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©1994-2004 All Rights Reserved. Online Journal of Veterinary Research. You may not store these pages in any form except for your own personal use. All other usage or distribution is illegal under international copyright treaties. Permission to use any of these pages in any other way besides the before mentioned must be gained in writing from the publisher. This article is exclusively copyrighted in its entirety to OJVR publications. This article may be copied once but may not be, reproduced or re-transmitted without the express permission of the editors. OJVR TM Online Journal of Veterinary Research © Volume 8:22-32, 2004. Peptide AS-48 (Enterococcus faecalis) for prevention and treatment of mastitis in dairy cows Davidse EKa, Balla Ea, Holzapfel WHb, Muller CJCc, Cloete SWPc, Dicks LMTa Department of Microbiology, Stellenbosch University, 7600 Stellenbosch, South Africa, bInstitute of Hygiene and Toxicology, Federal Research Centre for Nutrition, Haid-und-Neu-Str. 9, 76131, Karlsruhe, Germany, cDepartment of Agriculture, Private Bag X1, 7607 Elsenburg, South Africa a ABSTRACT Davidse ek, Balla E, Holzapfel WH, Muller CJC, Cloete SWP, Dicks LMT Peptide AS-48, a cyclic antimicrobial peptide from Enterococcus faecalis, may be used in the prevention and treatment of mastitis in dairy cows, Online Journal of Veterinary Research 8:22-32, 2004. Peptide AS-48, produced by Enterococcus faecalis FAIRE 92, inhibited the growth of a Staphylococcus aureus strain isolated from mastitic milk. Peptide AS-48 was isolated from the cell-free supernatant by using a combination of Triton X-114 phase partitioning and cation exchange 2 chromatography. The partially purified peptide was liposomeencapsulated at a yield of 400 AU (arbitrary units)/ml and injected into infection-free quarters of healthy Holstein cows. These quarters were then infected by injecting 2 ml of the S. aureus pathogen (3.3 x 103 cfu/ml) through the teat canals. Control udders not pretreated with AS-48 were also injected with the same level of S. aureus. From the second day after milking to day 7, the somatic cell count (SCC) in milk from udders that have been pre-treated with liposome-encapsulated peptide AS-48 decreased by 60%. The viable cell numbers of S. aureus in milk from pre-treated udders remained more-or-less the same over the 7-day period (1 x 102 cfu/ml), whereas the S. aureus cell numbers in milk from untreated udders increased to 8 x 102 cfu/ml. When S. aureus-infected udders with a SCC higher than 5 x 105/ml were injected together with liposome-encapsulated peptide AS-48 (6 400 AU/ml), the SCC in milk from these animals decreased by ca. 85% and the number of viable S. aureus by ca. 99%. Streptococcus agalactiae and Streptococcus dysgalactiae, isolated from mastitic milk, were also inhibited in in vitro tests, but not Escherichia coli. KEYWORDS: Mastitis, Treatment, Peptide AS-48 INTRODUCTION Staphylococcus aureus is one of the most common etiological agents of bovine mastitis in lactating dairy cows and contributes to significant economic losses in the dairy industry (Hébert et al 2000; Twomey et al 2000). In the Western Cape of South Africa, S. aureus is responsible for 40% of all mastitis cases reported. Despite mastitis management programs to reduce the incidence of intramammary udder infections (IMI), S. aureus remains a major pathogen (Twomey et al 2000). After entering the mammary gland through the teat canal, the bacteria multiply rapidly, leading to inflammation and tissue damage (Hébert et al 2000). Apart from infection, S. aureus secretes various toxins and enzymes, which may lead to food poisoning when the milk is consumed (Coetzer et al 1994). Staphylococci found in infected tissues are mainly located extracellularly (Onyeji et al 1994). However, virulent strains of S. aureus can penetrate phagocytic cells and survive within leukocytes (Bramley & Dodd 1984; Onyeji et al 1994; Watson 1994; Hébert et al 2000). Antibiotics are routinely administered at drying-off to treat sub clinical cases of mastitis and prevent further infection (Twomey et al 2000). However, animals infected with S. aureus respond poorly to antibiotic treatment (Watts 1990), probably due to the intracellular location of the bacterial cells in the alveoli 3 and/or macrophages (Bramley & Dodd 1984; Hébert et al 2000). Although many of the antibiotics penetrate neutrophils, S. aureus may survive (Francis 1989) and causes chronic intraphagocytic infections (Onyeji et al 1994). The emergence of antibiotic resistance in bacteria has led to a considerable debate in the use of antibiotics for prophylactic treatment, which in turn led to a search for alternative treatments (Twomey et al. 2000). Immunization against S. aureus does not prevent IMI, as evident from the lack of high concentrations of antibody and phagocytic cells in the milk (Nickerson 1985). Furthermore, inadequate knowledge of the pathogenesis of staphylococcal mastitis and the immune mechanisms protecting the mammary gland from infection has limited the scope for novel approaches to vaccination (Watson, 1992). Bacteriocins produced by lactic acid bacteria are generally considered safe and may present a cost-effective alternative to treat mastitis caused by strains of S. aureus, Streptococcus agalactiae and Streptococcus dysgalactiae. In a previous study with a combination of the bacteriocins nisin and lysostaphin, 66% of the S. aureus-infections could be cured (Reichelt et al 1984). In another study, which involved the incorporation of the bacteriocin lacticin 3147 into a teat seal (1280 AU/ml), 99.9% of S. aureus cells were killed (Ryan et al 1998). In the present study we have encapsulated the cyclic peptide AS48, produced by E. faecalis FAIRE 92, into liposomes and evaluated its effect against a pathogenic strain of S. aureus that causes mastitis in dairy cows. MATERIALS AND METHODS Bacterial strains and growth conditions: Enterococcus faecalis FAIRE 92, the producer of peptide AS-48, forms part of the EU FAIR-E collection at the BCCMTM/LMG Culture Collection, Laboratorium voor Microbiologie (LMG), University of Ghent (RUG) (K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium). Strains of S. aureus, S. agalactiae, S. dysgalactiae and Escherichia coli (Table 1) were isolated from mastitic milk and were obtained from the Diagnostic Veterinary Laboratory, Stellenbosch, South Africa. Other indicator strains used in this study (Table 1) were from the LMG at the University of Ghent. The Lactobacillus and Leuconostoc spp. were grown in MRS broth (Biolab Diagnostics, Midrand, South Africa). The other indicator bacteria were cultured in BHI broth (Biolab). 4 Table 1. Spectrum of antimicrobial activity of peptide AS-48 Organism Bacillus cereus Escherichia coli Lactobacillus acidophilus Lactobacillus casei Lactobacillus curvatus Lactobacillus fermentum Lactobacillus plantarum Lactobacillus reuteri Lactobacillus sakei Leuconostoc cremoris Pediococcus pentosaceus Staphylococcus aureus Staphylococcus carnosus Streptococcus agalactiae Streptococcus dysgalactiae Strain(s) LMG 13569 MKB EC1 LMG 13550 LMG 13552 LMG 13553 LMG 13554 LMG 13556 LMG 13557 LMG 13558 LMG 13562, 13563 LMG 13560, 13561 MKB 38 LMG 13567 MKB SA1 MKB SD1 Sensitivity* ++ ++ ++ ++++ ++ + + ++ ++ +++ + ++ ++ ++ *+, ++, +++, ++++ reflects the degree of sensitivity to peptide AS-48. Production and inhibitory activity of peptide AS-48: An overnight culture (10 ml) of E. faecalis FAIRE 92 was inoculated into 1 litre of BHI-G broth (BHI broth supplemented with 1%, wt/vol, glucose and 0.15 M NaH2PO4H2O) and incubated at 37oC without aeration until stationary growth. On an hourly basis 1 ml was sampled, serially diluted and plated onto BHI Agar (Biolab) and the number of viable cells (CFU/ml) determined. At the same time 1 ml of the culture was centrifuged and the cell-free supernatant adjusted to pH 6.0 to 7.0 with 1 N sterile NaOH. The sample was concentrated ten-fold by freeze-drying and tested for activity against S. aureus by using the spot-on-lawn method (Van Reenen et al. 1998). One arbitrary unit (AU) of peptide AS-48 is defined as the reciprocal of the highest dilution that produced an inhibition zone of at least 2 mm in diameter. From the antimicrobial activity, expressed as AU/ml, and the cell count (log10 cfu/ml), the specific antimicrobial activity (AU/log10 cfu) was determined. Isolation, purification and concentration: Peptide AS-48 was isolated and purified according to the method described by Métivier 5 et al (2000). One litre of BHI-G broth was inoculated with 10 ml of an actively growing culture of E. faecalis FAIRE 92 and incubated for 7 h at 37oC. Cells were harvested by centrifugation (9000 x g, 4oC) and Triton TX-114 added to the supernatant to obtain a final concentration of 2% (wt/vol). The sample was adjusted to pH 5.5 with concentrated HCl, heated to 25 to 30oC, and incubated at this temperature for 1 to 2 h, after which the upper-phase was removed and replaced with the same volume of cold Millipore Q water (18.2) containing Triton TX-114 (0.2%, wt/vol). The Triton TX114 was dissolved by careful stirring. The mixture was heated to 25 to 30oC and left to separate into two phases. The lower detergentrich phase was recovered, diluted five-fold with cold Millipore Q water, and loaded onto a 15 ml SP-Sepharose Fast flow column (Amersham Pharmacia Biotech, Uppsala, Sweden). The column was washed with cold Millipore Q water until a constant baseline absorbance at 280 nm was reached. Loading and washing was done at 8oC to avoid phase partitioning. The bacteriocin was eluted with an ammonium acetate step-gradient of 0.1 to 1.6 mol liter-1 (pH 6.0). Fractions of 4 ml were collected, concentrated by freezedrying, dissolved in 100 l Millipore Q water and tested for activity against S. aureus, S. agalactiae, S. dysgalactiae and E. coli, as described before. The active samples were pooled and stored at 80oC. Sensitivity to proteolytic enzymes: Purified peptide AS-48 (3200 AU/ml) was used in these tests. Resistance of peptide AS-48 to proteolytic enzymes was determined by incubation in the presence of proteinase K (20 U/mg of peptide AS-48), pronase (7 U/mg of peptide AS-48), pepsin (2500 U/mg of peptide AS-48), papain (30 U/mg of peptide AS-48), -chymotrypsin (90 U/mg of peptide AS-48) and trypsin (110 U/mg of peptide AS-48) at 37oC for 1 h. All enzymes were from Boehringer-Mannheim (Howard Place, Midrand, South Africa) and tested at their optimum activity pH. After incubation, the enzymes were heat-inactivated for 3 min at 100oC and peptide AS-48 tested for antimicrobial activity against S. aureus. The activity of the treated samples was compared to that of a control sample, i.e. peptide AS-48 that had not been treated with proteolytic enzymes. Molecular mass determination: A sample collected after separation in SP-Sepharose was subjected to tricine-SDSpolyacrylamide gel electrophoresis (SDS-PAGE), according to the method described by Schägger & Von Jagow (1987). A protein marker with sizes ranging from 2.35 to 46 kDa (Rainbow Marker; Amersham Pharmacia Biotech, Uppsala, Sweden) was used. One half of the gel was stained with Coomassie Brilliant Blue R250. The position of the active peptide AS-48 in the gel was determined by 6 overlaying the other half of the gel, prewashed as described by Van Belkum et al (1991), with cells of an overnight grown culture of S. aureus, embedded in BHI agar (0.75% agar, wt/vol). In another experiment, approximately 100 pmol of purified sample containing the bacteriocin was diluted in 10 l of 10:90 acetonitrile-water containing 0.01% formic acid and injected via the Rheodyne injection port of a Quattro triple quadropole mass spectrometer (Micromass, Manchester, United Kingdom). The carrier solvent was 10:90 acetonitrile-water at a flow rate of 20 l/min, delivered by a Pharmacia-LKB 2249 high-pressure liquid chromatography pump. The capillary voltage and the cone voltage were set at 3.5 kV and 60 V, respectively. Data were collected by scanning from 400 to 1500 m/z at 2 s/scan. The multiple charged spectra were deconvoluted to obtain the accurate mass of the peptides. Calibration was done by using horse heart myoglobin (Sigma, St. Louis, Mo). Preparation of liposomes and determination of encapsulation efficiency: The method of Degnan & Luchansky (1992) was used. Multilamellar vesicles were prepared using phosphatidyl choline (Sigma) at 100 mg/ml chloroform. Four ml methanol/chloroform (1:1) and 2 ml phosphatidylcholine/chloroform mixture was placed into a 500 ml round-bottom flask and the solvent removed through rotary evaporation (37oC, 60 rpm, 30 min, under vacuum). After evaporation, a thin, opaque lipid film was formed on the inner surface of the flask. Five ml of peptide AS-48 (6400 AU/ml) and eight glass beads (4 mm in diameter; Fisher Scientific, Pittsburgh, PA) were added to the contents of the flask and rotated for a further 1 h at atmospheric pressure. This suspension was incubated at 37oC for 2 h to complete swelling of the liposomes. The peptide AS-48/liposome preparation was then transferred to a sterile 15 ml polypropylene test tube and stored at 4oC for 7 d. The encapsulation efficiency (E%) was determined by adding 45 l of the peptide AS-48/liposome suspension to each of two microcentrifuge tubes. Five l proteinase K (10 mg/ml) was added to one of the tubes to inactivate free, i.e. non-encapsulated, peptide AS-48. Sterile distilled water was added to a second tube. Both samples were incubated at 37oC for 1 to 2 h and then heated for 3 min at 100oC to inactivate proteinase K and, at the same time, release encapsulated peptide AS-48 from the intact liposomes. The antimicrobial activity of the released peptide AS-48 was determined by using the spot-on-lawn method, as described before. The E% of encapsulated peptide AS-48 was calculated as follows: E% = activity (AU/ml) of peptide AS-48 in the liposome mixture treated with proteinase K, divided by the activity (AU/ml) of peptide AS-48 in the liposome mixture to which sterile distilled water was added (x100). 7 In vitro antimicrobial activity tests with encapsulated peptide AS-48: One ml of encapsulated peptide AS-48 (3200 AU/ml) was added to a 100 ml culture of S. aureus at the beginning of the lag and mid-exponential growth phases, respectively. Sterile demineralised water (1 ml) was added to the control flask. Changes in the turbidity of the cultures were recorded at 600 nm, and the number of viable cells (cfu/ml) was determined by plating onto BHI Agar (Biolab). The activity (AU/ml) of the encapsulated peptide was determined by using the spot-on-lawn method, as described before. Prevention and treatment of S. aureus-infections in udders: For the prevention experiment, 10 quarters of five mastitis-free cows with SCC of less than 500 000 per ml milk were selected. Milk from these quarters contained no viable cells of S. aureus, as determined by plating onto Baird-Parker Agar (Biolab). Five of the quarters were infused with 1 ml encapsulated peptide AS-48 (6400 AU/ml), followed by 2 ml of S. aureus (3.3 x 103 cells/ml) injected through the teat canal. The other five quarters served as controls and received the same treatment, except that peptide AS-48 was replaced by 1ml sterile saline (0.75%, w/v, NaCl). Injection was immediately after milking, thus on day 1 of milk collection. The AS48 peptide, saline and S. aureus cells were gently massaged upwards into the teat canal. Milk samples were collected daily, (up to day 7), from which the SCC and viable cell numbers of S. aureus were determined. The number of replications depended on the number of available mastitis-free cows at a specific stage of lactation. Since the microbial challenge with S. aureus was expected to result in marked changes in SCC and S. aureus counts, five replicates were regarded as sufficient for the purposes of this study. Moreover, since only a marked response to the treatment with peptide AS-48 would be of therapeutic value, it was argued that a high number of replicates was not needed. All counts were expressed as log10 values. Where no S. aureus was detected, a value of 100 was allocated to each count prior to analysis. The analyses were also complicated by the fact that ten different quarters of five individual cows were sampled. The data could thus not be described as uncorrelated, as assumed for analysis of variance. This complication was accounted for by the estimation of the intra-class correlation depicting all possible correlations between repeated samples obtained from the cows (Harvey, 1990). Co-variation arising from the repeated samples from specific cows was not only accounted for by this procedure, but it was also possible to estimate the repeatability of the SCC and 8 S. aureus cell numbers (Turner & Young 1969). Repeatability (t) was calculated as follows: t = b2/ b2 + e2 = b2 the intra-class correlation between cows = e2 the residual variation Apart from the random effect of cows in the models used to analyse the data, fixed effects of treatment and days post treatment were included. The interaction between treatment and days post treatment was also estimated. For the treatment experiment, six quarters from the udders of three lactating Holstein cows from the dairy herd of the Elsenburg Research Center suffering from mastitis and with a SCC higher than 2.5 x 106/ml milk, were selected. Milk collected from these quarters tested positive for the presence of S. aureus (≥ 1 x 104 CFU/ml). Three of these quarters were injected with 1ml encapsulated peptide AS-48 (6400 AU/ml), as described before. The other three quarters served as controls and were injected with 1ml sterile saline solution (0.75%, w/v, NaCl). As in the prevention experiment, injection was done immediately after milking and the encapsulated peptide and saline gently massaged upwards into the teat canals. Milk samples were collected daily (up to day 7) from which the SCC and number of viable S. aureus were determined. The number of replications conducted in this experiment was also relatively low. Again, the experiment depended on the number of mastitic udders available at a specific stage of lactation. The number of replicates was regarded sufficient for the purposes of this experiment. The data were analysed as a 2 (treatments) x 7 (day) factorial design. As for the previous experiment, the analysis was complicated by the fact that the same quarter was sampled repeatedly. The same basic procedure was followed to account for repeated sampling. The difference was that individual quarters were confounded with treatments in this case. The appropriate analysis was thus to nest individual quarters within treatments. Repeatability was estimated from between quarter variance components as described previously. RESULTS The highest antimicrobial activity levels recorded for peptide AS-48 was 1593 AU/ml during late exponential growth, i.e. after 7h at 37°C (Fig. 1). This corresponded to a specific antimicrobial activity 9 of 148 AU/log10 cfu (Fig. 1). Isolation and further purification of peptide AS-48 with Triton TX-114 and SP-Sepharose resulted in an increase of antimicrobial activity (3200 to 12800 AU/ml; data not shown), which corresponds to a two to eight-fold increase in specific antimicrobial activity (296 to 1184 AU/log10 cfu). In in vitro tests, all strains of Bacillus, Lactobacillus, Staphylococcus and Streptococcus were inhibited, but not E. coli (Table 1). Figure 1. Production of peptide AS-48 during the growth of E. faecalis FAIRE 92., Growth of E. faecalis FAIRE 92, expressed as CFU/ml; and , specific antimicrobial activity (AU/log10 CFU) of peptide AS-48 against S. aureus. = highest activity recorded as 1593 AU/ml. 11.5 10 9.5 80 9 log10 cfu/ml 100 8.5 60 7.5 20 7 8 40 0 0 1 2 3 4 5 6 7 8 9 Time ( hours) Peptide AS-48 was completely inhibited by treatment with proteinase K, pronase, pepsin, papain, -chymotrypsin and trypsin (results not shown). Mass spectrometry analysis indicated that the active samples, collected from the SP-Sepharose column and pooled, contained a single peptide with a molecular mass of 7.150 kDa (Fig. 2). Figure 2. Molecular mass of peptide AS-48 calculated from the electro-spray ionization-mass spectrometry multiple charged spectra. 10 Specific antimicrobial activity ( AU/log 10 cfu) 11 120 10 140 10.5 160 11 7150.00 100 % 7476.26 7490.24 0 3000 4000 5000 6000 7000 8000 12 Separation on tricine-SDS-PAGE yielded only one active peptide band in the range of 6.4 kDa (Fig. 3). 6.5 46 kDa 1 2 3.4 3 30 21.5 14.5 Figure 3. Separation of peptide AS-48 by tricine-SDSPAGE. Lane 1 , Rainbow protein size markers; lane 2, peptide AS-48 stained with Coomassie brilliant blue R250; lane 3, peptide AS-48 overlaid with a strain of S. aureus isolated from mastitic milk and embedded in BHI Agar (0.75% agar, wt/vol). The active peptide band is indicated by an arrow. The titer of the liposome-encapsulated peptide AS-48 varied between 400 AU/ml after proteinase K treatment, compared to 3200 AU/ml for the control sample (not treated with proteinase K), to 1600 AU/ml after proteinase K treatment compared to 6 400 AU/ml for the control sample. This compares to an encapsulation efficiency (E%) of between 12.5% and 25%. In general, encapsulation efficiencies varied between 10 and 25%. Addition of encapsulated peptide AS-48 (1000 AU/ml) to S. aureus in lag-phase resulted in a decrease in viable cell numbers from 3 x 108 cfu/ml to 1 x 104 cfu/ml within 30 min, followed by a further decrease over the next 210 min (3.5h) to below the detection limit 13 of 10 cfu/ml (Fig. 4). The cell numbers of the control, i.e. without peptide AS-48 added, increased from 3 x 108 cfu/ml to 1 x 1011 cfu/ml over 300 min (5h). The optical density readings of cells that received peptide AS-48 in lag-phase remained constant at approximately 0.2, whereas the density of the culture increased to approx. 6.0 in the absence of peptide AS-48 (Fig. 4). Encapsulated peptide AS-48 (1000 AU/ml) added to a mid-exponential growth phase culture of S. aureus (2.5 x 1010 cfu/ml) resulted in no growth inhibition, as evident from the steady increase in viable cell counts and optical density readings, respectively (Fig. 4). 6.5 13 6 12 Addition of peptide AS-48 5.5 11 10 9 4 8 3.5 7 3 6 2.5 5 2 4 Addition of peptide AS-48 1.5 3 1 2 0.5 1 0 0 0 30 60 90 120 150 180 211 240 270 300 Time ( minutes) Figure 4. Effect of encapsulated peptide AS-48 (1000 AU/ml) on the growth of S. aureus. Cell counts in the absence of peptide AS-48 (), in the presence of peptide AS-48 added at the beginning of the lag phase (), and added during mid-exponential growth (). The optical density (at 600 nm) was determined for cells growing in the absence of peptide AS-48 (), in the presence of peptide AS-48 added at the beginning of the lag phase (), and added during mid-exponential growth (). Treatment of healthy, uninfected udders with liposomeencapsulated peptide AS-48 (400 AU/ml), followed by infection with S. aureus, resulted in variable SCC and S. aureus cell counts (Figs. 5a and b). The SCC on day 1, directly after milking, was 6.3 x 330 360 log10 cfu/ml O.D. (600 nm) 5 4.5 14 104/ml and 4.9 x 104/ml for udders not treated with peptide AS-48 and udders treated with the peptide, respectively (Fig. 5a). Twentyfour hours later (day 2), the SCC in milk from untreated and treated udders increased to 1.3 x 106/ml and 1 x 106/ml, respectively (Fig. 5a). During the next 5 days, the SCC in milk from untreated udders decreased slightly to 1.2 x 106, whereas the SCC in milk from treated udders decreased from 1 x 106/ml to 4 x 105/ml, representing a 60% decrease in SCC. Log10 SCC/ml 7 6 Not treated Treated 5 4 1 2 3 4 5 6 7 Time (days) . Figure 5(a). The effect of liposome-encapsulated peptide AS48 on the somatic cell count (SCC) in milk sampled from mastitis-free udders that were infected with S. aureus relative to an untreated control (i.e. no peptide AS-48 injected into the udders). Each mean is based on five replicates. The experimental design involved five cows with one teat randomly assigned to the treated and control groups respectively. Vertical bars denote standard errors. 15 4 Log10 cfu/ml 3.5 3 Not treated Treated 2.5 2 1.5 1 2 3 4 5 6 7 Time ( Days) Figure 5(b). The effect of liposome-encapsulated peptide AS48 on the cell numbers of S. aureus in milk sampled from mastitis-free udders that were infected with S. aureus relative to an untreated control. Each mean is based on five replicates. The experimental design involved five cows with one teat randomly assigned to the treated and control groups respectively. Vertical bars denote standard errors The variation accounted for by the repeated sampling from different animals was significant in the analysis involving SCC (P < 0.001) and S. aureus counts (P < 0.05). Repeatability estimates (SE) derived from these analyses were 0.26 0.19 and 0.13 0.13, respectively. Although these estimates could not be proven as significant (P < 0.05) from zero, the variation between animals controlled significant (P < 0.05) portions of the overall variation in both analyses. It was thus decided to retain this effect in the models used. The interaction between treatment and days post treatment was not significant (P = 0.79) as far as SCC is concerned. A sharp increase (P < 0.05) in SCC was recorded on day 2 (Fig. 5a). In quarters treated with peptide AS-48 the SCC was generally lower than in the control (untreated) quarters, with a significant (P < 0.05) difference on day 6. When the overall mean values for treated and untreated quarters were compared, a significant difference (P < 0.01) was recorded in favour of the treated quarters. The viable cell numbers recorded for S. aureus in milk sampled from untreated udders increased from 1 x 102 cfu/ml (day 1) to 2.6 x 103 cfu/ml (day 4), followed by a decrease to 8 x 102 cfu/ml on 16 day 7 (Fig. 5b). The S. aureus count in milk collected from udders pre-treated with peptide AS-48 increased from 1 x 102 cfu/ml (day 1) to 2.4 x 102 cfu/ml (day 4), but decreased to 1 x 102 cfu/ml towards the end of the experiment, i.e. day 7 (Fig. 5b). Since no significant interaction was recorded between treatment and days post treatment (P < 0.05), it is appropriate to give overall mean values for treated and control quarters. These were (on the log10 scale) 2.79 0.18 for control quarters and 2.18 0.18 for treated quarters. Transformed back to the normal scale (with 100 subtracted) these mean values correspond to respective S. aureus counts of 509 and 51, i.e. a near to 90% reduction in treated quarters. The SCC in milk sampled from untreated S. aureus-infected quarters remained more-or-less the same and varied from 2.9 x 106/ml to 7.4 x 106/ml over a period of 7 d (Fig. 6a). When injected with liposome-encapsulated peptide AS-48 (1 600 AU/ml), the SCC in milk from S. aureus-infected quarters decreased from approx. 7 x 106/ml to 1.0 x 106/ml over 7 d (Fig. 6a). The viable cell count of S. aureus in infected milk from untreated udders increased from 1 x 104 cfu/ml to 3.9 x 104 cfu/ml over 7 d, whereas in milk from udders that have been treated with encapsulated peptide AS-48, the cell counts decreased from 3.2 x 104 cfu/ml to 2.3 x 102 cfu/ml over the same period (Fig. 6b). The between quarter variance was significant (P < 0.01) in analyses on SCC and S. aureus counts. The respective repeatability estimates derived from the variance components were 0.40 0.25 and 0.41 0.25. For SCC the interaction between treatment and days post treatment did not reach significance in this case (P = 0.06). Overall, the SCC was reduced (P < 0.05) in treated quarters relative to control quarters (Fig. 6a). Overall mean values (SE) for SCC were 6.70 0.12 for control quarters, compared to 6.01 0.12 for treated quarters. Expressed relative to the control treatment, the reduction in treated quarters amounted to approximately 99%. . 17 Log10 SCC/ml 8 7 6 5 4 1 2 3 4 5 6 7 Time (days) Figure 6(a). The effect of liposome-encapsulated peptide AS48 on mastitis-infected udders. SCC counts were determined in milk sampled from peptide AS-48-treated and not treated udders, respectively. Each mean is based on three replicates. The experimental design involved three cows with on teat randomly assigned to the treated and control groups respectively. Vertical bars denote standard errors Log10 cfu/ml 5.5 4.5 Not treated Treated 3.5 2.5 1.5 1 2 3 4 5 Time ( days) 6 7 18 Figure 6(b). The effect of liposome-encapsulated peptide AS48 on mastitis-infected udders. Cell counts of S. aureus were determined in milk sampled from peptide AS-48-treated and not treated udders, respectively. Each mean is based on three replicates. The experimental design involved three cows with on teat randomly assigned to the treated and control groups respectively. Vertical bars denote standard errors. In the case of S. aureus, treatment interacted (P < 0.01) with the days of post treatment. In the case of untreated quarters, S. aureus counts basically remained on the same level throughout the monitoring period (Fig. 6b). In treated quarters there was a significant (P < 0.01) decline in S. aureus counts, to reach levels not significantly different (P < 0.05) from two, which corresponds to zero for back transformed values. This decline was observed from day 4. Differences between mean values for treated and control quarters were significant (P < 0.05) from day 2 onwards. Overall means (SE) were 4.1 0.3 for control quarters, compared to 2.91 0.26 for treated quarters. Transformed back to the arithmetic scale (with 100 subtracted) these means correspond to respective S. aureus counts of 12 530 and 715, i.e. a reduction of up to 94% in treated quarters. DISCUSSION Peptide AS-48 exerts its antimicrobial action by incorporating into the cytoplasmic membrane of sensitive bacteria. This is followed by pore formation, and leads to the leakage of potassium ions and inorganic phosphate (Gálvez et al 1986, 1989; Samyn et al 1994). In contrast to previous reports on the antimicrobial activity spectrum of peptide AS-48 (Gálvez et al. 1986, 1989), we did not observe inhibition of E. coli (Table 1). It may very well be that higher concentrations of the peptide have to be used to effectively penetrate the outer membrane of Gram-negative bacteria, as suggested by other authors (Gálvez et al 1986, 1989; Joosten et al 1996). Peptide AS-48 did, however, inhibit the growth of S. aureus, S. agalactiae and S. dysgalactiae which are considered to be major pathogens involved in mastitis infection, especially in the Western Cape region of South Africa. Production of peptide AS-48 during late exponential growth is typical of most bacteriocins described for lactic acid bacteria (De Vuyst and Vandamme 1994). The molecular mass of peptide AS-48 (7.150 kDa; Fig. 2) is as previously reported for the peptide (Martínez-Bueno et al. 1994). A clear inhibition zone which surrounded a well-separated single peptide band of approx. 6.5 kDa 19 on a tricine-SDS gel (Fig. 3), confirmed that strain FAIRE 92 produces peptide AS-48. Peptide AS-48 was successfully encapsulated into liposomes. Although the antimicrobial activity recorded for the encapsulated peptide is much lower than for the non-encapsulated peptide, the encapsulation efficiency obtained (12 – 25% E) compared well with encapsulation results previously reported for this specific method (Degnan & Luchansky 1992). When encapsulated in liposomes, the antimicrobial peptides are delivered to phagocytic cells to accumulate intra-cellularly (Francis 1989; Onyeji et al 1994). Upon phagocytosis of the liposomes, the antimicrobial compound is released into the phagolysosome (Francis 1989). Virulent cells of S. aureus may be situated in phagocytic cells and survive within leukocytes (Bramley & Dodd 1984; Watson 1992; Onyeji et al 1994, Hébert et al 2000). Although non-encapsulated peptide AS48 would be more active in eliminating extra-cellularly located S. aureus, encapsulation of the peptide would ensure a slow release of antimicrobial activity and the deposition of the peptide to localised areas within the udder. The release of peptide AS-48 from encapsulated liposomes was clearly demonstrated when added to lag-phase cells of S. aureus (Fig. 4). The lack of inhibition recorded when encapsulated peptide AS-48 was added to a mid-exponential phase culture of S. aureus suggests that older cells are either less sensitive to the peptide, or that the peptide was present at sub-lethal concentrations, as previously reported for lacticin 3147 in similar experiments (Twomey et al 2000). Based on results obtained in the prevention and treatment experiments with encapsulated peptide AS-48 injected into the udders of healthy and mastitis-infected cows, respectively, better results were obtained when the peptide was used to treat S. aureus infection than when the peptide was used to prevent S. aureus infection. However, in none of the two experiments, S. aureus was completely eliminated. 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