DNA-protein immunization against the GapB and GapC proteins of a mastitis isolate of Staphylococcus aureus

One of the most economically important diseases that affect the dairy industry is bovine mastitis caused by strains of S. aureus. The development of an effective vaccine has been hampered by the antigenic diversity of the bacterium. Immunization with plasmid DNAs, encoding S. aureus antigens either as single molecule or as chimeric products containing at least two antigens, has been proposed as a novel strategy to prevent this costly disease. We continued our studies on a chimeric protein composed of the surface-located GapB and GapC proteins of S. aureus and in this work we tested the effects of DNA vaccination with plasmids encoding the individual antigens as well as the GapC/B protein with or without a boost with the recombinant proteins. The results showed that DNA vaccination alone was unable to elicit a significant humoral response and barely able to elicit a detectable cell-mediated response to the recombinant antigens. These effects were overcome by boosting with the proteins indicating that these DNA vaccines alone were not sufficient to mount an immune response against the S. aureus GapB and GapC proteins.     

Characterization of two proteins of Staphylococcus aureus isolated from bovine clinical mastitis with homology to glyceraldehyde-3-phosphate dehydrogenase

Staphylococcus aureus is the most common causative agent of bovine mastitis and vaccines developed to control this disease showed limited protection due in part to the lack of common antigens among the mastitis isolates. We isolated and identified two genes encoding proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from a S. aureus strain isolated from bovine clinical mastitis. The GapB and GapC proteins share considerable homology to the GapB and GapC products of human strains of S. aureus. These two proteins could be distinguished by their different GAPDH activities and binding to bovine transferrin properties. Both gapB and gapC genes were conserved in 11 strains tested, and the GapC protein was present on the surface of all S. aureus strains.     

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.     

Efficacy of Staphylococcus aureus vaccines for bovine mastitis: a systematic review

Bovine mastitis is the main cause of economic loss in milk production worldwide and Staphylococcus aureus is the agent most frequently associated with the disease. The aim of this systematic review was to assess the efficacy of vaccines for bovine mastitis caused by S. aureus and suggest the immunotherapeutic protocols that have achieved the best and/or most promising results. An electronic search was made of the PubMed and Web of Science databases in November 2009. Only studies that tested vaccines in vivo in cows were included. The experimental design, methodological quality, type of vaccine and results of the studies were analyzed. Twenty-four papers were selected for this review. In general, bacterin-toxoid vaccines, vaccines of DNA-recombinant protein and recombinant protein alone were investigated in the studies selected. This systematic review suggests that vaccines that employ new technologies (DNA and/or recombinant protein vaccines) and some long-standing bacterins have achieved good results, which supports their use in the prevention and control of bovine mastitis caused by S. aureus. However, methodological differences and in some cases, a lack of more severe scientific criteria (such as double blind protocols) hinder the assessment of the effectiveness of these vaccines.     

Experimental trial in heifers vaccinated with Staphylococcus aureus avirulent mutant against bovine mastitis

Staphylococcus aureus, is the most frequently isolated pathogen from cases of bovine mastitis. Vaccination against S. aureus seems to be a rational approach for the control of staphylococcal mastitis. In the present work we evaluate the response of heifers vaccinated with a S. aureus avirulent mutant to the intramammary challenge with a S. aureus virulent strain. Clinical signs, production of milk, shedding of S. aureus cells, somatic cell count (SCC) and antigen-specific IgG in blood and milk, were determined. Two subcutaneous doses of a culture of the mutant, used as vaccine, was administered to four pregnant heifers 30 and 10 days before calving. The vaccinated heifers and four non-vaccinated were challenged 10 days after calving with the homologous virulent S. aureus strain, which was inoculated by intramammary route into two quarters of each animal. No local tissue damage was observed due to the administration of the vaccine. A significantly increase of specific IgG to S. aureus RC122 was detected in blood and milk of vaccinate heifers as well as a slight increase in daily milk yield during the trial. No significant difference on shedding of bacteria in milk and SCC were found among groups. In conclusion, vaccination of heifers before calving by an avirulent mutant vaccine of S. aureus, induced specific and significant antibody responses and provide better post-challenge conditions in vaccinated heifers.     

Variations in immunoglobulin isotype produced during the antibody response to Brucella abortus and Staphylococcus aureus vaccines in sheep

Experiments in sheep were carried out to examine factors modifying the immunoglobulin (Ig) isotype of the antibody response to Brucella abortus. Live B abortus (S19) stimulated higher titres of agglutinating antibody and IgG1 and IgG2 antibody than did killed B abortus. Live B abortus stimulated a more protracted synthesis of IgG2 antibody during the primary and secondary responses than did the killed S19 vaccine. In a second experiment, the capacity of live and killed Staphylococcus aureus to modify the antibody response to killed B abortus was examined. Both live and killed S aureus enhanced production of anti-brucella antibodies; this response was attributed to the adjuvant properties of S aureus. Killed S aureus enhanced production of anti-brucella antibody to a greater extent than live S aureus. Live S aureus did not preferentially enhance production of IgG2 anti-brucella antibody. The results suggested that the enhanced production of IgG2 antibody induced by live vaccines does not depend solely on a pyogenic lesion at the vaccination site.     

如何进行奶牛乳房炎金黄色葡萄球菌疫苗的合理评估

奶牛乳房炎是造成全球奶业经济损失的主要原因,而金黄色葡萄球菌是最主要的病原菌。本文系统、直观地对研究奶牛乳房炎金黄色葡萄球菌疫苗的科研结论进行量化总结和评估,目的是为奶牛乳房炎疫苗的研发提供新思路,为设计合理的奶牛乳房炎疫苗给动物评估试验方案提供科学依据。本文在Pubmed、Science数据库和CNKI数据库对＂奶牛乳房炎疫苗＂类的文章进行了合理的电子检索,然后对细菌疫苗、细菌类毒素疫苗、DNA-重组蛋白疫苗、单一的重组蛋白疫苗等方面的科研论文从试验设计、方法、疫苗类型和研究结果 4个方面进行了全面的分析。结果表明,采用DNA、重组蛋白新技术的疫苗和传统的细菌疫苗已取得良好效果,疫苗可以成为预防和控制金黄色葡萄球菌性奶牛乳房炎的最好或最有前景的一种途径。但是研究方法差异和双盲试验的缺乏阻碍对疫苗效果的合理评估。     

Staphylococcus aureus vaccine against mastitis in dairy cows,composition and evaluation of its immunogenicity in a mouse model

Bovine mastitis caused by Staphylococcus aureus (S. aureus) is a most important infection disease that affects both the quality and the quantity of milk production. Antibiotic therapies formulated for intramammary use are generally unsuccessful in eliminating existing S. aureus infections. Vaccination is a logical approach to the control of S. aureus udder infections. However, to date commercially available S. aureus vaccine have shown limited efficacy under field conditions, mainly due to the paucity of information regarding relevant antigens which will induce a broad spectrum immunization. In the present paper the attempt to develop a new vaccine designated MASTIVAC I is described. MASTIVAC I is composed of three strains of S. aureus namely: VLVL8407; ZO3984 and BS449 which were isolated from clinical and sub-clinical cases of bovine mastitis. A mouse model was used to evaluate the S. aureus specific antibody production and protection of mice against virulent S. aureus strains. The results obtained showed that this vaccine exhibits a broad spectrum of antigenic and immunogenic properties that protects mice from homologues and hetrologous S. aureus challenge.     

Protective effect of recombinant staphylococcal enterotoxin A entrapped in polylactic-co-glycolic acid microspheres against Staphylococcus aureus infection

ABSTRACT: Staphylococcus aureus is an important cause of nosocomial and community-acquired infections in humans and animals, as well as the cause of mastitis in dairy cattle. Vaccines aimed at preventing S. aureus infection in bovine mastitis have been studied for many years, but have so far been unsuccessful due to the complexity of the bacteria, and the lack of suitable vaccine delivery vehicles. The current study developed an Escherichia coli protein expression system that produced a recombinant staphylococcal enterotoxin A (rSEA) encapsulated into biodegradable microparticles generated by polylactic-co-glycolic acid (PLGA) dissolved in methylene chloride and stabilized with polyvinyl acetate. Antigen loading and surface properties of the microparticles were investigated to optimize particle preparation protocols. The prepared PLGA-rSEA microspheres had a diameter of approximately 5 μm with a smooth and regular surface. The immunogenicity of the PLGA-rSEA vaccine was assessed using mice as an animal model and showed that the vaccine induced a strong humoral immune response and increased the percent survival of challenged mice and bacterial clearance. Histological analysis showed moderate impairment caused by the pathogen upon challenge afforded by immunization with PLGA-rSEA microspheres. Antibody titer in the sera of mice immunized with PLGA-rSEA microparticles was higher than in vaccinated mice with rSEA. In conclusion, the PLGA-rSEA microparticle vaccine developed here could potentially be used as a vaccine against enterotoxigenic S. aureus.     

Effect of a trivalent vaccine against Staphylococcus aureus mastitis lymphocyte subpopulations, antibody production, and neutrophil phagocytosis

The effect of a novel bovine mastitis trivalent vaccine, containing Staphylococcus aureus capsular polysaccharide type 5 (T5), 8 (T8), and 336 (T336), on lymphocyte subpopulations, antibody production, and neutrophil phagocytosis was evaluated. Twenty pregnant heifers were immunized with either the trivalent alone, trivalent emulsified in Freund's incomplete adjuvant (FICA), trivalent in aluminum hydroxide, or adjuvant only (FICA). Immunization was done 30 d before the expected calving date followed by 2 boosts in a 2-week interval. Compared to FICA, serum antigen-specific immunoglobulin (Ig)G1 and IgG2 were significantly increased in all the vaccinated groups before parturition and sustained until 3 wk postpartum. In comparison with the trivalent alone, formulation with either adjuvant enhanced production of IgG2, but not IgG1. Immune sera, which contained the highest amount of antibodies, slightly increased neutrophil phagocytosis to the 3 serotypes of killed S. aureus, but most of the differences were not significant due to large variation between the cows. The percentage of CD4+ lymphocyte was significantly higher in vaccinated groups than that of FICA 4 wk after the primary immunization. In comparison with FICA, cows inoculated with trivalent vaccine and adjuvants had an increased percentage of CD8+ lymphocytes at 2 time points, 2 wk before and after calving. Our results indicated that the whole cell trivalent vaccine, with or without adjuvants, is able to elicit antibody responses specific to the 3 capsular polysaccharide antigens. The increase of T8-specific IgG2 was more noticeable when the vaccine was emulsified with adjuvants.     

Immunopotentiation of cattle vaccinated with a soluble Brucella abortus antigen with low LPS content: an analysis of cellular and humoral immune responses.

The adjuvant effects of dimethyl dioctadecyl ammonium bromide (DDA) alone or in combination with trehalose dimycolate (TDM) or muramyl dipeptide (MDP) on bovine humoral and cellular responses to a soluble protein extract of gamma irradiated Brucella abortus strain 19 (SPEBA) were investigated. Thirty-five beef steers were randomly allotted to nine groups. Three of these groups received SPEBA (2 mg protein per dose) subcutaneously in combination with adjuvants, one group received the reduced dose of B. abortus strain 19 (S19), and one group received SPEBA alone. Controls included groups receiving adjuvant preparations only or no vaccine. Immune responses to the various immunizations were assessed sequentially for 56 days using various in vitro and in vivo assays. The humoral response to B. abortus was measured using standard serologic tests, an enzyme-linked immunosorbent assay, and a quantitative fluorometric immunoassay. The cell-mediated immune (CMI) response was measured by antigen-specific lymphoproliferation (LP), interleukin 2 (IL 2) production, and soluble suppressor factor release. Skin testing at day 35 for delayed-type hypersensitivity (DTH) to SPEBA was also performed. Minimal humoral responses were induced with SPEBA alone. The highest and most sustained serum antibody responses to B. abortus antigens were elicited by the S19 vaccine. A combination of SPEBA with DDA + TDM induced higher antibody levels than SPEBA with DDA or SPEBA with DDA + MDP. Responses to DTH among the groups receiving SPEBA were most notable in the SPEBA with DDA + TDM groups. Increased IL 2 production was greatest in the S19 and SPEBA with DDA + TDM vaccinates. The results indicated that a combination of DDA + TDM best potentiated immune responses to a soluble B. abortus antigen preparation and may be useful as adjuvants for future vaccines.     

Effects of Staphylococcus aureus mastitis on bovine mammary gland plasma cell populations and immunoglobin concentrations in milk

Bovine mammary tissue and milk samples were examined to determine effects of chronic Staphylococcus aureus mastitis on the humoral immune response. Parenchymal and teat end tissues from lactating bovine mammary glands were stained immunohistochemically to determine distribution of immunoglobulin (Ig) G1-, IgG2-, IgA-, and IgM-producing plasma cells. Numbers of all Ig-producing plasma cells tended to be higher in tissues from S. aureus infected quarters compared with controls, but most differences were not statistically different. Numbers of IgG1-producing plasma cells at the Furstenberg's rosette area of infected quarters were significantly (P less than 0.05) higher than uninfected quarters. There were no significant differences in concentrations of Ig isotypes in milk from S. aureus infected and uninfected quarters. Data suggest that the antigenic effect of chronic S. aureus infection on the humoral immune response of the bovine mammary gland is minimal. Persistency of S. aureus infection may result, in part, from suboptimal stimulation or immunosuppression of the mammary immune system.     

人参皂甙Rb1的免疫佐剂作用

分别将人参皂甙Rbl(人参主要成分之一)以及氢氧化铝胶作为佐荆和金黄色葡萄球菌菌体抗原混合免疫豚鼠，观察免疫前后血清抗体滴度变化；并用Rbl和奶牛乳房炎金黄色葡萄球菌疫苗混合免疫奶牛，观察免疫前后血清抗体滴度变化，以及血液淋巴细胞在刀豆蛋白A(ConA)、美洲商陆(PWM)和金黄色葡萄球菌抗原刺激下的体外细胞增殖反应。结果表明，Rbl和抗原混合物免疫动物后，未见任何不良反应；Rbl组豚鼠血清抗体滴度比对照组和氢氧化铝胶佐剂组滴度增加快，幅度显著增高；Rbl组奶牛血清抗体滴度比对照组奶牛显著增加，ConA、PWM和金黄色葡萄球菌抗原诱导的血液淋巴细胞体外细胞增殖反应比对照组显著提高。因此，Rbl是一种有效的免疫佐剂。     

Vaccination against experimental staphylococcal mastitis in dairy heifers

Vaccination-challenge experiments were carried out with dairy heifers using new, killed cell-toxoid-adjuvant Staphylococcus aureus vaccines. The organisms in the vaccines were cultured under conditions which simulated in vivo growth and induced expression of a pseudocapsule. Dextran sulphate which promotes synthesis of IgG2 antibody was included in the vaccines as the primary adjuvant. Vaccinated heifers developed very high levels of both IgG1 and IgG2 anti-pseudocapsule antibody in serum, however, titres of neutralising antibody against toxoided haemolysins were generally low. Vaccinated and unvaccinated control heifers were challenged by intramammary infusion of three virulent strains of S aureus in four experiments. Vaccinated heifers were more resistant to clinical mastitis following challenge than were controls, and the vaccinates had significantly greater milk production than controls following challenge. The most promising vaccine had dextran sulphate combined with mineral oil as the adjuvant injected intramuscularly.     

Serological proteome analysis of Staphylococcus aureus isolated from sub-clinical mastitis

Staphylococcus aureus is the most common aetiologic agent of contagious bovine mastitis. Studies of the molecular epidemiology of S. aureus strongly suggest that some genetic subsets of strains are particularly well adapted for causing infections in cattle. This communication reports the setup of experimental protocols to identify the immunogenic proteins expressed by one of the most common field isolated strain of S. aureus responsible for sub-clinical mastitis cases. The serological proteome analysis (SERPA) approach applied consists of three main steps: two-dimensional electrophoresis-based separation of the proteins contained in field isolated S. aureus extracts enriched for surface proteins, detection of immunogenic spots using anti-serum collected from sub-clinical mastitis cases and identification of antigens by mass spectrometric-based methodologies. The study allowed to identify three immunogenic proteins: DNAase translocase FtsK, ribosomal proteins S1 and a Tell-like protein.     

Quantitation of bovine immunoglobulin G2 antibodies binding Staphylococcus aureus, using a murine monoclonal antibody

A murine monoclonal antibody specific for bovine immunoglobulin (Ig) G2 was used to develop an enzyme-linked immunosorbent assay (ELISA) to measure serum antibodies against the bovine pathogen Staphylococcus aureus. Anion exchange chromatography was used to prepare IgG2 from serum taken from a mastitic cow infected with S aureus. Specific-IgG2 antibodies binding S aureus were purified with an affinity column, using a heat-killed, low protein A strain of S aureus (M-10). Purified antibodies did not contain IgG1 or IgM and were composed of greater than 95% heavy and light chains determined on the basis of polyacrylamide gel electrophoresis. Purified IgG2 anti-S aureus antibody was used as a reference in an ELISA that (i) could detect 5.0 ng of IgG2, (ii) was specific for IgG2 antibodies binding S aureus, (iii) was precise within and among different assays, (iv) yielded 112% recovery of the purified standard, and (v) when diluted in nonspecific IgG2, generated a curve that was parallel to the standard when a sample was serially diluted. A field study with cows having elevated California mastitis test scores showed evidence of infection with S aureus, as judged by a 59% increase (P less than 0.01) in IgG2 S aureus-specific antibodies and a 25% increase (P less than 0.05) in total IgG2 antibodies. There were no differences in IgG1 concentrations in plasma. Using indirect immunofluorescence, we also confirmed that bovine polymorphonuclear cells bound IgG2 preferentially over IgG1 or IgM. Measurement of antigen-specific IgG2 antibodies may therefore be useful as an index of specific antibody immunity to mastitis-causing organisms.     

Altered leukocyte responsiveness in dairy cows with naturally occurring chronic Staphylococcus aureus mastitis

Changes in inflammatory parameters, leukocyte surface markers, functional responses and cytokine mRNA expression of leukocytes of dairy cows with naturally occurring chronic Staphylococcus aureus (S. aureus) mastitis and healthy cows were determined to elucidate the leukocyte responses to S. aureus infection of the mammary gland. Increased values in inflammatory parameters and matrix metalloproteinase activities in milk revealed the characteristics of cows with chronic mastitis. Expression of L-selectin and CD18 molecules on neutrophils and proportion of CD8 cells in milk from cows with S. aureus mastitis were significantly (P<0.05) increased compared with those found in healthy cows. The FcR-stimulated CL response of blood neutrophils was significantly (P<0.05) decreased in cows with S. aureus mastitis. Significantly (P<0.05) decreased mitogenic responses of lymphocytes were found in cows with S. aureus mastitis; however, the values were not restored to those of healthy cows when stimulated with both mitogens and the cytokine IL-1β. The mRNA expression of TNF-α, IL-1β and IL-8 on milk leukocytes from cows with S. aureus was found to be increased compared with that of healthy cows. The changes of immune responses found in cows with S. aureus mastitis appear to be influenced by the severity and duration of inflammation in infected quarters. The down-regulation of the leukocyte functions found in cows with S. aureus mastitis appears to be associated with the progress of the chronic stage of S. aureus mastitis.     

Effects of autogenous toxoid-bacterin in lactating cows with Staphylococcus aureus subclinical mastitis

To evaluate clinical effects of autogenous toxoid-bacterin treatment for Staphylococcus aureus subclinical mastitis in lactating cows, 22 cows which had at least one S. aureus infected quarter were selected from among cows at a S. aureus prevalent dairy farm. Eleven cows were injected with their own autogenous toxoid-bacterin and the others were maintained as non-injected control. In the toxoid-bacterin injected group, 27% of infected quarters were cured during the 12-week trial, compared to 5% in the control group. New intramammary infections with S. aureus were only detected in 3 quarters of the control group. Mean IgG antibody titer against S. aureus somatic antigens and alpha-toxin in serum and milk were significantly increased in the toxoid-bacterin injected group (p<0.05) and remained higher than those of the control group which showed no significant changes (p<0.05). In contrast to the control group, from 3 weeks after the second injection of the toxoid-bacterin injected group, mean S. aureus cfu/ml in milk samples from injected quarters with S. aureus was significantly decreased until the end of the study (p<0.05). In the toxoid-bacterin injected group, significant decreases of mean SCC were detected from milk samples from infected quarters with S. aureus from week 7 to week 10 (p<0.05). These data show that autogenous toxoid-bacterin treatment against S. aureus subclinical mastitis in lactating cows may increase the cure rate of the infections, reduce the severity of the infections and also prevent occurrence of the new infections.     

Dynamics of Staphylococcus aureus infections during vaccination with an autogenous bacterin in dairy cattle

The effect of an autogenous vaccine against Staphylococcus aureus on S. aureus prevalence and mastitis, as well as on somatic cell count (SCC), was studied in a dairy herd with a high prevalence of S. aureus. The vaccination group (n = 35; 22 cows and 13 heifers) and the control group (n = 36; 23 cows and 13 heifers) received the vaccine or a placebo, respectively, according to the following protocol: all animals: basic immunization (twice, 3 weeks apart); cows: booster dose at the time of drying off, 5 and 2 weeks before calculated calving date; heifers: booster dose 2 and 5 weeks before calculated calving date. The vaccine or the placebo was administered subcutaneously in the area of the supramammary lymph nodes. Quarter milk samples were collected monthly and subjected to SCC and bacteriological evaluation. At this time, the animals were also checked for signs of clinical mastitis. Non-clinical S. aureus mastitis diagnoses were based on udder quarter SCC and a positive S. aureus culture. In order to compare the SCC in individual whole milk samples, records from the monthly milk quality testing were evaluated. Cow and udder quarter prevalence of S. aureus intramammary infections calculated for the experimental animals and quarters, respectively, did not differ between groups. However, during the lactation period following the boostcr dose, the prevalence of S. aureus increased in both groups (P < 0.05). The cumulative incidence of various mastitis diagnoses (clinical, subclinical, latent infection) due to S. aureus on an animal basis did not differ between groups. On an udder quarter basis, the cumulative incidence of subclinical mastitis was higher in vaccinated animals than in control animals (33.8 versus 26.0%; P < 0.05). This was mainly due to a higher cumulative incidence of subclinical mastitis in vaccinated than control heifers. The SCC in composite milk samples did not differ between groups, but increased as lactation progressed. The herd prevalence of S. aureus differed considerably throughout the study period, but declined consistently to below 10% at the end of the study period. Recent herd checks revealed a prevalence of S aureus infections of < 5%. It is concluded that the autogenous bacterin tested in this study did not have the desired effect on the prevalence of S. aureus infections and mastitis or SCC. The decline in S. aureus prevalence was very probably due to other factors than specific immunization against S. aureus.     

Changes in blood and milk lymphocyte sub-populations during acute and chronic phases of Staphylococcus aureus induced bovine mastitis

Staphylococcus aureus (S. aureus) often causes long-lasting chronic sub-clinical udder infections in dairy cows. To investigate if this can be due to a negative impact of S. aureus on lymphocytes important for the immune defence, alterations in proportions and expression intensity of CD4+, CD8+, WC1+, B and IL-2R+ lymphocytes was studied in blood and milk, as S. aureus mastitis developed from acute clinical to chronic sub-clinical form. Six healthy dairy cows were inoculated with S. aureus in one udder quarter per cow, and one quarter per cow acted as an uninfected control. Blood samples, and milk samples from infected and non-infected quarters were collected before infection and for five weeks after infection. All infected quarters developed acute clinical mastitis, of which five turned into chronic sub-clinical mastitis. In infected quarters, the proportions of all lymphocyte sub-sets, except WC1+ cells, differed in acute phase compared to pre-infection, while the dominant finding in the chronic phase was increased expression intensities per cell. An impact on blood lymphocytes and milk lymphocytes in non-infected quarters also occurred, mainly during the chronic phase. The most prominent finding was the increased proportion and expression of B-lymphocytes in blood, infected and non-infected quarters during chronic sub-clinical mastitis. As S. aureus can invade and survive intracellularly, a preferential stimulation of B-cells, suggesting development of a humoral response, may not be sufficient to eliminate intracellular bacteria, which could explain the persistence of the infection.