In a subsequent study, we showed that incubation of S. uberis with extracellular matrix proteins, particularly collagen, increased adherence and internalization of S. uberis to mammary epithelial cells. In the present report, the potential mechanism by which S. uberis exploits the presence of collagen to enhance adherence to bovine mammary epithelial cells was evaluated. Adherence assays were conducted with S. uberis pre-treated with and without collagen and co-cultured in medium supplemented with or without collagen. Pre-incubation with collagen followed by co-culture in medium containing collagen up-regulated ligands that enhanced adherence of S. uberis to mammary epithelial cells. Collagen-up-regulated ligand(s) also increased adherence of S. uberis to mammary epithelial cells in the absence of collagen, but adherence was lower than when collagen was present during the adherence assay. Chloramphenicol was added to the culture medium to inhibit bacterial protein synthesis. Adherence decreased significantly in chloramphenicol-treated S. uberis pre-treated or co-cultured in the presence of collagen. These results suggest that S. uberis expresses ligands with affinity for collagen that are up-regulated by collagen. We hypothesize that these ligands increase adherence by using collagen as a bridge between the bacterium and host cell and/or by direct interaction with host cell receptor(s).     

Effects of lactoferrin and milk on adherence of Streptococcus uberis to bovine mammary epithelial cells

OBJECTIVE: To determine whether lactoferrin (LF) or milk influenced adherence of Streptococcus uberis to bovine mammary epithelial cells. SAMPLE POPULATION: Three strains of S uberis from cows with mastitis, pooled milk samples from 3 clinically healthy Jersey cows early in the lactation period, and bovine mammary epithelial cells from a clonal cell line. PROCEDURES: Adherence of S uberis to bovine mammary epithelial cells in the presence of various concentrations of LF or milk and after pretreatment of bacteria with LF or milk was tested. Bacteria were cultured with mammary epithelial cell monolayers for 1 hour. The culture supernatant was removed, and the epithelial cells were lysed. Adherence index was calculated as number of colony-forming units (CFU) in the cell lysate divided by number of CFU in the supernatant times 10,000. RESULTS: All 3 strains of S uberis were found to bind to purified LF and LF in milk. Addition of LF to the culture medium enhanced adherence of all 3 strains to mammary epithelial cells, whereas addition of milk enhanced adherence of 2 strains and decreased adherence of the third. Pretreatment of bacteria with LF or milk increased adherence of 1 of the strains but decreased adherence of the other 2. Increased adherence was antagonized by rabbit antibovine LF antibody. CONCLUSIONS: Results suggest that LF may function as a bridging molecule between S uberis and bovine mammary epithelial cells, facilitating adherence of the bacteria to the cells.     

乳铁素参与乳房链球菌对乳腺上皮细胞的粘附

免疫印迹试验表明，试验用的3株乳房链球菌（Streptococcus uberis)菌株均可与乳铁素（Lactoferrin)结合。培养基中加入乳铁素或乳清可以显著促进细菌与乳腺上皮细胞之间的粘附，抗乳铁素抗体可以特异性地抑制乳铁素或乳清预处理细菌与乳腺上皮细胞的粘附。乳铁素在细菌和细胞之间起着桥梁分子作用。有助于细菌与乳腺上皮细胞之间的粘附和乳腺感染的建立。     

pGh9:ISS1 transpositional mutations in Streptococcus uberis UT888 causes reduced bacterial adherence to and internalization into bovine mammary epithelial cells

Streptococcus uberis is an important mastitis pathogen that affects dairy cows worldwide. In spite of the economic impact caused by the high prevalence of S. uberis intramammary infections (IMI) in many well-managed dairy herds, pathogenic strategies and associated virulence factors of S. uberis are not well understood. It has been shown that S. uberis attaches to and internalizes into mammary epithelial cells and can survive inside cells for extended periods of time. We hypothesize that early attachment to and internalization into mammary epithelial cells is a critical step for the establishment of intramammary infection. The aim of this study is to identify and characterize chromosomally encoded virulence factors of S. uberis that allow early bacterial attachment to and internalization into mammary epithelial cells. A common approach used to identify virulence factors is by generating random insertion mutants that are defective in adherence to and internalization into mammary epithelial cells using pGh9:ISS1 mutagenesis system. A random insertion mutant library of S. uberis strain UT888 was created using a thermo-sensitive plasmid pGh9:ISS1 carrying ISS1 insertion sequence. Integration of the insertion sequence into the chromosome of these mutant clones was confirmed by PCR and Southern blot. Southern blot analysis of mutant clones also showed that insertional integration was random. Of 1000 random chromosomal insertion mutants of S. uberis strain UT888 screened, 32 had significantly reduced ability to adhere to and internalize into mammary epithelial cells. Chromosomal mapping of insertion sequence integration sites in some of these defective mutants showed integration into penicillin binding protein 2A (pbp2A), sensor histidine kinase, tetR family regulatory protein, phosphoribosylaminoimidazole carboxylase catalytic subunit (purE), lactose phosphotransferase, phosphoribosylamine glycine ligase (purD), and other genes involved in metabolic activities. These proteins may have a significant role in early bacterial colonization of the mammary gland during infection.     

Involvement of connective tissue growth factor (CTGF) in insulin-like growth factor-I (IGF1) stimulation of proliferation of a bovine mammary epithelial cell line

The objective of this study was to determine the mechanism by which insulin-like growth factor-I (IGF1) stimulates proliferation of mammary epithelial cells, using the bovine mammary epithelial cell line MAC-T as a model. IGF1 significantly up- or down-regulated the expression of 155 genes in MAC-T cells. Among the most significantly suppressed was the gene for connective tissue growth factor (CTGF), a secretory protein that has both proliferative and apoptotic effects and is also a low-affinity binding protein of IGF1. IGF1 inhibited CTGF expression through the PI3K-Akt signaling pathway. Administration of growth hormone (GH), a strong stimulator of IGF1 production in vivo, decreased mammary CTGF mRNA in cattle; however, GH did not affect CTGF expression in MAC-T cells, suggesting that IGF1 may also inhibit CTGF expression in the mammary gland. Added alone CTGF stimulated proliferation of MAC-T cells, but in combination with IGF1 it attenuated IGF1's stimulation of proliferation of MAC-T cells. Excess IGF1 reversed this attenuating effect of CTGF. Despite being an IGF binding protein, CTGF did not affect IGF1-induced phosphorylation of IGF1 receptor (IGF1R) or IGF1R expression in MAC-T cells, indicating that the attenuating effect of CTGF on IGF1 stimulated proliferation of MAC-T cells was not mediated by decreasing IGF1's ability to bind to IGF1R or by decreasing IGF1R expression. Overall, these results suggest a novel biochemical and functional relationship between CTGF and IGF1 in the bovine mammary gland, where IGF1 may inhibit CTGF expression to reduce the attenuating effect of CTGF on IGF1 stimulated proliferation of epithelial cells.     

Role of GapC in the pathogenesis of Staphylococcus aureus

Staphylococcus aureus is recognized worldwide as a major pathogen causing clinical or subclinical intramammary infections in lactating cows, sheep and goats. S. aureus produces a wide arsenal of cell surface and extracellular proteins involved in virulence. Among these are two conserved proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity named glyceraldehyde-3-phosphate dehydrogenase-B (GapB) and -C (GapC). In this study, we used the S. aureus wild type strain RN6390 and its isogenic gapC mutant H330 in in vitro and in vivo studies and determined that the S. aureus GapC protein plays a role on adherence to and internalization into bovine mammary epithelial (MAC-T) cells. In addition, we found that S. aureus H330 did not caused mastitis after an experimental infection of ovine mammary glands. Together, these results show that GapC is important in the pathogenesis of S. aureus mastitis.     

Cytotoxic effects of peroxynitrite, polymorphonuclear neutrophils, free-radical scavengers, inhibitors of myeloperoxidase, and inhibitors of nitric oxide synthase on bovine mammary secretory epithelial cells

OBJECTIVE: To determine cytotoxic effects of activated polymorphonuclear neutrophils (PMN) and peroxynitrite on bovine mammary secretory epithelial cells before and after addition of nitric oxide synthase inhibitors, myeloperoxidase (MPO) inhibitors, and free-radical scavengers. SAMPLE POPULATION: Polymorphonuclear neutrophils from 3 lactating cows. PROCEDURE: Cells from the bovine mammary epithelial cell line MAC-T were cultured. Monolayers were treated with activated bovine PMN, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), 3-morpholino-sydnonimine (SIN-1), 4-amino-benzoic acid hydrazide (ABAH), NG-monomethyl-L-arginine, histidine, and superoxide dismutase (SOD). At 24 hours, activity of lactate dehydrogenase in culture medium was used as a relative index of cell death. Tyrosine nitration of proteins in MAC-T cell lysates was determined by visual examination of immunoblots. RESULTS: Lipopolysaccharide, PMA, and < or = 0.1 mM SIN-1 were not toxic to MAC-T cells. Activated PMN, > or = 6 mg of histidine/ml, and 0.5 mM SIN-1 were toxic. Together, histidine and 500,000 activated PMN/ml also were toxic. NG-monomethyl-L-arginine did not have an effect, but ABAH decreased PMN-mediated cytotoxicity. Ten and 50 U of SOD/ml protected MAC-T cells from cytotoxic effects of 0.5 mM SIN-1. Compared with control samples, nitration of MAC-T tyrosine residues decreased after addition of 500,000 PMN/ml or > or = 6 mg of histidine/ml. Superoxide dismutase increased and SIN-1 decreased tyrosine nitration of MAC-T cell proteins in a dose-responsive manner. CONCLUSIONS AND CLINICAL RELEVANCE: Peroxynitrite, MPO, and histidine are toxic to mammary secretory epithelial cells. Superoxide dismutase and inhibition of MPO activity mitigate these effects. Nitration of MAC-T cell tyrosine residues may be positively associated with viability.     

Elucidation of the DNA sequence of Streptococcus uberis adhesion molecule gene (sua) and detection of sua in strains of Streptococcus uberis isolated from geographically diverse locations

Streptococcus uberis is an important environmental pathogen that causes subclinical and clinical mastitis in lactating and nonlactating cows throughout the world. S. uberis adhesion molecule (SUAM) was identified recently by our laboratory and we hypothesize that SUAM is a potential virulence factor involved in the pathogenesis of S. uberis mastitis. The first objective of the present study was to clone and sequence the SUAM gene (sua) from S. uberis UT888. The second objective was to determine the prevalence of sua in strains of S. uberis isolated from geographically diverse locations. The 20 amino acid N-terminal sequence of purified SUAM was utilized to identify a single open reading frame (ORF) in the S. uberis O140J (ATCC BAA-854) genome database. Three sets of primers were identified from this sequence for amplification of sub-fragments and the complete gene encoding SUAM. Restriction fragment analysis of the largest polymerase chain reaction (PCR) product confirmed the desired fragment had been amplified. This 2970bp PCR fragment was cloned into plasmid pCR-XL-TOPO and sequenced. The S. uberis UT888 sua sequence (NCBI Accession no. DQ232760) was 99% similar to the S. uberis O140J database sequence. The three pairs of PCR primers were used in a subsequent experiment to identify sua in 12 strains of S. uberis isolated in milk from dairy cows with mastitis in Tennessee (n=6), Colorado (n=1), Washington (n=1), New Zealand (n=1) and from the American Type Culture Collection (n=3). Primer pairs yielded the expected 2970, 2639 and 2362bp PCR fragments in all strains evaluated. In conclusion, we cloned and sequenced sua, which codes for the first described S. uberis adhesin, SUAM. sua was detected in all strains of S. uberis evaluated suggesting that it is conserved.     

Adherence as a Prerequisite for Infection of the Bovine Mammary Gland by Bacteria

Using a simple in vitro test it was demonstrated that staphylococci, Streptococcus agalactiae, and micrococci, the species of bacteria which are commonly isolated from udder infections, adhered to mammary gland epithelial cells readily and in large numbers. Some strains of organisms which are associated with sporadic outbreaks or occur less commonly, like Str. dysgalactiae and Str. uberis, adhered moderately. Escherichia coli, Klebsiella spp., Corynebacterium pyogenes, C. bovis, Str. bovis, and Str. faecalis, species which are isolated occasionally, adhered poorly. From these studies, it appears that selective adherence of bacteria to the epithelial cells is a factor contributing to the ability of organisms to infect the mammary gland and may, therefore, be considered an important stage in the pathogenesis of bovine mastitis.     

蜂胶对细菌脂多糖刺激下奶牛乳腺上皮细胞炎症相关基因mRNA转录水平和紧密连接蛋白的影响

本试验旨在研究中国蜂胶乙醇提取物（ethanol extract of Chinese propolis，EECP）对细菌脂多糖（lipopolysaccharide，LPS）刺激下体外培养奶牛乳腺上皮细胞炎症相关基因mRNA转录水平和紧密连接渗透性的影响。EECP中总酚酸和总黄酮含量测定采用福林酚法和硝酸铝法，并建立LPS诱导奶牛乳腺上皮细胞（bovine mammary epithelial cells，MAC-T）炎症模型，采用CCK-8法测定EECP对MAC-T相对增殖率的影响，利用实时荧光定量PCR（RT-qPCR）评估EECP对LPS诱导的MAC-T细胞炎症相关因子（IL-6、IL-8、TNF-α和IL-1β）相对mRNA转录水平；以及对紧密连接蛋白（occludin、ZO-1）相对mRNA转录水平进行检测，并进一步利用免疫荧光技术对紧密连接膜蛋白进行定位，确定EECP对LPS诱导MAC-T细胞炎症紧密连接渗透性的影响。结果显示：EECP中总酚酸含量为106.35 mg没食子酸当量（GAE）·g^-1、总黄酮含量为320.85 mg芦丁当量（RE）·g^-1；CCK-8结果显示EECP的安全浓度为0~15 μg·mL^-1，并可有效提高LPS刺激下MAC-T的活力；LPS刺激显著增加了细胞炎症相关因子IL-6、IL-8、TNF-α和IL-1β mRNA的转录量（P<0.001）；但2.5~15.0 μg·mL^-1 EECP预处理显著降低了IL-6、IL-8、TNF-α和IL-1β mRNA的转录量；与此类似，LPS刺激显著抑制了紧密连接蛋白基因（occludin、ZO-1）mRNA的转录量（P<0.01），而EECP预处理后紧密连接蛋白基因（occludin和ZO-1）mRNA的转录量显著增加（P<0.05）；免疫荧光染色试验也证实EECP能通过上调紧密连接蛋白（occludin、ZO-1）的表达，缓解LPS诱导的乳腺上皮细胞屏障功能紊乱。该结果证实，EECP对细菌脂多糖诱导奶牛乳腺上皮细胞炎症具有良好的保护作用，这为利用中国蜂胶预防奶牛乳腺炎提供了试验基础。     

Cholecalciferol (vitamin D) differentially regulates antimicrobial peptide expression in bovine mammary epithelial cells: Implications during Staphylococcus aureus internalization

Vitamin D has immunomodulatory functions regulating the expression of host defense genes. The aim of this study was to determine the effect of cholecalciferol (vitamin D3) on S. aureus internalization into bovine mammary epithelial cells (bMEC) and antimicrobial peptide (AP) mRNA expression. Cholecalciferol (1-200nM) did not affect S. aureus growth and bMEC viability; but it reduced bacterial internalization into bMEC (15-74%). Also, bMEC showed a basal expression of all AP genes evaluated, which were induced by S. aureus. Cholecalciferol alone or together with bacteria diminished tracheal antimicrobial peptide (TAP) and bovine neutrophil β-defensin (BNBD) 5 mRNA expression; while alone induced the expression of lingual antimicrobial peptide (LAP), bovine β-defensin 1 (DEFB1) and bovine psoriasin (S100A7), which was inhibited in the presence of S. aureus. This compound (50nM) increased BNBD10 mRNA expression coinciding with the greatest reduction in S. aureus internalization. Genes of vitamin D pathway (25-hydroxylase and 1 α-hydroxylase) show basal expression, which was induced by cholecalciferol or bacteria. S. aureus induced vitamin D receptor (VDR) mRNA expression, but not in the presence of cholecalciferol. In conclusion, cholecalciferol can reduce S. aureus internalization and differentially regulates AP expression in bMEC. Thus, vitamin D could be an effective innate immunity modulator in mammary gland, which leads to a better defense against bacterial infection.     

Evaluation of a replacement method for mammary gland biopsies by comparing gene expression in udder tissue and mammary epithelial cells isolated from milk

Somatic cells isolated from milk offer an attractive non-invasive replacement of invasive udder biopsies for monitoring bovine mammary gland metabolism. However, for metabolic gene expression studies the mammary gland epithelial cells (MEC) isolated from milk have to be purified from the non-epithelial leukocyte fraction in milk samples. In our study, enrichment of MEC by using anti-cytokeratin peptide 18 (KRT18) antibody coated magnetic beads was evaluated. MEC showed a substantially increased expression of the epithelial-cell-specific KRT18 gene compared to udder tissue. The expression levels of genes specific for mammary gland epithelial cells (CSN3 and LALBA) showed a significant positive correlation in MEC and also in udder tissue. However, no significant correlation of the expression of a specific gene was found between udder and MEC samples. Therefore, MEC isolated from total milk samples via KRT18 antibodies probably do not reflect the true metabolic situation of the bovine udder. Thus, quantitative gene expression profiling of MEC isolated via KRT18 antibodies has to be interpreted carefully with respect to the situation in the udder.     

Use of an immortalized bovine mammary epithelial cell line (MAC-T) to measure the mitogenic activity of extracts from heifer mammary tissue: effects of nutrition and ovariectomy

The objectives of the experiment were (1) to determine whether MAC-T cells would accurately mimic the previously observed proliferative responses of primary mammary epithelial cells (MEC) to mammary tissue extracts from high and low-fed heifers and (2) to determine whether mammary tissue extracts from ovariectomized (OVX) heifers would have lower mitogenic activity than intact controls. Addition of mammary tissue extracts to cell culture media of MAC-T cells plated on plastic or collagen-coated plastic to a range of concentrations between 1 and 8% resulted in dose-dependent increases in cell proliferation. Furthermore, mammary tissue extracts from low-fed prepubertal heifers aged 9 months, stimulated significantly more proliferation of MAC-T cells, as measured by 3H-thymidine incorporation into DNA than mammary tissue extracts from high-fed heifers (40.6 cpm x 10(3) per well versus 21.9+/-1.8 cpm x 10(3) per well). These observations suggested that MAC-T cells would be a suitable alternative to primary MECs for measuring the mitogenic activity of mammary tissue extracts. Conversely, no difference was observed in the mitogenic activity of mammary tissue extracts from OVX or control heifers. Possibly, MAC-T cells provide a good model for nutrition- but not ovarian-induced changes in mammary growth. Alternatively, that reduction of in vivo mammary development following OVX did not result in reduced mitogenic activity of the mammary tissue extracts emphasizes that heifer mammary development is the result of complex interactions between local growth factors and systemic hormones.     

牛源坏死梭杆菌43K外膜蛋白的黏附特性研究

旨在明确牛源坏死梭杆菌43K OMP的黏附特性。将43K OMP基因克隆连接至pET-32a载体,转化入大肠杆菌BL21 DE3中,通过IPTG诱导进行原核表达,应用黏附试验、天然蛋白竞争试验、抗体抑制试验和蛋白酶水解试验,明确牛源坏死梭杆菌43K OMP的黏附性,同时将纯化的重组蛋白和提取的天然43K OMP与牛子宫内膜细胞和牛乳腺上皮细胞共孵育,观察43K OMP对细胞的黏附作用。结果显示：43K OMP基因克隆到pET-32a载体中,随后在大肠杆菌BL21 DE3以包涵体形式成功表达;携带重组质粒(H2019)的大肠杆菌经IPTG诱导后,与空载体对照相比,与宿主细胞的结合显著增强(P<0.05);天然43K OMP与细胞共孵育后,黏附细胞的细菌数量显著降低(P<0.05);H2019与43K OMP多抗或单抗预孵育后,显著降低黏附宿主细胞的细菌数量(P<0.05);经不同浓度蛋白酶K处理H2019后,黏附细胞的细菌数量显著降低(P<0.05),且与蛋白酶K浓度呈现剂量依赖关系。同时,43K OMP天然蛋白和重组蛋白能黏附于牛子宫内膜细胞和乳腺上皮细胞表面。...