Technical note: Validation of candidate reference genes for normalization of quantitative PCR in bovine mammary epithelial cells responding to inflammatory stimuli.

Mammary epithelial cells (MEC) participate in the first line of defense of the mammary gland to invading pathogens. In vitro culture of MEC is widely used as a model to study the capacity of these cells to sense and respond to mastitis-causing bacteria. Analysis of gene expression by quantitative PCR (qPCR) following exposure to bacteria or bacterial constituents is a powerful tool to assess responses of MEC to pathogens. Although internal standards such as reference genes are required for qPCR to yield valid data, the validation of proper genes to quantify mRNA transcripts in MEC exposed to pro-inflammatory stimuli has never been reported. In this study, 10 commonly used reference genes belonging to different functional classes (ACTB, ATP5B, EIF2B2, GAPDH, PPIA, SDHA, SUZ12, UXT, YWHAZ, and 18s rRNA) were analyzed by qPCR to determine the most stable in bovine MEC unstimulated and stimulated with mastitis pathogens (Staphylococcus aureus or Escherichia coli), microbial agonists of the innate immune system (lipoteichoic acid and muramyl dipeptide, or lipopolysaccharide), or proinflammatory cytokines (IL-17A and tumor necrosis factor-α). An M value was used as a measure of gene stability as determined using the geNorm application. This study demonstrated that the expression of the 10 reference genes was stable under the different experimental conditions. These data will be useful for bovine mastitis research in selecting reference genes and validating reverse transcription-qPCR data.

Induction of surface-associated proteins of Streptococcus uberis by cultivation with extracellular matrix components and bovine mammary epithelial cells.

Surface-associated protein expression by Streptococcus uberis was influenced by the presence of collagen, laminin and bovine mammary epithelial cells in the culture medium. After electrophoresis and silver staining, four proteins stained more intensively in samples from S. uberis cultivated with epithelial cells and extracellular matrix components than in samples from S. uberis cultivated alone. Induction of these proteins was more obvious after multiple bacterial passages. The correlation between the phenotype of S. uberis and its potential virulence status as illustrated by an immunoblotting study with sera obtained from infected cows revealed that these proteins are probably expressed in vivo during infection.

Purification, characterization, and seroactivity of a 20-kilodalton Brucella protein antigen.

An internal protein was purified from cell extracts of Brucella melitensis B115 by a combination of preparative isoelectric focusing and high-performance size exclusion chromatography. The protein has an apparent molecular mass of 230 kDa as determined by size exclusion chromatography. The protein was resolved to a single band of 20 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native protein had an isoelectric point of 4.9. The N-terminal sequence of the 20-kDa protein was determined. The 20-kDa protein has been identified as antigen A-2 with a previously described anti-antigen A-2 serum (B. Stemshorn, K. Nielsen, and B. Samagh, Can. J. Comp. Med. 45:77-81, 1981). Antigen A-2 reacted with sera from infected sheep in immunoblotting and may be useful in developing diagnostic tests for brucellosis.

Immunogenicity in cows of Staphylococcus aureus type 5 capsular polysaccharide-ovalbumin conjugate.

Six dairy cows were immunized subcutaneously with purified type 5 capsular polysaccharide (CP5) of Staphylococcus aureus or CP5-ovalbumin conjugate in Freund's incomplete adjuvant. The CP5 antibodies elicited were measured in sera and analysed with regard to isotypes by enzyme-linked immunosorbent assay. At the doses tested, the purified CP5 did not induce a humoral response in the cows. Immunization of two cows with the CP5-ovalbumin conjugate elicited a CP5 antibody response mainly of the IgG2 isotype, which culminated 4 weeks later. A second injection of conjugate, 3 months after the first one, resulted in a rapid and durable anti-CP5 response without exceeding the first antibody peak value. Intramammary infusion of purified CP5 failed to provoke an inflammatory response in the milk of the immunized cows. In contrast, a marked recruitment of cells was recorded in the milk of the sensitized cows after intramammary infusion of ovalbumin. These results demonstrate that injection of CP5-protein carrier conjugate in cows entails both antibody response against CP5 and carrier-specific recruitment of cells in milk of immunized animals.

Immunopotentiation of Staphylococcus aureus type 5 capsular polysaccharide co-entrapped in liposomes with alpha-toxin.

Immunopotentiation of Staphylococcus aureus type 5 capsular polysaccharide (CP5) by use of liposomes as an alternative to protein-polysaccharide conjugates was investigated. Mice were immunized twice with cationic liposomes containing CP5 alone or CP5 co-entrapped with alpha-toxin or heat-detoxified alpha-toxin. Immunogenicity of these different antigens was compared with CP5-alpha-toxin conjugates. Antibodies against CP5 were elicited in mice immunized with conjugates or liposomes containing co-entrapped CP5 and alpha-toxin. Liposomes containing CP5 alone or co-entrapped CP5 and alpha-toxoid failed to induce antibodies against CP5. All the preparations entailed an antibody response against alpha-toxin and highest antibody and neutralizing activity titers were obtained with liposomes. These results show that liposomes can be used to immunopotentiate CP5, however, a co-incorporated protein able to insert lipids bilayers is probably required.

Effects of culture conditions on production of type 5 capsular polysaccharide by human and bovine Staphylococcus aureus strains.

Two Staphylococcus aureus strains, the prototype human Reynolds strain and a bovine isolate, were grown in different complex media and in a synthetic medium (D. Taylor and K. T. Holland, J. Appl. Bacteriol. 66:319-329, 1989) and compared for their ability to produce type 5 capsular polysaccharide. Cell-bound and cell-free type 5 capsular polysaccharide were measured by a new one-step competition enzyme-linked immunosorbent assay. The total production and the proportion of cell-bound type 5 capsular polysaccharide were dependent on the nature of the medium, the duration of the culture, and the strain. Both strains produced more type 5 capsular polysaccharide when cultivated in the synthetic medium than when cultivated in complex media. The best yield of type 5 capsular polysaccharide, about 300 micrograms/ml of medium, was obtained with strain Reynolds grown for 48 h with shaking in the synthetic broth containing glucose as a carbon source.

Immune recruitment and bactericidal activity of neutrophils in milk of cows vaccinated with staphylococcal alpha-toxin.

The ability of Staphylococcus aureus alpha-toxin to recruit neutrophils in the milk of vaccinated cows and the bactericidal efficiency of these neutrophils were evaluated. Six lactating Holstein cows that were free of intramammary infection received systemic immunization by subcutaneous injection of Freund's incomplete adjuvant with alpha-toxin (n = 2), alpha-toxin mixed with type 5 capsular polysaccharide (n = 2), or a conjugate of these two antigens (n = 2). Controls (n = 4) and vaccinated cows (n = 6) received intramammary infusions of alpha-toxin. No increase in somatic cell count was recorded in quarter milk samples from unimmunized cows; however, 10 micrograms of alpha-toxin induced a local inflammatory reaction in vaccinated cows that was characterized by early and massive cellular recruitment into the mammary gland. More than 90% of the recruited cells were neutrophils. The speed and magnitude of the cellular recruitment were dose-dependent; the threshold dose was 0.6 microgram. Milk samples showed significant bactericidal activity against the type 5 S. aureus strain, regardless of the vaccine used, and showed a decrease in bacterial count of about 2 log10 from the initial inoculum. The best efficiency was recorded during the early phase of cellular recruitment with concomitant activation of blood-derived neutrophils. This study demonstrates that a bacterial virulence factor, alpha-toxin, is able to induce immune recruitment of neutrophils for efficient bactericidal activity in milk when cows are immunized with alpha-toxin that is used either as a nonconjugate vaccinate or as a carrier protein in a conjugate vaccine. The study also suggests that neutrophils that are recruited from blood are activated during inflammation in response to specific antigens.

Assessment of the opsonic activity of purified bovine sIgA following intramammary immunization of cows with Staphylococcus aureus.

The phagocytosis of Staphylococcus aureus by bovine polymorphonuclear neutrophils (PMN) requires the presence of antibodies. Among the major isotypes of bovine antibodies, IgG2 and IgM are considered opsonic for bovine PMN. However, the role of purified bovine secretory IgA (sIgA) as an opsonin has not been assessed. In the present study, IgG2 were obtained from serum and sIgA, IgG1, and IgM were purified from the colostrums of three cows intramammarily immunized with heat-killed Staphylococcus aureus. The Ig preparations were assayed for specific antibodies, and the opsonic capacity of every isotype was investigated. Despite the presence of antibodies, we observed no distinct chemiluminescence response of PMN stimulated with sIgA- or IgG1-opsonized S. aureus, whereas IgM or IgG2 bound to bacteria induced a marked chemiluminescence response. Moreover, the counting of internalized bacteria per PMN after phagocytosis revealed a low uptake of S. aureus opsonized with sIgA or IgG1, in contrast to IgM or IgG2, which triggered efficient ingestion of bacteria. Priming of neutrophils by TNF-alpha, IFN-gamma, or C5adesArg did not promote an oxidative burst or uptake of sIgA-opsonized S. aureus to a greater extent than with IgG1-opsonized bacteria. Furthermore, analysis of uningested bacteria by flow cytometry after incubation with PMN showed a preferential uptake of IgM-opsonized S. aureus by PMN and only few sIgA-positive stained bacteria were PMN-associated. These experiments indicate that sIgA, like IgG1 and unlike IgM or IgG2, could not be considered as a major opsonin for phagocytosis of S. aureus by bovine blood PMN.