Intramammary immunization with live-attenuated Staphylococcus aureus: microbiological and immunological studies in a mouse mastitis model

Abstract Mammary infection was induced in lactating mice by intramammary injection of Staphylococcus aureus . Histopathological analysis revealed infiltration and lesions of varying magnitude that were still apparent 21 days after the challenge. Concomitantly, viable S. aureus was recovered from infected mammary glands. Mice were immunized by the intramammary route with 5 × 10⁶ colony forming units of a temperature-sensitive mutant of S. aureus and subsequently received a boosting injection seven days later. On day 14 mice were challenged by the intramammary route with the wild-type strain. Intramammary immunization induced a significant increase in milk IgA ( P < 0.05), serum IgG ( P < 0.05) and serum IgA ( P < 0.05) on the day of the challenge, when compared with non-immunized mice. Immunization decreased significantly ( P < 0.01) the number of S. aureus colony forming units recovered 96 h after intramammary challenge. In conclusion, the feasibility of immunizing locally with temperature-sensitive S. aureus to induce immunity in the mouse mammary gland was demonstrated. The mouse model of mastitis is proposed as a useful system for screening temperature-sensitive S. aureus strains to be utilized in the development of a vaccine.     

Evaluation of quality changes in udder quarter milk from cows with low-to-moderate somatic cell counts

Much emphasis has been put on evaluating alterations in milk composition caused by clinical and subclinical mastitis. However, little is known about changes in milk composition during subclinical mastitis in individual udder quarters with a low-to-moderate increase in milk somatic cell count (SCC). This information is needed to decide whether milk from individual udder quarters with a moderate-to-high increase in milk SCC should be separated or not. The aim of this study was to determine how milk composition in separate udder quarters is affected when cow composite milk has low or moderately increased SCC levels. Udder quarter and cow composite milk samples were collected from 17 cows on one occasion. Milk yield was registered and samples were analyzed for SCC, fat, total protein, whey proteins, lactose, citric acid, non-protein nitrogen (NPN), lactoferrin, protein profile, free fatty acids (FFAs), lactate dehydrogenase (LDH), proteolysis, sodium and potassium. Bacteriological samples were collected twice from all four quarters of all cows. The cows were divided into three groups depending on their SCC at udder quarter level. The first group comprised healthy cows with four udder quarters with low SCC, <50 000 cells/ml; composition was equal when opposite rear and front quarters were compared. In the second and the third groups, cows had one udder quarter with 101 000 cells/ml < SCC < 600 000 cells/ml and SCC > 700 000 cells/ml, respectively. The remaining udder quarters of these cows had low SCC (<100 000 cells/ml). Despite the relatively low average cow composite SCC = 100 000 cells/ml of Group 2, milk from affected udder quarters exhibited lower casein number, content of lactose and β-casein (β-CN), while the content of whey protein, sodium, LDH and α-lactoalbumin (α-la) were higher compared to healthy opposite quarters. In addition to these changes, milk from affected udder quarters in Group 3 also exhibited lower values of potassium and αs1-casein (αs1-CN) and higher values of lactoferrin when compared to milk from opposite healthy quarters. This indicates that even when the SCC in cow composite milk is low, there might exist individual quarters for which milk composition is changed and milk quality impaired.     

The release of bradykinin in bovine mastitis.

The kinin peptides are released during inflammation and are amongst the most potent known mediators of vasodilatation, pain and oedema. Despite early reports of the presence of kinins in milk, no previous study has investigated the role of the kinin system in bovine mastitis. The present study indicated that mastitis was accompanied by raised levels of bradykinin (BK) in milk and the increased levels of BK correlated with the severity of mastitis. Raised BK levels in mastitic milk were not dependent on the presence of inflammatory cells, nor were they secondary to changes in blood levels of BK. In milk from sub-clinically inflamed quarters, BK was raised in those milks where Staphylococcus aureus (S. aureus) was isolated but not in those milks where no pathogen was isolated. Increasing S. aureus artificially, also caused an increase in the milk BK. Increases in milk BK were not restricted only to the mastitic quarters of the udder. In udders in which mastitis was detected in one or more quarters, BK increases were also detected in the apparently uninvolved quarters.     

Secretion composition during bovine mammary involution and the relationship with mastitis

1. Bacteriological analysis revealed that 30% of quarters contained coagulase-negative staphylococci, Staphylococcus aureus, Corynebacterium bovis, or streptococci. 2. As involution progressed, somatic cell counts, percent protein, pH, and concentrations of serum albumin, lactoferrin, and immunoglobulin G increased while percent fat, concentrations of citrate, and the citrate to lactoferrin molar ratio decreased. 3. Mammary secretion from infected quarters had significantly higher numbers of somatic cells, percent polymorphonuclear leukocytes, and pH, but lower percentage lymphocytes, fat, and lactoferrin concentrations compared to uninfected quarters. 4. Results suggest intramammary infection altered normal secretion composition during bovine involution and lactogenesis. 5. Lower levels of antibacterial components in bovine mammary secretion during the peripartum period may have reduced the natural defense potential of the gland.     

Characterization of hemolytic activity of Staphylococcus aureus strains isolated from bovine mastitic milk

Beta (beta) and delta (delta)-hemolysin of Staphylococcus aureus strains were cultured in vitro in milk lactoserum (whey) prepared from both healthy and mastitis bovine milk. Production of beta- and delta-hemolysins were detected in 12 out of 50 strains studied. The association between N-acetyl-beta-D-glucosaminidase (NAGase) activity, plasmin activity (PL) and trypsin inhibitory capacity (TIC), known as inflammatory indicators for mastitis, and hemolytic activity were also studied. Mastitic milk decreased directly the lytic effect of both beta-and delta-hemolysins of S. aureus on hemolytical blood agar plates. S. aureus in healthy milk samples produced more beta-hemolysin (3 times) and delta-hemolysin (2 times) when compared to S. aureus supernatants in milk from infected quarters. Furthermore, beta- and delta-hemolysis correlated negatively with TIC and NAGase and PL activities. Addition of reduced glutathione (GSH) or beta-mercaptoethanol into the artificial medium enhanced hemolysins activity.     

Purification of camel milk lysozyme and its lytic effect on Escherichia coli and Micrococcus lysodeikticus

1. Camel milk lysozyme was purified using heparin-Sepharose 4B, Sephadex G-75 and hydroxyapatite chromatography. By this procedure lysozyme was separated from lactoferrin and a low molecular weight protein. 2. The lytic effect of camel milk lysozyme was assayed using Escherichia coli and Micrococcus lysodeikticus and its activity was compared with that of lysozyme from human milk and egg white. 3. The specific activity of camel milk lysozyme was found to be lower than that of lysozyme from human milk or from egg white. 4. Camel milk lactoferrin did not show a lytic effect on bacteria, while the low molecular weight protein showed lytic activity.     

Nisin F in the treatment of respiratory tract infections caused by Staphylococcus aureus

Aims:  To determine the antimicrobial activity of nisin F against Staphylococcus aureus in the respiratory tract.
Methods and Results:  The respiratory tract of nonimmunosuppressed and immunosuppressed Wistar rats were colonized with 4 × 10⁵ viable cells of S. aureus K and then treated by administering 8192 arbitrary units (AU) nisin F intranasal. Symptoms of pneumonia were detected in the trachea and lungs of immunosuppressed rats that had not been treated with nisin F. The trachea and lungs of immunosuppressed rats treated with nisin F were healthy. No significant differences were recorded in blood cell indices. The antimicrobial activity of low concentrations nisin F (80–320 AU ml⁻¹) was slightly stimulated by lysozyme and lactoferrin.
Conclusions:  Nisin F inhibited the growth of S. aureus K in the respiratory tract of immunocompromised rats. Treatment with nisin F at 8192 AU proofed safe, as the trachea, lungs, bronchi and haematology of the rats appeared normal.
Significance and Impact of the Study:  Nisin F is nontoxic and may be used to control respiratory tract infections caused by S. aureus . This is, however, a preliminary study with an animal model and need to be confirmed with studies on humans.     

The Effect of UV-C Pasteurization on Bacteriostatic Properties and Immunological Proteins of Donor Human Milk

Background

Human milk possesses bacteriostatic properties, largely due to the presence of immunological proteins. Heat treatments such as Holder pasteurization reduce the concentration of immunological proteins in human milk and consequently increase the bacterial growth rate. This study investigated the bacterial growth rate and the immunological protein concentration of ultraviolet (UV-C) irradiated, Holder pasteurized and untreated human milk.

Methods

Samples (n=10) of untreated, Holder pasteurized and UV-C irradiated human milk were inoculated with E. coli and S. aureus and the growth rate over 2 hours incubation time at 37°C was observed. Additionally, the concentration of sIgA, lactoferrin and lysozyme of untreated and treated human milk was analyzed.

Results

The bacterial growth rate of untreated and UV-C irradiated human milk was not significantly different. The bacterial growth rate of Holder pasteurized human milk was double compared to untreated human milk (p<0.001). The retention of sIgA, lactoferrin and lysozyme after UV-C irradiation was 89%, 87%, and 75% respectively, which were higher than Holder treated with 49%, 9%, and 41% respectively.

Conclusion

UV-C irradiation of human milk preserves significantly higher levels of immunological proteins than Holder pasteurization, resulting in bacteriostatic properties similar to those of untreated human milk.     

Relations Between Udder Infection and Somatic Cells in Camel (Camelus dromedarius) Milk

Quarter milk samples (n = 391) from 101 camels were examined to study the occurrence and causes of mastitis in traditionally managed camels in eastern Sudan and to evaluate the value of the California Mastitis Test (CMT), somatic cell count (SCC) and adenosine triphosphate (ATP) in the detection of subclinical mastitis in the camel. One hundred and seventy (43.5%) of the quarter milk samples yielded pathogenic bacteria. Streptococcus agalactiae, other Streptococcus spp., Staphylococcus aureus, coag–ulase–negative staphylococci, and Escherichia coli were isolated from milk. Thirty–two (8.2%) quarter milk samples yielded mixed cultures, and 189 (48.3%) yielded no growth. Mean values for CMT, SCC and ATP were higher for quarters infected with major pathogens. However, a significant number of quarter milk samples had elevated values in these tests but were from quarters from which no bacteria were isolated. The ability of the tests to predict a positive bacteriology increased slightly when 2 or 3 tests were combined. kw|Keywords|k]inflammation; k]diagnostic tests; k]Mastitis; k]CMT; k]ATP; k]bacteriology; k]Sudan     

Detection of indicator pathogens from pharmaceutical finished products and raw materials using multiplex PCR and comparison with conventional microbiological methods

A multiplex PCR assay was devised and compared with standard conventional methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples which were artificially contaminated with <10 colony forming units of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella species and possibly contaminated samples were incubated for 16 h with different enrichment media. Primers that deduce 559 bp fragment of the 16S rRNA gene was employed in amplifying E. coli species, similarly invasion protein gene with 275 bp fragment size was used as target for detecting Salmonella spp., in case of S. aureus a 461 bp amplicon from m-RNA nuclease gene, and an 709 bp fragment from oprL gene was used for amplifying P. aeruginosa. The detection limits for artificially contaminants by multiplex PCR was 1 CFU/g, where as in case of conventional method the detection limit was >2 CFU/g. Similarly, when tested with possibly contaminated samples, 35% were detected for E. coli, Salmonella spp., S. aureus and P. aeruginosa species with multiplex PCR, while only 21% were detected with standard conventional microbial methods. Multiplex PCR assay provides sensitive and reliable results and allows for the cost-effective detection of all four bacterial pathogens in single reaction tube.     

Interactions between cationic liposomes and bacteria: the physical-chemistry of the bactericidal action.

The bactericidal effect of dioctadecyldimethylammonium bromide (DODAB), a liposome forming synthetic amphiphile, is further evaluated for Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Staphylococcus aureus in order to establish susceptibilities of different bacteria species towards DODAB at a fixed viable bacteria concentration (2.5 x 10(7) viable bacteria/mL). For the four species, susceptibility towards DODAB increases from E. coli to S. aureus in the order above. Typically, cell viability decreases to 5% over 1 h of interaction time at DODAB concentrations equal to 50 and 5 microm for E. coli and S. aureus, respectively. At charge neutralization of the bacterial cell, bacteria flocculation by DODAB vesicles is shown to be a diffusion-controlled process. Bacteria flocculation does not yield underestimated counts of colony forming units possibly because dilution procedures done before plating cause deflocculation. The effect of vesicle size on cell viability demonstrates that large vesicles, due to their higher affinity constant for the bacteria (45.20 m(-)) relative to the small vesicles (0.14 m(-)), kill E. coli at smaller DODAB concentrations. For E. coli and S. aureus, simultaneous determination of cell viability and electrophoretic mobility as a function of DODAB concentration yields a very good correlation between cell surface charge and cell viability. Negatively charged cells are 100% viable whereas positively charged cells do not survive. The results show a clear correlation between simple adsorption of entire vesicles generating a positive charge on the cell surfaces and cell death.     

Production of Enterotoxin A in Milk

Enterotoxin A production in milk was studied by use of variables of milk quality, initial numbers of enterotoxigenic staphylococci, incubation temperature, and time. In both raw and pasteurized milks having a low total viable count, enterotoxin was detected in minimal incubation times of 6 to 9 hr at 35 C, 9 to 12 hr at 30 C, 18 hr at 25 C, and 36 hr at 20 C, after inoculation with 10(6)Staphylococcus aureus cells per ml. When similar milks were inoculated with 10(4)S. aureus cells per ml, enterotoxin was detected in 12 hr at 35 C, 18 hr at 30 C, 24 to 36 hr at 25 C, and 48 to 96 hr at 20 C. In high-count raw milk, enterotoxin was detected only in samples inoculated with 10(6)S. aureus cells per ml and incubated at 35 C. Generally, a concentration of 5 x 10(7)S. aureus cells per ml of milk was reached before enterotoxin A was detected.     

A lysozyme isolated from rainbow trout acts on mastitis pathogens

The antibacterial effects of two lysozymes purified from rainbow trout kidney (type I and II) were tested on eight bacterial strains isolated from cases of clinical mastitis (staphylococci, streptococci and coliforms). Three other lytic agents were included in the experiments as controls: hen egg-white lysozyme, lysostaphin and mutanolysin. Proliferating bacteria were incubated with the various lytic agents, either in hearts infusion broth or in milk. The type II rainbow trout lysozyme decreased the number of live bacteria (colony forming units) of all the strains tested, but was most efficient against staphylococci. The other two lysozymes had little effect.     

A lysozyme isolated from rainbow trout acts on mastitis pathogens

Abstract The antibacterial effects of two lysozymes purified from rainbow trout kidney (type I and II) were tested on eight bacterial strains isolated from cases of clinical mastitis (staphylococci, streptococci and coliforms). Three other lytic agents were included in the experiments as controls: hen egg-white lysozyme, lysostaphin and mutanolysin. Proliferating bacteria were incubated with the various lytic agents, either in hearts infusion broth or in milk. The type II rainbow trout lysozyme decreased the number of live bacteria (colony forming units) of all the strains tested, but was most efficient against staphylococci. The other two lysozymes had little effect.     

Isolation of yeasts from bovine milk in Belgium

A total of 436 milk samples from non-infected and 80 from infected quarters were investigated: 24.5% of the samples collected from non-infected and 55% of those collected from infected quarters were positive. Normal milk yielded not less than 16 different species and among them many potentially pathogenic yeast species such as C. parapsilosis, C. guilliermondii, C. tropicalis, C. glabrata and T. asahii, all five able to grow at 40 ° C. In contrast, the yeasts isolated from infected quarters were from 3 species: C. kefyr, C. catenulata and C. lambica, which were also among the yeasts species recovered from normal milk. Among the three species, only one i.e. Candida kefyr is able to grow above 40 ° C and from there can be considered as potentially pathogenic, even if bacterial association is necessary to cause mastitis.     

重组人乳铁蛋白和重组人溶菌酶对小鼠溃疡性结肠炎的改善作用研究

溃疡性结肠炎（ulcerative colitis,UC）是一种发生于直肠和结肠的慢性非特异性炎症疾病,近年来发病率明显上升。为探究转基因牛乳中提取的重组人乳铁蛋白和重组人溶菌酶对改善UC的作用,采用葡聚糖硫酸钠（dextran sulfate sodium salt,DSS）构建小鼠UC模型,30只雄性C57BL/6N小鼠随机分为空白对照组（CON组）、模型对照组（DSS组）、低浓度乳铁蛋白组（L-rLF组,50 mg·kg^-1·BW^-1）、高浓度乳铁蛋白组（H-rLF组,100 mg·kg^-1·BW^-1）、低浓度溶菌酶组（L-rLZM组,50 mg·kg^-1·BW^-1）和高浓度溶菌酶组（H-rLZM组,100 mg·kg^-1·BW^-1）。造模后用重组乳铁蛋白和溶菌酶分别灌胃1周,取小鼠血清及结肠,观察器官病理变化,测定炎症因子以及肠道菌群等相关指标。研究结果显示,与模型组相比,低浓度乳铁蛋白组和低浓度溶菌酶组小鼠疾病活动指数（disease activity index,DAI）、结肠缩短量、组织病理学评分均显著降低,且结肠组织中促炎因子（IL-6、lL-1β、TNF-α）表达量、血清和肝脏中脂多糖（lipopolysaccharide,LPS）浓度显著降低,高浓度乳铁蛋白处理组小鼠肠道菌群结构显著改善。表明重组人乳铁蛋白和重组人溶菌酶均可以不同程度地改善小鼠UC,为重组人乳铁蛋白和重组人溶菌酶的未来应用提供了新的思路和理论支持。     

Surface antibacterial characteristics of plasma-modified polyethylene

The antibacterial characteristics of triclosan- or bronopol-coated and plasma-modified polyethylene (PE) are investigated. The modified PE samples exhibit excellent bactericidal effects against Escherichia coli and Staphylococcus aureus even when the bacteria concentration in the suspension is 10(6) colony forming units (CFU)/mL. However, when the concentration exceeds 10(8) CFU/mL, the materials fail to develop noticeable resistance to large amount of bacteria because of the formation of a bacterial biofilm on their surfaces. The PE treated by this relatively simple technique possesses excellent antimicrobial properties and is useful in biomedical and disinfection applications because the bacteria concentrations in most situations are well below 10(6) CFU/mL.     

Consumption of lysozyme-rich milk can alter microbial fecal populations

Human milk contains antimicrobial factors such as lysozyme and lactoferrin that are thought to contribute to the development of an intestinal microbiota beneficial to host health. However, these factors are lacking in the milk of dairy animals. Here we report the establishment of an animal model to allow the dissection of the role of milk components in gut microbiota modulation and subsequent changes in overall and intestinal health. Using milk from transgenic goats expressing human lysozyme at 68%, the level found in human milk and young pigs as feeding subjects, the fecal microbiota was analyzed over time using 16S rRNA gene sequencing and the G2 Phylochip. The two methods yielded similar results, with the G2 Phylochip giving more comprehensive information by detecting more OTUs. Total community populations remained similar within the feeding groups, and community member diversity was changed significantly upon consumption of lysozyme milk. Levels of Firmicutes (Clostridia) declined whereas those of Bacteroidetes increased over time in response to the consumption of lysozyme-rich milk. The proportions of these major phyla were significantly different (P < 0.05) from the proportions seen with control-fed animals after 14 days of feeding. Within phyla, the abundance of bacteria associated with gut health (Bifidobacteriaceae and Lactobacillaceae) increased and the abundance of those associated with disease (Mycobacteriaceae, Streptococcaceae, Campylobacterales) decreased with consumption of lysozyme milk. This study demonstrated that a single component of the diet with bioactivity changed the gut microbiome composition. Additionally, this model enabled the direct examination of the impact of lysozyme on beneficial microbe enrichment versus detrimental microbe reduction in the gut microbiome community.     

Bioluminescent assay of total bacterial contamination of raw milk]

A rapid (30-35 min) bioluminescence assay of total bacterial contamination (TBC) of raw milk was optimized. This method includes incubation of milk samples in the presence of Neonol-10 and medical purity grade pancreatin with further removal of nonbacterial ATP by filtration through a membrane filter, cell disruption by treatment with dimethyl sulfoxide, and measurement of ATP concentration in a reaction with the bioluminescent reagent Immolum. The TBC detection threshold is 0.5 x 10(5) colony-forming units (CFU) per ml milk. Coefficients of correlation between the standard plate count method and bioluminescence assay (R) and residual standard deviations (Sxy) in raw milk samples (n = 140) were 0.83 and 0.54, respectively. In sterilized milk samples artificially contaminated with pure cultures of the main representatives of milk microflora (coli-forms, Staphylococcus aureus, Streptococcus thermophilus, and Streptococcus group D), these values were 0.89-0.99 and 0.09-0.29, respectively. The specific content of ATP was found to be (0.8 +/- 0.1) x 10(-18) mol/CFU in coli-forms; (12.0 +/- 8.1) x 10(-18) mol/CFU in S. aureus; (35.2 +/- 16.9) x 10(-18) mol/CFU in S. thermophilus; and (42.5 +/- 1.3) x 10(-18) in Streptococcus group D.     

Viable Staphylococcus aureus quantitation using ¹⁵N metabolically labeled bacteriophage amplification coupled with a multiple reaction monitoring proteomic workflow

A multiple reaction monitoring liquid chromatography method with tandem mass spectrometric detection for quantitation of Staphylococcus aureus via phage amplification detection is described. This phage amplification detection method enables rapid and accurate quantitation of viable S. aureus by detecting an amplified capsid protein from a specific phage. A known amount of metabolically labeled (15)N reference bacteriophage, utilized as the input phage and as the internal standard for quantitation, was spiked into S. aureus samples. Following a 2-h incubation, the sample was subjected to a 3-min rapid trypsin digest and analyzed by high-throughput liquid chromatography tandem mass spectrometric detection targeting peptides unique to both the (15)N (input phage) and (14)N (progeny phage) capsid proteins. Quantitation was achieved by comparing peak areas of target peptides from the metabolically labeled (15)N bacteriophage peptide internal standard with that of the wild-type (14)N peptides that were produced by phage amplification and subsequent digestion when the host bacteria was present. This approach is based on the fact that a labeled species differs from the unlabeled one in terms of its mass but exhibits almost identical chemical properties such as ion yields and retention times. A 6-point calibration curve for S. aureus concentration was constructed with standards ranging from 5.0 × 10(4) colony forming units (CFU) ml(-1) to 2.0 × 10(6) CFU ml(-1), with the (15)N reference phage spiked at a concentration of 1.0 × 10(9) plaque forming units (PFU) ml(-1). Amplification with (15)N bacteriophage coupled with LC-MS/MS detection offers speed (3 h total analysis time), sensitivity (LOD: < 5.0 × 10(4) CFU ml(-1)), accuracy, and precision for quantitation of S. aureus.