I.
The Bovine Alveolar Macrophage In vitro Studies with Pasteurella haemolytica M. L. Benson, R. G. Thomson and V. E. 0. VaIIi* ABSTRACT
Bovine alveolar macrophages were cultured in vitro and challenged with suspensions of live and dead bacteria. These cells showed severe cytotoxic morphological changes and a low rate of phagocytosis after exposure to live Pasteurella haemolytica type I whereas heat killed P. haemolytica type I was readily phagocytosed and produced only mild cytotoxic changes.
RESUME
Cette experience visait a cultiver, in vitro, des macrophages alveolaires bovins et a les mettre ensuite en contact avec des suspensions de bacteries vivantes ou mortes. Ces macrophages manifesterent des changements morphologiques cytotoxiques marques et une faible activit4 phagocytaire, lorsqu'ils vinrent en contact avec une souche vivante de Pasteurella haemolytica du type I. Par ailleurs cette meme bacterie, prealablement tue ae l' aide de la chaleur, subit une phagocytose rapide et ne provoqua que de minimes changements cytotoxiques.
A previous report described the isolation,
in vitro culture, ultrastructure and phagocytic function of bovine alveolar macrophages (4). This background was intended to support in vitro studies of the interaction of Pasteurella haemolytica and bovine alveolar macrophages (BAM) as part of an ongoing investigation of the pathogenesis of pneumonic pasteurellosis in cattle (5) in which alveolar macrophages are a prominent component of the microscopic lesion
(3). *Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ontario NlG 2W1. Submitted November 15, 1976.
368
Cultures of alveolar macrophages were prepared as described previously (4). Calves from which macrophages were collected varied in age from two to 26 weeks. One 18 hr colony of P. haemolytica type I from a calf blood agar plate was added to 50 ml of brain heart infusion broth1 and was grown for 4.5 hr in a 250 ml Erlenmeyer flask in a shaking water bath at 370C. The culture was centrifuged at 10,000 g for ten minutes, washed twice in Hanks balanced salt solution (HBSS) and resuspended in 20 ml of HBSS. This was diluted to the desired concentration, generally 2 x 108 colony forming units per ml in medium 199 (Hanks base)2 which contained 10 to 20% fetal calf serum (FCS). A culture was prepared in 20 ml of HBSS as above and then heated at 60°C for 30 minutes to kill the bacteria. This was diluted in medium 199 and FCS to yield a suspension corresponding in total number of bacteria to the live suspension. The following live cultures were tested: P. haemolytica types I, II, VII and XII, P. multocida type B, P. multocida bovine lung (untyped), Serratia marsescens, Listeria monocytogenes and Staphylococcus aureus FDA 209P type 420. To infect the alveolar macrophages, 1.5 ml of the final bacterial suspension was added to Leighton tubes (in duplicate) after the tissue culture medium was decanted. Infected cultures were incubated at 37°C for 30 or 45 min and then fixed and stained. Appropriate controls were tested simultaneously. Coverslips were rinsed in HBSS and fixed for five minutes in methanol for May-Grunwald Giemsa (MGG) stain or overnight in 95% ethanol for H and E stain. An estimate of cellular toxicity was made based on cellular rounding (withdrawal of pseudopodia), nuclear condensation, nuclear membrane irregularities and decrease in cell numbers compared to controls. A range of 1+ to 2+ Becton 1BBL Ontario.
Dickinson
and Co.,
Canada Ltd.,
Clarkson,
2Microbiological Associates, Bethesda, Maryland.
Can. J. comp. Med.
to 3+ was used to grade the toxicity from mild to moderate to severe. In MGG-stained preparations of cells infected with live P. haemolytica type I, there was a very low rate of phagocytosis < 20% and a toxicity grade of 2+ or 3+ (moderate or severe) but those challenged with dead P. haemolytica type I had a high rate of phagocytosis (>89%) and a toxicity grade of 1 + (mild). When cells of calves more than 16 wk of age were infected with live P. haemolytica type I under similar conditions the rate of phagocytosis was 25-75% but the toxicity was never greater than 1+. When infected with four serotypes of live P. haemolytica type I, the rate of phagocytosis by BAM varied from 16 wk old may be related to phagocytic ability alone
Volume 42
-
July, 1978
or may involve different susceptibilities to the toxic factor in cells from different age groups. Cells from the two age groups were not tested simultaneously although the results for each age group were reproduced at several times during the study. To more accurately assess the toxic effects of P. haemolytica type I it will be necessary to use the more refined functional and biochemical tests which are available to measure the responses of the alveolar macrophages and to purify the toxic principle present in the filtrates of the bacterial cultures.
ACKNOWLEDGMENTS The authors acknowledge the technical assistance of Mrs. Heather Rand. The senior author wishes to thank Dr. H. Vance and Dr. R. Christian of the Veterinary Services Division, Alberta Department of Agriculture, for their cooperation in making available to her the facilities of the 0. S. Longman Laboratory, Edmonton, Alberta. The work was supported by National Research Council of Canada grant A5770 and by the Ontario Ministry of Agriculture and Food.
REFERENCES
1. BIBERSTEIN, E. L., M. E. MYER and P. C. KENNEDY. Colonial variation of Pasteurella haemolytica isolated from sheep. J. Bact. 76: 445-452. 1958. 2. BIBERSTEIN, E. L., M. GILLS and H. KNIGHT. Serological types of Pasteurella haemolytica. Cornell Vet. 50: 283-300. 1960. 3. FRIEND, S. C., R. G. THOMSON and B. N. WILKIE. Pulmonary lesions induced by Pasteurella hemolytica in cattle. Can. J. comp. Med. 41: 219-223. 1977. 4. FOX, M. L. The bovine alveolar macrophage. I.Isolation, in vitro cultivation, ultrastructure and phagocytosis. Can. J. Microbiol. 19: 1207-1210. 1973. 5. THOMSON, R. G., S. CHANDER, M. SAVAN and M. L. FOX. Investigations of factors of probable significance in the pathogenesis of pneumonic pasteurellosis of cattle. Can. J. comp. Med. 39: 194-207. 1975.
369
The Bovine Alveolar Macrophage In vitro Studies with Pasteurella haemolytica M. L. Benson, R. G. Thomson and V. E. 0. VaIIi* ABSTRACT
Bovine alveolar macrophages were cultured in vitro and challenged with suspensions of live and dead bacteria. These cells showed severe cytotoxic morphological changes and a low rate of phagocytosis after exposure to live Pasteurella haemolytica type I whereas heat killed P. haemolytica type I was readily phagocytosed and produced only mild cytotoxic changes.
RESUME
Cette experience visait a cultiver, in vitro, des macrophages alveolaires bovins et a les mettre ensuite en contact avec des suspensions de bacteries vivantes ou mortes. Ces macrophages manifesterent des changements morphologiques cytotoxiques marques et une faible activit4 phagocytaire, lorsqu'ils vinrent en contact avec une souche vivante de Pasteurella haemolytica du type I. Par ailleurs cette meme bacterie, prealablement tue ae l' aide de la chaleur, subit une phagocytose rapide et ne provoqua que de minimes changements cytotoxiques.
A previous report described the isolation,
in vitro culture, ultrastructure and phagocytic function of bovine alveolar macrophages (4). This background was intended to support in vitro studies of the interaction of Pasteurella haemolytica and bovine alveolar macrophages (BAM) as part of an ongoing investigation of the pathogenesis of pneumonic pasteurellosis in cattle (5) in which alveolar macrophages are a prominent component of the microscopic lesion
(3). *Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ontario NlG 2W1. Submitted November 15, 1976.
368
Cultures of alveolar macrophages were prepared as described previously (4). Calves from which macrophages were collected varied in age from two to 26 weeks. One 18 hr colony of P. haemolytica type I from a calf blood agar plate was added to 50 ml of brain heart infusion broth1 and was grown for 4.5 hr in a 250 ml Erlenmeyer flask in a shaking water bath at 370C. The culture was centrifuged at 10,000 g for ten minutes, washed twice in Hanks balanced salt solution (HBSS) and resuspended in 20 ml of HBSS. This was diluted to the desired concentration, generally 2 x 108 colony forming units per ml in medium 199 (Hanks base)2 which contained 10 to 20% fetal calf serum (FCS). A culture was prepared in 20 ml of HBSS as above and then heated at 60°C for 30 minutes to kill the bacteria. This was diluted in medium 199 and FCS to yield a suspension corresponding in total number of bacteria to the live suspension. The following live cultures were tested: P. haemolytica types I, II, VII and XII, P. multocida type B, P. multocida bovine lung (untyped), Serratia marsescens, Listeria monocytogenes and Staphylococcus aureus FDA 209P type 420. To infect the alveolar macrophages, 1.5 ml of the final bacterial suspension was added to Leighton tubes (in duplicate) after the tissue culture medium was decanted. Infected cultures were incubated at 37°C for 30 or 45 min and then fixed and stained. Appropriate controls were tested simultaneously. Coverslips were rinsed in HBSS and fixed for five minutes in methanol for May-Grunwald Giemsa (MGG) stain or overnight in 95% ethanol for H and E stain. An estimate of cellular toxicity was made based on cellular rounding (withdrawal of pseudopodia), nuclear condensation, nuclear membrane irregularities and decrease in cell numbers compared to controls. A range of 1+ to 2+ Becton 1BBL Ontario.
Dickinson
and Co.,
Canada Ltd.,
Clarkson,
2Microbiological Associates, Bethesda, Maryland.
Can. J. comp. Med.
to 3+ was used to grade the toxicity from mild to moderate to severe. In MGG-stained preparations of cells infected with live P. haemolytica type I, there was a very low rate of phagocytosis < 20% and a toxicity grade of 2+ or 3+ (moderate or severe) but those challenged with dead P. haemolytica type I had a high rate of phagocytosis (>89%) and a toxicity grade of 1 + (mild). When cells of calves more than 16 wk of age were infected with live P. haemolytica type I under similar conditions the rate of phagocytosis was 25-75% but the toxicity was never greater than 1+. When infected with four serotypes of live P. haemolytica type I, the rate of phagocytosis by BAM varied from 16 wk old may be related to phagocytic ability alone
Volume 42
-
July, 1978
or may involve different susceptibilities to the toxic factor in cells from different age groups. Cells from the two age groups were not tested simultaneously although the results for each age group were reproduced at several times during the study. To more accurately assess the toxic effects of P. haemolytica type I it will be necessary to use the more refined functional and biochemical tests which are available to measure the responses of the alveolar macrophages and to purify the toxic principle present in the filtrates of the bacterial cultures.
ACKNOWLEDGMENTS The authors acknowledge the technical assistance of Mrs. Heather Rand. The senior author wishes to thank Dr. H. Vance and Dr. R. Christian of the Veterinary Services Division, Alberta Department of Agriculture, for their cooperation in making available to her the facilities of the 0. S. Longman Laboratory, Edmonton, Alberta. The work was supported by National Research Council of Canada grant A5770 and by the Ontario Ministry of Agriculture and Food.
REFERENCES
1. BIBERSTEIN, E. L., M. E. MYER and P. C. KENNEDY. Colonial variation of Pasteurella haemolytica isolated from sheep. J. Bact. 76: 445-452. 1958. 2. BIBERSTEIN, E. L., M. GILLS and H. KNIGHT. Serological types of Pasteurella haemolytica. Cornell Vet. 50: 283-300. 1960. 3. FRIEND, S. C., R. G. THOMSON and B. N. WILKIE. Pulmonary lesions induced by Pasteurella hemolytica in cattle. Can. J. comp. Med. 41: 219-223. 1977. 4. FOX, M. L. The bovine alveolar macrophage. I.Isolation, in vitro cultivation, ultrastructure and phagocytosis. Can. J. Microbiol. 19: 1207-1210. 1973. 5. THOMSON, R. G., S. CHANDER, M. SAVAN and M. L. FOX. Investigations of factors of probable significance in the pathogenesis of pneumonic pasteurellosis of cattle. Can. J. comp. Med. 39: 194-207. 1975.
369
