Patent Application: US-57601004-A

Abstract:
the disclosure relates to the live cultures of probiotic bacteria to treat infectious diseases in humans and animals . food - grade or non - pathogenic cultures are used to treat localised infections .

Description:
lactococcus lactis dpc3147 was isolated previously from a kefir grain . it was routinely propagated at 30 ° c . in m17 broth ( difco laboratories , detroit , usa ) supplemented with 0 . 5 % glucose or lactose . this culture ( 3 ml , ˜ 10 9 cfu ml − 1 ) was either used directly , or an infusion mixture was prepared in the following way : 2 ml of an overnight culture of l . lactis dpc3147 were mixed with 3 ml sterile water for injection ( antigen pharmaceuticals ) to produce a working culture concentration of approximately 10 9 cfu ml − 1 . control infusion mixtures included uninoculated broth , incubated overnight at 30 ° c . and then diluted in a similar fashion to the culture . the diluted l . lactis dpc3147 cultures were infused directly into the teat sinus via the streak canal . the culture was inoculated to a depth of 17 mm using a syringe with a blunted smoothed tip to prevent injury to the teat . infusion of the mixture was usually performed after the evening milking . for the six chronic disease cases , a single application of 3 mls of undiluted culture was performed . for the nine clinical mastitis cases , infusion was performed twice at a 24 h interval for seven quarters ( cow 1163rh , 14lh , 717rf , 1154lf , 1850rf , 1184rf , 1176lh ), twice with a 72 h interval for one quarter ( 1178lh ) and four times for cow 264lf , with 24 h between and first and second infusions , 84 h between the second and third infusion and 48 h between the third and fourth infusions ( appendix 1 - 9 ). 5 ml of the infusion mixture prepared as described above were used when infusing the quarters with clinical mastitis . before in vivo experiments commenced , foremilk quarter samples were collected in an aseptic manner from all prospective cows and these were screened for mastitis - causing pathogens by streaking 10 μl on separate quadrants on the surface of aba plates and incubating for 16 h at 37 ° c . somatic cell counts ( scc ) or cmt results were also determined for each quarter before treatment . previous history of infection was also considered during selection . six udder quarters from 4 cows with a history of chronic infection were initially selected for treatment . nine quarters from 9 cows with newly acquired clinical infections were also treated subsequently . eighteen hours post - infusion , foremilk samples were taken in an aseptic manner for microbiological analysis from all of the treated quarters . one hundred microlitres of each milk sample was streaked on the surface of an aesculin blood agar plate ( aba ) containing blood agar base no . 2 ( oxoid ), supplemented with 7 % citrated whole calf blood and 0 . 1 % aesculin ( sigma , st . louis , mo ., usa ) and incubated for 24 h at 37 ° c . following incubation , colonies were enumerated and identified on the basis of haemolytic activity and colony appearance on aba . one hundred microlitres of each milk sample was also streaked on the surface of a m17 agar plate supplemented with 0 . 5 % glucose or lactose and incubated overnight at 30 ° c . the antimicrobial activity of l . lactis colonies was assessed against l . lactis hp , using the agar well diffusion assay described previously ( parente and hill , 1992 ). additionally , colonies isolated on gm17 or lm17 from milk were selected randomly , purified and assayed to confirm isolation of l . lactis dpc3147 from udder quarters . two independent studies were performed to investigate the effect of l . lactis dpc 3147 on the immunity of the cow . in the first study , two cows were used , an uninfected animal ( cow 273 ) and an infected animal with high scc ( cow 1850 ). one quarter of cow 273 was infused with sterile broth as a control ( left hind , lh ) and one quarter was infused with the l . lactis culture ( right hind , rh ). one quarter of cow 1850 was also infused with the l . lactis culture ( rf ). milk samples were taken immediately prior to intramammary infusion ( day 0 ) to determine baseline levels of leukocyte subpopulations . milk samples were also collected 16 hours following treatment ( day 1 ). in a second study , an infection - free cow with scc counts in all udder quarters of & lt ; 100 , 000 / ml was chosen for immunological studies . one quarter ( rh ) was inoculated with the l . lactis dpc3147 preparation , a second quarter ( lh ) was infused with cell - free supernatant from an overnight culture of l . lactis dpc3147 , a third quarter ( lf ) was infused with the contents of one intra - mammary antibiotic syringe containing 250 mg of neomycin sulphate , 100 mg of procaine penicillin , 10 mg of oxytetracycline hydrochloride and 10 mg of prednisolone ( multimast l . c ., bimeda ltd ., dublin ) and one quarter ( rf ) was left untreated . milk samples were taken just prior to intra - mammary injections ( day 0 ) and also at 24 and 48 hours following treatment . all samples from both trials were stored at room temperature following milking and were analysed within three hours of collection . neutrophils and lymphocytes were identified using a combination of bovine - specific antibodies ( bn15 . 6 and cd3 respectively ) and precise gating techniques in flow cytometry . superoxide anion production assays were performed to assess the functional activity of neutrophils . in the second trial , milk samples were also taken every 24 h for up to 7 days to monitor the somatic cell count ( scc ). an experiment was performed to investigate the effect of infusing dead l . lactis dpc3147 cells on the immune response of cows . three low scc cows were selected ( cow 1852 , 1135 , and 1570 ) and the teats were randomised and infused with either live lactococci , dead lactococci or sterile saline ( 0 . 85 % nacl { w / v }). the live lactococci infusion mixtures were prepared as described above . dead cells were prepared by growing l . lactis dpc3147 in lm17 overnight as described above , followed by boiling at 100 ° c . for 10 min . following boiling , the bacteria were plated on lm17 agar and incubated overnight to confirm lack of viability . the dead culture was mixed with sterile water for injection in a ratio of 2 : 3 , and this mixture was used for infusion as described above . similarly , sterile saline was also mixed with sterile water for injection and infused as a control . a fourth quarter was left untreated in each cow as a negative control . milk samples were taken just prior to intra - mammary injections ( day 0 ) and on days 1 , 3 , 6 , 7 and 10 following treatment . all samples were then analysed for immunological activity as described above . lactobacillus plantarum dpc4922 was grown anaerobically at 37 ° c . in mrs medium . l . lactis dpc5329 , a derivative of l . lactis dpc3147 , which is incapable of producing bacteriocin ( bac -), was grown in an identical manner to l . lactis dpc3147 ( as described above ). three cows ( cow 1163 , cow 1171 and cow 1181 ) were selected to investigate the effects of infusing potential probiotic bacterial strains on the immune response of the mammary gland of cows with low somatic cell counts . three cows were used to monitor the immune responses amongst different animals . each quarter received a different treatment , with the treatments randomised amongst teats . milk samples were taken just prior to intramammary injections ( day 0 ), to determine baseline levels of leukocyte subpopulations . milk samples were also collected 1 , 2 , 3 , 7 and 10 days following treatment . all samples were stored at room temperature following milking and were analysed within three hours of collection . neutrophils and lymphocytes were identified using a combination of bovine - specific antibodies and flow cytometry . lm17 ( 200 ml ) was inoculated with 1 % ( v / v ) l . lactis dpc3147 and incubated overnight at 30 ° c . the cells were then harvested by centrifugation at 4 ° c . and 4500 rpm in a sorvall rc 3cplus centrifuge . the supernatant was removed and the cells were resuspended in ˜ 150 ml sterile distilled water . the cells were then harvested again by centrifugation and the pellet was resuspended in ˜ 100 ml sterile distilled water . the resuspended cells were then freeze - dried to a powder preparation overnight using a modulyo freeze dryer ( edwards ). a sample of the resulting powder was then resuspended in sterile distilled water as a 10 % solution and bacteria were enumerated by plating dilutions on lm17 agar and incubating overnight at 30 ° c . appropriate amounts of powder were then added to 5 ml of sterile water for injection such that the final concentration was equivalent to 10 9 cfu ml − 1 approximately . powder resuspended in this way was then used as an infusion mixture . an infusion mixture containing resuspended freeze - dried l . lactis dpc3147 cells was prepared as described above . this mixture , and a standard infusion mixture ( diluted broth culture ) were then infused randomly into teats of three different cows . three different cows were used to allow for variation between different cows in immunological response . one quarter in each cow was left untreated as a negative control . milk samples were taken just prior to intramammary injections ( day 0 ), to determine baseline levels of leukocyte subpopulations . milk samples were also collected at 24 h and 48 h following treatment . all samples were stored at room temperature following milking and were analysed within three hours of collection . neutrophils and lymphocytes were identified as outlined above . comparison of l . lactis treatment using broth cultures with antibiotic therapy a small - scale trial was performed to assess the efficacy of treatment with l . lactis dpc3147 in comparison to a commonly used intra - mammary antibiotic containing amoxycillin ( 200 mg ) and clavulanic acid ( 50 mg ), ( synulox , pfizer animal health ). 24 infected quarters in 12 cows were used , and quarters were infused with either l . lactis dpc3147 ( 285lh , 370rh , 400lh , 598lf , 1157lf , 1170lf , 1183lh , 1658rf , 1807lf , 1827rh , 1867lh , 1868lf ) or synulox ( 285rf , 370rf , 400rh , 598rf , 1157rf , 1157lh , 1170lh , 1183lf , 1807rh , 1807lh , 1867rh , 1868rf ). the antibiotic was administered three times , at 12 hour intervals , as per the manufacturer &# 39 ; s instructions . the l . lactis dpc3147 infusion mixture was administered twice , with a 24 h interval between infusions . scc and standard microbiological analysis were performed before and after infusion , and samples were also taken 7 days post - infusion and 12 days post - infusion . the treatment of clinical mastitis using a resuspended freeze - dried preparation of l . lactis dpc3147 in a comparison with a positive control ( antibiotic therapy ) this study was performed over a 6 - month period . 50 cases of clinical mastitis in 48 cows were detected by farm staff during routine milking and were selected for the trial . the quarters were classified as having either mild ( c1 / c2 mastitis , 25 quarters ) or severe ( c3 / c4 mastitis , 25 quarters ) clinical mastitis . quarters were treated with the antibiotic leo yellow milking cow ® ( penethamate hydriodide 150 mg , dihydrostreptomycin 150 mg , framycetin sulphate 5 mg , leo laboratories ltd ., dublin , ireland ) or with a resuspended freeze - dried culture of l . lactis dpc 3147 prepared as described above . infusions of the culture or the antibiotic were administered three times , with a 24 h interval between each infusion . milk samples were taken on day 1 prior to treatment and on day 7 and day 14 post - treatment . pathogens were enumerated and the scc or cmt was determined for all samples as described previously . quarters were also assessed at every milking during the 14 - day trial period to detect any adverse effects of treatment . analysis of the day 14 sample allowed quarters to be classified as a “ cure ” or “ no cure ”. cured quarters were defined as having a “ clinical cure ” if the milk had no visible clots or flakes , and the scc was & lt ; 9 × 10 6 cells ml − 1 . the presence or absence of pathogens was not taken into account when classifying as a “ clinical cure ”. quarters were defined as having a “ bacteriological cure ” if the scc of the day 14 sample was & lt ; 1 × 10 6 cells ml − 1 and the pathogen count was & lt ; 0 . 5 cfu μl − 1 in the milk sample . six udder quarters from 4 cows with a history of chronic infection were selected for treatment . eighteen hours after infusing the l . lactis dpc3147 culture , milk samples were taken from each udder . samples were taken at intervals up to approximately 30 days post - infusion and bacteria enumerated as described above . colonies were identified as l . lactis dpc3147 by the production of lacticin 3147 . staphylococci and streptococci were identified on the basis of their characteristic haemolysis on blood agar . in three of the quarters ( 714rh , 714lf and 96rf ), infusion of l . lactis dpc3147 was followed by a sharp rise in scc , and a concomitant reduction / elimination of the pathogen ( staphylococci ) ( fig1 a ). in three of the quarters ( 714lh , 700rh and 408rh ) the infection persisted despite infusion of the lactococcal culture ( fig1 b ). however , interestingly , in the latter three udder quarters , the lactococcal culture did not appear to colonise the udder quarter ( fig1 b ), whereas in the “ cured ” quarters , the presence of l . lactis dpc 3147 was evident ( fig1 a ). the lactococcal culture did not survive long - term in any of the udder quarters . somatic cell counts returned rapidly to , and remained at acceptable levels in all quarters . the above results prompted us to investigate the effect of infusing of l . lactis dpc3147 into clinically affected quarters . nine quarters from 9 cows with newly acquired clinical signs of mastitis were treated . after treatment , milk samples were collected daily for up to 14 days and intermittently for up to 55 days . bacterial cultures were enumerated on aba or gm17 as described above . in all cases the quality and appearance of the milk improved dramatically following the infusion of the lactococcal culture ( fig2 - 9 ). in some cases , despite the clinical nature of the milk , no pathogen was cultured prior to infusion . where a pathogen was identified , however , infusion of the l . lactis dpc3147 culture resulted in the elimination / reduction of the pathogen . pathogens eliminated included staphylococcus epidermidis ( cow 14 lh ), s . aureus ( cow 1184 rf ) non - haemolytic e . coli ( cow 1163 rh ), and strep . uberis ( cow 1154 lf and cow 264 lf ) ( table 1 ). in two cases , while treatment resulted in an improved appearance and quality in the milk , the pathogen was not eliminated . these cases included one strep . uberis infection ( cow 1176lh ) and one s . aureus infection ( cow 1850rf ) ( table 1 ). the data from this trial , including historical data on all the cows used can be viewed in tables 1 - 7 and fig1 a and 1 b . the effect of the probiotic l . lactis dpc3147 on the immune systems of cows was investigated by analysing leukocyte levels and phenotypes in milk . in an initial pilot trial , the effect of l . lactis on the immune response of two cows was investigated . both an infected animal and an infection - free cow were used . the results ( fig1 ) indicated that infusion of the l . lactis dpc3147 , but not the infusion of sterile broth , resulted in a massive recruitment of pmn to the udder , indicating the l . lactis may be a specific trigger of the mammary immune response and elicits pmn migration and accumulation . superoxide anion production assays were also performed and indicated that the newly recruited neutrophils had a higher respiratory burst capacity than resident neutrophils , thus providing the mammary gland with an effective mechanism for the elimination of mastitis pathogens . in light of these results , a second trial was performed . an uninfected animal ( cow 1803 ) was chosen to investigate the effects of the l . lactis culture . as controls , one quarter ( lf ) was infused with a lactating - cow antibiotic ( multimast l . c .) and one quarter ( rf ) was left untreated . a third quarter ( hl ) was infused with cell - free supernatant from an overnight culture of l . lactis dpc3147 , and the final quarter ( rh ) was infused with the diluted l . lactis dpc3147 culture . milk samples were collected pre - and post - infusion and analysed for scc and differential cell ( leukocyte ) count . fig1 presents neutrophil ( pmn ) and lymphocyte ( cd3 ) proportions in milk samples before treatment ( 0 hour ) and after treatment ( 24 and 48 hours ). actual values were calculated using the percentage of positive cells from live / dead flow cytometry analysis and results from the bentley somacount somatic cell counter . pmn levels in the control quarter ( rf ) remained unchanged during the trial period . the probiotic - injected quarter ( rh ) experienced a dramatic increase in neutrophils over the first 24 h period from 2 . 85 × 10 2 cells / ml before treatment to 1 . 46 × 10 4 cells / ml at 24 h after treatment ( fig1 and table 2 ). the supernatant and antibiotic treatments also induced an increase , albeit not as pronounced ( from 4 . 29 × 10 2 cells / ml to 1 . 68 × 10 3 cells / ml and 5 . 5 × 10 2 to 1 . 3 × 10 3 respectively ), in pmn levels in milk ( table 2 ). at 48 hours the pmn levels appeared to decrease in the l . lactis treated and in the multimast - treated quarter , but continued to rise in the supernatant - treated quarter ( table 2 ). considering these results , it can be concluded that the injection of the l . lactis culture resulted in a massive recruitment of pmn to the udder in the 24 hour period following treatment . the culture supernatant also induced a recruitment of pmn to the udder and this was a more sustained induction increasing over 24 hours and continuing to rise , up to 48 hours after treatment . the antibiotic multimast generated a weaker transient recruitment of pmn to the udder . these results suggest that both the l . lactis culture and the culture supernatant could be specific triggers of the mammary immune system response and elicit pmn migration and accumulation . it is possible that the factor responsible for the immune response could be released into the growth medium , which would explain the significant pmn migration in response to the culture supernatant . levels of lymphocytes were also investigated . the l . lactis culture , and the culture supernatant to a lesser extent , triggered an influx of lymphocytes to the udder ; the antibiotic , however , did not alter the level of lymphocytes present when compared to the control values ( table 2 ). the functional activity of pmns in the quarter milk samples was investigated for all samples before and after injection . the results of the superoxide anion production assays are presented in fig1 a and 12b . the fold increase refers to the proportional increase in superoxide anion production by pmn , from a resting state ( t 0 ) to an activated state following activation by phorbol myristate acetate ( pma ; incubation for 10 minutes , { t 10 }). the most obvious activation occurred in the lh quarter which was treated with cell - free supernatant , with a massive activation of neutrophils at 24 hours . surprisingly , treatment with the l . lactis culture did not result in a huge fold activation of neutrophils ( fig1 a ). this can be explained , however , by results of analysis of the fluorescence intensity of all samples ( fig1 b ). the relative fluorescence intensity is a measurement of the fluorescence emitted by the cells ; a stronger fluorescence indicates a higher capacity to generate superoxide anion . the resting resident neutrophils in the l . lactis - treated quarter ( t 0 ) already possessed a very high superoxide anion production capacity ( elevated fluorescence intensity ) at 24 hours , and , therefore , could not exhibit a marked increase in activation following pma treatment . in conclusion , l . lactis treatment resulted in a massive recruitment of pmn to the udder which were in a highly activated state . the multimast treatment did not alter superoxide anion production in the treated quarter and the control quarter did not change significantly over the trial period ( fig1 a and 12b ). the pmns recruited in response to culture supernatant treatment also possessed an elevated superoxide anion production capacity , which appeared to peak over the first 24 - hour period following treatment . the results indicated that intramammary treatment with l . lactis dpc3147 or with the cell - free supernatant generated from this culture , activated the mammary immune response by triggering the influx of neutrophils to the mammary gland ( fig1 ). these newly recruited neutrophils appeared to possess a higher respiratory burst capacity than resident neutrophils ( fig1 ). the somatic cell count was monitored every 24 hours up to 7 days following treatment and cell counts are presented in fig1 and table 3 . from fig1 and table 3 , it is clear that l . lactis dpc3147 elicits an enormous cellular response by 24 hours resulting in an elevated scc , which peaks after 48 hours resulting in a mild clinical infection and gradually drops back to normal over the course of 3 - 4 days . treatment with the culture supernatant appeared to elicit a similar response . our analysis of leukocyte populations and neutrophil activity levels confirm these findings up to 48 hours . in order to investigate if viable l . lactis dpc3147 were required to produce the immune response generated above , infusion mixtures containing either live or dead cells were prepared and infused randomly into the teats of three cows as outlined in table 4 . both live and dead cells generated a rise in scc ( data not shown ), and , as can be seen from fig1 , the dead cells elicited a weak influx of both pmn in each of the cows . an increase in lymphocyte numbers was also observed ( data not shown ). this recruitment of pmn and lymphocytes in the quarters treated with the dead culture , however , was insignificant compared to the influx in response to the live culture . thus , it would appear that viable l . lactis , but not a killed culture , can specifically elicit recruitment of pmn and lymphocytes to the mammary gland . on analysis of results , the question arose as to whether the phenomenon of pmn recruitment was limited to l . lactis dpc3147 itself , or if other bacterial strains could also exert this effect . it was decided , therefore , to examine the effect of infusing other food - grade , non - pathogenic bacteria into the udder of lactating cows . a bacterial strain , lb . plantarum dpc4922 was selected on the basis of its evolutionary divergence from l . lactis ( quite distantly related ) as well as the fact that as it was originally isolated from a food source , it can , like l . lactis dpc3147 , be regarded as a gras organism . a third strain , l . lactis 5329 ( a bac - derivative of l . lactis dpc3147 ) was also used because of its close similarity to l . lactis dpc3147 . the infusion mixtures were prepared as described in the materials and methods and the mixtures were then infused randomly into the teats of three cows as outlined in table 5 . fig1 and 16 present neutrophil ( pmn ) proportions in milk samples from the three cows over the 10 day trial period . actual values were calculated using the percentage of positive cells from live / dead flow cytometry analysis and the somacount readings . the response in the three cows was variable but a similar trend was observed in each case . pmn levels in all the untreated quarters remained relatively unchanged over the trial period ( fig1 ). treatment with lb . plantarum dpc4922 resulted in a slight increase in pmn in all quarters , approaching similar levels to that resulting from l . lactis dpc3147 treatment by day 3 in cow 1181 ( fig1 ). however , the l . lactis dpc3147 response in cow 1181 was somewhat reduced compared to the other two animals ( fig1 ). infusion of l . lactis dpc3147 in each animal resulted in a dramatic increase in neutrophils in the first 24 - hour period after treatment . the bac - culture ( l . lactis dpc5399 ) also induced an increase in all treated quarters , with particularly higher levels of pmn obtained in milk from cow 1163 . however , if the proportional increases in pmn relative to d 0 are compared ( fig1 ), it can be seen that there is a significant proportional increase in pmn in the l . lactis dpc3147 - treated quarter compared to the l . lactis dpc5399 - treated quarter . the pmn influx seems to occur earlier in the l . lactis dpc3147 - treated quarters ( by day 1 ) compared to the quarters treated with l . lactis dpc5399 ( bac -) or lb . plantarum dpc4922 ( fig1 ). in the quarters treated with the latter two treatments , a significant increase was only observed on day 2 ( fig1 ). thus , it appears that l . lactis dpc 3147 can elicit a stronger and more rapid immune response than either a bacteriocin negative derivative of the same strain or another lab strain , though the latter strains may also elicit a weaker response . in order to investigate if different preparations of l . lactis dpc3147 , other than the standard overnight culture ( broth preparation ) could produce the immune response generated above , infusion mixtures containing either freeze - dried cells , or broth cultures were prepared . the mixtures were then infused randomly into the teats of three cows as outlined in table 6 . the results ( fig1 ) show an increase in pmn by day 1 in both treated quarters compared to the untreated quarter in each animal tested . there seems to be a greater influx in two of the overnight culture - treated quarters compared to quarters treated with the freeze - dried culture ( cow 275 and cow 1134 ) with a higher number of pmn elicited by the freeze - dried culture in the remaining animal ( cow 2810 ). the variation in response is due to the typical variations in immune response between different animals . thus , both “ fresh ” and freeze - dried preparations of l . lactis dpc3147 are capable of eliciting an immune response in the mammary gland . the effects of using l . lactis dpc3147 treatment versus using a commonly used antibiotic treatment both for treatment and prevention of intramammary infections caused by s . aureus are shown in table 7 . as can be seen in the table , by day 7 , the l . lactis results were very promising , with staphylococci isolated from only two quarters of the 7 quarters originally infected with this organism . in comparison , the quarters treated with synulox were still shedding s . aureus from 6 of the 8 quarters originally infected . however , by day 12 , two more of the quarters infused with l . lactis were also shedding s . aureus , giving a total of 3 / 7 “ cured ” by l . lactis treatment as opposed to 2 / 8 “ cured ” by the synulox treatment . these data indicate that the l . lactis dpc3147 treatment is as effective at eliminating infections as the synulox treatment . in this study , the efficacy of infusing a resuspended freeze - dried preparation of l . lactis dpc3147 ( approximately 10 9 cfu ml − 1 ) for treatment of clinical mastitis was compared to the efficacy of using an established intramammary antibiotic . overall , 18 of the 25 cases treated with the antibiotic were defined as having a “ clinical cure ” on day 14 . of these 18 quarters , nine were defined as having a “ bacteriological cure ”— i . e ., the scc was & lt ; 1 × 10 6 cells ml − 1 and the bacteriological count was & lt ; 0 . 5 cfu μl − 1 in the milk sample . this corresponds to an overall clinical cure rate of 72 % and a bacteriological cure rate of 36 % for the antibiotic treated quarters . for the quarters treated with the resuspended freeze - dried preparation of l . lactis dpc3147 , 16 ( out of 25 ) cases had a “ clinical cure ” and 7 ( out of 25 ) cases had a “ bacteriological cure ”. this corresponds to a clinical cure rate of 64 % and a bacteriological cure rate of 28 % for the probiotic - treated quarters . comparing the two treatments , the clinical cure rate was 72 % versus 64 % and the bacteriological cure rate was 36 % versus 28 % for the antibiotic versus l . lactis treatments , respectively . when these values were compared using fischer &# 39 ; s exact probability test , no statistical difference was found between treatments . this indicates that infusion of a resuspended freeze - dried preparation of l . lactis dpc3147 was as effective as an antibiotic in the treatment of clinical mastitis . when the treatment groups were subdivided according to the severity of the mastitis in the quarter ( i . e . c1 / c2 subgroups or c3 / c4 subgroups ) a similar trend was observed . in the c1 / c2 group , 8 out of 12 cases treated with the antibiotic had a clinical cure ( 66 . 7 %). of these , 4 cases had a “ bacteriological cure ” ( 33 . 3 %). thirteen quarters with c1 / c2 mastitis were treated with l . lactis dpc3147 , and these quarters achieved a clinical cure rate of 76 . 9 % and a bacteriological cure rate of 30 . 8 %. in the c3 / c4 group , 10 out of 13 cases treated with the antibiotic had a clinical cure ( 76 . 9 %). of these , 5 cases had a “ bacteriological cure ” ( 38 . 5 %). of the twelve quarters with c3 / c4 mastitis that were treated with l . lactis dpc3147 , 6 achieved a clinical cure ( 50 %) and 3 were defined as having a bacteriological cure ( 25 %). comparison of all these values using fischer &# 39 ; s exact probability test indicated that there was no significant difference in either the clinical or bacteriological cure rate , regardless of treatment in either the quarters with c1 / c2 mastitis or the quarters with c3 / c4 mastitis . thus we can conclude that intramammary infusion of a resuspended freeze - dried preparation of l . lactis dpc 3147 is as effective as an intramammary antibiotic in the treatment of clinical mastitis . from these results several conclusions may be drawn . firstly , it is apparent that intramammary infusion of l . lactis dpc3147 into cows with chronic infections results in a rapid rise in scc , often followed by eradication of infection . additionally , infusion into cows with newly acquired clinical mastitis results in a rapid improvement in milk quality . l . lactis dpc3147 treatment has also been shown to be as effective as using a widely used commercial intra - mammary antibiotic in the treatment and prevention of intramammary infections caused by s . aureus . it is possible , therefore , that the infusion of l . lactis acts as a stimulus which induces release of proinflammatory factors and a prompt recruitment of neutrophils to the mammary gland . the results of immunological studies highlight a number of important findings that may shed some light on the mechanism of action of the probiotic bacteria l . lactis in the mammary gland . these findings include the following : intramammary treatment with either l . lactis culture or the culture supernatant activates the mammary immune response by triggering the influx of neutrophils to the mammary gland . the l . lactis culture and culture supernatant appear to be specific in eliciting recruitment of pmn to the udder , when compared to the antibiotic multimast l . c . these newly recruited neutrophils possess a higher respiratory burst capacity than resident neutrophils thus providing the mammary gland with an effective mechanism for the elimination of mastitis pathogens . the factor ( s ) responsible for eliciting an immune response in the udder may be a soluble factor ( s ) released into the growth medium , this factor ( s ) must be either be heat labile or destroyed / utilised rapidly when cells are killed , as dead cells plus supernatant did not elicit an immune response . the l . lactis culture must be viable to elicit an adequate immune response , although a freeze dried preparation is also effective at stimulating the immune system in the mammary gland . other lab may also be capable of eliciting an immune response in the mammary gland , in a similar fashion to l . lactis dpc 3147 , though possibly not to the same extent as l . lactis dpc 3147 . this implies that other lab may also be capable of curing clinical mastitis in dairy cows and other animals if infected quarters were infused with these cultures . treatment with l . lactis culture or a resuspended freeze - dried preparation of l . lactis dpc3147 is as effective at eliminating infections as intra - mammary antibiotic treatment . the words “ comprises / comprising ” and the words “ having / including ” when used herein with reference to the present invention are used to specify the presence of stated features , integers , steps or components but does not preclude the presence or addition of one or more other features , integers , steps , components or groups thereof . alvarez , s ., herrero , c ., bru , e . and g . perdigon ( 2001 ). effect of lactobacillus casei and yoghurt administration on prevention of pseudomonas aeruginosa infection in young mice . j . food prot . 64 : 1768 - 1774 . alvarez - olmos , m . i . and r . a . oberhelman . 2001 . probiotic agents and infectious diseases : a modern perspective on a traditional therapy . clin . infect . diseases 32 : 1567 - 1576 . blackburn , p . projan , s . j . and e . b . goldberg . 1994 . pharmaceutical bacteriocin compositions and methods for doing the same . united states patent 5 : 304 , 540 . cross , m . l . 2002 . microbes versus microbes : immune signals generated by probiotic lactobacilli and their role in protection against microbial pathogens . fems immun . med . microbiol . 1442 : 1 - 9 . galvin , m ., hill , c . and r . p . ross . 1999 . lacticin 3147 displays activity in buffer against gram - positive bacterial pathogens which appear sensitive in standard plate assays . lett . appl . microbiol . 28 : 355 - 358 . gardiner , g . e ., heinmann , c ., bruce , a . w ., beuerman , d . and g . reid . 2002 . peristence of lactobacillus fermentum rc - 14 and lactobacillus rhamnosus gr - 1 but not l . rhamnosus gg in the human vagina as demonstrated by randomly amplified polymorphic dna . clin . diagn . lab . immunol . 9 : 92 - 96 . morgan , s . m ., galvin , m ., kelly , j ., ross , r . p . and c . hill . 1999 . development of a lacticin 3147 enriched whey powder with inhibitory activity against foodborne pathogens . j . food protection . 62 : 1011 - 1016 . parente , e ., and c . hill . 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