it would be of interest to determine whether tissue and causing clinical symptoms, thus large inocula (106 to 108 CFU) are required to

Transcription

1 INFECTION AND IMMUNrIy, June 1973, p Copyright American Society for Microbiology Vol. 7, No. 6 Printed in U.SA. Virulence of Two Mastitis Strains of Staphylococcus aureus in Bovine Skin: Enhancement by Growth in High Carbohydrate-High Salt Medium or in Raw Milk J. H. BROCK, A. TURVEY, AND B. REITER National Institute for Research in Dairying, University of Reading, Reading, England Received for publication 12 December 1972 Cows were inoculated intradermally with two strains of Staphylococcus aureus, and the severity of the resulting skin lesions was assessed on the basis of size, degree of necrosis, and in some cases by measuring the area of inflammation in histological preparations of excised skin. At least 106 colony forming units were required to produce a detectable skin lesion, indicating that the cow has a resistance to staphylococcal skin infection comparable to man and rabbit. The severity of the lesions produced by the two strains correlated with their known virulence in the bovine udder as mastitis pathogens. A comparison between lesions produced by cocci grown in a stock laboratory medium and those grown in the high carbohydrate-high salt modified 110 medium showed that the virulence of cocci grown in the latter medium was enhanced. The comparable effect of milk to the modified 110 medium is discussed. The bovine udder is very susceptible to infection by Staphylococcus aureus (and other pathogens) since infusion of less than 10 colony forming units (CFU) into the teat sinus can cause mastitis (11). To a varying degree, the infectious process causes the production of milk which is abnormal in appearance, increased leukocyte count, transfer of serum proteins into the udder, and extensive tissue damage (3, 13, 16), and can even lead to severe systemic infection necessitating slaughter of the animal (15). The virulence of a strain can, therefore, be judged by the severity of the reaction in the udder. Strains of low virulence do not evoke clinical signs of the infection, although organisms are shed in the milk and the leukocyte count is increased. Recently it was shown that the virulence of some strains of S. aureus in mice can be increased by culturing them in modified 110 medium (17), which contains high concentrations of carbohydrate and sodium chloride. When staphylococci grown in this medium were injected intraperitoneally in mice, the mean lethal dose was reduced by as much as a factor of 10 as compared with the mean lethal dose of staphylococci grown in brain heart infusion broth. The enhanced virulence was shown to be 865 due to the production of extracellular slime, since removal of the slime by washing decreased the virulence of these organisms (17). Therefore, it would be of interest to determine whether bovine staphylococci would also show an -increase in virulence in the udder when grown in this medium. Such experiments cannot be made by intramammary challenge because the number of organisms required to infect an udder is already extremely low, and organisms probably multiply in the milk prior to invading the tissue and causing clinical symptoms, thus nullifying any effect of the original growth medium. However, as it is well known that very large inocula (106 to 108 CFU) are required to produce local skin infections in man (6), rabbits (8, 12), or mice (7), it was worthwhile to investigate the effect of staphylococci in the bovine skin, since this would provide a more suitable model for investigating the virulence of bovine staphylococci grown in different media. This paper shows that the bovine skin is as resistant to staphylococcal infection as that of other animals, but that the virulence of two strains in the udder can be correlated with the reaction in the bovine skin and that culturing in modified 110 medium or in raw milk increases their virulence.

2 866 BROCK, TURVEY, AND REITER INFECT. IMMUNITY MATERIALS AND METHODS Strains of S. aureus. Strain BB was provided by J. B. Derbyshire, Agricultural Research Council, Institute for Research on Animal Diseases, Compton, Berkshire. It was originally isolated from a mastitic cow and is of high virulence, since infusion of 10 CFU into an udder quarter invariably gives rise to acute mastitis within 48 h, which, without antibiotic treatment, can lead to death of the animal (Brock and Reiter, unpublished data). Strain Mexi was isolated from a mastitic cow at the National Institute for Research in Dairying. It has been used in a number of experiments in which organisms were infused into the udder and usually produces only subclinical mastitis. Some quarters infected with strain Mexi may occasionally become clinical, but the severe type of reaction occurring with BB is virtually never observed. Media. Dextrose-Lemco broth (DLB) consisted of 1% peptone (Evans), 1% Lab-Lemco (Oxoid), 0.5% sodium chloride, and 1% glucose. Modified 110 medium was prepared by the method of Yoshida and Ekstedt (17). Essentially, this medium contains 3% NaCl, 0.5% K2HPO4, 5% of 1 M phosphate buffer (ph 7.6), 1% mannitol, 0.2% lactose, and a dialysate of 1% peptone (Oxoid) and 2.5% yeast extract (Difco). Raw milk was obtained by aseptic cannulation of an udder quarter of a lactating cow known to be free of udder infection. Cows. Friesian cows of varying age were used. All had completed at least one lactation. Preparation of challenge doses. Eighteen-hour cultures of S. aureus in 10 ml of the appropriate medium were centrifuged and the organisms were suspended in 5 ml of ice-cold one-fourth-strength Ringers solution containing 0.5% of the appropriate medium (Ringer broth). Further washing was avoided to prevent loss of extracellular slime. These suspensions had a viable count of approximately 109 CFU/ ml. Dilutions were made in Ringer broth to give 103, 105, and 107 CFU/ml. To ensure that the inocula were comparable, the actual viable count of each culture was determined by the method of Miles and Misra (10). In the case of milk cultures it was not possible to sediment the organisms by centrifugation; to obtain comparable inocula both the broth and milk cultures were plated and kept at 4 C overnight. The broth cultures were then diluted appropriately (without prior centrifugation) in one-fourth-strength Ringers solution to give the same count as the corresponding milk cultures. All suspensions were kept in an ice bath up to the time of use. Inoculation of cows. The hair was clipped from patches of skin approximately 5 cm by 5 cm along the back of each of the cows. When skin samples were to be excised for histological examination, the areas were also shaved. Cows were inoculated intradermally with 0.1 ml of the appropriate suspension by using a Maclintock tuberculin syringe. As control, 0.1 ml of sterile Ringer broth or sterile raw milk was injected. One inoculation was made per clipped or shaved area. Examination of skin. The injected sites were examined for swelling and necrosis 3 h after inoculation and daily thereafter for at least 3 days. Lesions were scored according to size and degree of necrosis. For histological examination, specimens of infected and normal adjoining skin were excised, the size of the specimens ranging from 5 mm by 5 mm to approximately 50 mm by 20 mm, depending on the size of the lesion. After fixation in phosphate-buffered Formalin, the skin was sectioned at 5 Aim and the sections were stained with hematoxylin and eosin. Bacteria were stained by the Twort modification of the Gram stain. Estimation of area of inflammation in sections of skin lesions. Sections of skin in which lesions were visible were examined, and for each lesion the section showing the largest area of inflammation or necrosis, or both, was selected. A x 10-magnified image of the section was projected onto a sheet of thin card, and the area of inflammation and necrosis was marked with a pencil. The marked area was cut out and weighed, and a factor for calculating the actual lesion area was obtained by weighing 1 cm2 of the card. RESULTS Variation of lesions due to size of challenge dose. To establish the effect of the size of the inoculum, four cows were each inoculated with doses of S. aureus strain BB ranging from 102 to 108 CFU. Observation after 48 h (Table 1) showed that sites inoculated with 104 CFU or less showed little or no swelling. Sites inoculated with 106 CFU showed slight or moderate swelling. Sites inoculated with 108 CFU mostly showed large swellings and, in two of the four cows, necrosis of the skin was observed. Specimens of skin from one of the cows (161) were excised 2 days after inoculation and examined histologically. No evidence of inflammation was seen at the site inoculated with 104 CFU; no bacteria were seen, and the skin at the site of inoculation was indistinguishable from normal skin (Fig. 1). With 106 CFU, a small area showing large numbers of inflammatory cells was visible (Fig. 2) and a few bacteria were present (Fig. 3). The lesion produced by 108 CFU showed extensive inflammation at the surface, and this extended into the collagen layer and muscle in the form of an inflammatory "ring" (Fig. 4). Bacteria were widespread within and at the edge of this ring (Fig. 5). These results showed that at least 106 CFU of S. aureus BB were needed to cause any reaction in the bovine skin, smaller inocula being almost without any detectable effect. Subsequently, 106 or more CFU were used in experiments to compare the virulence of different strains and the effect of growth in different media on virulence. Comparison of skin lesions produced by strains Mexi and BB. Appearance of lesions. Ten cows were inoculated intradermally with 108 CFU of S. aureus Mexi and BB. Swellings

3 VOL. 7, 1973 VIRULENCE OF BOVINE STAPHYLOCOCCI TABLE 1. Appearance of swellings in four cows 2 days after intradermal inoculation with varying doses of S. aureus BB No. of cows Inoculum Size of swelling" Degree of necrosisb Mean (CFU) ~~~~~~~~~~~~~~~~ Very ~~~~~~~~~~~~~~~~~~ Nil Vlight Slight Moderate Large Absent Slight Moderate a Size, diameter, and score, respectively: very slight, < 1.5 cm, 0.5; slight, 1.5 to 2.4 cm, 1.0; moderate, 2.5 to 3.4 cm, 2.0; large, >3.5 cm, 3.0. b Necrosis, appearance, additional score, respectively: none, normal, 0.0; slight, skin surface puckered but not depressed (normally <0.5 cm in diameter), 1.0; moderate, central depressed area of puckered skin (>0.5 cm diameter), 2.0. / 4. _ -, *.-Imi --. FIG. 1. Section of skin from cow injected intradermally with 104 CFU of S. aureus BB. No inflammation is visible and the skin appears normal. x5.5. ".: t V.-,.:o.1.., lki.:....u.. FIG. 2. Section of skin from cow injected intradermallv with 106 CFU of S. aureus BB. A small area of inflammation is present at the center of the section. x5.5. were observed at all the sites, and in some cases necrosis occurred. These results at 2 days postinoculation are shown in Table 2. The swellings produced by BB were generally larger than those produced by Mexi, and necrosis was observed in only three of the swellings due to Mexi but in nine of those due to BB. In none of the animals did Mexi produce a more severe reaction than BB. Two of the cows were also inoculated with 106 CFU. Both cows showed moderate swellings with 106 BB, but with neither cow was there any visible swelling with 106 Mexi. Effect of medium. To assess the effect of the growth medium on the virulence of S. aureus, nine cows were inoculated with 10' CFU of BB grown in DLB and in modified 110 medium. Examination of the inoculated sites after 48 h (Table 3) showed that the organisms grown in modified 110 medium generally produced larger swellings than the corresponding dose of organisms grown in DLB and, although necrosis occurred in nearly all cases, it was less severe at sites injected with DLB-grown organisms than at sites injected with modified 110-grown organisms. Thus, growth in modified 110 medium appeared to enhance the virulence of strain BB. Histological examination of infected skin. To provide further evidence for the difference in virulence between strains Mexi and BB and to

4 868 BROCK, TURVEY, AND REITER INFECT. IMMUNITY w *: 4' A~~~~~~~~~~~~~~~~~~~~~~ S ; 4.4 4'Syvp* 4* st A~~~~ r if 0, jb S * ~ * 9 #js* Wa ~ ~ ~ livb y $i%{t; > r i. *'. *>rc s -' 'I: S*.lo'.: #4.~~~~~~1 g: 4 t?:tix'! 4i tbp FIG. 3. Section of skin from cow injected intradermally with 106 CPU of S. aureus BB, numbers of inflammatory cells, and (arrowed) a few small groups of cocci. x52x. t histologically. In all cases some evidence of inflammation was found. The areas of inflammation were measured, and the presence or absence of a ring of inflammatory cells and degradation of the dermis were noted (Fig. 6). In almost every case a larger area of inflammation resulted from BB than from a corresponding dose of Mexi. The lesions produced by organisms grown in modified 110 medium generally showed a larger area of inflammation than those produced by the corresponding dose of orgashowing large Downloaded from FIG. 4. Section of skin from cow injected intradermally with 108 CFU of S. aureus BB. A ring of inflammatory cells extends almost through the entire thickness of the skin from the dermis to the muscle layver, and degradation of the dermis is apparent in the upper left part of the ring. x5.5. investigate further the effect of the growth medium on production of skin lesions, a histological examination of lesions produced by the two strains grown in the two media was made. Two cows (522, 533) were injected on two occasions with 106 and 108 CFU of Mexi and BB grown in both DLB and modified 110 medium, the second injections being given at separate sites 2 days after the first. On the day after the second series of injections, all the injected areas of skin were excised and sections were examined..::.....n!.-. on October 17, 2018 by guest

5 VOL. 7, 1973 VIRULENCE OF BOVINE STAPHYLOCOCCI 869 ".3r F' IF 4 4 f.a jc 4 i.e ss i 14 FIG. 5. Section of skin from cow injected intradermally with 10' CFUof S. aureus BB. Large clumps of cocci within the ring of inflammatorv cells are visible on the right. Large numbers of inflammatory cells at the inner edge of the ring (cf. Fig. 4) are visible on the left, and there are clumps of cocci among the cells. x 185. TABLE 2. Appearance of swellings in 10 cows 2 days after intradermal inoculation of 108 CFUof S. aureus Mexi and BB No. of cowsa Strain Size of swelling Degree of necrosis lmesioan Moder- Moder- score Slight ate Large Absent Slight ate BB Mexi asee footnotes, Table 1. nisms grown in DLB (e.g., Fig. 7). The only anomalous results occurred with strain BB in cow 533 at 1 day, and these can be partly explained by what appears to be a smaller lesion than was usually observed resulting from 10" CFU grown in modified 110 medium. The characteristic ring of inflammatory cells was seen in all lesions except some of those produced by 106 CFU of Mexi, in which only a small area showing high numbers of cells was seen. Within the inflammatory ring bacteria could be observed, and in the more severe 3-day-old lesions the tissue within the ring was showing loss of structure and degeneration. In the most severe lesions, degradation of the dermis could be observed. This was generally only seen with 108 CFU of BB; with 10' CFU of Mexi it occurred only with modified 110-grown organisms, and with 106 CFU of either strain the dermis remained more or less intact. There was generally little difference in the size of lesions at 1 and 3 days. Effect of growth in raw milk. Since organisms grown in modified 110 medium showed enhanced virulence for the bovine skin, it was decided to use the method of skin-inoculation to investigate the virulence of S. aureus grown in raw milk. Six cows were injected with strains Mexi and BB grown in DLB, modified 110 medium, and in raw milk. The doses used were approximately 2.3 x 107 CFU of Mexi and 2.9 x 107 CFU of BB. Examination of the inoculated sites after 48 h (Table 4) showed that, as expected, the response was greater to organisms grown in modified 110 medium than the corresponding dose grown in DLB and also that an even more severe response resulted from the same dose grown in raw milk. Because milk alone might cause an inflammatory response in the bovine TABLE 3. Appearance of swellings in nine cows 2 davs after intradermal inoculation with S. aureus. BB grown in DLB, and modified 110 medium Medium No. of cowsa Size of swelling Degree of necrosis Mean lesion Mdoder- Ab- Moder- Slight score ate Large sent Slight ate DLB Modified a See footnotes, Table 1.

6 870 BROCK, TURVEY, AND REITER INFECT. IMMUNITY *Inflammatory ring+ ++ +_ + Degradation of dermis. Mod-llOgrown organisms M DLB-grown organisms + =Present + =Doubtful - = Absent FIG. 6. Area of inflammation, inflammatory ring formation, and degradation of dermis in skin sections excised from two cows I or 3 days after intradermal injection with S. aureus Mexi and BB grown in dextrose-lemco Broth (DLB) or modified 110 medium (Mod-10). A~~~~~~~~~~ FIG. 7. Sections of skin from cow h after injection of 108 CFU of S. aureus Mexi grown irn dextrose-lemco broth (A) and modified 110 medium (B). The ring of inflammatory cells is larger in B than in A, and degradation of the dermis has started to occur in B but not in A. x3.7. skin, each cow was also given a control injection of sterile milk. Two of the six cows showed a very slight swelling at the control site, but in the remaining four there was no visible reaction. Thus, the relatively more severe response in the skin to inoculation of S. aureus grown in raw milk did not appear to be due to the presence of milk per se. DISCUSSION It is clear from the results of these experiments that the cow has a resistance to staphylococcal skin infection comparable to that of man (6) and the rabbit (8, 12). Intradermal inoculation of less than 106 CFU of even the highly virulent BB strain did not produce detectable skin lesions, and 108 CFU were required

7 VOL. 7, 1973 VIRULENCE OF BOVINE STAPHYLOCOCCI 871 TABLE 4. Appearance of swellings in six cows 2 days after intradermal inoculation with S. aureus BB and Mexi grown in DLB, modified 110 medium, and raw milk No. of cowsa Mean Strain Medium Size of swelling Degree of Necrosis lesion score Slight Moderate Large Absent Large Moderate BB DLB Modified Raw milk Mexi DLB Modified Raw milk a See footnotes, Table 1. to produce necrosis visible at the surface. This is in accord with the observation that cows are not known to suffer from natural staphylococcal skin infection such as boils or furunculosis. Derbyshire (4) found that subcutaneous inoculation of goats with 108 CFU of strain BB resulted in a febrile response lasting 2 days and abscess formation, but the abscesses appeared to be healing after 14 days. Although it has not been confirmed that very low challenge doses can infect the goat mammary gland, it is known that, in sheep, infusion of <50 CFU of a strain of S. aureus will cause mastitis (14). Furthermore, it is possible to infect the mammary gland of the mouse with a challenge dose containing the equivalent of only 3.5 CFU (2). Thus, it may be concluded that the mammary gland is unusually susceptible to staphylococcal infection. Although such extreme susceptibility is unusual it is not unique, since Kondo et al. (9) were able to infect the mouse kidney by direct intrarenal injection of very low numbers of staphylococci. The factors responsible for the differing virulence of strains of S. aureus are still poorly understood. Although toxins, coagulase, and leukocidin may all play some part, it is now generally agreed that none of these extracellular products is a virulence factor of primary importance. It is clear, however, that in the case of strains BB and Mexi their ability to produce skin lesions in the cow correlated with their virulence as udder pathogens. Preliminary experiments (unpublished data) with two other strains of S. aureus of known virulence as udder pathogens indicated that such a correlation appears to apply to other strains. Staphylococci grown in modified 110 medium showed increased virulence as measured by the reaction in the bovine skin, thus confirming and extending the findings of Yoshida and Ekstedt (17), who used intraperitoneal inoculation in mice as a challenge system. These workers found that extracellular slime produced by organisms growing in this high carbohydratehigh salt medium was responsible for the increase in virulence, and showed that the slime removed mouse protective antibody to the capsulated Smith diffuse strain from rabbit antisera (5). The composition of the slime from strain BB is being investigated, and a preliminary examination has shown it to contain ribitol teichoic acid, a mannan, and a serologically active basic polypeptide (1). When small numbers of staphylococci enter the teat cistern, it is highly probable that some multiplication initially takes place in milk within the udder prior to invasion of the tissue. If multiplication in milk (which is also a carbohydrate-rich medium containing -4.5% lactose) could enhance the virulence of staphylococci in a similar manner to modified 110 medium, this might go some way to explaining why invasion of the mammary tissue occurs so readily in staphylococcal mastitis. The results reported here (Table 4) appear to confirm that milkgrown staphylococci produced more severe lesions in the bovine skin than equivalent challenge doses of organisms grown in either DLB or modified 110 medium, thus supporting this hypothesis. It would, however, be desirable to confirm these results by histological examination of the lesions. Since invasion of the mammary tissue and abscess formation is considered the main reason for the poor response of staphylococcal udder infections to antibiotic therapy (3), such observations may lead to a better understanding of the mechanisms whereby invasion of mammary tissue occurs and eventually to a means of reducing the incidence of staphylococcal mastitis refractory to antibiotic therapy.

8 872 BROCK, TURVEY, AND REITER INFECT. IMMUNITY ACKNOWLEDGMENTS We thank H. L. Buttle and E. R. Jackson for performing skin biopsies, and Sandra Christopherson and Marjorie Crossfield for excellent technical assistance. LITERATURE CITED 1. Brock, J. H., and B. Reiter Virulence of bovine strains of Staphylococcus aureus. J. Gen. Microbiol. 71:v-vi. 2. Chandler, R. L Experimental bacterial mastitis in the mouse. J. Med. Microbiol. 3: Derbyshire, J. B The pathology of experimental staphylococcal mastitis in the goat. J. Comp. Pathol. 68: Derbyshire, J. B The immunization of goats against staphylococcal mastitis by means of experimental infection of the skin and udder. Res. Vet. Sci. 2: Ekstedt, R. D., and K. Yoshida Immunity to staphylococcal infection in mice: effect of living versus killed vaccine, role of circulating antibody, and induction of protection-inducing antigen(s) in vitro. J. Bacteriol. 100: Elek, S. D., and P. E. Conen The virulence of Staphylococcus pyogenes for man. A study of the problems of wound infection. Brit. J. Exp. Pathol. 38: James, R. C., and C. J. Macleod Induction of staphylococcal infections in mice with small inocula introduced on sutures. Brit. J. Exp. Pathol. 42: Johnson, I. J., L. E. Cluff, and K. Goshi Studies of the pathogenesis of staphylococcal infection. I. The effect of repeated skin infections. J. Exp. Med. 113: Kondo, I., S. Masuda, K. Kimura, K. Kurosaka, and N. Hasegawa Effects of intrarenal inoculation of Staphylococcus aureus on mice. Infect. Immunity 4: Miles, A. A., and S. S. Misra The estimation of the bactericidal power of the blood. J. Hyg. 38: Newbould, F. H. S., and F. K. Neave The response of the bovine mammary gland to an infusion of staphylococci. J. Dairy Res. 32: Panton, P. N., and F. C. 0. Valentine Staphylococcal infection and reinfection. Brit. J. Exp. Pathol. 10: Pattison, I. H The progressive pathology of bacterial mastitis. Vet. Rec. 70: Plommet, M Mammite staphylococcique de la brebis: infection experimentelle. Ann. Inst. Pasteur 98: Slanetz, L. W., C. H. Bartley, and F. E. Allen Evaluation of cell-toxoid vaccines for the vaccination of dairy cattle against staphylococcic mastitis. Amer. J. Vet. Res. 26: Stabenfeldt, G. H., and G. R. Spencer The lesions in bovine udders shedding hemolytic coagulase-positive staphylococci. Pathol. Vet. 2: Yoshida, K., and R. D. Ekstedt Relation of mucoid growth of Staphylococcus aureus to clumping factor reaction, morphology in serum-soft agar, and virulence. J. Bacteriol. 96: Downloaded from on October 17, 2018 by guest