Location of Staphylococcus aureus within the experimentally infected bovine udder and the expression of capsular polysaccharide type 5 in situ

The objective of this study was to locate Staphylococcus aureus in the bovine udder and to investigate the expression of capsular polysaccharide type 5 (CP5) in situ in both the early and chronic stages of experimental intramammary S. aureus infections. Bovine udder tissue was obtained in early and chronic stages of intramammary infection; i.e., 24 to 96 h and 122 d after experimental intramammary infection with S. aureus Newbould 305. The presence and location of S. aureus was investigated by Gram staining of tissue sections. The expression of CP5 by S. aureus in situ was investigated by immunochemical staining of tissue sections with specific antibodies against CP5. Both in the early and chronic stages of infection, S. aureus was located within the lumen of alveoli or lactiferous ducts, in association with the epithelium, and within phagocytic cells. The staphylococci were mainly observed in clusters and often in the presence of polymorphonuclear neutrophils. Expression of CP5 by S. aureus was observed both in the early and chronic stages of infection. In general, CP5-positive S. aureus were located in alveoli and in association with the mammary epithelium. In the chronic infection, CP5-positive S. aureus were also located deep in the interstitial tissue. These results indicate that--both in early and chronic stages of experimental S. aureus mastitis--colonization of the mammary epithelia and invasion into the interstitial tissue occurs and that CP5 is expressed by S. aureus Newbould 305 in situ. The invasion of S. aureus in the interstitial tissue and the expression of CP5 probably help the bacteria to withstand the host defense mechanisms.     

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.     

Phagocytic and postphagocytic activities of bovine neutrophils for pure and mixed bacterial cultures

A comparative study of phagocytosis and postphagocytic oxidative metabolic activity of bovine blood neutrophils incubated with pure and mixed cultures of Escherichia coli, Staphylococcus aureus, and Streptococcus agalactiae was preformed. Most neutrophils when incubated with mixed cultures showed preferential phagocytosis for one species and a smaller number phagocytized both species of microorganisms. Percent phagocytosis for E. coli in pure culture was similar to that of Strep. agalactiae in pure culture and higher than that for Staph. aureus in pure culture. Neutrophils incubated with mixed cultures of E. coli and Staph. aureus or E. coli and Strep. agalactiae showed greater than expected phagocytosis of each microorganisms alone and reduced phagocytosis of both microorganisms together. Postphagocytic oxidative metabolic activity of neutrophils, measured by percent nitroblue tetrazolium reduction, did not differ following phagocytosis of these three microorganisms in pure cultures. In comparison, a synergistic effect on nitroblue tetrazolium reductive activity was seen in mixed cultures as evidenced by higher percent nitroblue tetrazolium reduction following phagocytosis of both E. coli and Staph. aureus or E. coli and Strep. agalactiae. These observations indicate that the phagocytic and metabolic activities of neutrophils for bacteria in mixed cultures may not be identical to those in pure cultures.     

Production of antibodies to Staphylococcus aureus serotypes 5, 8, and 336 using poly(DL-lactide-co-glycolide) microspheres

Staphylococcus aureus is responsible for a major portion of the economic losses due to mastitis. Attempts to produce a vaccine to prevent S. aureus mastitis have been hampered by the low immunogenicity of the polysaccharide, which forms on the surface of the organism when it enters the mammary gland. The polysaccharide inhibits phagocytosis and destruction of the organism by neutrophils. This study was conducted to determine if S. aureus polysaccharide serotypes 5, 8, and 336 conjugated to a protein and incorporated in poly(DL-lactide-co-glycolide) microspheres would enhance the production of opsonizing antibodies to the polysaccharide. Cows were immunized with either polysaccharide conjugates emulsified in Freund's incomplete adjuvant or polysaccharide conjugates encapsulated in poly (DL-lactide-co-glycolide) microspheres emulsified in Freund's incomplete adjuvant. All cows produced sustained antibody titers to the three polysaccharide serotypes. Cows immunized with microspheres had higher antibody titers. Cows in both groups produced increased concentrations of IgG1 and IgG2 antibodies; neither group produced an increase in IgM. Immune sera from cows immunized with conjugates alone increased phagocytosis, which decreased at the end of the study. Sera from cows immunized with conjugates in microspheres increased phagocytosis, which was sustained at the end of the study. Immune sera from both groups decreased bacterial adherence to bovine mammary epithelial cells. These data showed that a single injection of antigen in microspheres produced higher titers and more sustained enhancement of phagocytosis, which could aid in the defense of the cow against S. aureus infections.     

Enhancement of neutrophil function by ultrafiltered bovine whey

The objective of this work was to evaluate a proprietary ultrafiltered bovine whey product for its in vitro influence on the function of neutrophils from normal and dexamethasone-treated cattle, and for its in vivo influence on neutrophil function in periparturient dairy cows. The ultrafiltered bovine whey was produced by hyperimmunizing cows to various bacterial pathogens by intramammary injection, collecting and pooling the colostrum and milk for the first 3 wk after parturition, then separating and processing the whey through a filter with a nominal molecular mass cut off of 10,000 Da. In vitro treatment of neutrophils from normal calves with ultrafiltered bovine whey significantly increased neutrophil random migration, cytochrome C reduction, iodination activity, antibody-dependent cell-mediated cytotoxicity, and antibody-independent cell-mediated cytotoxicity. Cytochrome C reduction was the only neutrophil function parameter significantly enhanced by the in vitro treatment of neutrophils from dexamethasone-treated cattle with ultrafiltered bovine whey. In vivo treatment of periparturient cows with ultrafiltered bovine whey did not alter the total or differential leukocyte counts in the animals but did significantly increase the total erythrocyte counts. In vivo treatment with ultrafiltered bovine whey also significantly increased neutrophil iodination activity in the periparturient cows. Neutrophil iodination activity (a measure of the myeloperoxidase/hydrogen peroxide/halide antibacterial system) is a very potent bactericidal mechanism of neutrophils and has previously been shown to be suppressed in periparturient cows.     

Phagocytosis and serum susceptibility of Escherichia coil cultured in iron-deplete and iron-replete media

The susceptibility of Escherichia coli cultured in either iron-deplete or iron-replete media to phagocytosis by bovine neutrophils and the bactericidal activity of bovine serum was tested in vitro. Fourteen E. coli isolates from naturally occurring intramammary infections (IMI) were cultured overnight at 37 degrees C in iron-replete media and iron-deplete media. The iron-replete media were trypticase soy broth or a chemically defined medium. The iron-deplete media were either trypticase soy broth plus 0.2 mM alpha, alpha' dipyridyl and 1mM citrate, or the chemically defined medium plus 0.2 mM alpha, alpha' dipyridyl, and 1 mM citrate. Iron-replenished medium was the chemically defined iron-deplete medium plus 40 mM ferric citrate. Bacteria grown in iron-deplete media were less susceptible to phagocytosis compared with bacteria grown in iron-replete media. Replenishing the chemically defined iron-deplete medium with ferric citrate obliterated the decreased susceptibility to phagocytosis observed in iron-deplete media. The iron availability in media used to culture E. coli before assay did not affect the bactericidal action of either the classical pathway of complement or the antibody independent alternative pathway of complement in serum. The growth of bacteria in iron-deplete medium did not alter the expression of capsule compared with growth in iron-replete medium. Iron availability during culture of E. coli altered the susceptibility of isolates to phagocytosis by neutrophils, but had no effect on the susceptibility of isolates to the bactericidal activity of serum.     

Antibacterial and antiviral activity of camel milk protective proteins.

Lysozyme (LZ), lactoferrin (LF), lactoperoxidase (LP), immunoglobulin G and secretory immunoglobulin A were extracted from camel milk. The activity of these protective proteins was assayed against Lactococcus lactis subsp. cremoris, Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and rotavirus. Comparative activities of egg white LZ, bovine LZ and bovine LF are also presented. The antibacterial activity spectrum of camel milk LZ was similar to that of egg white LZ, and differed from bovine milk LZ. Bovine and camel milk LF antibacterial activity spectra were similar. The camel milk LP was bacteriostatic against the Gram-positive strains and was bactericidal against Gram-negative cultures. The immunoglobulins had little effect against the bacteria but high titres of antibodies against rotavirus were found in camel milk. The LP system was ineffective against rotavirus.     

Effects of yeast expressed recombinant interleukin-2 and interferon-gamma on physiological changes in bovine mammary glands and on bactericidal activity of neutrophils

The physiological effects of intramammary infusions of recombinant bovine cytokines in six lactating dairy cows on the quality and yield of milk and the bactericidal activity of milk neutrophils were investigated. Recombinant bovine interleukin-2 (rboIL-2) and interferon-gamma (rboIFN-gamma) were produced in the yeast Pichia pastoris. Two animals were given rboIL-2 (2 x 10(5) units) in two quarters, two animals were given rboIFN-gamma (6.5 x 10(5) units) in two quarters, and the other two cows received a dose of rboIL-2 in one quarter and rboIFN-gamma in a second quarter. In addition, each animal was given phosphate-buffered saline (PBS) in the other two quarters as a control. Somatic cell counts and conductivity of the fore milk were monitored before and after infusion. Neutrophils were isolated from quarter milk samples 36 h after infusion of cytokine or PBS and their bactericidal activities against Staphylococcus aureus were measured in vitro with a colorimetric assay. Quarters infused with rboIL-2 or rboIFN-gamma showed significant but transitory increases in both milk somatic cell counts and conductivity when compared with preinfusion values and with control quarters. There were minimal effects on daily milk yield. Neutrophils isolated from milk from quarters infused with rboIL-2 showed enhanced bactericidal activity against Staph. aureus. The bacterial killing from rboIL-2 treated quarters was significantly greater, with a mean of 63.5% compared with a mean of 5.4% for neutrophils taken from uninfected quarters to which PBS had been administered. The bactericidal activities for quarters treated with rboIFN-gamma and infected quarters treated with PBS were 15.0 and 30.0% respectively. The results indicate that intramammary infusions of rboIL-2 and rboIFN-gamma to lactating cows are well tolerated, and that rboIL-2 can activate milk neutrophils and augment their bactericidal activity.     

Effect of anticapsular antibodies on neutrophil phagocytosis of Staphylococcus aureus.

One of the major virulence factors of Staphylococcus aureus is development of an exopolysaccharide capsule in vivo, which inhibits recognition of antibodies to highly antigenic cell wall by neutrophils. To circumvent this inhibition, an attempt was made to produce anticapsular antibodies. Three cows per group were immunized in midlactation by injections in the area of the supramammary lymph node and intramuscularly and were boosted on d 14, 42, and 70 with three variants of Smith S. aureus: compact, unencapsulated; diffuse, rigid capsule; and diffuse large clearing, exceptionally large flaccid capsule using dextran sulfate as adjuvant. Serum agglutination and ELISA titers of cows immunized with diffuse and diffuse large clearing increased after immunization and after each boost and remained elevated to the end of the experiment at 112 d. Phagocytosis of diffuse and diffuse large clearing, measured by flow cytometry, was enhanced by immunization with either organism. No antibody response to capsule or enhanced phagocytosis of diffuse developed in cows immunized with compact. However, anticompact antibodies were opsonic for diffuse large clearing. These data show that bovine antibodies to S. aureus capsule are opsonic for bovine neutrophils and that capsule plays a role in inhibition of cell-wall opsonization of S. aureus.     

Lysostaphin: use of a recombinant bactericidal enzyme as a mastitis therapeutic.

A recombinant mucolytic protein, lysostaphin, was evaluated as a potential intramammary therapeutic for Staphylococcus aureus mastitis in dairy cattle. Lysostaphin, a product of Staphylococcus simulans, enzymatically degrades the cell wall of Staphylococcus aureus and is bactericidal. Thirty Holstein-Freisian dairy cattle in their first lactation were infected with Staphylococcus aureus (Newbould 305, ATCC 29740) in all quarters. Infections were established and monitored for somatic cell counts and Staphylococcus aureus colony-forming units 3 wk prior to subsequent treatment. Infected animals were injected through the teat canal with a single dose of recombinant lysostaphin (dose response 1 to 500 mg) or after three successive p.m. milkings with 100 mg of recombinant lysostaphin in 60 ml of sterile phosphate-buffered saline. Animals were considered cured if the milk remained free of Staphylococcus aureus for a total of 28 milkings after last treatment. Kinetic analysis of immunologically active recombinant lysostaphin demonstrated that a minimum bactericidal concentration was maintained in the milk for up to 36 to 48 h after a single infusion of 100 mg of recombinant lysostaphin. The cure rate of quarters receiving recombinant lysostaphin (100 mg in sterile phosphate-buffered saline, administered over three consecutive p.m. milkings) was 20% compared with 29% for sodium cephapirin in saline and 57% for a commercial antibiotic formulation, respectively. An improved formulation of recombinant lysostaphin may prove to be an effective alternative to antibiotic therapy for bovine mastitis.     

Effect of cis-urocanic acid on bovine neutrophil generation of reactive oxygen species

Neutrophils play a fundamental role in the host innate immune response during mastitis and other bacterial-mediated diseases of cattle. One of the critical mechanisms by which neutrophils contribute to host innate immune defenses is through their ability to phagocytose and kill bacteria. The ability of neutrophils to kill bacteria is mediated through the generation of reactive oxygen species (ROS). However, the extracellular release of ROS can be deleterious to the host because ROS induce tissue injury. Thus, in diseases such as mastitis that are accompanied by the influx of neutrophils, the generation of large quantities of ROS may result in significant injury to the mammary epithelium. cis-Urocanic acid (cis-UCA), which is formed from the UV photoisomerization of the trans isoform found naturally in human and animal skin, is an immunosuppressive molecule with anti-inflammatory properties. Little is known about the effect of cis-UCA on neutrophils, although one report demonstrated that it inhibits human neutrophil respiratory burst activity. However, the nature of this inhibition remains unknown. Because of the potential therapeutic use that a molecule such as cis-UCA may have in blocking excessive respiratory burst activity that may be deleterious to the host, the ability of cis-UCA to inhibit bovine neutrophil production of ROS was studied. Further, because neutrophil generation of ROS is necessary for optimal neutrophil bactericidal activity, a response which is critical for the host innate immune defense against infection, the effects of cis-UCA on bovine neutrophil phagocytosis and bacterial killing were assayed. cis-Urocanic acid dose-dependently inhibited the respiratory burst activity of bovine neutrophils as measured by luminol chemiluminescence. Subsequently, the effect of cis-UCA on the production of specific oxygen radicals was investigated using more selective assays. Using 2 distinct assays, we established that cis-UCA inhibited the generation of extracellular superoxide. In contrast, cis-UCA had no effect on the generation of intracellular levels of superoxide or other ROS. At concentrations that inhibited generation of extracellular superoxide, bovine neutrophil phagocytosis and bacterial activity remained intact. Together, these data suggest that cis-UCA inhibits the tissue-damaging generation of extracellular ROS while preserving neutrophil bactericidal activity.     

Immunization with Staphylococcus aureus lysate incorporated into microspheres

Antibiotics are of limited value against Staphylococcus aureus due to development of resistant strains, scar tissue formation, and blockage of ducts due to inflammation. Though macrophages are the predominant cell type in the mammary gland, they are primarily scavenger cells and are not effective against bacteria entering the gland. Neutrophil phagocytosis is the bovine's primary defense against S. aureus mastitis. Attempts to develop vaccines that enhance neutrophil phagocytosis by stimulating production of opsonizing antibodies to S. aureus have met with limited success because of the low immunogenicity of the exopolysaccharide capsule surrounding S. aureus. Staphylococcus aureus can also adhere to and penetrate epithelial tissue. This study was conducted to determine whether lysates of S. aureus encapsulated in biodegradable microspheres would increase the production of opsonizing antibodies to capsule and block adherence. Four groups of four cows each were injected with 1 ml of the respective treatment in the area of the supramammary lymph node and 1 ml in the hip muscle. The treatments were: lysate in NaCl, lysate in Freund's incomplete adjuvant (FICA), lysate in microspheres in NaCl, and lysate in microspheres in FICA. Antigen in microspheres produced a similar antibody response to antigen emulsified in FICA, but to a lesser magnitude. Antigen in microspheres produced antibodies that were more opsonic for neutrophils at 20 and 52 wk postimmunization and inhibited S. aureus adherence to mammary epithelium. Ability to control antigen release and presentation, and the benefit of a single injection for long-term immunity using microspheres warrants additional studies.     

Functional activity of neutrophils from bovine mammary glands infected with Staphylococcus aureus

To test the effect of Staphylococcus aureus infection on mammary neutrophil function, intramammary neutrophils from S. aureus-infected quarters (n = 8), from adjacent uninfected quarters (n = 8) of S. aureus-infected cows, and from quarters (n = 8) of uninfected cows were collected and incubated with S. aureus in vitro. Mean percent neutrophils phagocytizing, number S. aureus per neutrophil, and log10 viable phagocytized S. aureus/ml were: 53.2, 6.4, and 4.72. Differences in function of neutrophils collected from infected and uninfected quarters were not statistically significant. Results indicate that function of neutrophils from S. aureus-infected quarters is similar to function of neutrophils from uninfected quarters.     

Use of bovine primary mammary epithelial cells for the comparison of adherence and invasion ability of Staphylococcus aureus strains

Adherence and invasion of epithelial cells are thought to play a role in the pathogenesis of Staphylococcus aureus mastitis. A cell culture model with primary mammary epithelial cells originating from the secretory tissue from the bovine udder was used to study adherence and invasion of S. aureus. The cells were characterized with antibodies against several cell markers that had been validated on histologic cryostat sections of bovine mammary tissue. All cells stained positively with the anticytokeratin antibodies, which are restricted to epithelial cells. The cell cultures contained a small number of alpha-smooth-muscle-actin positive cells (< 1%), probably myoepithelial cells. The use of bovine primary mammary epithelial cells and bovine S. aureus isolates, which were cultured in milk serum, results in a system similar to in vivo. Strain differences for adherence and invasion of S. aureus strains cultured in milk serum were studied. In addition, the correlation between adherence and invasion was evaluated. The number of adhered and invaded bacteria was strain dependent. The percentage of adherence after 5 min of incubation was correlated to the percentage of adherence after 3 h of incubation (r = 0.94; Pearson's correlation test). Fourteen of the 20 strains were able to invade epithelial cells. The percentage of invasion was correlated to the percentage of adherence after 5 min and to the percentage adherence after 3 h (r = 0.95 and 0.90, respectively; Pearson's correlation test). Results indicate that strain differences of adherence and invasion exist for S. aureus and that the invasion is a post adherence event.     

High pressure increases bactericidal activity and spectrum of lactoferrin, lactoferricin and nisin

We have studied the inactivation of a panel of eight test bacteria (two Escherichia coli strains, Salmonella enteritidis, Salmonella typhimurium, Shigella sonnei, Shigella flexneri, Pseudomonas fluorescens and Staphylococcus aureus) by high pressure in the presence of bovine lactoferrin (500 microg/ml), pepsin hydrolysate of lactoferrin (500 microg/ml), lactoferricin (20 microg/ml) and nisin (100 IU/ml). None of these compounds, at the indicated dosage, were bactericidal when applied at atmospheric pressure, except nisin, which caused a low level of inactivation of the bacteria. Under high pressure, lactoferrin, lactoferrin hydrolysate and lactoferricin displayed bactericidal activity against some of the test bacteria, however, the former had a narrower bactericidal spectrum than the two latter compounds. The bactericidal efficiency and spectrum of nisin were also enhanced under high pressure. The sensitisation of the test bacteria to these antimicrobials under pressure was transient, since no bactericidal activity was observed when bacteria were pressure treated before exposure to the compounds. We propose a mechanism of pressure-promoted uptake of these antimicrobial proteins and peptides in gram-negative bacteria to explain this sensitisation.     

Evaluation of serotypes of Staphylococcus aureus strains used in the production of a bovine mastitis bacterin

The five Staphylococcus aureus strains used in the manufacture of a commercially available bacterin were examined for capsular and surface polysaccharide serotypes. Double immunodiffusion assays of antigenic extracts of test and reference strains with monospecific typing sera to capsular serotypes 1, 2, 5, and 8 and to surface polysaccharide serotype 336 were performed to detect the specific reactivities and antigenic relationships of test samples. Antigenic extracts of two S. aureus strains reacted with antibodies to serotype 8, but not with antibodies to serotype 5, by producing specific precipitin lines. A third strain reacted with monospecific antibodies to serotype 5 and not with the antibodies to serotype 8. The extracts of two other strains failed to exhibit any detectable reaction with antiserum to serotypes 1, 2, 5, or 8. Antibodies to serotype 336, however, precipitated an identical, specific 336 antigen from the antigenic extracts of these two nontypeable strains. Thus, S. aureus bacterin includes one serotype 5, two serotype 8, and two serotype 336 strains, the three predominant serotypes responsible for bovine mastitis.     

Lytic and nonlytic mechanism of inactivation of gram-positive bacteria by lysozyme under atmospheric and high hydrostatic pressure

A different behavior was observed in three gram-positive bacteria exposed to hen egg white lysozyme by plate counts and phase-contrast microscopy. The inactivation of Lactobacillus johnsonii was accompanied by spheroplast formation, which is an indication of peptidoglycan hydrolysis. Staphylococcus aureus was resistant to lysozyme and showed no signs of peptidoglycan hydrolysis, and Listeria innocua was inactivated and showed indications of cell leakage but not of peptidoglycan hydrolysis. Under high hydrostatic pressure, S. aureus also became sensitive to lysozyme but did not form spheroplasts and was not lysed. These results suggested the existence of a nonlytic mechanism of bactericidal action of lysozyme on the latter two bacteria, and this mechanism was further studied in L. innocua. Elimination of the enzymic activity of lysozyme by heat denaturation or reduction with beta-mercaptoethanol eliminated this bactericidal mechanism. By means of a LIVE/DEAD viability stain based on a membrane-impermeant fluorescent dye, the nonlytic mechanism was shown to involve membrane perturbation. In the absence of lysozyme, high-pressure treatment was shown to induce autolytic activity in S. aureus and L. innocua.     

Oxygen concentration in milk of healthy and mastitic cows and implications of low oxygen tension for the killing of Staphylococcus aureus by bovine neutrophils

The partial pressure of O2 in milk from normal cows and from cows with mastitis was measured and the concentrations of O2 calculated. Oxygen levels of milk from normal cows were similar to those in venous plasma, but inflammation of the mammary gland led to a dramatic drop in O2 concentration to less than 10% of control values. Intracellular survival of Staphylococcus aureus strain M60 in bovine neutrophils was greater under anaerobic than aerobic conditions. The implications of low O2 concentrations in milk from infected mammary glands for the bactericidal activity of bovine neutrophils is discussed.     

Neutrophils in the war against Staphylococcus aureus: predator-prey models to the rescue

To address the question of whether a minimum concentration of blood neutrophils is necessary to decrease Staphylococcus aureus concentration in mastitic milk, literature was searched for studies in which neutrophils were incubated with Staph. aureus. Different mathematical models that describe the changes in Staph. aureus population as a function of neutrophilic concentrations were applied to the collected data. The best fitted model established (1) that the rate of bacterial killing depended on the ratio of neutrophils to bacteria with neutrophilic attack rate accelerating at first before decelerating as the ratio increases, and (2) that neutrophil concentration should be within a limited range to trigger a decline in the bacterial population. Outcomes of this model are supported by what is known about neutrophilic functions and laboratory findings in bovine and human neutrophils. These results may be of assistance in setting selection goals for a better resilience to Staph. aureus mastitis in dairy cattle. Indeed, an optimal neutrophilic concentration appears to exist for successful clearance of Staph. aureus infection, which is neither the lowest nor the highest one.     

Effectiveness of Some Natural Antimicrobial Compounds in Controlling Pathogen or Spoilage Bacteria in Lightly Fermented Chinese Cabbage

ABSTRACT: This study was designed to evaluate the bactericidal or bacteriostatic effect of chitosan, an allyl isothiocyanate (AIT) product, and nisin for the artificially inoculated pathogenic bacteria ( Escherichia coli , Salmonella Enteritidis, Staphylococcus aureus , and Listeria monocytogenes ) or natural microflora of fermented Chinese cabbage. Addition of 0.1% chitosan decreased the population of pathogens from 0.7 to 1.7 log colony-forming units (CFU)/g after 4 d of storage at 10 °C. The bactericidal activity of chitosan was found to be stronger than that of nisin (0.05 mg/g). Addition of 0.2% of the AIT product (containing AIT and hop extract) exhibited a bacteriostatic effect. However, a combination of AIT product and chitosan enhanced bactericidal efficacy against L. monocytogenes . The addition of chitosan or AIT product was observed to suppress the populations of mesophilic and coliform bacteria during storage at 10 °C for 4 d. Moreover, the use of chitosan or the AIT product did not change the sensory quality of the lightly fermented vegetable. Therefore, these results suggest that chitosan or the AIT product could be useful to improve the microbial safety and quality of lightly fermented vegetable.