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The effect of colonization of the mouse mammary gland by Staphylococcus epidermidis on subsequent infection with Staphylococcus aureus or Escherichia coli
1. ~:OMP.
P.lTII.
1978.
1.01..
88.
‘THE EFFECT OF COLONIZATION _\IX1fA,IARY GLAND BY STdPHl-LOCOCCL,-S OS SUBSEQUENT IKFECTION TVITH A 1’REl’S OR ESCHERICHI;l
OF
‘THE XIOUSE EPIDERXIDI~~ ,ST-iPH1‘LOC’OC(;l..~ COLI
J. C. .A NDERSOiY .-IRC’Indifute
for Researzhon .-lnimal Diseases.Con~pton. hembwy. Berkshire.1-.h‘. INTRODUCTIOS
1~1~~~ S’t~pf~~lo~~oc~~is uuwus \vas inoculated into the marnmar\: glands of‘ mice. the organisms multiplied rapidly in an acute reaction w.llich was characterized 1)~ epithelial necrosis. Hoavever in mice in which a neutrophil infiltration had lIeen induced in the mammary gland by intramammary inoculation of endotoxin 6 1~ before staphylococcal challenge, a degree of’ resistance to infrctiun was obscrvcd. The multiplication of staphylococci \vas inhibited iind the, This latter reaction was CWI~epithelium became hyperplastic. mammary sidercd to be chronic mastitis (Anderson, 1975, 1977). In this study cndotoxin is replaced by Stnphylococczts epidermidis as the induce1 of a modifying inflammatory reaction and the effect: is assessed by challen,q~c~ of mammary glands colonized 1~)’ S. ep~de~midis with ,C. muem or I.kfre~~~hin roli. MATERIALS
AbJD
METHODS
A\1ire. Lactating mice of the BSVS strain bred at Compton wcrc used 4 to 6 days after parturition. The offspring were removed at the first inoculation. Organisms. Strain M60 of Stap&ocorcus aureus coagulated rabbit plasma within -& 1) and produced alpha- and beta-haemolysis on 5 per cent ox blood agar. The strain was isolated from a case of chronic bovine mastitis. Strain 23 of Stap/$ococcus e~idermidis failed to coa,gulate rabbit plasma within 24 h and was non-haemoiytic. Strain 21 and E&zerichza coli strain P4 were obtained from Dr 0. Holmberg, Sational Vetcrinar!. Institute, Stockholm, Sweden and Dr A. J. Bramley. National Institute for Research in Dairying, Shinfield, Reading. respectively, who recovered the strains from bovine, mastitis and used them to reproduce the disease cxperimcntally in cattle (Holmberg. 1973 ; Bramley, 1976). For inoculation each strain was srown on ox blood agar for 18 h at 37 “C, and harvested and washed twice in sterile isotonic saline; finally, with the aid of a nephelometer, the concentration of organisms was adjusted to I to 2 10” colony forming units (cfu) per ml or 1 to 2 : lo4 cfu per ml. Endotoxir~. Rrcherichin coli 026 B6 endotoxin, prepared by the Boivin method (Difco I,abs, Detroit) was dissolved in isotonic saline at the rate of 250 pg per ml. Inoculations. Mice were anaesthetized with ether and 0.1 ml of organisms or rndotoxin was inoculated via the teat into the fourth mammary gland on rach side (R4 and L4) by the method described by Anderson (1976). Histological a& bacteriological procedures. At necropsy R4 was fixed in 12 per cent tleutral buffered formalin (NBF) and embedded in paraffin wax. Sections (5 pn~ were stained with Giemsa stain or by the Gram method. L4 was processed for viablt 0021-9975/78/04O-i4.5+09
$02.00/O
0
1978 Academic
Press Inc. (London1 Limitvtl
543
J. C. ANDERSON
bacterial-cell numbers and alpha-toxin was detected as previously described 1974). In cultures made from. serial dilutions of tissue homogenates (Anderson, prepared from glands inoculated with S. epidermidls and S. aweus, the latter was identified by its haemolytic zone, S. epidermidis being non. haemolytic; in cultures from glands inoculated with S. epidermidis and E. cali, the latter was identified by its large grey mucoid colonies that contrasted sharply with the small white dry colonies of S. epidermidis. Geometric m.eans arc used throughout. In the final experiment both the R4 and L4 glands were processed separately as follows. The glands were exposed by making a midline abdominal incision and reflecting the skin. Incisions were made with a sterile scalpel at 2 levels of each gland, and the incised tissue was sampled with a sterile wire loop. The samples were plated on blood agar and incubated for 24 h at 37 “C. and the organisms were identified as already described. The tissue between the incisions was fixed in NBF’ for histological examination if required. Design of 4 experiments. Thirty-five mice were inoculated (R4 and L4) with S. epidermidis (IO” organisms). Five mice were killed after 24 h and the remainder were killed in groups of IO at daily intervals thcrcafter. Of 15 additional mice, IO wcrc inoculated (R4 and L4) with S. epidermidis (IO3 organisms). Twenty-four hours later S. aureus (1 O5 organisms) was inoculated into R4 and L4 of all 15 mice. The 5 mice that received onlv S. aureus were killed after 24 h together with 5 of the mice that had received S. epiiermidis before S. aureus; the 5 remaining mice were killed after a further 24 h. The second experiment was repeated substituting E. coli strain P4 for S. aureus strain M60. In the fourth and final experiment 20 mice were inoculated (R4 and L4) with endotoxin (25 pg) and 6 h later with IO3 S. aureus (IO mice) or lOi E. coli (IO mice). A further 20 mice were inoculated (R4 and L4) with S. epidermidis ( IO3 organisms) and 24 h later with IO3 S. aureus (10 mice) or E. coli (IO mice). In this experiment the mice were killed 48 h after the injection of S. aurezt.r or E. coli. RESULTS
Response of nornzal mice to intramammary
inoculation
of S. epidermidis
strain 2.5
All the mice were clinically normal after inoculation of S. epidermidis except that one mouse in the group, killed after 3 days, had a poor coat and a sunken abdomen. The numbers of 5’. epidermidis recovered from mammary glands are shown in Fig. 1. An average of 8.19 x lo5 organisms was recovered 24 h after inoculation and the range of the numbers recovered was narrow. However, at 2, 3 and 4 days after inoculation the range was large; the greatest recovery, at 3 days, was from the only mouse that showed clinical signs of illness. The trend, as shown by the geometric mean, was towards elimination of the infection and at 4 days 2 of 10 glands were sterile. Histological examination of the mammary glands 24 h after inoculation of ,S. epidermidis showed that there was a neutrophil infiltration in the alveolar lumen. Almost all the staphylococci were within neutrophils though many ncutrophils did not contain staphylococci. There was cell debris and epithelial cells in the alveolar lumen and ducts. The epithelium was low and slightly vacuolated. In the glands of the 5 mice from which large numbers of S. epidermidis were recovered (> log,, 6-O) 2 days after inoculation, staphylococci were present in large numbers in the alveolar lumen, in neutrophils and in the epithelium.
MODIFICATION
OF MASTITIS
IN
.517
MICE
The alveoli were large and the epithelium was vacuolated (Fig. 2). Staphylococci were difficult to find in sections of mammary glands of mice from which low numbers of S. epi&nidis were recovered. There were neutrophils and degenerate neutrophils in the large alveoli.
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b’ig. 1. Response to intramammary inoculation of S. e&dermidis. Individual values for the numbers 01 organisms recovered from mammary glands are shown and the broken line joins the geometric mean of each group.
Fig. 2. Mammary gland from which a large number of S. epidermidir was recovered 48 h after intramammary inoculation. There are organisms in the alveolar lumen and in the epithelium which is only mildly damaged. Giemsa. x 480.
548
J. C. ANDERSON
There was liquefactive necrosis of the epithelium in the mammary gland from which the highest bacterial recovery was made 3 days after inoculation (clinically affected mouse). There were large numbers of staphylococci in the mammary glands of the other 4 mice from which large numbers were recovered; the staphylococci were predominantly in the alveolar lumen, but some were in neutrophils or in the epithelium. The alveoli were contracted and the epithelium had a hyperplastic appearance. Staphylococci were rarely found in sections of mammary gland from mice that yielded low numbers of S. epidermidis. The alveoli were contracted and hypcrplastic, and mononuclear cells and neutrophils were observed among the interalveolar fat. Only 2 mammary glands yielded high numbers of S. epidermidb 4 days after inoculation. The alveoli were contracted in these glands and staphylococci were detected in the lumen or epithelium of the contracted alveoli, and in neutrophils in the alveoli and ducts. There was a striking interalveolar infiltration of neutrophils and mononuclear cells. The mammary glands of 8 mice were sterile or yielded low numbers of S. epidermidis. The alveoli in these mammary glands were contracted and there was extensive interalveolar fat deposition. Apart from mast cells there waslittleinteralveolarcellinfiltration. Staphylococci were detected in neutrophils amongst the debris in the ducts and occasionally in the alveoli. Response of normal mice and of mice colonized b_yS. epidermidis inoculation of S. aureus strain A460 [Fig. 3(a)]
to intramammary
Four mice were dead and one was moribund 24 h after intramammary inoculation of S. aureus into normal mice. An average of 7.5 x lOlo organisms was recovered and alpha-toxin was detected in all 5 glands; there was 100 per cent haemolysis of 1 per cent rabbit red blood cells at 1 in 8 (3 glands) and 1 in 4 (2 glands) dilutions of mammary gland homogenate. Histological examination showed that there was liquefactive necrosis of the epithelium and staphylococci abounded in the alveolar lumen in the absence of neutrophils; occasionally staphylococci penetrated the epithelium but penetration was associated with degenerative changes to the epithelium. All the mice colonized by S. epidermidis and challenged 24 h later with S. aureus were clinically normal after challenge. The numbers of S. epidermidis recovered were similar to those found when it was the only infecting organism. At 24 h after inoculation the range was 7.50 x lo4 to 1.41 x lo8 and at 48 h, 2.13 x lo3 to 2.29 x 107. In contrast the numbers of S. aureus recovered were within a narrow range; 2.00 x lo6 to 3.50~ 10’ at 24 h and 1.64~ lo5 to 3.46 x lo6 at 48 h after inoculation and alpha-toxin was not detected. The geometric means are shown in Fig. 3 (a). Histological examination of the mammary glands from 2 mice from which high numbers of S. epidermidis were recovered 24 h after inoculation of S. aureus showed large numbers of staphylococci in the alveolar lumen and in the ducts. were within Neutrophils and cell debris were present; some staphylococci neutrophils and some were in the epithelium, but there was no liquefactive necrosis of the epithelium. In the glands from which low numbers of S. epidermidis
MODIFICATIOS
OF MASTITIS
IX
:X1
MICE
\verc recovered, the alveoli were contracted and the epithelium was h~,pcrplastic in appearance. Staphylococci were detected in neutrophils in the contracted alveoli and in the ducts. The epithelium of the mammary glands of all the mice killed 48 h alit-I. inoculation of 5’. azireu$ was hyperplastic in appearance, and there was an accumulation of interalveolar fat which vvas lightly infiltrated by neutrophils. mononuclear cells and mast cells. Staphylococci \\-ere detected within cells irr the contracted alveoli and in the ducts. f(b)
0
24
48 Time
oL---e-d (h)
i
IJig. 3. [ai S. aureus strain M60 or (b) E. coli strain I’4 was inoculated into the mammary mice at time 0 and the dotted line represents the geometric means (GM) of organisms from normal mice. The GM in mice whose mammary glands were colonized by S. 24 h before intrammary challenge with S. auretu or E. coli is indicated by the broken Ghl of S. ejv’dermidi.r recovered after challenge is shown by the solid line.
Response of normal mice and of mice colonized by S. epidermidis inoculation of E. coli strain P3 [Fig. 3(b)]
glands 01. recoverrd epidermidi\ lint. The
to intramammq
The 5 normal mice inoculated with E. coli by the intramammary rout< shovvcd only* a slightly roughened coat and sunken abdomen 24 h after inoculation. An average of69 x 1Ogorganisms was recovered from the mammar!. glands. Histological examination showed that E. coli was present in large numbers in the alveolar lumen and ducts. There lvere neutrophils in the alveoli. but most coliform organisms were free in the lumen. The cpithelium was cscessixely vacuolated, and in some areas there was liquefactive necrosis and fxnetration of organisms into the degenerate epithelium. -All the mice colonized by S. epidermidis and challenged 2-4 h later with E. coli were clinically normal after challenge. The numbers of S. epidermidis recovered again showed a large range: 7.50;; lo3 to 2.73 1 lo5 and 0 to 3.90 10” organisms 24 h and 48 h respectively after inoculation of E. coli. Recoveries of I?. coii were tvithin a narrow range; 2.14~ 105 to 3.14 ’ 105 and 1.81 lOA to 2.13 IO5 at 2-l h and 48 h respectively. The geometric means are shower iu Fig. 3(b).
J. C. ANDERSON
550
Histological examination of mammary glands 24 h after challenge with E. coli showed that there were neutrophils and neutrophil debris in the alveoli and ducts. The epithelium was vacuolated and occasional areas of liquefactive necrosis were noted. Coliform organisms were not detected but staphylococci were seen in neutrophils, in the alveoli and in the ducts. At 48 h after inoculation of E. coli the alveoli were contracted and the epithelium was hyperplaytic in appearance. There was deposition of interalveolar fat which was infiltrated 1~) neutrophils, mononuclear cells and mast cells. Coliform organisms were not seen, but staphylococci were detected in the contracted alveoli and in the ducts. Comparison qf the resistance of mice pre-treated with endotoxin and mice colonized 61 S. epidermidis to intramammary challenge with S. aureus or E. coli All the mice were clinically normal 48 h after injection of 5’. aureus or E. coli into endotoxin pre-treated mice, and all the bacteriological samples from the glands were sterile. TABLE 1 CHALLENGE
OF S.epidermidis-COLONIZED MAMMARY
InJammation inducer
Endotoxin S. epidermidis
GLANDS
WITH
s.
OR ENDOTOXIN-INOCULATED (LUI~~US OR E. CO&
Number of glands (out of 20) in which the stated organisms were present 48 h after challenge s. aureus E. coli
0 11 (55 per cent)
0 6 (30 per cent)
aVumber of glands (out of 40) from which S. epidermidis was recovered 72 h after colonization
26 (65 per cent)
Among the mice colonized by S. epidermidis, 2 had a poor coat 48 h after challenge with S. aureus and 2 were similarly affected in the group challenged with E. coli. The results of bacteriological sampling are shown in Table 1. In bacteriological terms and within the limits of the sampling technique 70 per cent of glands challenged with E. coli were sterile after 48 h and 45 per cent of glands challenged with S. aureus were sterile after 48 h. The histopathological changes in the glands from which S. aureus or E. coli was isolated were similar respectively to those seen 48 h after inoculation of lo5 organisms of S. aureus or E. coli into mammary glands colonized by S. epidermidis. DISCUSSION
S. epidermidis, along with other micrococci and Corynebacterium bouis, is generally considered to be a secondary pathogen in the bovine udder (Bramley, 1975). It can be isolated from the milk of cows that do not show clinical signs of mastitis and there may be no significant reduction in milk yield. However experimental infections (Holmberg, 1973) indicate that clinical mastitis can be caused by S. epidermidis though the histopathological changes are less severe
M0DIFICATION
OF MASTITIS
IN
MICE
53 1
than those caused by S. azlreus (Stabenfeldt and Spencer, 1966; Lee and Frost. 1970). In the present study in mice an inflammatory response was induced in the mammary glands inoculated with 5‘. epidermidis. S. epidermidis multiplied within the glands and at 24 h almost all staphylococci were within neutrophils. HoM-evcr, at 48 h after inoculation the neutrophils were unable to overcome the infection in a number of glands and unrestrained multiplication occurred in the alveolar lumen. Unlike multiplication of S. aureus, multiplication 01 ,Y. epidermidis was not accompanied by liquefactive necrosis of the epithelium or by general clinical signs of illness except in one mouse 3 days after inoculation. The results show that the average response was directed towards the elimination of 5’. epidermidis, but individual responses were erratic, thus demonstrating the. ability of S. epidermidis to cause a mastitis of moderate severity in an unpredictable number of mice. The results suggest that this comes about because of the inefficiency of the neutrophils rather than the possession by S. epidermidis of’ \irulcncc factors. When S. aureus or E. coli was inoculated into the mammary glands of normal mice an acute mastitis was induced. When S. aureus or E. coli was inoculated into mammary glands already colonized or infected by S. epidermidis in order to induce a mild inflammatory response, the rapid multiplication of S. artrezcs01 I:‘. coli was averted and a histopathological reaction vvas induced which was distinct from that of the acute reaction. The reaction was similar to the chronic mastitis produced when endotoxin was used to induce the inflammatory response (Anderson, 1977). It is therefore concluded that the effect of colonization of the mouse mammary gland by S. epidermidis on intramammary challenge I,).\‘. aureus or E. co/i is to prevent an acute reaction but to produce a chronic, mastitis. It was shown that mice that were inoculated with cndotoxin by the intramammary route were more resistant to intramammary challenge by S. aww.c or E. coli than mice whose mammary glands were colonized by S. epidermk’i.\. llammary glands colonized by S. epidermidis were more resistant to intramammary challenge by E. coli than by S. aureus. While it is appreciated that cndotoxin can induce biological changes other than neutrophil infiltration Seter. 1969), the superiority of endotoxin may lie in the fact that 1vherea.s some of the neutrophils evoked by S. epidermidis arc involved in phagocytosis of S. epidermidis, none is so involved following inoculation of endotoxin, vvhiclr is non-particulate and non-replicating. The greater resistance of A”. epidermidi.+ infected glands to E. coli than to S. aureus presumably reflects the ,greatcr \-irulence of S. aureus for the mammary gland. It has been suggested that benefit may be derived from colonization of tlrc udder by coagulase-negative staphylococci on the basis that a mild inflammatory reaction induced by organisms of low virulence might inhibit organisms of greater virulence (Spencer, Fluharty and Scwman, 1968). There is somt evidence from experimental work in cows to support this concept (Linde. Holmberg and Astrom, 1975; Bramley, 1978), but 3 points emerge from this study in mice which may be of relevance to the problem in lactating co\vs. First, S. epidermidis may itself be the cause of clinical or subclinical mastitis i Holmherg, 1973) with consequent loss of milk production (Satzke et al.,
552
J, C. ANDERSON
1972). Second, the results of the present experiments do not allow the conclusion that colonization of the mammary gland by S. epidermidis prevents infection by primary pathogens. In the experiment in which mammary glands which were colonized by ,S. epidermidis were challenged by lo3 organisms of S. aureus or E. coli, it cannot be concluded that S. epidermidis protected 70 per cent of glands against E. coli (or 45 per cent of glands against S. aureus). In fact, the infection by E. coli was eliminated from 70 per cent of glands within 48 h and this was accompanied by a mastitis reaction; all the glands were infected at the beginning of the experiment. Third, colonization of the mammary gland may, especially if the challenge by a primary pathogen is large, convert an otherwise acute response into a chronic reaction with persistent infection. In view of the refractiveness of chronic staphylococcal mastitis to antibiotic therapy, control of mastitis should consist in prevention by hygienic measures, including dry cow therapy, of invasion of the udder by primary and secondary pathogens, and prompt treatment of acute clinical cases. SUMMARY
A strain of Staphylococcus aureus and of Escherichia coli derived from bovine mastitis caused acute reactions when injected into the mammary glands of mice. The response of the mammary gland to the injection of 5’. epidermidis is described. When mouse mammary glands were colonized by S. epidermidis before challenge with S. aureus or E. coli a chronic mastitis was induced. In a comparison of S. epidermidis and endotoxin as inducers of an inflammatoq reaction that would eliminate pathogens, endotoxin \vas superior to S. epidermidis, and S. epidermidis was more effective against E. coli than against S. aureus. The results are discussed in relation to the control of bovine mastitis. ACKNOWLEDGMENTS
The author is indebted to Mrs G. Hill for technical and the Photography
assistance and to the Histology
Sections. REFERENCES
Anderson, .J. C. (1974). Experimental staphylococcal mastitis in the mouse: effects of extracellular products and whole bacterial cells from a high-virulence and a low-virulence strain of Stabhylncoccus aureus. Journal qf Medical Microbiology, * _ 7, 2055212. Anderson, J. C. (1975). Pathogenesis of experimental mastitis in the mouse caused by a strain of Staphylococcus aureus of low virulence and its modification by endotoxin. Journal of Comparative Patholo
MODIFICATION
OF MASTITIS
IN
MICE
Bramlcy,
n 5 13
A. J. (1976). Variations in the susceptibility of lactating and non-lactating bovine udders to infection when infused with Esrherichia coli. ~yohurnal qf‘Daiy Research, 43, 205-2 11. B!.amlcy, A. .J. (1978). The effect of subclinical Staphylococcus epidermidis infection of the lactating bovine udder on its susceptibility to infection with Streptorocczr\ agalactiae or Escherichia coli. British L’eterinar_y,Journal, 134, 146-l 5 1. Holmbrrg:, 0. (1973). Staphylococcus epidermidis isolated from bovine milk. .fc/n rle/erinaria .xandiuavica, Suppl. 45, 1~ 14.4. IR, <: . S. and Frost, A. .J. (1970). Mastitis in slaughtered dairy co~vs. 2. Pathologic~ observations. Australian Veterinary ~~oonmal,46, 2OG209. I,indc. (1.. Holmberg, O., and Astrom, G. (1975). An attempt to supcrinipo c ~Sta~~~~lococcus aureus, Streptococcus agalactiae and Streptocorws d,.~Cyalactine uprw .kap~~~lococcu.repidermidis infections in the cow’s udder. In I’rocepdiqc ojf‘,\‘eminco on .\lastiti.r Control, International Dairy Federation, 85, 39 l-39-1. Satzkc. R. P. P., Everett. R. LII., Guthrie, R. S., Keown.,J. F., Meek. r1. M., ^1Icrrill. \I’. G., Robert:;. S. J., and Schmidt, G. H. (1972). Mastitis control program: cffcct on milk production. Journal of Daky Science, 55, 1256-l 260. Setcs. E. (1969). Endotoxins and the immune response. Current TO/G-T in .\lic-robioiugl, and Immunolo~, 47, 82-l 23. Spencci-. G. li.: Fluharty, D. M., and Newman, B. (1968). Staphylococci of different virulcnccs infused successively into bovine udders. I’atholo,cia oeterinaria, 5, 7-2\5. Stabenfeldt, G. H., and Spencer, G. R. (1966). Th e 1esrons in bovine udders shcddin,g noit-hentolytic coagulasc-negative staphylococci. Patholo,$a zleterinaria, 3,27 ~-3’1.
[Receizledfor publication, Jvo?ember 21st, 19771
P.lTII.
1978.
1.01..
88.
‘THE EFFECT OF COLONIZATION _\IX1fA,IARY GLAND BY STdPHl-LOCOCCL,-S OS SUBSEQUENT IKFECTION TVITH A 1’REl’S OR ESCHERICHI;l
OF
‘THE XIOUSE EPIDERXIDI~~ ,ST-iPH1‘LOC’OC(;l..~ COLI
J. C. .A NDERSOiY .-IRC’Indifute
for Researzhon .-lnimal Diseases.Con~pton. hembwy. Berkshire.1-.h‘. INTRODUCTIOS
1~1~~~ S’t~pf~~lo~~oc~~is uuwus \vas inoculated into the marnmar\: glands of‘ mice. the organisms multiplied rapidly in an acute reaction w.llich was characterized 1)~ epithelial necrosis. Hoavever in mice in which a neutrophil infiltration had lIeen induced in the mammary gland by intramammary inoculation of endotoxin 6 1~ before staphylococcal challenge, a degree of’ resistance to infrctiun was obscrvcd. The multiplication of staphylococci \vas inhibited iind the, This latter reaction was CWI~epithelium became hyperplastic. mammary sidercd to be chronic mastitis (Anderson, 1975, 1977). In this study cndotoxin is replaced by Stnphylococczts epidermidis as the induce1 of a modifying inflammatory reaction and the effect: is assessed by challen,q~c~ of mammary glands colonized 1~)’ S. ep~de~midis with ,C. muem or I.kfre~~~hin roli. MATERIALS
AbJD
METHODS
A\1ire. Lactating mice of the BSVS strain bred at Compton wcrc used 4 to 6 days after parturition. The offspring were removed at the first inoculation. Organisms. Strain M60 of Stap&ocorcus aureus coagulated rabbit plasma within -& 1) and produced alpha- and beta-haemolysis on 5 per cent ox blood agar. The strain was isolated from a case of chronic bovine mastitis. Strain 23 of Stap/$ococcus e~idermidis failed to coa,gulate rabbit plasma within 24 h and was non-haemoiytic. Strain 21 and E&zerichza coli strain P4 were obtained from Dr 0. Holmberg, Sational Vetcrinar!. Institute, Stockholm, Sweden and Dr A. J. Bramley. National Institute for Research in Dairying, Shinfield, Reading. respectively, who recovered the strains from bovine, mastitis and used them to reproduce the disease cxperimcntally in cattle (Holmberg. 1973 ; Bramley, 1976). For inoculation each strain was srown on ox blood agar for 18 h at 37 “C, and harvested and washed twice in sterile isotonic saline; finally, with the aid of a nephelometer, the concentration of organisms was adjusted to I to 2 10” colony forming units (cfu) per ml or 1 to 2 : lo4 cfu per ml. Endotoxir~. Rrcherichin coli 026 B6 endotoxin, prepared by the Boivin method (Difco I,abs, Detroit) was dissolved in isotonic saline at the rate of 250 pg per ml. Inoculations. Mice were anaesthetized with ether and 0.1 ml of organisms or rndotoxin was inoculated via the teat into the fourth mammary gland on rach side (R4 and L4) by the method described by Anderson (1976). Histological a& bacteriological procedures. At necropsy R4 was fixed in 12 per cent tleutral buffered formalin (NBF) and embedded in paraffin wax. Sections (5 pn~ were stained with Giemsa stain or by the Gram method. L4 was processed for viablt 0021-9975/78/04O-i4.5+09
$02.00/O
0
1978 Academic
Press Inc. (London1 Limitvtl
543
J. C. ANDERSON
bacterial-cell numbers and alpha-toxin was detected as previously described 1974). In cultures made from. serial dilutions of tissue homogenates (Anderson, prepared from glands inoculated with S. epidermidls and S. aweus, the latter was identified by its haemolytic zone, S. epidermidis being non. haemolytic; in cultures from glands inoculated with S. epidermidis and E. cali, the latter was identified by its large grey mucoid colonies that contrasted sharply with the small white dry colonies of S. epidermidis. Geometric m.eans arc used throughout. In the final experiment both the R4 and L4 glands were processed separately as follows. The glands were exposed by making a midline abdominal incision and reflecting the skin. Incisions were made with a sterile scalpel at 2 levels of each gland, and the incised tissue was sampled with a sterile wire loop. The samples were plated on blood agar and incubated for 24 h at 37 “C. and the organisms were identified as already described. The tissue between the incisions was fixed in NBF’ for histological examination if required. Design of 4 experiments. Thirty-five mice were inoculated (R4 and L4) with S. epidermidis (IO” organisms). Five mice were killed after 24 h and the remainder were killed in groups of IO at daily intervals thcrcafter. Of 15 additional mice, IO wcrc inoculated (R4 and L4) with S. epidermidis (IO3 organisms). Twenty-four hours later S. aureus (1 O5 organisms) was inoculated into R4 and L4 of all 15 mice. The 5 mice that received onlv S. aureus were killed after 24 h together with 5 of the mice that had received S. epiiermidis before S. aureus; the 5 remaining mice were killed after a further 24 h. The second experiment was repeated substituting E. coli strain P4 for S. aureus strain M60. In the fourth and final experiment 20 mice were inoculated (R4 and L4) with endotoxin (25 pg) and 6 h later with IO3 S. aureus (IO mice) or lOi E. coli (IO mice). A further 20 mice were inoculated (R4 and L4) with S. epidermidis ( IO3 organisms) and 24 h later with IO3 S. aureus (10 mice) or E. coli (IO mice). In this experiment the mice were killed 48 h after the injection of S. aurezt.r or E. coli. RESULTS
Response of nornzal mice to intramammary
inoculation
of S. epidermidis
strain 2.5
All the mice were clinically normal after inoculation of S. epidermidis except that one mouse in the group, killed after 3 days, had a poor coat and a sunken abdomen. The numbers of 5’. epidermidis recovered from mammary glands are shown in Fig. 1. An average of 8.19 x lo5 organisms was recovered 24 h after inoculation and the range of the numbers recovered was narrow. However, at 2, 3 and 4 days after inoculation the range was large; the greatest recovery, at 3 days, was from the only mouse that showed clinical signs of illness. The trend, as shown by the geometric mean, was towards elimination of the infection and at 4 days 2 of 10 glands were sterile. Histological examination of the mammary glands 24 h after inoculation of ,S. epidermidis showed that there was a neutrophil infiltration in the alveolar lumen. Almost all the staphylococci were within neutrophils though many ncutrophils did not contain staphylococci. There was cell debris and epithelial cells in the alveolar lumen and ducts. The epithelium was low and slightly vacuolated. In the glands of the 5 mice from which large numbers of S. epidermidis were recovered (> log,, 6-O) 2 days after inoculation, staphylococci were present in large numbers in the alveolar lumen, in neutrophils and in the epithelium.
MODIFICATION
OF MASTITIS
IN
.517
MICE
The alveoli were large and the epithelium was vacuolated (Fig. 2). Staphylococci were difficult to find in sections of mammary glands of mice from which low numbers of S. epi&nidis were recovered. There were neutrophils and degenerate neutrophils in the large alveoli.
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b’ig. 1. Response to intramammary inoculation of S. e&dermidis. Individual values for the numbers 01 organisms recovered from mammary glands are shown and the broken line joins the geometric mean of each group.
Fig. 2. Mammary gland from which a large number of S. epidermidir was recovered 48 h after intramammary inoculation. There are organisms in the alveolar lumen and in the epithelium which is only mildly damaged. Giemsa. x 480.
548
J. C. ANDERSON
There was liquefactive necrosis of the epithelium in the mammary gland from which the highest bacterial recovery was made 3 days after inoculation (clinically affected mouse). There were large numbers of staphylococci in the mammary glands of the other 4 mice from which large numbers were recovered; the staphylococci were predominantly in the alveolar lumen, but some were in neutrophils or in the epithelium. The alveoli were contracted and the epithelium had a hyperplastic appearance. Staphylococci were rarely found in sections of mammary gland from mice that yielded low numbers of S. epidermidis. The alveoli were contracted and hypcrplastic, and mononuclear cells and neutrophils were observed among the interalveolar fat. Only 2 mammary glands yielded high numbers of S. epidermidb 4 days after inoculation. The alveoli were contracted in these glands and staphylococci were detected in the lumen or epithelium of the contracted alveoli, and in neutrophils in the alveoli and ducts. There was a striking interalveolar infiltration of neutrophils and mononuclear cells. The mammary glands of 8 mice were sterile or yielded low numbers of S. epidermidis. The alveoli in these mammary glands were contracted and there was extensive interalveolar fat deposition. Apart from mast cells there waslittleinteralveolarcellinfiltration. Staphylococci were detected in neutrophils amongst the debris in the ducts and occasionally in the alveoli. Response of normal mice and of mice colonized b_yS. epidermidis inoculation of S. aureus strain A460 [Fig. 3(a)]
to intramammary
Four mice were dead and one was moribund 24 h after intramammary inoculation of S. aureus into normal mice. An average of 7.5 x lOlo organisms was recovered and alpha-toxin was detected in all 5 glands; there was 100 per cent haemolysis of 1 per cent rabbit red blood cells at 1 in 8 (3 glands) and 1 in 4 (2 glands) dilutions of mammary gland homogenate. Histological examination showed that there was liquefactive necrosis of the epithelium and staphylococci abounded in the alveolar lumen in the absence of neutrophils; occasionally staphylococci penetrated the epithelium but penetration was associated with degenerative changes to the epithelium. All the mice colonized by S. epidermidis and challenged 24 h later with S. aureus were clinically normal after challenge. The numbers of S. epidermidis recovered were similar to those found when it was the only infecting organism. At 24 h after inoculation the range was 7.50 x lo4 to 1.41 x lo8 and at 48 h, 2.13 x lo3 to 2.29 x 107. In contrast the numbers of S. aureus recovered were within a narrow range; 2.00 x lo6 to 3.50~ 10’ at 24 h and 1.64~ lo5 to 3.46 x lo6 at 48 h after inoculation and alpha-toxin was not detected. The geometric means are shown in Fig. 3 (a). Histological examination of the mammary glands from 2 mice from which high numbers of S. epidermidis were recovered 24 h after inoculation of S. aureus showed large numbers of staphylococci in the alveolar lumen and in the ducts. were within Neutrophils and cell debris were present; some staphylococci neutrophils and some were in the epithelium, but there was no liquefactive necrosis of the epithelium. In the glands from which low numbers of S. epidermidis
MODIFICATIOS
OF MASTITIS
IX
:X1
MICE
\verc recovered, the alveoli were contracted and the epithelium was h~,pcrplastic in appearance. Staphylococci were detected in neutrophils in the contracted alveoli and in the ducts. The epithelium of the mammary glands of all the mice killed 48 h alit-I. inoculation of 5’. azireu$ was hyperplastic in appearance, and there was an accumulation of interalveolar fat which vvas lightly infiltrated by neutrophils. mononuclear cells and mast cells. Staphylococci \\-ere detected within cells irr the contracted alveoli and in the ducts. f(b)
0
24
48 Time
oL---e-d (h)
i
IJig. 3. [ai S. aureus strain M60 or (b) E. coli strain I’4 was inoculated into the mammary mice at time 0 and the dotted line represents the geometric means (GM) of organisms from normal mice. The GM in mice whose mammary glands were colonized by S. 24 h before intrammary challenge with S. auretu or E. coli is indicated by the broken Ghl of S. ejv’dermidi.r recovered after challenge is shown by the solid line.
Response of normal mice and of mice colonized by S. epidermidis inoculation of E. coli strain P3 [Fig. 3(b)]
glands 01. recoverrd epidermidi\ lint. The
to intramammq
The 5 normal mice inoculated with E. coli by the intramammary rout< shovvcd only* a slightly roughened coat and sunken abdomen 24 h after inoculation. An average of69 x 1Ogorganisms was recovered from the mammar!. glands. Histological examination showed that E. coli was present in large numbers in the alveolar lumen and ducts. There lvere neutrophils in the alveoli. but most coliform organisms were free in the lumen. The cpithelium was cscessixely vacuolated, and in some areas there was liquefactive necrosis and fxnetration of organisms into the degenerate epithelium. -All the mice colonized by S. epidermidis and challenged 2-4 h later with E. coli were clinically normal after challenge. The numbers of S. epidermidis recovered again showed a large range: 7.50;; lo3 to 2.73 1 lo5 and 0 to 3.90 10” organisms 24 h and 48 h respectively after inoculation of E. coli. Recoveries of I?. coii were tvithin a narrow range; 2.14~ 105 to 3.14 ’ 105 and 1.81 lOA to 2.13 IO5 at 2-l h and 48 h respectively. The geometric means are shower iu Fig. 3(b).
J. C. ANDERSON
550
Histological examination of mammary glands 24 h after challenge with E. coli showed that there were neutrophils and neutrophil debris in the alveoli and ducts. The epithelium was vacuolated and occasional areas of liquefactive necrosis were noted. Coliform organisms were not detected but staphylococci were seen in neutrophils, in the alveoli and in the ducts. At 48 h after inoculation of E. coli the alveoli were contracted and the epithelium was hyperplaytic in appearance. There was deposition of interalveolar fat which was infiltrated 1~) neutrophils, mononuclear cells and mast cells. Coliform organisms were not seen, but staphylococci were detected in the contracted alveoli and in the ducts. Comparison qf the resistance of mice pre-treated with endotoxin and mice colonized 61 S. epidermidis to intramammary challenge with S. aureus or E. coli All the mice were clinically normal 48 h after injection of 5’. aureus or E. coli into endotoxin pre-treated mice, and all the bacteriological samples from the glands were sterile. TABLE 1 CHALLENGE
OF S.epidermidis-COLONIZED MAMMARY
InJammation inducer
Endotoxin S. epidermidis
GLANDS
WITH
s.
OR ENDOTOXIN-INOCULATED (LUI~~US OR E. CO&
Number of glands (out of 20) in which the stated organisms were present 48 h after challenge s. aureus E. coli
0 11 (55 per cent)
0 6 (30 per cent)
aVumber of glands (out of 40) from which S. epidermidis was recovered 72 h after colonization
26 (65 per cent)
Among the mice colonized by S. epidermidis, 2 had a poor coat 48 h after challenge with S. aureus and 2 were similarly affected in the group challenged with E. coli. The results of bacteriological sampling are shown in Table 1. In bacteriological terms and within the limits of the sampling technique 70 per cent of glands challenged with E. coli were sterile after 48 h and 45 per cent of glands challenged with S. aureus were sterile after 48 h. The histopathological changes in the glands from which S. aureus or E. coli was isolated were similar respectively to those seen 48 h after inoculation of lo5 organisms of S. aureus or E. coli into mammary glands colonized by S. epidermidis. DISCUSSION
S. epidermidis, along with other micrococci and Corynebacterium bouis, is generally considered to be a secondary pathogen in the bovine udder (Bramley, 1975). It can be isolated from the milk of cows that do not show clinical signs of mastitis and there may be no significant reduction in milk yield. However experimental infections (Holmberg, 1973) indicate that clinical mastitis can be caused by S. epidermidis though the histopathological changes are less severe
M0DIFICATION
OF MASTITIS
IN
MICE
53 1
than those caused by S. azlreus (Stabenfeldt and Spencer, 1966; Lee and Frost. 1970). In the present study in mice an inflammatory response was induced in the mammary glands inoculated with 5‘. epidermidis. S. epidermidis multiplied within the glands and at 24 h almost all staphylococci were within neutrophils. HoM-evcr, at 48 h after inoculation the neutrophils were unable to overcome the infection in a number of glands and unrestrained multiplication occurred in the alveolar lumen. Unlike multiplication of S. aureus, multiplication 01 ,Y. epidermidis was not accompanied by liquefactive necrosis of the epithelium or by general clinical signs of illness except in one mouse 3 days after inoculation. The results show that the average response was directed towards the elimination of 5’. epidermidis, but individual responses were erratic, thus demonstrating the. ability of S. epidermidis to cause a mastitis of moderate severity in an unpredictable number of mice. The results suggest that this comes about because of the inefficiency of the neutrophils rather than the possession by S. epidermidis of’ \irulcncc factors. When S. aureus or E. coli was inoculated into the mammary glands of normal mice an acute mastitis was induced. When S. aureus or E. coli was inoculated into mammary glands already colonized or infected by S. epidermidis in order to induce a mild inflammatory response, the rapid multiplication of S. artrezcs01 I:‘. coli was averted and a histopathological reaction vvas induced which was distinct from that of the acute reaction. The reaction was similar to the chronic mastitis produced when endotoxin was used to induce the inflammatory response (Anderson, 1977). It is therefore concluded that the effect of colonization of the mouse mammary gland by S. epidermidis on intramammary challenge I,).\‘. aureus or E. co/i is to prevent an acute reaction but to produce a chronic, mastitis. It was shown that mice that were inoculated with cndotoxin by the intramammary route were more resistant to intramammary challenge by S. aww.c or E. coli than mice whose mammary glands were colonized by S. epidermk’i.\. llammary glands colonized by S. epidermidis were more resistant to intramammary challenge by E. coli than by S. aureus. While it is appreciated that cndotoxin can induce biological changes other than neutrophil infiltration Seter. 1969), the superiority of endotoxin may lie in the fact that 1vherea.s some of the neutrophils evoked by S. epidermidis arc involved in phagocytosis of S. epidermidis, none is so involved following inoculation of endotoxin, vvhiclr is non-particulate and non-replicating. The greater resistance of A”. epidermidi.+ infected glands to E. coli than to S. aureus presumably reflects the ,greatcr \-irulence of S. aureus for the mammary gland. It has been suggested that benefit may be derived from colonization of tlrc udder by coagulase-negative staphylococci on the basis that a mild inflammatory reaction induced by organisms of low virulence might inhibit organisms of greater virulence (Spencer, Fluharty and Scwman, 1968). There is somt evidence from experimental work in cows to support this concept (Linde. Holmberg and Astrom, 1975; Bramley, 1978), but 3 points emerge from this study in mice which may be of relevance to the problem in lactating co\vs. First, S. epidermidis may itself be the cause of clinical or subclinical mastitis i Holmherg, 1973) with consequent loss of milk production (Satzke et al.,
552
J, C. ANDERSON
1972). Second, the results of the present experiments do not allow the conclusion that colonization of the mammary gland by S. epidermidis prevents infection by primary pathogens. In the experiment in which mammary glands which were colonized by ,S. epidermidis were challenged by lo3 organisms of S. aureus or E. coli, it cannot be concluded that S. epidermidis protected 70 per cent of glands against E. coli (or 45 per cent of glands against S. aureus). In fact, the infection by E. coli was eliminated from 70 per cent of glands within 48 h and this was accompanied by a mastitis reaction; all the glands were infected at the beginning of the experiment. Third, colonization of the mammary gland may, especially if the challenge by a primary pathogen is large, convert an otherwise acute response into a chronic reaction with persistent infection. In view of the refractiveness of chronic staphylococcal mastitis to antibiotic therapy, control of mastitis should consist in prevention by hygienic measures, including dry cow therapy, of invasion of the udder by primary and secondary pathogens, and prompt treatment of acute clinical cases. SUMMARY
A strain of Staphylococcus aureus and of Escherichia coli derived from bovine mastitis caused acute reactions when injected into the mammary glands of mice. The response of the mammary gland to the injection of 5’. epidermidis is described. When mouse mammary glands were colonized by S. epidermidis before challenge with S. aureus or E. coli a chronic mastitis was induced. In a comparison of S. epidermidis and endotoxin as inducers of an inflammatoq reaction that would eliminate pathogens, endotoxin \vas superior to S. epidermidis, and S. epidermidis was more effective against E. coli than against S. aureus. The results are discussed in relation to the control of bovine mastitis. ACKNOWLEDGMENTS
The author is indebted to Mrs G. Hill for technical and the Photography
assistance and to the Histology
Sections. REFERENCES
Anderson, .J. C. (1974). Experimental staphylococcal mastitis in the mouse: effects of extracellular products and whole bacterial cells from a high-virulence and a low-virulence strain of Stabhylncoccus aureus. Journal qf Medical Microbiology, * _ 7, 2055212. Anderson, J. C. (1975). Pathogenesis of experimental mastitis in the mouse caused by a strain of Staphylococcus aureus of low virulence and its modification by endotoxin. Journal of Comparative Patholo
MODIFICATION
OF MASTITIS
IN
MICE
Bramlcy,
n 5 13
A. J. (1976). Variations in the susceptibility of lactating and non-lactating bovine udders to infection when infused with Esrherichia coli. ~yohurnal qf‘Daiy Research, 43, 205-2 11. B!.amlcy, A. .J. (1978). The effect of subclinical Staphylococcus epidermidis infection of the lactating bovine udder on its susceptibility to infection with Streptorocczr\ agalactiae or Escherichia coli. British L’eterinar_y,Journal, 134, 146-l 5 1. Holmbrrg:, 0. (1973). Staphylococcus epidermidis isolated from bovine milk. .fc/n rle/erinaria .xandiuavica, Suppl. 45, 1~ 14.4. IR, <: . S. and Frost, A. .J. (1970). Mastitis in slaughtered dairy co~vs. 2. Pathologic~ observations. Australian Veterinary ~~oonmal,46, 2OG209. I,indc. (1.. Holmberg, O., and Astrom, G. (1975). An attempt to supcrinipo c ~Sta~~~~lococcus aureus, Streptococcus agalactiae and Streptocorws d,.~Cyalactine uprw .kap~~~lococcu.repidermidis infections in the cow’s udder. In I’rocepdiqc ojf‘,\‘eminco on .\lastiti.r Control, International Dairy Federation, 85, 39 l-39-1. Satzkc. R. P. P., Everett. R. LII., Guthrie, R. S., Keown.,J. F., Meek. r1. M., ^1Icrrill. \I’. G., Robert:;. S. J., and Schmidt, G. H. (1972). Mastitis control program: cffcct on milk production. Journal of Daky Science, 55, 1256-l 260. Setcs. E. (1969). Endotoxins and the immune response. Current TO/G-T in .\lic-robioiugl, and Immunolo~, 47, 82-l 23. Spencci-. G. li.: Fluharty, D. M., and Newman, B. (1968). Staphylococci of different virulcnccs infused successively into bovine udders. I’atholo,cia oeterinaria, 5, 7-2\5. Stabenfeldt, G. H., and Spencer, G. R. (1966). Th e 1esrons in bovine udders shcddin,g noit-hentolytic coagulasc-negative staphylococci. Patholo,$a zleterinaria, 3,27 ~-3’1.
[Receizledfor publication, Jvo?ember 21st, 19771