Research in Veterinary Science 1992, 52, 123-125
Experimental infection of the mouse mammary gland with
Campylobacter coli K. S. DIKER, Department of Microbiology, Faculty of Veterinary Medicine, Ankara University, Ankara 06110, R. H A Z I R O G L U , Department of Pathology, Faculty of Veterinary Medicine, Ankara University, Ankara 06110, F. S. DIKER, Laboratory Animal Unit, Gazi Medical School, Ankara, Turkey
Campylobacter coli strains of bovine and avian origin were inoculated into the mammary gland of mice. A bovine strain isolated from a case of mastitis produced gross and histological changes in most of the glands; one bovine and one avian faecal isolate did not. Histologically, lesions were characterised by neutrophil inf'dtration in the alveolar spaces and necrosis and oedema in the interulveolar tissue. On bacteriological examination, the bovine mastitis strain could be isolated from most of the glands, but neither of the faecal strains. The mouse, therefore, appears to provide a convenient model for studying campylobacter mastitis. STUDIES on experimental infection of the bovine mammary gland with Campylobacter coli/jejuni and an aerotolerant campylobacter raised the possibility that these might be a naturally occurring campylobacter mastitis (Lander and Gill 1980, Logan et al 1982). Subsequently this was confirmed by the isolation of Cjejuni (Morgan et al 1985, Ismail et al 1988), C coli (Diker et al 1987b) and an aerotolerant campylobacter (Logan et al 1982) from field cases of bovine mastitis. As an alternative to experimental bovine mastitis, mouse mastitis models have been established by Chandler (1970) and by Anderson (1972). This experimental model has been extensively used in the study of staphylococcal and coliform mastitis (Chandler 1970, Anderson 1972, Bramley and Foster 1990). Macroscopic and histological observations of the mastitis produced in the mouse closely resembled those occurring in the cow (Chandler 1970). The purpose of the present study was to demonstrate that the mouse might be a useful model to describe the characteristics of bovine mastitis caused by C coli. C coli strains designated SM46, SB58 and TB231 were used to infect mouse mammary glands. C coli strain SM46 was isolated from a clinical case of bovine mastitis (Diker et al 1987b). Strains SB58 and TB231 were isolated from the faeces of a cow and a broiler chicken, respectively. All strains showed the phenotypic characteristics described for C coli by Smibert (1984). All C coli strains were grown in brucella broth (Oxoid)
in microaerobic conditions at 37°C for 48 hours. Before inoculation, total viable cell counts of cultures were made. Each culture used as inoculum contained approximately 107 colony forming units (cfu) m1-1. Lactating Swiss albino mice that had borne only one litter were used 10 to 14 days after parturition. The offspring were removed from the lactating mice and disposed of two hours before inoculation. Mice were anaesthetised with ether and 0-05 ml ofinoculum (5 × 105 cfu) was injected into the fourth mammary gland on each side (L4 and R4) by the method described by Chandler (1970). C coli SM46 was inoculated into 20 mice, strain SB58 into 12 mice and strain TB231 into 12 mice and sterile brucella broth (as a control) into six mice. The mice were killed with ether after 48 hours. The mice were necropsied and gross pathological changes were noted. For the histological examination, right mammary glands (R4) were fixed in 12 per cent neutral buffered formalin solution, and embedded in paraffin wax. Sections (5 ~m) were stained with haematoxylin and eosin and Gram's methods. For the bacteriological examination, left mammary glands (L4) were ground in Griffith's tubes with sterile saline and inoculated onto Preston selective medium (Oxoid). The media were incubated in a microaerobic atmosphere at 37°C for 76 hours. The livers of each mice were also cultured for C coli. All the mice were clinically normal 48 hours after inoculation. At the post mortem examination, 23 of 40 mammary glands inoculated with C coli strain SM46 showed gross pathological changes. The affected glands were enlarged and congested with a reddish appearance and associated with varying degrees of a subcutaneous gelatinous exudate. Uninoculated mammary glands remained normal in appearance. C coli strail~s SB58 and TB231 and sterile brucella broth did not produce any gross changes. At histological examination, all the glands (L4) inoculated with SM46 showed mild to severe pathological lesions. Only mild lesions were observed in two of 12 glands inoculated with strain SB58. Strain TB231 did
123
124
K. S. Diker, R. Haziroglu, F. S. Diker
FIG 1: (a) Control mouse mammary gland. (b) Histological changes in the mouse mammary gland 48 hours after the inoculation of C coli SM46; note the large number of polymorphs and cellular debris within the alveolar spaces. Bar = 50 I.tm
not produce histological changes in any of the glands. Control glands injected with brucella broth remained histologically unaltered (Fig la). Histologically, sections of mildly infected mammary tissue showed large numbers of neutrophils within the alveolar spaces (Fig lb). Some alveoli were packed with these cells; others appeared normal or contained only a few cells. The secretory epithelium exhibited degenerative changes but the alveolar structure was not lost. In severe cases, marked intra-alveolar infiltration of neutrophils and small areas of necrosis were observed and interacinar oedema was evident. There was cell debris in the alveoli, interalveolar tissue and teat duct. On bacteriological examination, C coliwas reisolated from 14 of 20 glands (R4), and from seven of 20 livers of mice infected with strain SM46. C coli could not be isolated from any of the mammary glands or rivers of mice infected with strains SB58 and TB231.
Although experimental infection of the bovine mammary gland with campylobacters has produced clinical mastitis, problems of management and cost cause difficulties in the use of cows for the study of bovine mastitis. The authors therefore used the mouse as a model. The results of these experiments indicate that C coli can establish an infection in the mammary gland of the mouse and produce mastitis with a comparatively small number of organisms. In this study C coli isolates from three sources were used for comparison. SM46 isolated from a case of mastitis caused mild to severe gross and histological changes in most of the glands whereas SB58 and TB231 did not produce any lesion except for mild histological changes in two glands. Severe histopathological changes appeared to be associated with the ability of the organism to multiply in the mammary gland; SM46 could be reisolated from most of the glands whereas SB58 and TB231 could not. In a previous study (Diker et al 1987a), the in vitro survival of strains SM46 and TB231 in bovine milk was compared and it was found that SM46 survived significantly longer than TB231 at 37°C. All these findings indicate that strains of C coli can differ in their effect on the mammary gland of the mouse in that SM46 is more virulent than SB58 or TB231. Therefore, it may be speculated that some strains of C coli, such as SM46, are adapted to colonisation of mammary glands. Histological changes observed in the mammary glands of mice in the present study resembled those seen in experimental coliform mastitis reported previously (Chandler 1970, Anderson 1979). In an experimental study, Lander and Gill (1980) also found that the clinical picture of bovine mastitis produced by C coli/jejuni resembled the mastitis caused by coliform organisms. Although no comparative data are available on the histological characteristics ofmastitis produced by C coli in cows and mice, the present findings and the literature suggest that the mouse provides a convenient model for fundamental studies on campylobacter mastitis. In particular, this mouse mastitis model may allow the identification of pathogenic campylobacter strains which can cause mastitis in cows. References ANDERSON,J. C. (1972)The effectof teat damageon the incidence ofexperimentalmastitisinthemouse.Researchin VeterinaryScience 13, 390-392 ANDERSON, J. C. (1979) The epidemiologyand pathogenesisof experimentalstaphylococcaland coliformmastitisin the mouse. British VeterinaryJournal 135, 163-171 BRAMLEY, A. J. & FOSTER, R. (1990) Effect of lysostaphinon Staphylococcus aureus infectionsof the mouse mammarygland. Research in VeterinaryScience 49, 120-121 CHANDLER, R. L. (1970) Experimentalbacterialmastitisin the mouse. Journal of Medical Microbiology 3, 273-282 DIKER, K. S., ERDEGER, J. & ARDA, M. (1987a) A study on the survivalof Campylobaetercoliin rawmilk.MikrobiyolojiBulteni 21, 200-205
Experimental C coli mastit& in mice DIKER, K. S., ERDEGER, J., KESKINTEPE, L. & ARDA, M. (1987b) Isolation of Campylobacter coli from a cow with mastitis. Ankara Oniversitesi Veteriner Fakultersi 34, 500-502 ISMAIL, M., HATEM, M. E. & EL-SEEDY, F. R. (1988) Campylobacter organisms as a bacterial cause ofmastitis in Egyptian cattle and buffaloes. Veterinary Medical Journal 36, 257-265 LANDER, K. P. & GILL, K. P. W. (1980) Experimental infection of the bovine udder with Campylobacter eoli/jejuni. Journal of Hygiene 84, 421-428 LOGAN, E. F., NEILL, S. D. & MACKIE, D. P. (1982) Mastitis in dairy cows associated with an aerotolerant campylobacter. Veterinary Record 110, 229-230
125
MORGAN, G., CHADWICK, P., LANDER, K. P. & GILL, K. P. W. (1985) Campylobacterjejuni mastitis in a cow: a zoonosis related incident. Veterinary Record 116, 111 SMIBERT, R. M. (1984) Bergey's Manual of Systematic Bacteriology: Campylobacter. Eds N. R. Krieg and J. G. Holt. Baltimore, Williams and Wilkins. pp 111-118
Received July 10, 1991 Accepted October 2, 1991
Experimental infection of the mouse mammary gland with
Campylobacter coli K. S. DIKER, Department of Microbiology, Faculty of Veterinary Medicine, Ankara University, Ankara 06110, R. H A Z I R O G L U , Department of Pathology, Faculty of Veterinary Medicine, Ankara University, Ankara 06110, F. S. DIKER, Laboratory Animal Unit, Gazi Medical School, Ankara, Turkey
Campylobacter coli strains of bovine and avian origin were inoculated into the mammary gland of mice. A bovine strain isolated from a case of mastitis produced gross and histological changes in most of the glands; one bovine and one avian faecal isolate did not. Histologically, lesions were characterised by neutrophil inf'dtration in the alveolar spaces and necrosis and oedema in the interulveolar tissue. On bacteriological examination, the bovine mastitis strain could be isolated from most of the glands, but neither of the faecal strains. The mouse, therefore, appears to provide a convenient model for studying campylobacter mastitis. STUDIES on experimental infection of the bovine mammary gland with Campylobacter coli/jejuni and an aerotolerant campylobacter raised the possibility that these might be a naturally occurring campylobacter mastitis (Lander and Gill 1980, Logan et al 1982). Subsequently this was confirmed by the isolation of Cjejuni (Morgan et al 1985, Ismail et al 1988), C coli (Diker et al 1987b) and an aerotolerant campylobacter (Logan et al 1982) from field cases of bovine mastitis. As an alternative to experimental bovine mastitis, mouse mastitis models have been established by Chandler (1970) and by Anderson (1972). This experimental model has been extensively used in the study of staphylococcal and coliform mastitis (Chandler 1970, Anderson 1972, Bramley and Foster 1990). Macroscopic and histological observations of the mastitis produced in the mouse closely resembled those occurring in the cow (Chandler 1970). The purpose of the present study was to demonstrate that the mouse might be a useful model to describe the characteristics of bovine mastitis caused by C coli. C coli strains designated SM46, SB58 and TB231 were used to infect mouse mammary glands. C coli strain SM46 was isolated from a clinical case of bovine mastitis (Diker et al 1987b). Strains SB58 and TB231 were isolated from the faeces of a cow and a broiler chicken, respectively. All strains showed the phenotypic characteristics described for C coli by Smibert (1984). All C coli strains were grown in brucella broth (Oxoid)
in microaerobic conditions at 37°C for 48 hours. Before inoculation, total viable cell counts of cultures were made. Each culture used as inoculum contained approximately 107 colony forming units (cfu) m1-1. Lactating Swiss albino mice that had borne only one litter were used 10 to 14 days after parturition. The offspring were removed from the lactating mice and disposed of two hours before inoculation. Mice were anaesthetised with ether and 0-05 ml ofinoculum (5 × 105 cfu) was injected into the fourth mammary gland on each side (L4 and R4) by the method described by Chandler (1970). C coli SM46 was inoculated into 20 mice, strain SB58 into 12 mice and strain TB231 into 12 mice and sterile brucella broth (as a control) into six mice. The mice were killed with ether after 48 hours. The mice were necropsied and gross pathological changes were noted. For the histological examination, right mammary glands (R4) were fixed in 12 per cent neutral buffered formalin solution, and embedded in paraffin wax. Sections (5 ~m) were stained with haematoxylin and eosin and Gram's methods. For the bacteriological examination, left mammary glands (L4) were ground in Griffith's tubes with sterile saline and inoculated onto Preston selective medium (Oxoid). The media were incubated in a microaerobic atmosphere at 37°C for 76 hours. The livers of each mice were also cultured for C coli. All the mice were clinically normal 48 hours after inoculation. At the post mortem examination, 23 of 40 mammary glands inoculated with C coli strain SM46 showed gross pathological changes. The affected glands were enlarged and congested with a reddish appearance and associated with varying degrees of a subcutaneous gelatinous exudate. Uninoculated mammary glands remained normal in appearance. C coli strail~s SB58 and TB231 and sterile brucella broth did not produce any gross changes. At histological examination, all the glands (L4) inoculated with SM46 showed mild to severe pathological lesions. Only mild lesions were observed in two of 12 glands inoculated with strain SB58. Strain TB231 did
123
124
K. S. Diker, R. Haziroglu, F. S. Diker
FIG 1: (a) Control mouse mammary gland. (b) Histological changes in the mouse mammary gland 48 hours after the inoculation of C coli SM46; note the large number of polymorphs and cellular debris within the alveolar spaces. Bar = 50 I.tm
not produce histological changes in any of the glands. Control glands injected with brucella broth remained histologically unaltered (Fig la). Histologically, sections of mildly infected mammary tissue showed large numbers of neutrophils within the alveolar spaces (Fig lb). Some alveoli were packed with these cells; others appeared normal or contained only a few cells. The secretory epithelium exhibited degenerative changes but the alveolar structure was not lost. In severe cases, marked intra-alveolar infiltration of neutrophils and small areas of necrosis were observed and interacinar oedema was evident. There was cell debris in the alveoli, interalveolar tissue and teat duct. On bacteriological examination, C coliwas reisolated from 14 of 20 glands (R4), and from seven of 20 livers of mice infected with strain SM46. C coli could not be isolated from any of the mammary glands or rivers of mice infected with strains SB58 and TB231.
Although experimental infection of the bovine mammary gland with campylobacters has produced clinical mastitis, problems of management and cost cause difficulties in the use of cows for the study of bovine mastitis. The authors therefore used the mouse as a model. The results of these experiments indicate that C coli can establish an infection in the mammary gland of the mouse and produce mastitis with a comparatively small number of organisms. In this study C coli isolates from three sources were used for comparison. SM46 isolated from a case of mastitis caused mild to severe gross and histological changes in most of the glands whereas SB58 and TB231 did not produce any lesion except for mild histological changes in two glands. Severe histopathological changes appeared to be associated with the ability of the organism to multiply in the mammary gland; SM46 could be reisolated from most of the glands whereas SB58 and TB231 could not. In a previous study (Diker et al 1987a), the in vitro survival of strains SM46 and TB231 in bovine milk was compared and it was found that SM46 survived significantly longer than TB231 at 37°C. All these findings indicate that strains of C coli can differ in their effect on the mammary gland of the mouse in that SM46 is more virulent than SB58 or TB231. Therefore, it may be speculated that some strains of C coli, such as SM46, are adapted to colonisation of mammary glands. Histological changes observed in the mammary glands of mice in the present study resembled those seen in experimental coliform mastitis reported previously (Chandler 1970, Anderson 1979). In an experimental study, Lander and Gill (1980) also found that the clinical picture of bovine mastitis produced by C coli/jejuni resembled the mastitis caused by coliform organisms. Although no comparative data are available on the histological characteristics ofmastitis produced by C coli in cows and mice, the present findings and the literature suggest that the mouse provides a convenient model for fundamental studies on campylobacter mastitis. In particular, this mouse mastitis model may allow the identification of pathogenic campylobacter strains which can cause mastitis in cows. References ANDERSON,J. C. (1972)The effectof teat damageon the incidence ofexperimentalmastitisinthemouse.Researchin VeterinaryScience 13, 390-392 ANDERSON, J. C. (1979) The epidemiologyand pathogenesisof experimentalstaphylococcaland coliformmastitisin the mouse. British VeterinaryJournal 135, 163-171 BRAMLEY, A. J. & FOSTER, R. (1990) Effect of lysostaphinon Staphylococcus aureus infectionsof the mouse mammarygland. Research in VeterinaryScience 49, 120-121 CHANDLER, R. L. (1970) Experimentalbacterialmastitisin the mouse. Journal of Medical Microbiology 3, 273-282 DIKER, K. S., ERDEGER, J. & ARDA, M. (1987a) A study on the survivalof Campylobaetercoliin rawmilk.MikrobiyolojiBulteni 21, 200-205
Experimental C coli mastit& in mice DIKER, K. S., ERDEGER, J., KESKINTEPE, L. & ARDA, M. (1987b) Isolation of Campylobacter coli from a cow with mastitis. Ankara Oniversitesi Veteriner Fakultersi 34, 500-502 ISMAIL, M., HATEM, M. E. & EL-SEEDY, F. R. (1988) Campylobacter organisms as a bacterial cause ofmastitis in Egyptian cattle and buffaloes. Veterinary Medical Journal 36, 257-265 LANDER, K. P. & GILL, K. P. W. (1980) Experimental infection of the bovine udder with Campylobacter eoli/jejuni. Journal of Hygiene 84, 421-428 LOGAN, E. F., NEILL, S. D. & MACKIE, D. P. (1982) Mastitis in dairy cows associated with an aerotolerant campylobacter. Veterinary Record 110, 229-230
125
MORGAN, G., CHADWICK, P., LANDER, K. P. & GILL, K. P. W. (1985) Campylobacterjejuni mastitis in a cow: a zoonosis related incident. Veterinary Record 116, 111 SMIBERT, R. M. (1984) Bergey's Manual of Systematic Bacteriology: Campylobacter. Eds N. R. Krieg and J. G. Holt. Baltimore, Williams and Wilkins. pp 111-118
Received July 10, 1991 Accepted October 2, 1991
