Br. vet .
_7.
(1991) . 147, 463
CHANGES IN THE BOVINE UDDER QUARTERS NATURALLY INFECTED BY CORYNEBACTERIUM BOVIS
T . A . NGATIA*, N . E . JENSENt and B . B . BERGt *Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, University of .Nairobi, P. 0. Box 29053, Nairobi, Kenya ; tStatens Veterinaere Serumlaboratorium, Odinsvej 4, Ringsted, 4100, Denmark
SUMMARY Of 272 bovine udder quarters studied for mastitis, 19 of them naturally infected with Corynebacterium bovis alone, were compared with 16 others infected by C . boots together with other bacteria and another 36 non-infected quarters . While there was no significant difference in milk somatic cell counts between the quarters infected by C. boats alone and those affected by C . boats together with other bacteria .28X10' .37±20 (33 and 33 .86±23 .18X 103 /ml of milk, respectively), there was a significant difference between these and the non-infected quarters (5 .60 ± 3 .23X 103/ml of milk) . Microscopically, quarters infected by either C. bovis alone or C. bovis in combination with other bacteria had inflammatory changes in the teat cisterns, Furstenberg's rosettes and/or mammary parenchyma . The non-infected quarters had no changes . In all 82 quarters no pathological changes could be seen in the teat canals .
INTRODUCTION Corynebacterium bovis is the organism most frequently recovered from bovine milk samples . Its prevalence is greatly reduced by routine teat disinfection and dry cow therapy (Black et al., 1972 ; Bramley, 1975 ; Bramley et al., 1976 ; Brooks et al., 1983 ; Brooks & Barnum, 1984a) . The organism is generally regarded as a harmless commensal, although it causes mild elevation in milk somatic cell counts (Forbes, 1970 ; Black et al., 1972 ; Brooks & Barnum, 1984a, b) . Occasionally it has also been suspected to cause mastitis in cattle (McEwen & Cooper, 1947 ; Cobb & Walley, 1962) . Despite these observations, C. bovis has received little attention from mastitis workers who have been more concerned with pathogenic microorganisms associated with significant loss . Recently, interest in C. bovis as a natural biological control mechanism against mastitis pathogens had developed (Black et al., 1972 ; Bramley, 1975 ; Linde et al., 1980 ; Brooks & Barnum, 1984b) . The present investigation was conducted to determine the influence of C. bovis on milk somatic cell counts (SCC) and to study the pathology of the bovine udder associated with this organism .
464
BRITISH VETERINARY JOURNAL, 147, 5
MATERIALS AND METHODS Dairy cows A herd of 68 dairy cows of the red Danish breed was involved in this study . The animals were maintained on an intensive production system in Zealand, Denmark . They were milked twice a day using a regularly maintained milking machine and equipment . Post milking teat disinfection was routinely practised using organic iodides . Since all the animals in this herd were to be slaughtered because the herd had tested positive for infectious bovine rhinotracheitis (IBR) virus infection, it was decided to study the various aspects of mastitis . Collection of milk samples Eight days before slaughter, a total of 18 milk samples was taken from each quarter as follows : on days 8, 7, 6, 3, 2, and 1, foremilk samples (after discarding the first few jets) were taken before the morning and afternoon milkings ; on days 6, 2 and 1 additional samples were taken immediately after the morning and afternoon milkings, while on days 5 and 4 no samples were taken as these were weekend days . In taking these samples aseptic procedures were followed as described by Schalm et al. (1971) . In addition to quarter milk samples (QMS), teat cistern puncture samples (TOPS) were taken immediately after killing each animal . This was achieved by the use of needle and syringe after disinfecting each teat twice with alcohol soaked udder cloths . In all cases, the samples were collected in sterile colour coded disposable tubes (NUNC, Roskilde, Denmark) . The samples were refrigerated (4 ° C) overnight and examined the following morning . Examination of milk samples Quarter milk samples were examined for bacteria and the results interpreted according to the Scandinavian recommendations (Klastrup & Schmidt-Madsen, 1974) . In the case of TCPS larger volumes (0.1 ml) than those for QMS (0 .02 ml) were used to inoculate the entire surface of an aesculin bovine blood agar (BA) plate . In all cases, standard bacteriological procedures were followed . Inoculated plates were incubated aerobically at 37°C and the readings made after 24 and 48 h . Whereas the Scandinavian recommendations were followed in interpreting the bacteriological results of QMS, in the case of TCPS, all bacteria were recorded with an estimate of the number of colonies of each . A quarter was considered infected if it yielded bacteria on one or more of the 19 sampling occasions . The number of somatic cells in each QMS was determined by the use of Fossomatic equipment (A/S N Foss Electic, Hillerod, Denmark) following the procedure described by Schmidt-Madsen (1975) . In the case of TCPS cellular contents were not determined due to suspected contamination with blood . The average cell count of 18 samples from each quarter was taken to represent the SCC for the particular quarter . Gross and histopathological procedures Immediately after killing each animal the mammary quarters were systematically examined for any morphological abnormalities . Tissues for histopathology were taken from different areas of the quarter (including the teat) and fixed in 10% formalin solution . After proper fixation, the tissues were processed routinely, sectioned at a thickness of 6µm and stained with haematoxylin and eosin (HE) .
CORi’XEBACTERIUM
BOVZS IN BOVINE
UDDER
465
RESULTS In this paper, only quarters infected by C. bovis and some non-infected quarters are considered. During the 19 sampling occasions, 36 quarters yielded no bacteria, 19 yielded C. bovis alone, while 16 yielded C. bovis together with other bacteria. The latter included Stup/zylococ~usaweus, 11 of 16; micrococci, 3 of 16 and Streptococcus dysglactiae, 2 of 16. In all these 71 quarters, no abnormalities could be detected with the naked eye and no microscopic alterations could be seen in the teat canals. However, microscopic examination showed that all infected quarters had inflammatory changes in one or more areas. No such changes could be seen in the non-infected quarters. The inflammatory reaction was characterized by proliferation of connective tissue, mononuclear cell infiltration and sometimes also neutrophil infiltration. Regarding the SCC of milk samples, the non-infected quarters had a mean of 5.60 &3.23X103/ml. Those infected by C. bovis alone had a mean SCC of 33.37 + 20.28X10”/ml of milk while those affected by C. bouts together with other bacteria had a mean SCC of 33.86 &23.18X103/ml of milk. Microscopically, 16 of 19 quarters infected by C. bovis alone had inflammatory changes affecting the teat cisterns and/or Furstenberg’s rosettes (Fig. 1). In some of these cases, there was also librinocellular exudate in the teat lumen and degenerative changes in the epithelium of the teat cistern. In addition to these changes, 13 of 16 quarters also showed inllammatory changes in the mammary parenchyma (Fig. 2). Besides the inflammation there were also degenerative changes affecting both the ductal and alveolar epithelium.
1. Section through the right front teat (cow no. 364) infected by Cotynebacteriumbovzs alone. showing leucocytic infiltration and proliferation of fibroblasts (arrows) in the subepithelial tissues of the teat cistern and/or Furstenberg’s rosettes. HE X75.
Fig.
In the other 3 of 16 quarters,
the mammary
parenchyma
had no microscopic
alterations
(Fig. 3). In the remaining 3 of 19 quarters the teat cisterns and Furstenberg’s rosettes were of normal appearance (Fig. 4), but the mammary parenchyma showed inflammatory changes.
466
BRITISH
VETERINARY
JOURNAL,
147, 5
Fig. 2.
Section through the left front quarter (cow no. 364) infected by Cotynebacteriumbovis alone showing leucocytes admixed with milk in the alveoli of the mammary gland (A). HE X75.
Fig. 3.
Section through the left hind quarter (cow no. 444) with no infection, showing normal mammary parenchyma. HE X300.
Sixteen quarters affected by C. bovis and other bacteria showed similar changes to those described above. While all these quarters had pathological changes in the teat cisterns and/or Furstenberg’s rosettes, 14 of them also had changes in the mammary parenchyma, but two had none.
DISCUSSION The results of this study indicated that quarters naturally infected by either C. bovis alone, or C. bouis in conjunction with other bacteria had significantly higher milk SCC (33.37 f 20.28X103/ml and 33.86 f 23.18X103/ml, respectively) than the non-infected
COR?WEBACTERIlJM
BOWS IN BOVINE
UDDER
467
quarters (5.60 + 3.23X103/ml). Although similar findings have been reported by earlier workers (Forbes, 1970; Black et al., 1972; Brooks & Barnum, 1984a, b) their values for both infected as well as non-infected quarters were higher than those reported here, a discrepancy which is difficult to explain.
Fig. 4. Section through the left front teat (cow no. 750) with no infection, showing normal teat cistern and/or Furstenberg’s rosettes, with keratinaceous material at the teat canal termination (arrows). Note the absence of inflammatory cells or proliferating t’ibroblasts in the subepithelial tissues. HE X75.
From our study, it is evident that elevation of milk SCC in the infected quarters is attributable to the inflammatory process, which was seen in one or more areas of such quarters. This observation confirms earlier suspicions that C. bouis causes bovine mastitis (McEwen & Cooper, 1947; Cobb & Walley, 1962). The latter workers had also suspected C. bouis to be responsible for a large proportion of the cases usually regarded as nonspecific mastitis. This suggestion might be true bearing in mind that during routine mastitis diagnosis, C. bouts is generally ignored even though it may be the only organism isolated from quarters with mastitic milk. Although some workers have shown that, though in not a well defined way, C. bovzs infection can protect the udder against major pathogens, for example, Stuphyfococus au7eus and Streptococcus agalactiae (Forbes, 1970; Black et al., 1972; Bramley, 1975; Linde et al., 1980) others did not observe such a beneficial effect (Kainard & Poutrel, 1982; Brooks et al., 1983). If such a beneficial effect really exists, it is more likely to be attributable to the products of inflammation rather than C. bouis itself. Furthermore the products of inflammation (i.e. leucocytes and blood proteins) in the mammary gland have been shown to be capable of killing pathogens or inhibiting their growth (Jones & Simms, 1930; Murphy & Stuart, 1952; Wilson & Roseblum, 1952; Auclair & Hirsch, 1953; Reiter & Oram, 1967; Schalm et al, 1971; Schanbacher & Smith, 1975; Hibbit & Hill, 1977; Lee et al., 1980).
468
BRITISH VETERINARY JOURNAL, 147, 5
ACKNOWLEDGEMENTS The authors wish to thank the staff members of SVS, Ringsted, Denmark, for their assistance in the collection and analysis of milk samples . A vote of thanks is also extended to Dr P . Krogh of SVS, Copenhagen, for allowing us to use his laboratory and the staff members in his laboratory in the preparation of histological slides . This research project was funded by the Nordic Council of Ministers, for which we are grateful .
REFERENCES AUCLAIR, J . E . & HIRSCH, A . (1953) . ,Journal of Dairy Research 20, 45 . BLACK, R . T ., MARSHALL, R . T . & BOURLAND, C . T . (1972) . ,Journal of Dairy Science 55, 413. BRAMLEY, A . J . (1975) . International Dairy Federation Doc. 85 . BRAMLEY, A . J ., KINGWILL, R . G ., GRIFFIN, T. K . & SIMPKIN, D . L . (1976). Veterinary Record 99, 275 . BROOKS, B . W . & BARNUM, D . A . (1984a) . Canadian journal of Comparative Medicine 48, 141 . BROOKS, B . W . & BARNUM, D. A . (1984b) . Canadian Journal of Comparative Medicine 48, 146 . BROOKS, B . W ., BARNUM, D . A . & MEEK, A . H . (1983) . Canadian journal of Comparative Medicine 47,
73 . COBB, R . W . & WALLEY, J . K. (1962) . Veterinary Record 74, 101 . FORBES, D . (1970) . British Veterinary journal 126, 260 . HIBBIT, K . G . & HILL, A . W . (1977) . In Antibiotics and Antibiosis in Agriculture, ed. M . Woodbine . London, UK : Butterworths. JONES, F. S . & SiMMs, H . S . (1930) . journal of Experimental Medicine 47, 877 . KLASTRUP, O. & SCHMIDT-MADSEN, P . (1974) . Nordic Veterinaere Medicin 26, 197 . LEE, C . S ., WOODING, F . P . & KEMP, P . (1980) . Journal of Dairy Research 47, 39 . LINDE, C ., HOLMBERG, O . & ASTROM, G . (1980) . Nordic Veterinaere Medicin 32, 552 . MCEwEN, A . D . & COOPER, M. B . (1947) . Veterinary Record 59, 655 . MURPHY, J . M . & STUART, O. M . (1952) . Cornell Veterinarian 42, 537 . RAINARD, P . & POUTREL, B . (1982) . American Journal of Veterinary Research 43, 2143 . REITER, B . & ORAM, J . D . (1967) . Nature 216, 328 . SCHALM, O . W ., CARROLL, E . J . & JAIN, N . C . (1971) . Bovine Mastitis. Philadelphia : Lea and Febiger. SCHANBACHER, F . L . & SMITH, K . L . (1975) . ,Journal of Dairy Science 58, 1049 . SCHMIDT-MADSEN, P . (1975) . Journal of Dairy Research 42, 227 . WILSON, C . D . & ROSEBLUM, H . (1952) . ,Journal of Experimental Medicine 95, 25 . (Accepted for publication 26 November 1990)
_7.
(1991) . 147, 463
CHANGES IN THE BOVINE UDDER QUARTERS NATURALLY INFECTED BY CORYNEBACTERIUM BOVIS
T . A . NGATIA*, N . E . JENSENt and B . B . BERGt *Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, University of .Nairobi, P. 0. Box 29053, Nairobi, Kenya ; tStatens Veterinaere Serumlaboratorium, Odinsvej 4, Ringsted, 4100, Denmark
SUMMARY Of 272 bovine udder quarters studied for mastitis, 19 of them naturally infected with Corynebacterium bovis alone, were compared with 16 others infected by C . boots together with other bacteria and another 36 non-infected quarters . While there was no significant difference in milk somatic cell counts between the quarters infected by C. boats alone and those affected by C . boats together with other bacteria .28X10' .37±20 (33 and 33 .86±23 .18X 103 /ml of milk, respectively), there was a significant difference between these and the non-infected quarters (5 .60 ± 3 .23X 103/ml of milk) . Microscopically, quarters infected by either C. bovis alone or C. bovis in combination with other bacteria had inflammatory changes in the teat cisterns, Furstenberg's rosettes and/or mammary parenchyma . The non-infected quarters had no changes . In all 82 quarters no pathological changes could be seen in the teat canals .
INTRODUCTION Corynebacterium bovis is the organism most frequently recovered from bovine milk samples . Its prevalence is greatly reduced by routine teat disinfection and dry cow therapy (Black et al., 1972 ; Bramley, 1975 ; Bramley et al., 1976 ; Brooks et al., 1983 ; Brooks & Barnum, 1984a) . The organism is generally regarded as a harmless commensal, although it causes mild elevation in milk somatic cell counts (Forbes, 1970 ; Black et al., 1972 ; Brooks & Barnum, 1984a, b) . Occasionally it has also been suspected to cause mastitis in cattle (McEwen & Cooper, 1947 ; Cobb & Walley, 1962) . Despite these observations, C. bovis has received little attention from mastitis workers who have been more concerned with pathogenic microorganisms associated with significant loss . Recently, interest in C. bovis as a natural biological control mechanism against mastitis pathogens had developed (Black et al., 1972 ; Bramley, 1975 ; Linde et al., 1980 ; Brooks & Barnum, 1984b) . The present investigation was conducted to determine the influence of C. bovis on milk somatic cell counts (SCC) and to study the pathology of the bovine udder associated with this organism .
464
BRITISH VETERINARY JOURNAL, 147, 5
MATERIALS AND METHODS Dairy cows A herd of 68 dairy cows of the red Danish breed was involved in this study . The animals were maintained on an intensive production system in Zealand, Denmark . They were milked twice a day using a regularly maintained milking machine and equipment . Post milking teat disinfection was routinely practised using organic iodides . Since all the animals in this herd were to be slaughtered because the herd had tested positive for infectious bovine rhinotracheitis (IBR) virus infection, it was decided to study the various aspects of mastitis . Collection of milk samples Eight days before slaughter, a total of 18 milk samples was taken from each quarter as follows : on days 8, 7, 6, 3, 2, and 1, foremilk samples (after discarding the first few jets) were taken before the morning and afternoon milkings ; on days 6, 2 and 1 additional samples were taken immediately after the morning and afternoon milkings, while on days 5 and 4 no samples were taken as these were weekend days . In taking these samples aseptic procedures were followed as described by Schalm et al. (1971) . In addition to quarter milk samples (QMS), teat cistern puncture samples (TOPS) were taken immediately after killing each animal . This was achieved by the use of needle and syringe after disinfecting each teat twice with alcohol soaked udder cloths . In all cases, the samples were collected in sterile colour coded disposable tubes (NUNC, Roskilde, Denmark) . The samples were refrigerated (4 ° C) overnight and examined the following morning . Examination of milk samples Quarter milk samples were examined for bacteria and the results interpreted according to the Scandinavian recommendations (Klastrup & Schmidt-Madsen, 1974) . In the case of TCPS larger volumes (0.1 ml) than those for QMS (0 .02 ml) were used to inoculate the entire surface of an aesculin bovine blood agar (BA) plate . In all cases, standard bacteriological procedures were followed . Inoculated plates were incubated aerobically at 37°C and the readings made after 24 and 48 h . Whereas the Scandinavian recommendations were followed in interpreting the bacteriological results of QMS, in the case of TCPS, all bacteria were recorded with an estimate of the number of colonies of each . A quarter was considered infected if it yielded bacteria on one or more of the 19 sampling occasions . The number of somatic cells in each QMS was determined by the use of Fossomatic equipment (A/S N Foss Electic, Hillerod, Denmark) following the procedure described by Schmidt-Madsen (1975) . In the case of TCPS cellular contents were not determined due to suspected contamination with blood . The average cell count of 18 samples from each quarter was taken to represent the SCC for the particular quarter . Gross and histopathological procedures Immediately after killing each animal the mammary quarters were systematically examined for any morphological abnormalities . Tissues for histopathology were taken from different areas of the quarter (including the teat) and fixed in 10% formalin solution . After proper fixation, the tissues were processed routinely, sectioned at a thickness of 6µm and stained with haematoxylin and eosin (HE) .
CORi’XEBACTERIUM
BOVZS IN BOVINE
UDDER
465
RESULTS In this paper, only quarters infected by C. bovis and some non-infected quarters are considered. During the 19 sampling occasions, 36 quarters yielded no bacteria, 19 yielded C. bovis alone, while 16 yielded C. bovis together with other bacteria. The latter included Stup/zylococ~usaweus, 11 of 16; micrococci, 3 of 16 and Streptococcus dysglactiae, 2 of 16. In all these 71 quarters, no abnormalities could be detected with the naked eye and no microscopic alterations could be seen in the teat canals. However, microscopic examination showed that all infected quarters had inflammatory changes in one or more areas. No such changes could be seen in the non-infected quarters. The inflammatory reaction was characterized by proliferation of connective tissue, mononuclear cell infiltration and sometimes also neutrophil infiltration. Regarding the SCC of milk samples, the non-infected quarters had a mean of 5.60 &3.23X103/ml. Those infected by C. bovis alone had a mean SCC of 33.37 + 20.28X10”/ml of milk while those affected by C. bouts together with other bacteria had a mean SCC of 33.86 &23.18X103/ml of milk. Microscopically, 16 of 19 quarters infected by C. bovis alone had inflammatory changes affecting the teat cisterns and/or Furstenberg’s rosettes (Fig. 1). In some of these cases, there was also librinocellular exudate in the teat lumen and degenerative changes in the epithelium of the teat cistern. In addition to these changes, 13 of 16 quarters also showed inllammatory changes in the mammary parenchyma (Fig. 2). Besides the inflammation there were also degenerative changes affecting both the ductal and alveolar epithelium.
1. Section through the right front teat (cow no. 364) infected by Cotynebacteriumbovzs alone. showing leucocytic infiltration and proliferation of fibroblasts (arrows) in the subepithelial tissues of the teat cistern and/or Furstenberg’s rosettes. HE X75.
Fig.
In the other 3 of 16 quarters,
the mammary
parenchyma
had no microscopic
alterations
(Fig. 3). In the remaining 3 of 19 quarters the teat cisterns and Furstenberg’s rosettes were of normal appearance (Fig. 4), but the mammary parenchyma showed inflammatory changes.
466
BRITISH
VETERINARY
JOURNAL,
147, 5
Fig. 2.
Section through the left front quarter (cow no. 364) infected by Cotynebacteriumbovis alone showing leucocytes admixed with milk in the alveoli of the mammary gland (A). HE X75.
Fig. 3.
Section through the left hind quarter (cow no. 444) with no infection, showing normal mammary parenchyma. HE X300.
Sixteen quarters affected by C. bovis and other bacteria showed similar changes to those described above. While all these quarters had pathological changes in the teat cisterns and/or Furstenberg’s rosettes, 14 of them also had changes in the mammary parenchyma, but two had none.
DISCUSSION The results of this study indicated that quarters naturally infected by either C. bovis alone, or C. bouis in conjunction with other bacteria had significantly higher milk SCC (33.37 f 20.28X103/ml and 33.86 f 23.18X103/ml, respectively) than the non-infected
COR?WEBACTERIlJM
BOWS IN BOVINE
UDDER
467
quarters (5.60 + 3.23X103/ml). Although similar findings have been reported by earlier workers (Forbes, 1970; Black et al., 1972; Brooks & Barnum, 1984a, b) their values for both infected as well as non-infected quarters were higher than those reported here, a discrepancy which is difficult to explain.
Fig. 4. Section through the left front teat (cow no. 750) with no infection, showing normal teat cistern and/or Furstenberg’s rosettes, with keratinaceous material at the teat canal termination (arrows). Note the absence of inflammatory cells or proliferating t’ibroblasts in the subepithelial tissues. HE X75.
From our study, it is evident that elevation of milk SCC in the infected quarters is attributable to the inflammatory process, which was seen in one or more areas of such quarters. This observation confirms earlier suspicions that C. bouis causes bovine mastitis (McEwen & Cooper, 1947; Cobb & Walley, 1962). The latter workers had also suspected C. bouis to be responsible for a large proportion of the cases usually regarded as nonspecific mastitis. This suggestion might be true bearing in mind that during routine mastitis diagnosis, C. bouts is generally ignored even though it may be the only organism isolated from quarters with mastitic milk. Although some workers have shown that, though in not a well defined way, C. bovzs infection can protect the udder against major pathogens, for example, Stuphyfococus au7eus and Streptococcus agalactiae (Forbes, 1970; Black et al., 1972; Bramley, 1975; Linde et al., 1980) others did not observe such a beneficial effect (Kainard & Poutrel, 1982; Brooks et al., 1983). If such a beneficial effect really exists, it is more likely to be attributable to the products of inflammation rather than C. bouis itself. Furthermore the products of inflammation (i.e. leucocytes and blood proteins) in the mammary gland have been shown to be capable of killing pathogens or inhibiting their growth (Jones & Simms, 1930; Murphy & Stuart, 1952; Wilson & Roseblum, 1952; Auclair & Hirsch, 1953; Reiter & Oram, 1967; Schalm et al, 1971; Schanbacher & Smith, 1975; Hibbit & Hill, 1977; Lee et al., 1980).
468
BRITISH VETERINARY JOURNAL, 147, 5
ACKNOWLEDGEMENTS The authors wish to thank the staff members of SVS, Ringsted, Denmark, for their assistance in the collection and analysis of milk samples . A vote of thanks is also extended to Dr P . Krogh of SVS, Copenhagen, for allowing us to use his laboratory and the staff members in his laboratory in the preparation of histological slides . This research project was funded by the Nordic Council of Ministers, for which we are grateful .
REFERENCES AUCLAIR, J . E . & HIRSCH, A . (1953) . ,Journal of Dairy Research 20, 45 . BLACK, R . T ., MARSHALL, R . T . & BOURLAND, C . T . (1972) . ,Journal of Dairy Science 55, 413. BRAMLEY, A . J . (1975) . International Dairy Federation Doc. 85 . BRAMLEY, A . J ., KINGWILL, R . G ., GRIFFIN, T. K . & SIMPKIN, D . L . (1976). Veterinary Record 99, 275 . BROOKS, B . W . & BARNUM, D . A . (1984a) . Canadian journal of Comparative Medicine 48, 141 . BROOKS, B . W . & BARNUM, D. A . (1984b) . Canadian Journal of Comparative Medicine 48, 146 . BROOKS, B . W ., BARNUM, D . A . & MEEK, A . H . (1983) . Canadian journal of Comparative Medicine 47,
73 . COBB, R . W . & WALLEY, J . K. (1962) . Veterinary Record 74, 101 . FORBES, D . (1970) . British Veterinary journal 126, 260 . HIBBIT, K . G . & HILL, A . W . (1977) . In Antibiotics and Antibiosis in Agriculture, ed. M . Woodbine . London, UK : Butterworths. JONES, F. S . & SiMMs, H . S . (1930) . journal of Experimental Medicine 47, 877 . KLASTRUP, O. & SCHMIDT-MADSEN, P . (1974) . Nordic Veterinaere Medicin 26, 197 . LEE, C . S ., WOODING, F . P . & KEMP, P . (1980) . Journal of Dairy Research 47, 39 . LINDE, C ., HOLMBERG, O . & ASTROM, G . (1980) . Nordic Veterinaere Medicin 32, 552 . MCEwEN, A . D . & COOPER, M. B . (1947) . Veterinary Record 59, 655 . MURPHY, J . M . & STUART, O. M . (1952) . Cornell Veterinarian 42, 537 . RAINARD, P . & POUTREL, B . (1982) . American Journal of Veterinary Research 43, 2143 . REITER, B . & ORAM, J . D . (1967) . Nature 216, 328 . SCHALM, O . W ., CARROLL, E . J . & JAIN, N . C . (1971) . Bovine Mastitis. Philadelphia : Lea and Febiger. SCHANBACHER, F . L . & SMITH, K . L . (1975) . ,Journal of Dairy Science 58, 1049 . SCHMIDT-MADSEN, P . (1975) . Journal of Dairy Research 42, 227 . WILSON, C . D . & ROSEBLUM, H . (1952) . ,Journal of Experimental Medicine 95, 25 . (Accepted for publication 26 November 1990)
