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Experimental staphylococcal mastitis in the mouse: The induction of chronic mastitis and its response to antibiotic therapy
J,
COMP.PATH.
1977.Vo~.
87.
611
EXPERIMENTAL STAPHYLOCOCCAL MASTITIS MOUSE: THE INDUCTION OF CHRONIC MASTITIS ITS RESPONSE TO ANTIBIOTIC THERAPY
IN
THE AND
BY
J. C. A
NDERSON
INTRODUCTION
In a previous study (Anderson, 1975) the pathogenesis of experimental staphylococcal mastitis in the mouse was modified by inoculation of endotoxin. When endotoxin was inoculated by the intraperitoneal route 6 h before or after intramammary challenge with Sta@ylococcus aureus and when endotoxin was inoculated by the intramammary route 6 h after intramammary challenge, the animals exhibited an increased susceptibility and large numbers of staphylococci were recovered from mammary glands after 48 h. This susceptibility was associated in each case with a defect in neutrophil function. However, when endotoxin was inoculated by the intramammary route 6 h before intramammary challenge there was a reduction in the number of staphylococci recovered from the mammary glands 48 h later. This resistance was attributed to the presence in the alveolar lumen of neutrophils that were able to phagocytose the staphylococci at the time of challenge. In this study, examination of the histopathological and bacteriological changes associated with the resistance induced by a pre-existant neutrophil response suggests that this is a chronic reaction. The susceptibility of this type of infection to antibiotic therapy is investigated. MATERIALS
AND
METHODS
&lice. Lactating mice of the BSVS strain bred at Compton were used 4 to 6 days after parturition. The offspring were removed at the first inoculation. Strains of Staphvlococcus. Strains M60 and M63 of Staphylococcus aureus (S. aureus) were used. Both strains coagulated rabbit plasma within 4 h and failed to coagulate cow and mouse plasma within 24 h. Strain M60 produced alpha- and betahaemolysins on 5 per cent ox blood agar and strain M63 was non-haemolytic. Both strains were isolated from clinical cases of chronic bovine mastitis. For inoculation each strain was grown on ox blood agar for 18 h at 37 ‘C, harvested in sterile isotonic saline and washed twice in saline and finally, with the aid of a nephelometer, the concentration of organisms was adjusted to 2 to 3 x 108 cfu/ml. Endotoxin. Escherichia coli 026:B6 endotoxin, prepared by the Boivin method (Difco Labs, Detroit), was dissolved in isotonic saline to contain 250 Kg/ml. Gelatine. The gelatine solution contained 8 per cent (w/v) gelatine (BDH Chemicals Ltd, Poole) and 0,2 per cent (w/v) brilliant green (G. T. Gurr Ltd, London) in isotonic saline and was held at 37 “C before inoculation.
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Antibiotic. Sodium cloxacillin (Orbenin veterinary injectable, Beecham Animal Health, Brentford) 250 mg was dissolved in saline to a concentration of 10 mg/ml. The antibiotic sensitivity of strains M60 and M63 in vitro was demonstrated by adding 0.1 ml of the strain (10’ organisms) to each of 2 tubes containing 4.8 ml Hartley’s broth. 0.1 ml (1 mg) of cloxacillin solution was added to one tube and O-1 ml of saline was added to the other tube. After 18 h at 37 “C the tota number of viable bacterial cells was determined (Miles, Misra and Irwin, 1938). Inoculations. Mice were anaesthetized with ether and according to the experimental design 0.1 ml of endotoxin (25 pg) or staphylococci (1 O7 organisms) or cloxacillin (1 mg) was inoculated via the teat into the fourth mammary gland on each side (R4 and L4) by the method described by Anderson (1976a) ~ In the case of gelatine 0.2 ml was inoculated into each gland. Histological and bacteriological procedures. At necropsy R4 was fixed in 12 per cent neutral buffered formalin (NBF) and embedded in paraffin wax. Sections (5 p) were stained with Giemsa’s stain and by Gram’s method. Mammary glands inoculated with gelatine were fixed in cold (4 “C) NBF in which the gelatine is irreversibly solidified, processed thereafter in the normal manner and stained additionally with Crossman’s stain for connective tissue. L4 was processed for total viable bacterial cell numbers and alpha-toxin detection as previously described (Anderson, 1974). Geometric means of bacterial cell numbers are used throughout. Clinical assessment. A mouse was described as “malaised” if its coat stood on end and if it was not as alert as a normal mouse. The term “ill” signified a mouse with a very poor coat and a disinclination to move. Experimental design. Forty-eight mice were removed from their offspring and 32 were inoculated (R4 and L4) with endotoxin. Six hours later all the mice received 107 staphylococci by the intramammary route. Of the 16 mice that had not received endotoxin, 8 were killed 18 h and eight 28 h after inoculation of staphylococci. Of the 32 mice that had received endotoxin before staphylococci, 8 were killed 18 h and 8 48 h after bacterial challenge; at 48 h gelatine (0.2 ml) was inoculated into R4 of 4 of the 8 mice immediately before killing to assess the patency of the duct system. Cloxacillin (1 mg) was inoculated into R4 and L4 of 8 of the remaining 16 mice and all 16 were killed 18 h later. The whole experiment (see Fig. 1) was carried out for each strain of S. aureus. An additional control group consisted of 4 mice that received endotoxin in R4 and 54 h later gelatine in both R4 and L4 immediately before killing, to assess the effect of endotoxin and of natural involution on the distribution of gelatine within the
mammary gland. RESULTS
Kesfonse of Normal Mice to Intramammav of S. aureus (Fig I)
InouJation of Strain lvf60 or Strain A463
Two mice were dead, 3 were moribund and 3 were ill 18 h after intramammary inoculation of strain M60. An average of 3.8 x lOlo organisms was recovered from each mammary gland and alpha-toxin was detected in all 8 mammary gland homogenates (100 per cent haemolysis of 1 per cent red blood cells (rbc) at l/4 (5 glands) and l/S (3 glands) dilution of mammary gland homogenate). At 28 h after inoculation 5 mice were dead, one was moribund and 2 were ill. An average of l-9 x 1Oro organisms was recovered from each gland and alpha-toxin was detected in 5 of 8 glands (100 per cent haemolysis of 1 per cent rabbit rbc at l/4 (one gland) and l/2 (4 glands) dilution of mammary gland homogenates). Histological examination of mammary glands 18 h after inoculation of
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strain M60 showed the organisms present in large numbers in the alveolar lumen. There were few neutrophils and the epithelium, though low, vacuolated and in some areas reduced to the basement membrane, retained its basophilia (Fig. ‘2). At 28 h many staphylococci were present, often as rafts in the alveolar lumen. There were no neutrophils in the alveolar lumen and the epithelium was low and vacuolated or lost. In areas of complete liquefactive necrosis the epithelial cytoplasm was eosinophilic.
Fig. 1. Strain M60 (a) or M63 (b) of Staphylococcus aureu~ was inoculated into mammary glands of mice at time 0. The broken line indicates the response in normal mice. The response in mice given endotoxin by the intramammary route 6 h before staphylococci is indicated by the solid line and the effect of sodium cloxacillin on this chronic reaction at 48 h is indicated by the dotted line.
Three mice were dead, one was moribund and 4 were ill 18 h after inoculation of strain M63. The average number of staphylococci recovered from each gland was 1.8 x 10s and alpha-toxin was detected in 2 of 8 mammary gland homogenates (100 per cent haemolysis of 1 per cent rbc at l/2 dilution of homogenate). At 28 h after inoculation 2 mice were dead, 5 were ill and one was normal. An average of 1*5 x log organisms was recovered from each gland and alpha-toxin was detected in none of the mammary gland homogenates. The histopathological changes seen 18 and 28 h after inoculation of strain 1463 were indistinguishable from those observed at these times following inoculation of strain M60.
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Fig. 2. The acute reaction to strain M60 of S. HUMUS18 h after intramammary inoculation of normal mice. There are degenerative changes in the epithelium and staphylococci, but no neutrophils abound in the alveoli. Giemsa. s 315.
Response of Endotoxin-treated Mice to Intramammary Inoculation of the Strain M60 of S. aureus (Fig. 1) It has been established that the intramammary inoculation of 25 pg endotoxin induces a neutrophil response such that by 6 h after inoculation neutrophils are present in the alveolar lumen (Anderson, 1975). All the mice were clinically normai i8 h after inoculation and an average of 7.5 x lo6 organisms was recovered from infected glands. Alpha-toxin was not detected in any of the mammary gland homogenates. At 48 h after bacterial challenge all the mice were clinically normal, an average of 3.2 x lo6 organisms was recovered from each gland and alpha-toxin was not detected in any of the mammary gland homogenates. Histological examination 18 h after inoculation showed that there were many neutrophils and detached epithelial cells in the alveolar lumen. Most of the neutrophils were morphologically normal and many contained staphylococci; only rarely were staphylococci seen free in the alveolar lumen. The epithelium was basophilic and mildly hyperplastic in appearance due to the combination of reduction in alveolar diameter and early degenerative changes in the epithelial cells (Fig. 3). At 48 h after bacterial challenge the ducts contained cells and cell debris and staphylococci were identified within neutrophils. The epithelial hyperplasia was advanced to such an extent that, together with the interalveolar cell infiltration, adjacent alveoli formed
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confluent masses of cells (Fig. 4). Staphylococci were identified in neutrophils in the centre of contracted alveoli and some staphylococci appeared to be in macrophages or epithelial cells. Neutral fat surrounded groups of contracted alveoli, fibrosis was limited to major septae and there were no abscesses.
Fig. 3. The chronic reaction to strain M60 of S. aweus 18 h after intramammary inoculation of mice in which a neutrophil response had been induced prior to stapbylococcal challenge. There is early hyperplasia of the epithelium and there are neutrophils and shed epithelial cells in the alveoli. Giemsa. x 315.
There was resistance to inoculation of gelatine 48 h after bacterial challenge and there was leakage of gelatine when the needle was withdrawn from the teat. When counterstained with Crossman’s stain for connective tissue the gelatine appeared as an amorphous green material. Histological examination showed that the distribution of gelatine was severely restricted; gelatine was absent from the centre of alveoli that were contracted to a knot of cells and in these circumstances the gelatine was diverted through interalveolar and subcapsular spaces (Fig. 5). In the control group O-2 ml of gelatine was easily inoculated into normal mammary glands 54 h after removal of offspring. The gelatine was distributed to all alveolar lumens and was not present in interalveolar spaces (Fig. 6). It is known that the intramammary inoculation of endotoxin results in a neutrophil infiltration which is followed by involution of the gland (Anderson, 1976b). Thus, at 54 h after intramammary inoculation of endotoxin, the distribution of gelatine was restricted when compared with normal mammary glands. However, the restriction was not as great as that seen following bacterial challenge of endotoxin-treated mammary glands.
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Fig. 4. The chronic reaction to strain M60 of S. ~ZLWUS 48 h after intramammary inoculation of mice in which a neutrophil response had been induced prior to staphylococcal challenge. The alveoli are contracted and the hyperplastic epithelium impinges upon the neutrophils and cell debris in the alveolar lumen. Giemsa. s 315.
Resfionse of Endotoxin-treated Mice to Intramammary Inoculation of Strain -\63 S. aureus (Fig. 1)
oj’
Two mice showed signs of malaise and 6 were normal 18 h after inoculation. An average of 1.9 x 10’ organisms was recovered from each gland and alphatoxin was not detected in any mammary gland homogenates. At 48 h all the mice were clinically normal, an average of 6.7 x lo6 organisms was recovered from each mammary gland and alpha-toxin was not detected in the mammary gland homogenates. The histopathological changes following inoculation were indistinguishable from those observed following inoculation of strain M60 and there was also the same restriction in the distribution of gelatine. Resportseof Endotoxirz-treated Mice to Intramammary Inoculation of Sodium Cloxacillirt 48 h ilfter Intramammary Inoculation of S. aureus Strain A!60 or A163 (Fig. I) Eighteen hours after intramammary inoculation of 1 mg sodium cloxacillin into R4 and L4 all the mice were clinically normal. Staphylococci were recovered from all mammary glands and the average number of organisms was 1.3 x 105; alpha-toxin was not detected.
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Fig. 5. Distribution of gelatine following intramammary inoculation 48 h after establishment of chronic mastitis. The g&tine is present in a duct (D) but instead of reaching the alveolar lumen it has been diverted to septae (S). Crossman. x 126.
Fig. 6. Distribution of gelatine in uninfectkd mammary gland following intramammary The gelatine is seen in terminal alveolar lumens. Grossman. x 126.
inoculation.
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Histological examination showed neutrophils and cell debris in the ducts. The alveoli were contracted into knots of cells which, with interalveolar infiltration of mononuclear cells, created confluent cell masses which were surrounded by neutral fat. Staphylococci were identified in neutrophils in the ducts and in the centre of contracted alveoli. In this latter site staphylococci were also associated with macrophages or epithelial cells. All the 8 mice in the control group not treated with sodium cloxacillin were clinically normal 66 h after intramammary inoculation of strain M60; the average number of organisms recovered was 2.0 x IO5 and alpha-toxin was not detected. The histopathological changes were indistinguishable from those in the group treated with antibiotic. There was a similar lack of response to sodium cloxacillin therapy in the group of mice infected with strain M63. In the treated group an average of 9.2 x lo4 organisms was recovered from each gland 18 h after therapy and in the control group the average number of organisms recovered was 1.1 x 10”; alpha-toxin was not detected in mammary gland homogenates of mice from either group. The histopathological changes were also similar to those in mammary glands infected with strain M60. Response of S. aureus Strain M60 and M63 to Sodium Cloxacillin In Vitro The results showed that 1 mg sodium cloxacillin killed 100 per cent of 2 to 3 x 10’ organisms of S. aureus strain M60 or M63 within 18 h. Eighteen hours after the addition of O-1 ml saline instead of 0.1 ml antibiotic to the culture medium, there were 4.37 x lOlO and 1.95 x lOlo organisms of strains M60 and M63 respectively. DISCUSSION
In this study 2 strains of S. aureus recovered from chronic bovine mastitis multiplied rapidly when inoculated into the mammary glands of mice with consequent histopathological and clinical changes typical of an acute reaction. It is of interest that both strains of S. aureus induced an acute mastitis in mice despite the fact that only one of the strains produced haemolysins on blood agar. It is known that staphylococci may produce toxins in vivo that are not detected in vitro (Gladstone and Glencross, 1960) and in this study traces of alpha-toxin were detected in 2 of 8 mammary gland homogenates after inoculation of the non-haemolytic strain M63. However, the culminating histopathological change in glands infected with either strain was liquefactive necrosis in contrast to coagulative necrosis which is typical of a strongly alpha-toxigenic strain of S. aureus (Anderson and Chandler, 1975). Thus, although the production of alpha-toxin by strain M60 may account for the slightly greater multiplication in the mouse mammary gland, there seem to be factors other than alpha-toxin that cause necrosis (Anderson, 1976c). These findings emphasize the importance .of non-haemolytic S. aureus (as well as haemolytic S. aureus) in the aetiology of mastitis. An alternative to the acute reaction was induced in the mammary glands of mice by intramammary inoculation of endotoxin 6 h before staphylococci.
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The endotoxin induced a migration of neutrophils into the alveolar lumen so that they were present to phagocytose staphylococci at the time of challenge. However, the advantage conferred on the mice was not sufficient to sterilize the mammary gland and histopathological and bacteriological changes occurred that were different from those of the acute reaction. Instead of extensive extracellular bacterial multiplication the organisms were intracellular and multiplication was inhibited, and instead of necrosis of mammary epithelium there was hyperplasia and involution. On the basis of the time course and the distinct histopathological reaction this alternative response was considered to be chronic mastitis. Thus, in the mouse, acute staphylococci mastitis was converted to chronic mastitis by inducing an alveolar neutrophil population in advance of staphylococcal challenge. The significance of the leucocyte barrier of the udder to infection by coliform bacteria and by Streptococcusagalactiae has been demonstrated (Schalm, 1970) but the significance of the barrier with regard to staphylococci is less certain. The leucocyte barrier appears to be efficient in that it prevents acute mastitis (Blobel and Katsube, 1964) but there is evidence that the barrier may lead to persistent or chronic infection rather than to elimination of the staphylococci (Blobel and Katsube, 1964; Newbould and Neave, 1965). The critical role of the neutrophil in chronic mastitis in cattle was emphasized by Schalm, Lasmanis and Jain (1976) who converted chronic staphylococcal mastitis to acute gangrenous mastitis by administration of equine anti-bovine leucocyte serum. The consequences of chronic staphylococcal mastitis for the duct system were demonstrated by the inoculation of gelatine into the mammary glands of mice. The distribution of gelatine to all parts of the gland was apparent in uninfected glands; there was some restriction in distribution in glands inoculated only with endotoxin, but in endotoxin-pretreated infected glands the gelatine failed to reach the alveoli and was diverted into the interalveolar tissue. The significance of this observation is that the patency of the duct system and hence of the continuity of a milk stream in which antibiotics could diffuse, was interrupted only by the accumulation of cells in the alveolar lumen and the contraction (involution) of the alveoli. This is in contrast to the findings of several workers (Funke, 1961; Platonow and Blobel, 1963) who, from pathological studies of bovine udders, have argued that fibrosis and oedema played the major role in occluding the duct system. The refractiveness of staphylococcal mastitis in the cow to antibiotic therapy has been noted since the advent of antibiotics (Schalm and Ormsbee, 1949; Edwards, 1954; Newbould, 1974) including sodium cloxacillin (Wilson, Westgarth, Kingwill, Griffin, Neave and Dodd, 1972). Because of the intracellular location of the staphylococci and the collapse of the duct system it seemed possible that the infection in the mouse mammary gland might not be amenable to antibiotic therapy. This hypothesis was tested by the inoculation of 1 mg sodium cloxacillin via the teat into the mammary glands of mice in w-hich a chronic infection had been established. It was shown in vitro that the dose of cloxacillin was sufficient to bring about 100 per cent kill of the number of staphylococci known to be present in the mammary gland at the time of
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therapy. However, in vivo, there was no difference between the number of staphylococci recovered from cloxacillin-treated glands and from untreated glands. There are 4 points of significance in these observations. First, strains of S. aureus recovered from cases of chronic bovine mastitis caused acute mastitis when inoculated into mouse mammary glands in which there was no alveolar neutrophil population. This suggests that there may be a predisposition for these strains of S. aureus to cause acute reactions in udders in which the somatic cell content of the milk is particularly low, Second, the demonstration of failure of therapy in experimental chronic mastitis in the mouse means that an in vivo test is now available in which the efficacy of new antibiotics and formulations can be tested. Third, the reasons for failure to respond to therapy can be investigated. The results of this investigation indicate that the intracellular location of the staphylococci together with the contraction of the alveoli (rather than abscess formation, deep tissue penetration of organisms or duct occlusion by fibrosis and oedema which are all later manifestations of chronic infection) are sufficient to prevent effective therapy. Fourth, the induction of a neutrophil response in the alveoli prior to staphylococcal chahenge not only converted an acute mastitis into a chronic infection but also created the situation in which the infection failed to respond to therapy. This implies that the somatic cell content of milk should be kept as low as possible in order to avoid the establishment of a chronic staphylococcal mastitis which may fail to respond to therapy. SUMMARY
Two strains of Staphylococcus aureus derived from chronic bovine mastitis caused an acute reaction when inoculated into the mammary glands of mice. The response in mice was converted to a chronic mastitis if a neutrophil population was elicited in the alveolar lumen by intramammary inoculation of endotoxin 6 h before staphylococcal challenge. Chronic mastitis was established in this way with each of the strains of S. aureus and in each case the infection failed to respond to intramammary therapy with sodium cloxacillin. The results are discussed in relation to bovine mastitis. ACKNOWLEDGMENTS
The author is indebted to Mrs G. Hill for technical assistance, to Mr P. F. Dennis for histological preparations and to the Photography Section. 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 Staphylococcus aureus. Journal of Medical Microbiology, 7, 205-212. 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 Pathology, 85, 531-538.
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Anderson, J. C. (1976a). The contribution of the mouse mastitis model to our understanding of staphylococcal infection. zentralblatt fur Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygien? (Abteilung I) &~pPl. 5, 783-790. Anderson, J. C. (197613). The increased resistance of mice to experimental staphylococcal mastitis following inoculation of endotoxin. Research zn Veterinary Science, 21, 64-68. Anderson, J. C. (1976~). Mechanisms of staphylococcal virulence in relation to bovine mastitis. British Veterinary Journal, 132, 229-245. Anderson, J. C., and Chandler, R. L. (1975). E x p erimental staphylococcsl mastitis in the mouse : histological, ultrastructural and bacteriological changes caused by a virulent strain of Staphylococcus aureus. Journal of Comparative Pathology, 85, 499-5 10. Blobel, H., and Katsube, Y. (1964). Effects of experimentally induced leucocytosis in bovine mammary glands upon infections with Staphylococcus aureus, Streptococcus agalactiae and Aerobacter aerogznes. American Journal of Veterinary Research, 25, 1085-1089. Edwards, S. J. (1954). Antibiotic therapy in staphylococcal infection of the bovine udder. Journal of Comparative Pathology and Therapeutics, 64, 275-281. Funke, H. (1961). The distribution of S35-labelled benzyl-penicillin in normal and mastitic mammary glands of cows and goats after local and systemic administration. Acta veterinaria scandinauica, 2 (Suppl. I), I-88. Gladstone, G. P., and Glencross, E. J. G. (1960). Growth and toxin production of staphylococci in cellophane sacs in vivo. British Journal of Experimental Pathology. 41,313-333. Miles, A. A., Misra, S. S., and Irwin, J. 0. (1938). The estimation of the bactericidal power of the blood. Journal of Hygiene, 38, 732-749. Newbould, F. H. S. (1974). Antibiotic treatment of experimental Staphylococcus aureus infections of the bovine mammary gland. CanadianJournal of Comparative Medicine. 38, 41 I-416. Newbould, F. H. S., and Neave, F. K. (1965). Th e recovery of small numbers of Staphylococcus aureus infused into the bovine teat cistern. Journal of Dairy Research, 32, 157-162. Platonow, I., and Blobel, H. (1963). Therapeutic failures in chronic staphylococcic mastitis. Journal of the American Veterinary Medical Association, 142, 1097-l 101. Schalm, 0. W. (1970). Response of the cow to udder infection. 6th International Conference on Cattle Diseases, Philadelphia, pp. 25-37. Schalm, 0. W. and Ormsbee, R. W. (1949). Effects of management and therapy on staphylococcic mammary infections. Journal of the American Veterinary Medical Association, 115, 464-473. Schalm, 0. W., Lasmanin, J., and Jain, N. C. (1976). Conversion of chronic staphylococcal mastitis to acute gangrenous mastitis after neutropenia in blood and bone marrow produced by an equine anti-bovine leucocyte serum. American Journal of Veterinary Research, 37, 885-890. Wilson, C. D., Westgarth, D. R., K’ mgwill, R. G., Griffin, T. K., Neave, F. K., and Dodd, F. H. (1972). The effect of infusion of sodium cloxacillin in all infected quarters of lactating cows in sixteen herds. British Veterinary Journal, 128, 71-86. [Receivedfor publication, February 8th, 19771
COMP.PATH.
1977.Vo~.
87.
611
EXPERIMENTAL STAPHYLOCOCCAL MASTITIS MOUSE: THE INDUCTION OF CHRONIC MASTITIS ITS RESPONSE TO ANTIBIOTIC THERAPY
IN
THE AND
BY
J. C. A
NDERSON
INTRODUCTION
In a previous study (Anderson, 1975) the pathogenesis of experimental staphylococcal mastitis in the mouse was modified by inoculation of endotoxin. When endotoxin was inoculated by the intraperitoneal route 6 h before or after intramammary challenge with Sta@ylococcus aureus and when endotoxin was inoculated by the intramammary route 6 h after intramammary challenge, the animals exhibited an increased susceptibility and large numbers of staphylococci were recovered from mammary glands after 48 h. This susceptibility was associated in each case with a defect in neutrophil function. However, when endotoxin was inoculated by the intramammary route 6 h before intramammary challenge there was a reduction in the number of staphylococci recovered from the mammary glands 48 h later. This resistance was attributed to the presence in the alveolar lumen of neutrophils that were able to phagocytose the staphylococci at the time of challenge. In this study, examination of the histopathological and bacteriological changes associated with the resistance induced by a pre-existant neutrophil response suggests that this is a chronic reaction. The susceptibility of this type of infection to antibiotic therapy is investigated. MATERIALS
AND
METHODS
&lice. Lactating mice of the BSVS strain bred at Compton were used 4 to 6 days after parturition. The offspring were removed at the first inoculation. Strains of Staphvlococcus. Strains M60 and M63 of Staphylococcus aureus (S. aureus) were used. Both strains coagulated rabbit plasma within 4 h and failed to coagulate cow and mouse plasma within 24 h. Strain M60 produced alpha- and betahaemolysins on 5 per cent ox blood agar and strain M63 was non-haemolytic. Both strains were isolated from clinical cases of chronic bovine mastitis. For inoculation each strain was grown on ox blood agar for 18 h at 37 ‘C, harvested in sterile isotonic saline and washed twice in saline and finally, with the aid of a nephelometer, the concentration of organisms was adjusted to 2 to 3 x 108 cfu/ml. Endotoxin. Escherichia coli 026:B6 endotoxin, prepared by the Boivin method (Difco Labs, Detroit), was dissolved in isotonic saline to contain 250 Kg/ml. Gelatine. The gelatine solution contained 8 per cent (w/v) gelatine (BDH Chemicals Ltd, Poole) and 0,2 per cent (w/v) brilliant green (G. T. Gurr Ltd, London) in isotonic saline and was held at 37 “C before inoculation.
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Antibiotic. Sodium cloxacillin (Orbenin veterinary injectable, Beecham Animal Health, Brentford) 250 mg was dissolved in saline to a concentration of 10 mg/ml. The antibiotic sensitivity of strains M60 and M63 in vitro was demonstrated by adding 0.1 ml of the strain (10’ organisms) to each of 2 tubes containing 4.8 ml Hartley’s broth. 0.1 ml (1 mg) of cloxacillin solution was added to one tube and O-1 ml of saline was added to the other tube. After 18 h at 37 “C the tota number of viable bacterial cells was determined (Miles, Misra and Irwin, 1938). Inoculations. Mice were anaesthetized with ether and according to the experimental design 0.1 ml of endotoxin (25 pg) or staphylococci (1 O7 organisms) or cloxacillin (1 mg) was inoculated via the teat into the fourth mammary gland on each side (R4 and L4) by the method described by Anderson (1976a) ~ In the case of gelatine 0.2 ml was inoculated into each gland. Histological and bacteriological procedures. At necropsy R4 was fixed in 12 per cent neutral buffered formalin (NBF) and embedded in paraffin wax. Sections (5 p) were stained with Giemsa’s stain and by Gram’s method. Mammary glands inoculated with gelatine were fixed in cold (4 “C) NBF in which the gelatine is irreversibly solidified, processed thereafter in the normal manner and stained additionally with Crossman’s stain for connective tissue. L4 was processed for total viable bacterial cell numbers and alpha-toxin detection as previously described (Anderson, 1974). Geometric means of bacterial cell numbers are used throughout. Clinical assessment. A mouse was described as “malaised” if its coat stood on end and if it was not as alert as a normal mouse. The term “ill” signified a mouse with a very poor coat and a disinclination to move. Experimental design. Forty-eight mice were removed from their offspring and 32 were inoculated (R4 and L4) with endotoxin. Six hours later all the mice received 107 staphylococci by the intramammary route. Of the 16 mice that had not received endotoxin, 8 were killed 18 h and eight 28 h after inoculation of staphylococci. Of the 32 mice that had received endotoxin before staphylococci, 8 were killed 18 h and 8 48 h after bacterial challenge; at 48 h gelatine (0.2 ml) was inoculated into R4 of 4 of the 8 mice immediately before killing to assess the patency of the duct system. Cloxacillin (1 mg) was inoculated into R4 and L4 of 8 of the remaining 16 mice and all 16 were killed 18 h later. The whole experiment (see Fig. 1) was carried out for each strain of S. aureus. An additional control group consisted of 4 mice that received endotoxin in R4 and 54 h later gelatine in both R4 and L4 immediately before killing, to assess the effect of endotoxin and of natural involution on the distribution of gelatine within the
mammary gland. RESULTS
Kesfonse of Normal Mice to Intramammav of S. aureus (Fig I)
InouJation of Strain lvf60 or Strain A463
Two mice were dead, 3 were moribund and 3 were ill 18 h after intramammary inoculation of strain M60. An average of 3.8 x lOlo organisms was recovered from each mammary gland and alpha-toxin was detected in all 8 mammary gland homogenates (100 per cent haemolysis of 1 per cent red blood cells (rbc) at l/4 (5 glands) and l/S (3 glands) dilution of mammary gland homogenate). At 28 h after inoculation 5 mice were dead, one was moribund and 2 were ill. An average of l-9 x 1Oro organisms was recovered from each gland and alpha-toxin was detected in 5 of 8 glands (100 per cent haemolysis of 1 per cent rabbit rbc at l/4 (one gland) and l/2 (4 glands) dilution of mammary gland homogenates). Histological examination of mammary glands 18 h after inoculation of
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strain M60 showed the organisms present in large numbers in the alveolar lumen. There were few neutrophils and the epithelium, though low, vacuolated and in some areas reduced to the basement membrane, retained its basophilia (Fig. ‘2). At 28 h many staphylococci were present, often as rafts in the alveolar lumen. There were no neutrophils in the alveolar lumen and the epithelium was low and vacuolated or lost. In areas of complete liquefactive necrosis the epithelial cytoplasm was eosinophilic.
Fig. 1. Strain M60 (a) or M63 (b) of Staphylococcus aureu~ was inoculated into mammary glands of mice at time 0. The broken line indicates the response in normal mice. The response in mice given endotoxin by the intramammary route 6 h before staphylococci is indicated by the solid line and the effect of sodium cloxacillin on this chronic reaction at 48 h is indicated by the dotted line.
Three mice were dead, one was moribund and 4 were ill 18 h after inoculation of strain M63. The average number of staphylococci recovered from each gland was 1.8 x 10s and alpha-toxin was detected in 2 of 8 mammary gland homogenates (100 per cent haemolysis of 1 per cent rbc at l/2 dilution of homogenate). At 28 h after inoculation 2 mice were dead, 5 were ill and one was normal. An average of 1*5 x log organisms was recovered from each gland and alpha-toxin was detected in none of the mammary gland homogenates. The histopathological changes seen 18 and 28 h after inoculation of strain 1463 were indistinguishable from those observed at these times following inoculation of strain M60.
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Fig. 2. The acute reaction to strain M60 of S. HUMUS18 h after intramammary inoculation of normal mice. There are degenerative changes in the epithelium and staphylococci, but no neutrophils abound in the alveoli. Giemsa. s 315.
Response of Endotoxin-treated Mice to Intramammary Inoculation of the Strain M60 of S. aureus (Fig. 1) It has been established that the intramammary inoculation of 25 pg endotoxin induces a neutrophil response such that by 6 h after inoculation neutrophils are present in the alveolar lumen (Anderson, 1975). All the mice were clinically normai i8 h after inoculation and an average of 7.5 x lo6 organisms was recovered from infected glands. Alpha-toxin was not detected in any of the mammary gland homogenates. At 48 h after bacterial challenge all the mice were clinically normal, an average of 3.2 x lo6 organisms was recovered from each gland and alpha-toxin was not detected in any of the mammary gland homogenates. Histological examination 18 h after inoculation showed that there were many neutrophils and detached epithelial cells in the alveolar lumen. Most of the neutrophils were morphologically normal and many contained staphylococci; only rarely were staphylococci seen free in the alveolar lumen. The epithelium was basophilic and mildly hyperplastic in appearance due to the combination of reduction in alveolar diameter and early degenerative changes in the epithelial cells (Fig. 3). At 48 h after bacterial challenge the ducts contained cells and cell debris and staphylococci were identified within neutrophils. The epithelial hyperplasia was advanced to such an extent that, together with the interalveolar cell infiltration, adjacent alveoli formed
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confluent masses of cells (Fig. 4). Staphylococci were identified in neutrophils in the centre of contracted alveoli and some staphylococci appeared to be in macrophages or epithelial cells. Neutral fat surrounded groups of contracted alveoli, fibrosis was limited to major septae and there were no abscesses.
Fig. 3. The chronic reaction to strain M60 of S. aweus 18 h after intramammary inoculation of mice in which a neutrophil response had been induced prior to stapbylococcal challenge. There is early hyperplasia of the epithelium and there are neutrophils and shed epithelial cells in the alveoli. Giemsa. x 315.
There was resistance to inoculation of gelatine 48 h after bacterial challenge and there was leakage of gelatine when the needle was withdrawn from the teat. When counterstained with Crossman’s stain for connective tissue the gelatine appeared as an amorphous green material. Histological examination showed that the distribution of gelatine was severely restricted; gelatine was absent from the centre of alveoli that were contracted to a knot of cells and in these circumstances the gelatine was diverted through interalveolar and subcapsular spaces (Fig. 5). In the control group O-2 ml of gelatine was easily inoculated into normal mammary glands 54 h after removal of offspring. The gelatine was distributed to all alveolar lumens and was not present in interalveolar spaces (Fig. 6). It is known that the intramammary inoculation of endotoxin results in a neutrophil infiltration which is followed by involution of the gland (Anderson, 1976b). Thus, at 54 h after intramammary inoculation of endotoxin, the distribution of gelatine was restricted when compared with normal mammary glands. However, the restriction was not as great as that seen following bacterial challenge of endotoxin-treated mammary glands.
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Fig. 4. The chronic reaction to strain M60 of S. ~ZLWUS 48 h after intramammary inoculation of mice in which a neutrophil response had been induced prior to staphylococcal challenge. The alveoli are contracted and the hyperplastic epithelium impinges upon the neutrophils and cell debris in the alveolar lumen. Giemsa. s 315.
Resfionse of Endotoxin-treated Mice to Intramammary Inoculation of Strain -\63 S. aureus (Fig. 1)
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Two mice showed signs of malaise and 6 were normal 18 h after inoculation. An average of 1.9 x 10’ organisms was recovered from each gland and alphatoxin was not detected in any mammary gland homogenates. At 48 h all the mice were clinically normal, an average of 6.7 x lo6 organisms was recovered from each mammary gland and alpha-toxin was not detected in the mammary gland homogenates. The histopathological changes following inoculation were indistinguishable from those observed following inoculation of strain M60 and there was also the same restriction in the distribution of gelatine. Resportseof Endotoxirz-treated Mice to Intramammary Inoculation of Sodium Cloxacillirt 48 h ilfter Intramammary Inoculation of S. aureus Strain A!60 or A163 (Fig. I) Eighteen hours after intramammary inoculation of 1 mg sodium cloxacillin into R4 and L4 all the mice were clinically normal. Staphylococci were recovered from all mammary glands and the average number of organisms was 1.3 x 105; alpha-toxin was not detected.
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Fig. 5. Distribution of gelatine following intramammary inoculation 48 h after establishment of chronic mastitis. The g&tine is present in a duct (D) but instead of reaching the alveolar lumen it has been diverted to septae (S). Crossman. x 126.
Fig. 6. Distribution of gelatine in uninfectkd mammary gland following intramammary The gelatine is seen in terminal alveolar lumens. Grossman. x 126.
inoculation.
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Histological examination showed neutrophils and cell debris in the ducts. The alveoli were contracted into knots of cells which, with interalveolar infiltration of mononuclear cells, created confluent cell masses which were surrounded by neutral fat. Staphylococci were identified in neutrophils in the ducts and in the centre of contracted alveoli. In this latter site staphylococci were also associated with macrophages or epithelial cells. All the 8 mice in the control group not treated with sodium cloxacillin were clinically normal 66 h after intramammary inoculation of strain M60; the average number of organisms recovered was 2.0 x IO5 and alpha-toxin was not detected. The histopathological changes were indistinguishable from those in the group treated with antibiotic. There was a similar lack of response to sodium cloxacillin therapy in the group of mice infected with strain M63. In the treated group an average of 9.2 x lo4 organisms was recovered from each gland 18 h after therapy and in the control group the average number of organisms recovered was 1.1 x 10”; alpha-toxin was not detected in mammary gland homogenates of mice from either group. The histopathological changes were also similar to those in mammary glands infected with strain M60. Response of S. aureus Strain M60 and M63 to Sodium Cloxacillin In Vitro The results showed that 1 mg sodium cloxacillin killed 100 per cent of 2 to 3 x 10’ organisms of S. aureus strain M60 or M63 within 18 h. Eighteen hours after the addition of O-1 ml saline instead of 0.1 ml antibiotic to the culture medium, there were 4.37 x lOlO and 1.95 x lOlo organisms of strains M60 and M63 respectively. DISCUSSION
In this study 2 strains of S. aureus recovered from chronic bovine mastitis multiplied rapidly when inoculated into the mammary glands of mice with consequent histopathological and clinical changes typical of an acute reaction. It is of interest that both strains of S. aureus induced an acute mastitis in mice despite the fact that only one of the strains produced haemolysins on blood agar. It is known that staphylococci may produce toxins in vivo that are not detected in vitro (Gladstone and Glencross, 1960) and in this study traces of alpha-toxin were detected in 2 of 8 mammary gland homogenates after inoculation of the non-haemolytic strain M63. However, the culminating histopathological change in glands infected with either strain was liquefactive necrosis in contrast to coagulative necrosis which is typical of a strongly alpha-toxigenic strain of S. aureus (Anderson and Chandler, 1975). Thus, although the production of alpha-toxin by strain M60 may account for the slightly greater multiplication in the mouse mammary gland, there seem to be factors other than alpha-toxin that cause necrosis (Anderson, 1976c). These findings emphasize the importance .of non-haemolytic S. aureus (as well as haemolytic S. aureus) in the aetiology of mastitis. An alternative to the acute reaction was induced in the mammary glands of mice by intramammary inoculation of endotoxin 6 h before staphylococci.
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The endotoxin induced a migration of neutrophils into the alveolar lumen so that they were present to phagocytose staphylococci at the time of challenge. However, the advantage conferred on the mice was not sufficient to sterilize the mammary gland and histopathological and bacteriological changes occurred that were different from those of the acute reaction. Instead of extensive extracellular bacterial multiplication the organisms were intracellular and multiplication was inhibited, and instead of necrosis of mammary epithelium there was hyperplasia and involution. On the basis of the time course and the distinct histopathological reaction this alternative response was considered to be chronic mastitis. Thus, in the mouse, acute staphylococci mastitis was converted to chronic mastitis by inducing an alveolar neutrophil population in advance of staphylococcal challenge. The significance of the leucocyte barrier of the udder to infection by coliform bacteria and by Streptococcusagalactiae has been demonstrated (Schalm, 1970) but the significance of the barrier with regard to staphylococci is less certain. The leucocyte barrier appears to be efficient in that it prevents acute mastitis (Blobel and Katsube, 1964) but there is evidence that the barrier may lead to persistent or chronic infection rather than to elimination of the staphylococci (Blobel and Katsube, 1964; Newbould and Neave, 1965). The critical role of the neutrophil in chronic mastitis in cattle was emphasized by Schalm, Lasmanis and Jain (1976) who converted chronic staphylococcal mastitis to acute gangrenous mastitis by administration of equine anti-bovine leucocyte serum. The consequences of chronic staphylococcal mastitis for the duct system were demonstrated by the inoculation of gelatine into the mammary glands of mice. The distribution of gelatine to all parts of the gland was apparent in uninfected glands; there was some restriction in distribution in glands inoculated only with endotoxin, but in endotoxin-pretreated infected glands the gelatine failed to reach the alveoli and was diverted into the interalveolar tissue. The significance of this observation is that the patency of the duct system and hence of the continuity of a milk stream in which antibiotics could diffuse, was interrupted only by the accumulation of cells in the alveolar lumen and the contraction (involution) of the alveoli. This is in contrast to the findings of several workers (Funke, 1961; Platonow and Blobel, 1963) who, from pathological studies of bovine udders, have argued that fibrosis and oedema played the major role in occluding the duct system. The refractiveness of staphylococcal mastitis in the cow to antibiotic therapy has been noted since the advent of antibiotics (Schalm and Ormsbee, 1949; Edwards, 1954; Newbould, 1974) including sodium cloxacillin (Wilson, Westgarth, Kingwill, Griffin, Neave and Dodd, 1972). Because of the intracellular location of the staphylococci and the collapse of the duct system it seemed possible that the infection in the mouse mammary gland might not be amenable to antibiotic therapy. This hypothesis was tested by the inoculation of 1 mg sodium cloxacillin via the teat into the mammary glands of mice in w-hich a chronic infection had been established. It was shown in vitro that the dose of cloxacillin was sufficient to bring about 100 per cent kill of the number of staphylococci known to be present in the mammary gland at the time of
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therapy. However, in vivo, there was no difference between the number of staphylococci recovered from cloxacillin-treated glands and from untreated glands. There are 4 points of significance in these observations. First, strains of S. aureus recovered from cases of chronic bovine mastitis caused acute mastitis when inoculated into mouse mammary glands in which there was no alveolar neutrophil population. This suggests that there may be a predisposition for these strains of S. aureus to cause acute reactions in udders in which the somatic cell content of the milk is particularly low, Second, the demonstration of failure of therapy in experimental chronic mastitis in the mouse means that an in vivo test is now available in which the efficacy of new antibiotics and formulations can be tested. Third, the reasons for failure to respond to therapy can be investigated. The results of this investigation indicate that the intracellular location of the staphylococci together with the contraction of the alveoli (rather than abscess formation, deep tissue penetration of organisms or duct occlusion by fibrosis and oedema which are all later manifestations of chronic infection) are sufficient to prevent effective therapy. Fourth, the induction of a neutrophil response in the alveoli prior to staphylococcal chahenge not only converted an acute mastitis into a chronic infection but also created the situation in which the infection failed to respond to therapy. This implies that the somatic cell content of milk should be kept as low as possible in order to avoid the establishment of a chronic staphylococcal mastitis which may fail to respond to therapy. SUMMARY
Two strains of Staphylococcus aureus derived from chronic bovine mastitis caused an acute reaction when inoculated into the mammary glands of mice. The response in mice was converted to a chronic mastitis if a neutrophil population was elicited in the alveolar lumen by intramammary inoculation of endotoxin 6 h before staphylococcal challenge. Chronic mastitis was established in this way with each of the strains of S. aureus and in each case the infection failed to respond to intramammary therapy with sodium cloxacillin. The results are discussed in relation to bovine mastitis. ACKNOWLEDGMENTS
The author is indebted to Mrs G. Hill for technical assistance, to Mr P. F. Dennis for histological preparations and to the Photography Section. 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 Staphylococcus aureus. Journal of Medical Microbiology, 7, 205-212. 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 Pathology, 85, 531-538.
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Anderson, J. C. (1976a). The contribution of the mouse mastitis model to our understanding of staphylococcal infection. zentralblatt fur Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygien? (Abteilung I) &~pPl. 5, 783-790. Anderson, J. C. (197613). The increased resistance of mice to experimental staphylococcal mastitis following inoculation of endotoxin. Research zn Veterinary Science, 21, 64-68. Anderson, J. C. (1976~). Mechanisms of staphylococcal virulence in relation to bovine mastitis. British Veterinary Journal, 132, 229-245. Anderson, J. C., and Chandler, R. L. (1975). E x p erimental staphylococcsl mastitis in the mouse : histological, ultrastructural and bacteriological changes caused by a virulent strain of Staphylococcus aureus. Journal of Comparative Pathology, 85, 499-5 10. Blobel, H., and Katsube, Y. (1964). Effects of experimentally induced leucocytosis in bovine mammary glands upon infections with Staphylococcus aureus, Streptococcus agalactiae and Aerobacter aerogznes. American Journal of Veterinary Research, 25, 1085-1089. Edwards, S. J. (1954). Antibiotic therapy in staphylococcal infection of the bovine udder. Journal of Comparative Pathology and Therapeutics, 64, 275-281. Funke, H. (1961). The distribution of S35-labelled benzyl-penicillin in normal and mastitic mammary glands of cows and goats after local and systemic administration. Acta veterinaria scandinauica, 2 (Suppl. I), I-88. Gladstone, G. P., and Glencross, E. J. G. (1960). Growth and toxin production of staphylococci in cellophane sacs in vivo. British Journal of Experimental Pathology. 41,313-333. Miles, A. A., Misra, S. S., and Irwin, J. 0. (1938). The estimation of the bactericidal power of the blood. Journal of Hygiene, 38, 732-749. Newbould, F. H. S. (1974). Antibiotic treatment of experimental Staphylococcus aureus infections of the bovine mammary gland. CanadianJournal of Comparative Medicine. 38, 41 I-416. Newbould, F. H. S., and Neave, F. K. (1965). Th e recovery of small numbers of Staphylococcus aureus infused into the bovine teat cistern. Journal of Dairy Research, 32, 157-162. Platonow, I., and Blobel, H. (1963). Therapeutic failures in chronic staphylococcic mastitis. Journal of the American Veterinary Medical Association, 142, 1097-l 101. Schalm, 0. W. (1970). Response of the cow to udder infection. 6th International Conference on Cattle Diseases, Philadelphia, pp. 25-37. Schalm, 0. W. and Ormsbee, R. W. (1949). Effects of management and therapy on staphylococcic mammary infections. Journal of the American Veterinary Medical Association, 115, 464-473. Schalm, 0. W., Lasmanin, J., and Jain, N. C. (1976). Conversion of chronic staphylococcal mastitis to acute gangrenous mastitis after neutropenia in blood and bone marrow produced by an equine anti-bovine leucocyte serum. American Journal of Veterinary Research, 37, 885-890. Wilson, C. D., Westgarth, D. R., K’ mgwill, R. G., Griffin, T. K., Neave, F. K., and Dodd, F. H. (1972). The effect of infusion of sodium cloxacillin in all infected quarters of lactating cows in sixteen herds. British Veterinary Journal, 128, 71-86. [Receivedfor publication, February 8th, 19771