Differential induction of complement fragment C5a and inflammatory cytokines during intramammary infections with Escherichia coli and Staphylococcus aureus

The prompt recruitment of neutrophils to the site of infection is essential for the defense of the bovine mammary gland against invading pathogens and is determinant for the outcome of the infection. Escherichia coli is known to induce clinical mastitis, characterized by an intense neutrophil recruitment leading to the eradication of the bacteria, whereas Staphylococcus aureus induces subclinical mastitis accompanied by a moderate neutrophil recruitment and the establishment of chronic mastitis. To elicit the neutrophil recruitment into the udder, inflammatory mediators must be produced after recognition of the invading pathogen. To our knowledge, those mediators have never been studied during S. aureus mastitis, although understanding of the neutrophil recruitment mechanisms could allow a better understanding of the differences in the pathogeneses elicited by E. coli and S. aureus. Therefore, we studied, at several time points, the accumulation of neutrophils and the presence of the chemoattractant complement fragment C5a and of the cytokines interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and IL-8 in milk after inoculation of E. coli or S. aureus in lactating bovine udders. The low levels of C5a and the absence of cytokines in milk from S. aureus-infected cows, compared to the high levels found in milk from E. coli-infected animals, mirror the differences in the severities of the two inflammatory reactions. The cytokine deficit in milk after S. aureus inoculation in the lactating bovine mammary gland could contribute to the establishment of chronic mastitis. This result could help in the design of preventive or curative strategies against chronic mastitis.     

Role of biofilm-associated protein bap in the pathogenesis of bovine Staphylococcus aureus

Staphylococcus aureus is a common cause of intramammary infections, which frequently become chronic, associated with the ability of the bacteria to produce biofilm. Here, we report a relationship between the ability to produce chronic bovine mastitis and biofilm formation. We have classified bovine mastitis S. aureus isolates into three groups based on the presence of particular genetic elements required for biofilm formation: group 1 (ica(+) bap(+)), group 2 (ica(+), bap negative), and group 3 (ica negative, bap negative). Overall, animals naturally infected with group 1 and 2 isolates had a lower milk somatic cell count than those infected with isolates of group 3. In addition, Bap-positive isolates were significantly more able to colonize and persist in the bovine mammary gland in vivo and were less susceptible to antibiotic treatments when forming biofilms in vitro. Analysis of the structural bap gene revealed the existence of alternate forms of expression of the Bap protein in S. aureus isolates obtained under field conditions throughout the animal's life. The presence of anti-Bap antibodies in serum samples taken from animals with confirmed S. aureus infections indicated the production of Bap during infection. Furthermore, disruption of the ica operon in a bap-positive strain had no effect on in vitro biofilm formation, a finding which strongly suggested that Bap could compensate for the deficiency of the PIA/PNAG product (a biofilm matrix polysaccharide). Altogether, these results demonstrate that, in the bovine intramammary gland, the presence of Bap may facilitate a biofilm formation connected with the persistence of S. aureus.     

Opsonization of Streptococcus agalactiae of bovine origin by complement and antibodies against group B polysaccharide.

The contribution of bovine complement and antibodies (Ab) against the group B polysaccharidic antigen (GBA) to the opsonization of Streptococcus agalactiae isolated from bovine mastitis cases was investigated by using affinity-purified Ab. GBA-specific Ab were not opsonic by themselves, but in the presence of complement (precolostral calf serum) with an opsonization time of 15 min, they exhibited a dose-dependent opsonic activity in a polymorphonuclear leukocyte chemiluminescence assay. Kinetic studies of the deposition of complement component C3 on protein X-bearing nontypeable (NT/X) strains with an enzyme-linked immunosorbent assay showed that C3 was deposited on bacteria in the absence of Ab but that GBA-specific Ab markedly accelerated the process by reducing the lag phase, which extended up to 15 min when Ab were absent. In the absence of Ab, C3 deposition was inhibited by 5 mM salicylaldoxime or heat treatment at 56 degrees C for 3 min and necessitated Mg2+ ions but not Ca2+ ions, suggesting that activation of complement was effected by the alternative pathway only. When GBA-specific Ab were added to complement, the inhibitory treatments lost much of their efficacy, suggesting that the classical pathway was recruited. Deposition of C3 on NT/X strains in the absence of Ab induced chemiluminescence and phagocytic killing. With the addition of GBA-specific Ab, the numbers of surviving bacteria were halved (P < 0.05) compared with killing in the presence of complement alone. It can be concluded that NT/X strains are activators of the alternative pathway of complement and that GBA-specific Ab reinforce the opsonic efficiency of serum by recruiting the classical pathway and slightly enhancing phagocytic killing.     

The hyaluronic acid capsule of Streptococcus uberis is not required for the development of infection and clinical mastitis

The frequency at which the genes responsible for capsule biosynthesis occurred in field isolates of Streptococcus uberis was determined. Of the two genotypes detected (hasABC and hasC), the capsular genotype (hasABC) was more common. This genotype was present at a higher frequency in a population isolated from mastitis cases than in a population isolated from cattle bedding. The virulence of a mutant strain of S. uberis (TRF0-6) that lacked the ability to produce a hyaluronic acid capsule due to an insertion within its single copy of hasA (P. N. Ward, T. R. Field, W. G. F. Ditcham, E. Maguin, and J. A. Leigh, Infect. Immun. 69:392-399, 2001) was compared to that of the capsular parental strain (0140J). Strains TRF0-6 and 0140J infected all mammary gland quarters following experimental challenge. The wild type and the mutant induced overt signs of disease in four out of four and in six out of eight mammary gland quarters, respectively. Both the wild type and the hasA mutant were resistant to killing by bovine neutrophils following cultivation in bovine milk. The ability to withstand the bactericidal action of neutrophils following growth in milk was therefore independent of the capsule and coincided with the ability of supernatants from such cultures to prevent the bactericidal action of neutrophils. This investigation revealed that, in the absence of the capsule, S. uberis is able to withstand the bactericidal effect of bovine neutrophils and induce mastitis in dairy cows.     

Differential Induction of Complement Fragment C5a and Inflammatory Cytokines during Intramammary Infections with Escherichia coli and Staphylococcus aureus

The prompt recruitment of neutrophils to the site of infection is essential for the defense of the bovine mammary gland against invading pathogens and is determinant for the outcome of the infection. Escherichia coli is known to induce clinical mastitis, characterized by an intense neutrophil recruitment leading to the eradication of the bacteria, whereas Staphylococcus aureus induces subclinical mastitis accompanied by a moderate neutrophil recruitment and the establishment of chronic mastitis. To elicit the neutrophil recruitment into the udder, inflammatory mediators must be produced after recognition of the invading pathogen. To our knowledge, those mediators have never been studied during S. aureus mastitis, although understanding of the neutrophil recruitment mechanisms could allow a better understanding of the differences in the pathogeneses elicited by E. coli and S. aureus. Therefore, we studied, at several time points, the accumulation of neutrophils and the presence of the chemoattractant complement fragment C5a and of the cytokines interleukin-1β (IL-1β), tumor necrosis factor alpha, and IL-8 in milk after inoculation of E. coli or S. aureus in lactating bovine udders. The low levels of C5a and the absence of cytokines in milk from S. aureus-infected cows, compared to the high levels found in milk from E. coli-infected animals, mirror the differences in the severities of the two inflammatory reactions. The cytokine deficit in milk after S. aureus inoculation in the lactating bovine mammary gland could contribute to the establishment of chronic mastitis. This result could help in the design of preventive or curative strategies against chronic mastitis.     

Isolation of Staphylococcus aureus L forms from experimentally induced bovine mastitis.

Bacterial L forms were isolated from milk samples of dairy cattle infected experimentally with Staphylococcus aureus. Initially, bacterial L forms were induced in vitro from 12 of 44 S. aureus strains isolated from bovine mastitis. Cows were experimentally infected in two experiments with strains shown in vitro to be easily inducible to L form and with S. aureus Newbould 305. Each quarter of the mammary gland was infected with either 10(3) or 10(6) CFU of the test strains. Treatment was initiated with 100,000 U of penicillin G per quarter at the first signs of clinical mastitis. Milk samples were collected daily and cultured on bovine blood agar and PPLO agar (Difco Laboratories, Detroit, Mich.) with 10% horse serum and 5% NaCl. Staphylococcal L forms were isolated from milk samples collected from infected glands in both experiments after antibiotic therapy. Glands with the highest concentrations of leukocytes and bacteria were most likely to yield L forms in milk samples after treatment was initiated. Cows harboring L forms typically yielded parental organisms after cessation of antibiotic therapy. No detectable changes occurred in antibiotic susceptibilities, coagulase production, or biochemical activities in strains induced to L form followed by reversion to the parental form. These results demonstrated that L forms can occur during treatment of bovine mastitis and that L forms may be one explanation for the poor response of staphylococcal bovine mastitis to antibiotic therapy.     

Capsule expression by bovine isolates of Staphylococcus aureus from Argentina: genetic and epidemiologic analyses

Staphylococcus aureus is an important cause of bovine mastitis worldwide, and effective preventive or therapeutic modalities are lacking. Although most human S. aureus isolates produce capsular polysaccharides (CPs), few reports have described the prevalence of capsules on bovine isolates. This information is important for the rational design of a vaccine for the prevention of staphylococcal mastitis. We serotyped 195 S. aureus strains isolated between 1989 and 1997 from the milk of mastitic cows in Argentina. Only 14 (7.1%) of the strains were serotype 5, and all were recovered between 1989 and 1992. Thirteen serotype 8 strains were identified, and 12 of these were isolated between 1991 and 1994. The remaining 168 isolates were nonreactive (NR) with CP serotype 5 (CP5)- or CP8-specific antibodies. Hybridization studies performed with genomic DNA from eight NR strains revealed that only three of them carried the capsule genes. Pulsed-field gel electrophoresis (PFGE) performed with 127 of the 195 S. aureus isolates revealed that most (86%) strains belonged to one of four major PFGE groups. Although 8 of 14 CP5 isolates showed a common PFGE pattern (arbitrarily defined as A1), 31 other A1 isolates from the same time period (1989 to 1992) were not CP5 positive. In contrast, only nine PFGE type B3 isolates were recovered between 1990 and 1994, and eight of these were positive for CP8 (P < 0.0003). The results of this study underscore the variability in capsule expression by S. aureus strains isolated from different geographical regions and cast doubt on the roles of CP5 and CP8 in the pathogenesis and immunoprophylaxis of bovine mastitis in Argentina.     

Ability of Staphylococcus aureus coagulase genotypes to resist neutrophil bactericidal activity and phagocytosis.

This study investigated the functional capabilities of neutrophils against different Staphylococcus aureus genotypes isolated from cows with mastitis. Six strains of S. aureus were chosen for use in the study, two with a common genotype, two with an intermediate genotype, and two with a rare genotype. The interaction between bacteria and neutrophils was measured by phagocytosis and bactericidal effect. The average percent killing of bacteria was lowest (40.0%) with strains belonging to the most common genotype, medium (50%) with strains belonging to the intermediate type, and highest (64.2%) with strains belonging to the rare type (P < or = 0.001). Statistically significant differences (P < or = 0.001) in the numbers of phagocytized bacteria were also found between the most prevalent type (6.27 bacteria per cell) and the other two types (intermediate type, 9.26/cell; rare type, 10.5/cell). These findings suggest that one of the reasons for the variation in prevalence of different genotypes of S. aureus in the mammary gland is due to the superior ability of some types to resist phagocytosis and/or killing by bovine neutrophils.     

Genetic and serologic evaluation of capsule production by bovine mammary isolates of Staphylococcus aureus and other Staphylococcus spp. from Europe and the United States

Bovine mastitis caused by Staphylococcus aureus is responsible for major economic losses to the dairy industry, and more-effective therapeutic or preventive approaches are sorely needed. The predominance of staphylococcal capsular polysaccharide types 5 and 8 among human isolates from many sources is well documented, but there seems to be a greater variation in the distribution of capsular serotypes among isolates from cows. A total of 636 isolates of S. aureus from cases of bovine mastitis in Sweden, Denmark, Finland, Iceland, Ireland, and the United States were investigated for production of capsular polysaccharide types 5 and 8. Approximately half of all the European isolates tested were of serotype 8, although variation among countries and among isolates of clinical and subclinical origin was observed. Sweden had the highest frequency (87%) of serotypeable isolates, and Finland had the lowest (48%). Capsule types 5 and 8 accounted for only 42% of the U.S. isolates tested. A few isolates showed weak reactivity with CP5 antiserum in a colony blot assay, and an enzyme-linked immunosorbent assay inhibition method confirmed that the levels of capsule produced by these strains were <10% of those produced by control strains. Fifty isolates that failed to react with capsular antisera all possessed the genes for production of capsular polysaccharide type 5 or 8. These results underscore the variability in capsule production by bovine isolates of S. aureus from different geographic regions. This information is important for the rational design of a capsule-based vaccine to prevent S. aureus bovine mastitis.     

Differential abilities of capsulated and noncapsulated Staphylococcus aureus isolates from diverse agr groups to invade mammary epithelial cells

Staphylococcus aureus is the bacterium most frequently isolated from milk of bovines with mastitis. Four allelic groups, which interfere with the regulatory activities among the different groups, have been identified in the accessory gene regulator (agr) system. The aim of this study was to ascertain the prevalence of the different agr groups in capsulated and noncapsulated S. aureus bacteria isolated from mastitic bovines in Argentina and whether a given agr group was associated with MAC-T cell invasion and in vivo persistence. Eighty-eight percent of the bovine S. aureus strains were classified in agr group I. The remainder belonged in agr groups II, III, and IV (2, 8, and 2%, respectively). By restriction fragment length polymorphism analysis after PCR amplification of the agr locus variable region, six agr restriction types were identified. All agr group I strains presented a unique allele (A/1), whereas strains from groups II, III, and IV exhibited more diversity. Bovine S. aureus strains defined as being in agr group I (capsulated or noncapsulated) showed significantly increased abilities to be internalized within MAC-T cells, compared with isolates from agr groups II, III, and IV. agr group II or IV S. aureus strains were cleared more efficiently than agr group I strains from the murine mammary gland. The results suggest that agr group I S. aureus strains are more efficiently internalized within epithelial cells and can persist in higher numbers in mammary gland tissue than S. aureus strains classified in agr group II, III, or IV.     

Capsule-negative Staphylococcus aureus induces chronic experimental mastitis in mice

Staphylococcus aureus capsular polysaccharides (CP) have been shown to enhance staphylococcal virulence in numerous animal models of infection. Although serotype 5 CP (CP5) and CP8 predominate among S. aureus isolates from humans, most staphylococcal isolates from bovines with mastitis in Argentina are capsule negative. This study was designed to evaluate the effects of CP5 and CP8 expression on the pathogenesis of experimental murine mastitis. Lactating mice were challenged by the intramammary route with one of three isogenic S. aureus strains producing CP5, CP8, or no capsule. Significantly greater numbers of acapsular mutant cells were recovered from the infected glands 12 days after bacterial challenge compared with the encapsulated strains. Histopathological analyses revealed greater polymorphonuclear and mononuclear leukocyte infiltration and congestion in the mammary glands of mice infected with the encapsulated strains compared with the acapsular mutant, and the serotype 5 strain elicited more inflammation than the serotype 8 strain. In vitro experiments revealed that the acapsular S. aureus strain was internalized by MAC-T bovine epithelial cells in significantly greater numbers than the CP5- or CP8-producing strain. Taken together, the results suggest that S. aureus lacking a capsule was able to persist in the murine mammary gland, whereas encapsulated strains elicited more inflammation and were eliminated faster. Loss of CP5 or CP8 expression may enhance the persistence of staphylococci in the mammary glands of chronically infected hosts.     

Effect of specific antibody on adherence of Staphylococcus aureus to bovine mammary epithelial cells.

Staphylococcus aureus is a major pathogen in the bovine mammary gland. The ability of S. aureus to adhere to epithelial cells in the ductules and alveoli of the bovine mammary gland is believed to add greatly to its virulence and may be necessary for colonization. Two in vitro methods were developed for the purposes of quantifying adherence and of determining the effect which specific antibody may have in inhibiting the adherence of this organism. Both methods utilize bovine mammary epithelial primary cells as targets for labeled bacteria. In one assay, the bacteria are labeled with [methyl-3H]thymidine and incubated on the primary epithelial monolayers. The second assay involves labeling the bacteria with biotin. An enzyme-linked immunosorbent assay is then performed with streptavidin conjugated to horseradish peroxidase. Both methods have proven to be reliable and allow for the testing of many criteria in one assay. Cows were immunized with a whole-cell vaccine, and immune serum and milk were collected. The bacteria were then incubated in the presence of serum or milk as a test for antiadherent capability. By using the methods described, distinct antiadherent activity in both serum and milk was demonstrated.     

Enzyme-linked immunosorbent assay for detection of leukocidin toxin from Staphylococcus aureus in bovine milk samples.

An enzyme-linked immunosorbent assay was developed for the detection of leukocidin toxin from Staphylococcus aureus. The minimum concentration of leukocidin detectable with the assay was 30 ng/ml. The enzyme-linked immunosorbent assay was found to be a more sensitive method, by a mean of 45-fold, for leukocidin detection than was observation of cytolytic effects of the toxin on bovine neutrophils. A mean toxin concentration of 974 ng/ml was required to produce observable cytolytic effects on neutrophils. Although the enzyme-linked immunosorbent assay was able to detect leukocidin in milk samples from toxin-infused mammary glands, the toxin was detectable in only 2 of 27 S. aureus-infected milk samples (7%) from cows with chronic staphylococcal mastitis. To determine whether leukocidin antibodies in the mastitic milk samples were preventing toxin detection, leukocidin was mixed with milk with a high antileukocidin antibody titer (from a vaccinated cow) and evaluated with the immunoassay. Leukocidin was readily detected in this sample, indicating that milk antileukocidin antibodies were not sufficient to prevent detection of any leukocidin present in the mastitic milk samples. Failure to detect leukocidin in most mastitic milk samples with this assay indicated that, if leukocidin is produced in the bovine mammary gland during chronic staphylococcal mastitis, the concentration of the toxin may be too low to produce cytolytic effects on neutrophils.     

In vivo and in vitro demonstration that Staphylococcus aureus is an intracellular pathogen in the presence or absence of fibronectin-binding proteins

Staphylococcus aureus is the most significant bacterial pathogen associated with bovine mastitis. However, the relevance of intracellular infection to mastitis pathogenesis is poorly understood. We used in vitro assays and a mouse model of mastitis to demonstrate the intracellular component of the infection and to identify the importance of fibronectin-binding proteins in the processes of colonization and internalization. In vitro, a mutant strain, lacking fibronectin-binding protein (FnBPs(-)), had a reduced ability to bind fibronectin and to infect epithelial cells when compared to its parental wild type strain. After 2 h of infection, the internalization of the mutant bacteria into epithelial cell cultures was reduced by 60% compared with the wild type. After in vivo infection, microscopic examination using the FnBPs(-) strain revealed that production of a high density of live bacteria within the mammary gland epithelial cells was delayed. Both parental and mutant strains were identified within neutrophils, macrophages and epithelial cells suggesting a close similarity between the mouse mastitis model and bovine mastitis. These results demonstrate that S. aureus was able to cause an intracellular infection in the mouse model of mastitis and that the elimination of one adhesion protein delayed, but did not prevent, infection.     

Roles of alpha-toxin and beta-toxin in virulence of Staphylococcus aureus for the mouse mammary gland.

Mutants of Staphylococcus aureus which fail to express alpha-toxin (Hly), beta-toxin (Hlb), or both have been constructed by site-specific mutagenesis. The virulence of the mutants was compared with that of wild-type toxigenic strains by intramammary inoculation of lactating mice. A bovine strain, M60, and a laboratory strain, 8325-4, caused acute mastitis and death within 48 h for 60% of the mice inoculated. Animals inoculated with Hly mutants also developed acute mastitis, but no deaths occurred. Comparisons of Hly- or Hlb-positive strains with the double mutation Hly Hlb showed that both toxins led to a significantly higher recovery of S. aureus from the gland 48 h postinfection. Histopathological examination of mammary glands showed that phagocytosis of bacteria occurred irrespective of toxigenicity, but toxigenic strains, particularly those which were Hly+, continued to multiply, invaded the interalveolar tissues, and produced severe lesions. Stimulation of an inflammatory response by inoculation of the mammary gland with endotoxin prior to challenge with S. aureus reduced recovery of the bacteria 10- to 100-fold and, under these conditions, neither alpha-toxin nor beta-toxin contributed significantly to growth and survival.     

Reactivity of coagulase-negative staphylococci isolated from cow and goat milk with monoclonal antibodies to Staphylococcus aureus capsular polysaccharide types 5 and 8.

Monoclonal antibodies to Staphylococcus aureus capsular polysaccharide types 5 and 8 were used in an enzyme-linked immunosorbent assay to serotype 74 and 42 coagulase-negative isolates from cow and goat milk, respectively. Eighteen (15.5%) isolates were typable: 13 Staphylococcus haemolyticus, 1 S. hyicus, 1 S. simulans, and 1 S. warneri from bovine origin and 2 S. lentus from caprine origin. Type 5 was predominant, accounting for about 89% of typable isolates. Reactivity with monoclonal antibodies varied considerably according to isolates. The significance and the potential importance of these findings are discussed.     

Leucotoxic activities of Staphylococcus aureus strains isolated from cows,ewes, and goats with mastitis: importance of LukM/LukF'-PV leukotoxin

Among the toxins that Staphylococcus aureus is able to secrete, bi-component toxins named leukotoxins target specifically leukocytes, mainly phagocytic cells. Isolates from cows, goats and ewes with mastitis were selected on the basis of the presence or not of the genes encoding the recently described LukM/LukF'-PV leukotoxin. Of the 128 isolates tested, 126 had moderate to high leukotoxic activity to bovine polymorphonuclear cells (PMN). The supernatants of lukM-positive isolates were much more leukotoxic than the supernatants of lukM-negative isolates: mean leukotoxic titers were 122 versus 20 and 581 versus 26 for isolates of bovine and caprine origin, respectively. Among lukM/lukF'-PV positive isolates, those of caprine and ovine origins were more leukotoxic than were isolates of bovine origin (P < 0.01). The two most abundant proteins in the culture supernatant of a highly toxic isolate were purified and identified as the two components of LukM (LukM and LukF'-PV) on the basis of their molecular mass, N-terminal amino acid sequence, and high synergistic activity. LukM/LukF'-PV induced the flattening of bovine PMN at a concentration as low as 3.6 ng/ml (0.1 nM). A higher concentration (18 ng/ml) was necessary to produce the same effect on caprine or ovine PMN. Affinity-purified antibodies to LukM or to LukF'-PV neutralized the leukotoxic effect of all the culture supernatants. They neutralized with the same efficiency the toxic activity of supernatants from lukM/lukF'-PV positive or negative isolates. These results establish that LukM/LukF'-PV is very active on PMN of ruminants and suggest that this leukotoxin could be the most active leukotoxin produced by mastitis isolates. They prompt further studies to delineate the contribution of LukM/LukF'-PV to the pathogenesis of mastitis in ruminants and the protective effect of antibodies to this leukotoxin.     

Beta-hydroxybutyrate abrogates formation of bovine neutrophil extracellular traps and bactericidal activity against mammary pathogenic Escherichia coli

Escherichia coli is an important bacterial species isolated from bovine mastitis. The rate of neutrophil recruitment into the mammary gland and their bactericidal activity largely affect the severity and outcome of the disease. Ketosis is a common metabolic disease, and affected dairy cows are known to have increased risk for mastitis and other infectious conditions. The disease is associated with high blood and milk levels of beta-hydroxybutyrate (BHBA), previously shown to negatively affect neutrophil function by unknown mechanisms. We show here that the mammary pathogenic E. coli strain P4 activates normal bovine neutrophils to form neutrophil extracellular traps (NETs), which are highly bactericidal against this organism. Preincubation of these neutrophils with increasing concentrations (0.1 to 8 mmol/liter) of BHBA caused a fivefold decrease of E. coli P4 phagocytosis, though intracellular killing was unaffected. Furthermore, BHBA caused a 10-fold decrease in the NETs formed by E. coli P4-activated neutrophils and a similar decrease in NET bactericidal activity against this organism. These negative effects of BHBA on bovine neutrophils might explain the increased susceptibility of ketotic cows to mastitis and other infectious conditions.     

A periodate-sensitive anti-phagocytic surface structure, induced by growth in milk whey, on Staphylococcus aureus isolated from bovine mastitis.

The phagocytic and chemiluminescent activity of purified bovine neutrophils in response to two Staphylococcus aureus strains isolated from mastitic bovine milk and grown in milk whey was studied. The activity was significantly reduced compared with the response elicited by the same strains grown in tryptic soy broth (TSB). A mild periodate treatment of the milk whey-grown strains resulted in a significant increase of both chemiluminescence and phagocytosis, whereas trypsin, subtilisin or papain treatment had no effect. The decreased binding of complement factor C3 to milk-whey-grown bacteria was restored to the level of TSB-grown homologous organisms by periodate treatment. Moreover, this treatment, but not treatment with trypsin, increased the surface hydrophobicity of milk-whey-grown bacteria. The chemiluminescent activity was as high towards heat-killed as towards live bacteria. Also, incubation of heat-killed TSB-grown bacteria in milk whey did not alter the chemiluminescent response, indicating that the reduced neutrophil activity towards milk-whey-grown bacteria was not due to binding of milk components to the microorganisms. These results strongly suggest that bovine mastitis S. aureus strains grown in milk whey produce an anti-phagocytic surface structure. This structure is heat- and protease-resistant and renders the bacterial surface hydrophilic. The anti-phagocytic material is altered or, more likely, released from the bacterial surface on periodate treatment and is probably of carbohydrate nature.     

Evaluation of a novel chimeric B cell epitope-based vaccine against mastitis induced by either Streptococcus agalactiae or Staphylococcus aureus in mice

To construct a universal vaccine against mastitis induced by either Streptococcus agalactiae or Staphylococcus aureus, the B cell epitopes of the surface immunogenic protein (Sip) from S. agalactiae and clumping factor A (ClfA) from S. aureus were analyzed and predicted. sip-clfA, a novel chimeric B cell epitope-based gene, was obtained by overlap PCR, and then the recombinant Sip-ClfA (rSip-ClfA) was expressed and purified. rSip-ClfA and inactivated S. agalactiae and S. aureus were formulated into different vaccines with mineral oil as the adjuvant and evaluated in mouse models. The rSip-ClfA vaccination induced immunoglobulin G (IgG) titers higher than those seen in groups immunized with inactivated bacteria. Furthermore, the response to rSip-ClfA immunization was characterized as having a dominant IgG1 subtype, whereas both bacterial immunizations produced similar levels of IgG1 and IgG2a. The antiserum capacities for opsonizing adhesion and phagocytosis were significantly greater in the rSip-ClfA immunization group than in the killed-bacterium immunization groups (P < 0.05). The immunized lactating mice were challenged with either S. agalactiae or S. aureus via the intramammary route. At 24 h postinfection, the numbers of bacteria recovered from the mammary glands in the rSip-ClfA group were >5-fold lower than those in both inactivated-bacterium groups (P < 0.01). Histopathological examination of the mammary glands showed that rSip-ClfA immunization provided better protection of mammary gland tissue integrity against both S. agalactiae and S. aureus challenges. Thus, the recombinant protein rSip-ClfA would be a promising vaccine candidate against mastitis induced by either S. agalactiae or S. aureus.