INFECTION AND IMMUNITY, JUlY 1976, p. 271-276 Copyright © 1976 American Society for Microbiology
Vol. 14, No. 1 Printed in U.S.A.
Effect of Systemic BCG Infection in Syrian Golden Hamsters BRUCE S. ZWILLING* AND GARY W. DAVIS Departments of Microbiology,* College of Biological Sciences and Veterinary Pathobiology, Ohio State University, Columbus, Ohio 43210
Received for publication 23 March 1976
The response of Syrian golden hamsters to systemic infection with several doses of Mycobacterium bovis (strain BCG) was assessed. Large numbers of organisms (107), injected intravenously, were lethal for hamsters, whereas all animals survived infection with 104 colony-forming units of BCG. Animals responded immunologically to purified protein derivative as assessed by increased footpad swelling and splenic lymphocyte proliferation. The immediate cause of death was a diffuse granulomatous interstitial pneumonia. tive of tuberculin (PPD) came from Connaught Medical Research Laboratories, Toronto, Canada. Method of inoculation. Vials containing 1.2 x 108 CFU of BCG/ml were diluted with phosphatebuffered saline (pH 7.4) containing 0.1% gelatin. Hamsters, 12 weeks of age, were anesthesized by intraperitoneal injection of sodium brevitol (Eli Lilly & Co., Indianapolis, Ind.) and injected with 107, 106, 10", or 104 CFU of BCG intravenously via the tongue vein with a 27-gauge needle. Delayed cutaneous hypersensitivity to tuberculin. To assess the delayed cutaneous hypersensitivity reaction of hamsters infected with BCG, three animals at each time point were injected with 25 ,.tg of PPD into the right hind footpad. Saline injected into the left hind footpad served as a control. After 24 h the thickness of each footpad was measured with a dial thickness gauge. Lymphocyte proliferative response. Spleen cell suspensions were prepared from each of three hamsters by gently teasing the spleens with forceps to release the cells. The cell suspensions were centrifuged at 1,100 x g in a PR-6000 centrifuge and washed in Hanks balanced salt solution. The viability was determined by trypan blue exclusion, and the cells were adjusted to 10"/ml in RPMI 1640 medium (Microbiological Associates, Inc., Bethesda, Md.) supplemented with 10% pooled human serum, MATERIALS AND METHODS 100 U of penicillin per ml, and 100 Ag of streptomyAnimals. Male Syrian golden hamsters were ob- cin per ml. One milliliter of the cell suspension was tained from Sprague Dawley, Inc., Madison, Wis. added to test tubes (no. 2063, measuring 12 by 75 Animals were housed five per cage and given food mm; Falcon Plastics, Oxnard, Calif.). Preliminary and water ad libitum. experiments indicated that optimum stimulation of Mycobacterial antigens. Phipps strain M. bovis, hamster spleen cells by PPD occurred at 3 days. strain BCG (TMC no. 1029), was obtained from the Cultures therefore were incubated for 72 h in the Trudeau Institute, Saranac Lake, N.Y. This prepa- presence or absence of 25 jig of PPD in a humidified ration containing 6.2 x 107 unsonicated colony-form- atmosphere containing 5% CO2. After a 48-h incubaing units (CFU) or 1.2 x 108 sonicated units in 1.0 ml tion, cultures were pulsed with 2.5 yCi of tritiated of Middlebrook 7H9 medium was stored at -70 C thymidine ([3H]TdR) (specific activity, 1.9 Ci/mmol; until use. The number of unsonicated viable units Schwarz/Mann, Orangeburg, N.Y.) and incubated corresponded with the total number of units. The for an additional 24 h. After the 72-h incubation the vaccine as prepared by the Trudeau Institute was cultures were centrifuged at 2,300 x g, precipitated, derived from log-phase cultures grown in Middle- and washed three times with 5% trichloroacetic brook 7H9 medium as submerged cultures with acid. The trichloroacetic acid precipitates were disdaily intermittent shaking. Purified protein deriva- solved in NCS solubilizer (Amersham/Searle Corp., 271
The use ofMycobacterium bovis (strain BCG) has received much attention as an approach for the treatment of certain neoplastic diseases by immunotherapy (5, 7). We are studying the use of BCG as an immunotherapeutic approach for the treatment of lung cancer in Syrian golden hamsters. Conflicting reports have appeared in the literature concerning the virulence and/or toxicity of BCG in Syrian hamsters (1, 2, 3, 6). We therefore decided to (i) investigate the effect this microorganism had on the survival of hamsters, (ii) investigate the histopathology after infection, and (iii) assess the ability of animals to respond immunologically to tubercular antigens. The results of this investigation indicate that (i) hamsters are capable of mounting an immune response to BCG; (ii) large numbers of microorganisms are lethal to the hamster; and (iii) systemic infection leads to the production of granulomatous lesions primarily in the spleen, liver, and lungs. The immediate cause of death was a diffuse granulomatous interstitial pneumonia.
272
ZWILLING AND DAVIS
Des Plaines, Ill.), transferred into scintillation vials containing 10 ml of toluene-Liquiflor scintillation fluid (New England Nuclear Corp., Boston, Mass.), and assessed for radioactivity in a Packard liquid scintillation spectrometer (Packard Instrument Co., Inc., Downers Grove, Ill.). Pathology. Every 14 days, three hamsters, selected at random from each of the dosage groups, were killed and examined for the presence of gross lesions induced by the administration of BCG. In addition, all dead or moribund animals were examined. All tissues were fixed in 10% neutral buffered formalin. Selected sections were then embedded in paraffin, sectioned at 6 /Lm, and stained with hematoxylin and eosin. Sections of liver, lung, and spleen and any gross lesions from other organs were examined. Hepatic lesions were quantitated by counting five microscopic fields in livers from three hamsters in each group.
RESULTS Survival of hamsters inoculated with BCG. Large amounts of BCG injected intravenously were fatal to the Syrian hamsters. The median survival time of 25 animals infected with 107 CFU of BCG was 112 days (Fig. 1). Decreasing the dose of BCG increased the survival time. None of the animals receiving 104 CFU of BCG have died after 300 days of observation. Delayed cutaneous hypersensitivity. Infection of hamsters with BCG sensitizes the animals to tuberculin. The data presented in Fig. 2 represent the percentage of increase in footpad thickness of the right hind footpad injected with
INFECT. IMMUN.
20
z
20
B
w 60
-
40
-
Z
a
Q< 20 Co
-
o 60 c Z 40-
w 20-
o 60 D 4020
0
5
10
15
20
25
30
TIME (weeks)
FIG. 2. Tuberculin reactivity of hamsters infected with M. bovis (strain BCG). Infection of animals injected with 107 CFU (A), 106 CFU (B), 105 CFU (C), and 104 CFU (D).
PPD in comparison with that of the left hind footpad injected with saline. An increase of 20% or greater was considered a positive footpad response. Footpad reactions of uninfected animals were minimal, and increases in thickness of less than 5% were observed in footpads injected with PPD. Animals infected with 107 300 CFU of BCG had a positive reaction to PPD when first tested 3 weeks after infection. The response remained positive, increasing to 48% by 8 weeks and then gradually declining. Hamsters injected with 10" CFU of BCG had positive footpad reactivity 5 weeks after infection, and 200 those infected with 105 CFU had positive reactions at 6 weeks, whereas animals receiving 104 CFU did not become positive to tuberculin until n the 8th week. Animals in the latter three groups remained positive until the 19th week and eventually became positive again at the 29th week. 100 Effect of BCG on the lymphoproliferative response to PPD. The lymphoproliferative response to PPD of spleen cells from hamsters infected with BCG was evaluated over a 6month period. The results in Fig. 3 represent the response of spleen cells stimulated by 25 ,g of PPD. The data are expressed as the stimulation index or counts per minute in the presence BCG COLONY FORMING UNITS INJECTED of PPD divided by counts per minute obtained FIG. 1. Median survival time of hamsters infected in the absence of PPD. Little stimulation of with M. bovis (strain BCG). Vertical lines represent spleen cells obtained from animals infected the range. with 107 and 106 CFU of BCG was observed w
BCG INFECTION IN HAMSTERS
VOL. 14, 1976
until the 17th week postinfection. Cells from animals receiving 10O or 104 CFU of BCG were stimulated to undergo blastogenesis by PPD within 3 weeks. Responses continued to be observed throughout the study. Generally cells incubated in the presence of PPD were stimulated 5 to 15 times more than were control cells incubated without PPD. Cells from uninfected animals were not stimulated by PPD. Pathology. Granulomatous lesions were present in multiple tissues of all the infected hamsters. Since lesions were consistently present in lung and liver, these organs were selected as sites to quantitate the morphological response. In general, the frequency and size of the lesions decreased with dose of organisms and time. Figure 4 compares the mean number of hepatic granuloma of each inoculum group. More severe lesions developed, and developed earlier, in animals given 107 and 106 CFU of BCG than in animals given 106 or 104 CFU of BCG. Similarly, the size of individual lesions was greater with greater doses of BCG. 20
* 107cfu BCG A 106 cfu BCG a 10 5cf u BCG
40
o3
35
-
30
-
Ocfu BOG X \
0
25
-
\ I E
20
-
0
m s
Vol. 14, No. 1 Printed in U.S.A.
Effect of Systemic BCG Infection in Syrian Golden Hamsters BRUCE S. ZWILLING* AND GARY W. DAVIS Departments of Microbiology,* College of Biological Sciences and Veterinary Pathobiology, Ohio State University, Columbus, Ohio 43210
Received for publication 23 March 1976
The response of Syrian golden hamsters to systemic infection with several doses of Mycobacterium bovis (strain BCG) was assessed. Large numbers of organisms (107), injected intravenously, were lethal for hamsters, whereas all animals survived infection with 104 colony-forming units of BCG. Animals responded immunologically to purified protein derivative as assessed by increased footpad swelling and splenic lymphocyte proliferation. The immediate cause of death was a diffuse granulomatous interstitial pneumonia. tive of tuberculin (PPD) came from Connaught Medical Research Laboratories, Toronto, Canada. Method of inoculation. Vials containing 1.2 x 108 CFU of BCG/ml were diluted with phosphatebuffered saline (pH 7.4) containing 0.1% gelatin. Hamsters, 12 weeks of age, were anesthesized by intraperitoneal injection of sodium brevitol (Eli Lilly & Co., Indianapolis, Ind.) and injected with 107, 106, 10", or 104 CFU of BCG intravenously via the tongue vein with a 27-gauge needle. Delayed cutaneous hypersensitivity to tuberculin. To assess the delayed cutaneous hypersensitivity reaction of hamsters infected with BCG, three animals at each time point were injected with 25 ,.tg of PPD into the right hind footpad. Saline injected into the left hind footpad served as a control. After 24 h the thickness of each footpad was measured with a dial thickness gauge. Lymphocyte proliferative response. Spleen cell suspensions were prepared from each of three hamsters by gently teasing the spleens with forceps to release the cells. The cell suspensions were centrifuged at 1,100 x g in a PR-6000 centrifuge and washed in Hanks balanced salt solution. The viability was determined by trypan blue exclusion, and the cells were adjusted to 10"/ml in RPMI 1640 medium (Microbiological Associates, Inc., Bethesda, Md.) supplemented with 10% pooled human serum, MATERIALS AND METHODS 100 U of penicillin per ml, and 100 Ag of streptomyAnimals. Male Syrian golden hamsters were ob- cin per ml. One milliliter of the cell suspension was tained from Sprague Dawley, Inc., Madison, Wis. added to test tubes (no. 2063, measuring 12 by 75 Animals were housed five per cage and given food mm; Falcon Plastics, Oxnard, Calif.). Preliminary and water ad libitum. experiments indicated that optimum stimulation of Mycobacterial antigens. Phipps strain M. bovis, hamster spleen cells by PPD occurred at 3 days. strain BCG (TMC no. 1029), was obtained from the Cultures therefore were incubated for 72 h in the Trudeau Institute, Saranac Lake, N.Y. This prepa- presence or absence of 25 jig of PPD in a humidified ration containing 6.2 x 107 unsonicated colony-form- atmosphere containing 5% CO2. After a 48-h incubaing units (CFU) or 1.2 x 108 sonicated units in 1.0 ml tion, cultures were pulsed with 2.5 yCi of tritiated of Middlebrook 7H9 medium was stored at -70 C thymidine ([3H]TdR) (specific activity, 1.9 Ci/mmol; until use. The number of unsonicated viable units Schwarz/Mann, Orangeburg, N.Y.) and incubated corresponded with the total number of units. The for an additional 24 h. After the 72-h incubation the vaccine as prepared by the Trudeau Institute was cultures were centrifuged at 2,300 x g, precipitated, derived from log-phase cultures grown in Middle- and washed three times with 5% trichloroacetic brook 7H9 medium as submerged cultures with acid. The trichloroacetic acid precipitates were disdaily intermittent shaking. Purified protein deriva- solved in NCS solubilizer (Amersham/Searle Corp., 271
The use ofMycobacterium bovis (strain BCG) has received much attention as an approach for the treatment of certain neoplastic diseases by immunotherapy (5, 7). We are studying the use of BCG as an immunotherapeutic approach for the treatment of lung cancer in Syrian golden hamsters. Conflicting reports have appeared in the literature concerning the virulence and/or toxicity of BCG in Syrian hamsters (1, 2, 3, 6). We therefore decided to (i) investigate the effect this microorganism had on the survival of hamsters, (ii) investigate the histopathology after infection, and (iii) assess the ability of animals to respond immunologically to tubercular antigens. The results of this investigation indicate that (i) hamsters are capable of mounting an immune response to BCG; (ii) large numbers of microorganisms are lethal to the hamster; and (iii) systemic infection leads to the production of granulomatous lesions primarily in the spleen, liver, and lungs. The immediate cause of death was a diffuse granulomatous interstitial pneumonia.
272
ZWILLING AND DAVIS
Des Plaines, Ill.), transferred into scintillation vials containing 10 ml of toluene-Liquiflor scintillation fluid (New England Nuclear Corp., Boston, Mass.), and assessed for radioactivity in a Packard liquid scintillation spectrometer (Packard Instrument Co., Inc., Downers Grove, Ill.). Pathology. Every 14 days, three hamsters, selected at random from each of the dosage groups, were killed and examined for the presence of gross lesions induced by the administration of BCG. In addition, all dead or moribund animals were examined. All tissues were fixed in 10% neutral buffered formalin. Selected sections were then embedded in paraffin, sectioned at 6 /Lm, and stained with hematoxylin and eosin. Sections of liver, lung, and spleen and any gross lesions from other organs were examined. Hepatic lesions were quantitated by counting five microscopic fields in livers from three hamsters in each group.
RESULTS Survival of hamsters inoculated with BCG. Large amounts of BCG injected intravenously were fatal to the Syrian hamsters. The median survival time of 25 animals infected with 107 CFU of BCG was 112 days (Fig. 1). Decreasing the dose of BCG increased the survival time. None of the animals receiving 104 CFU of BCG have died after 300 days of observation. Delayed cutaneous hypersensitivity. Infection of hamsters with BCG sensitizes the animals to tuberculin. The data presented in Fig. 2 represent the percentage of increase in footpad thickness of the right hind footpad injected with
INFECT. IMMUN.
20
z
20
B
w 60
-
40
-
Z
a
Q< 20 Co
-
o 60 c Z 40-
w 20-
o 60 D 4020
0
5
10
15
20
25
30
TIME (weeks)
FIG. 2. Tuberculin reactivity of hamsters infected with M. bovis (strain BCG). Infection of animals injected with 107 CFU (A), 106 CFU (B), 105 CFU (C), and 104 CFU (D).
PPD in comparison with that of the left hind footpad injected with saline. An increase of 20% or greater was considered a positive footpad response. Footpad reactions of uninfected animals were minimal, and increases in thickness of less than 5% were observed in footpads injected with PPD. Animals infected with 107 300 CFU of BCG had a positive reaction to PPD when first tested 3 weeks after infection. The response remained positive, increasing to 48% by 8 weeks and then gradually declining. Hamsters injected with 10" CFU of BCG had positive footpad reactivity 5 weeks after infection, and 200 those infected with 105 CFU had positive reactions at 6 weeks, whereas animals receiving 104 CFU did not become positive to tuberculin until n the 8th week. Animals in the latter three groups remained positive until the 19th week and eventually became positive again at the 29th week. 100 Effect of BCG on the lymphoproliferative response to PPD. The lymphoproliferative response to PPD of spleen cells from hamsters infected with BCG was evaluated over a 6month period. The results in Fig. 3 represent the response of spleen cells stimulated by 25 ,g of PPD. The data are expressed as the stimulation index or counts per minute in the presence BCG COLONY FORMING UNITS INJECTED of PPD divided by counts per minute obtained FIG. 1. Median survival time of hamsters infected in the absence of PPD. Little stimulation of with M. bovis (strain BCG). Vertical lines represent spleen cells obtained from animals infected the range. with 107 and 106 CFU of BCG was observed w
BCG INFECTION IN HAMSTERS
VOL. 14, 1976
until the 17th week postinfection. Cells from animals receiving 10O or 104 CFU of BCG were stimulated to undergo blastogenesis by PPD within 3 weeks. Responses continued to be observed throughout the study. Generally cells incubated in the presence of PPD were stimulated 5 to 15 times more than were control cells incubated without PPD. Cells from uninfected animals were not stimulated by PPD. Pathology. Granulomatous lesions were present in multiple tissues of all the infected hamsters. Since lesions were consistently present in lung and liver, these organs were selected as sites to quantitate the morphological response. In general, the frequency and size of the lesions decreased with dose of organisms and time. Figure 4 compares the mean number of hepatic granuloma of each inoculum group. More severe lesions developed, and developed earlier, in animals given 107 and 106 CFU of BCG than in animals given 106 or 104 CFU of BCG. Similarly, the size of individual lesions was greater with greater doses of BCG. 20
* 107cfu BCG A 106 cfu BCG a 10 5cf u BCG
40
o3
35
-
30
-
Ocfu BOG X \
0
25
-
\ I E
20
-
0
m s
