Effect of Systemic BCG Infection in Syrian Golden Hamsters
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INFECTION AND IMMUNITY, JUlY 1976, p. 271-276 Vol. 14, No. 1
Copyright © 1976 American Society for Microbiology 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
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responded immunologically to purified protein derivative as assessed by in-
creased footpad swelling and splenic lymphocyte proliferation. The immediate
cause of death was a diffuse granulomatous interstitial pneumonia.
The use ofMycobacterium bovis (strain BCG) tive of tuberculin (PPD) came from Connaught Med-
has received much attention as an approach for ical Research Laboratories, Toronto, Canada.
the treatment of certain neoplastic diseases by Method of inoculation. Vials containing 1.2 x 108
immunotherapy (5, 7). We are studying the use CFU of BCG/ml were diluted with phosphate-
of BCG as an immunotherapeutic approach for buffered saline (pH 7.4) containing 0.1% gelatin.
Hamsters, 12 weeks of age, were anesthesized by
the treatment of lung cancer in Syrian golden intraperitoneal injection of sodium brevitol (Eli
hamsters. Lilly & Co., Indianapolis, Ind.) and injected with
Conflicting reports have appeared in the lit- 107, 106, 10", or 104 CFU of BCG intravenously via
erature concerning the virulence and/or toxic- the tongue vein with a 27-gauge needle.
ity of BCG in Syrian hamsters (1, 2, 3, 6). We Delayed cutaneous hypersensitivity to tubercu-
therefore decided to (i) investigate the effect lin. To assess the delayed cutaneous hypersensitiv-
this microorganism had on the survival of ham- ity reaction of hamsters infected with BCG, three
sters, (ii) investigate the histopathology after animals at each time point were injected with 25 ,.tg
infection, and (iii) assess the ability of animals of PPD into the right hind footpad. Saline injected
into the left hind footpad served as a control. After
to respond immunologically to tubercular anti- 24 h the thickness of each footpad was measured
gens. The results of this investigation indicate with a dial thickness gauge.
that (i) hamsters are capable of mounting an Lymphocyte proliferative response. Spleen cell
immune response to BCG; (ii) large numbers of suspensions were prepared from each of three ham-
microorganisms are lethal to the hamster; and sters by gently teasing the spleens with forceps to
(iii) systemic infection leads to the production release the cells. The cell suspensions were centri-
of granulomatous lesions primarily in the fuged at 1,100 x g in a PR-6000 centrifuge and
spleen, liver, and lungs. The immediate cause washed in Hanks balanced salt solution. The viabil-
of death was a diffuse granulomatous intersti- ity was determined by trypan blue exclusion, and
the cells were adjusted to 10"/ml in RPMI 1640 me-
tial pneumonia. dium (Microbiological Associates, Inc., Bethesda,
Md.) supplemented with 10% pooled human serum,
MATERIALS AND METHODS 100 U of penicillin per ml, and 100 Ag of streptomy-
Animals. 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 incuba-
ing 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 dis-
daily intermittent shaking. Purified protein deriva- solved in NCS solubilizer (Amersham/Searle Corp.,
271272 ZWILLING AND DAVIS INFECT. IMMUN.
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., 20
Inc., Downers Grove, Ill.).
Pathology. Every 14 days, three hamsters, se- z
lected at random from each of the dosage groups, 20 B
w 60 -
were killed and examined for the presence of gross -
lesions induced by the administration of BCG. In Z
a
40
addition, all dead or moribund animals were exam- Q< 20 -
ined. All tissues were fixed in 10% neutral buffered Co
formalin. Selected sections were then embedded in o 60 c
paraffin, sectioned at 6 /Lm, and stained with hema- Z 40-
toxylin and eosin. Sections of liver, lung, and spleen w
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and any gross lesions from other organs were exam- 20-
ined. Hepatic lesions were quantitated by counting
five microscopic fields in livers from three hamsters o 60 D
in each group. 40-
20
RESULTS
Survival of hamsters inoculated with BCG. 0 5 10 15 20 25 30
TIME (weeks)
Large amounts of BCG injected intravenously
were fatal to the Syrian hamsters. The median FIG. 2. Tuberculin reactivity of hamsters infected
survival time of 25 animals infected with 107 with M. bovis (strain BCG). Infection of animals
CFU of BCG was 112 days (Fig. 1). Decreasing injected with 107 CFU (A), 106 CFU (B), 105 CFU
the dose of BCG increased the survival time. (C), and 104 CFU (D).
None of the animals receiving 104 CFU of BCG
have died after 300 days of observation. PPD in comparison with that of the left hind
Delayed cutaneous hypersensitivity. Infec- footpad injected with saline. An increase of 20%
tion of hamsters with BCG sensitizes the ani- or greater was considered a positive footpad
mals to tuberculin. The data presented in Fig. 2 response. Footpad reactions of uninfected ani-
represent the percentage of increase in footpad mals were minimal, and increases in thickness
thickness of the right hind footpad injected with of less than 5% were observed in footpads in-
jected 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. Ham-
sters injected with 10" CFU of BCG had positive
footpad reactivity 5 weeks after infection, and
200 those infected with 105 CFU had positive reac-
tions at 6 weeks, whereas animals receiving 104
n
CFU did not become positive to tuberculin until
w
the 8th week. Animals in the latter three
groups remained positive until the 19th week
and eventually became positive again at the
100 29th week.
Effect of BCG on the lymphoproliferative
response to PPD. The lymphoproliferative re-
sponse to PPD of spleen cells from hamsters
infected with BCG was evaluated over a 6-
month period. The results in Fig. 3 represent
the response of spleen cells stimulated by 25 ,g
of PPD. The data are expressed as the stimula-
tion 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 observedVOL. 14, 1976 BCG INFECTION IN HAMSTERS 273
until the 17th week postinfection. Cells from 40 * 107cfu BCG
animals receiving 10O or 104 CFU of BCG were A 106 cfu BCG
stimulated to undergo blastogenesis by PPD a 10 5cf u BCG
within 3 weeks. Responses continued to be ob- 35 - Ocfu BOG
served throughout the study. Generally cells
incubated in the presence of PPD were stimu- X
lated 5 to 15 times more than were control cells o3 30 -
incubated without PPD. Cells from uninfected \
animals were not stimulated by PPD.
Pathology. Granulomatous lesions were 0 25 -
present in multiple tissues of all the infected \
hamsters. Since lesions were consistently pres- I
ent in lung and liver, these organs were se- E 20 -
lected as sites to quantitate the morphological 0
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response. In general, the frequency and size of m <
the lesions decreased with dose of organisms s 155
and time. Figure 4 compares the mean number z
of hepatic granuloma of each inoculum group. z
More severe lesions developed, and developed w IV
earlier, in animals given 107 and 106 CFU of 2 I
BCG than in animals given 106 or 104 CFU of
BCG. Similarly, the size of individual lesions 5
was greater with greater doses of BCG.
20 o I
A 0 5 l0 15 20 25
TIME (weeks)
10
FIG. 4. Effect of dose of M. bovis (strain BCG) on
o _ / 4
the number of hepatic granulomas.
20 B
The morphological characteristics of the le-
'- sions also varied with the dose of BCG. Tissues
/ from hamsters immunized with 1O' BCG orga-
E o J nisms contained numerous, sometimes coalesc-
u
30 c ing, granulomas that replaced up to one-half of
the normal parenchyma (Fig. 5A). These were
ffi 20 _ densely cellular containing primarily viable
_Q 4macrophages with a few scattered lymphocytes.
x i \ Specific granulomas were sometimes sur-
0 l\ rounded by a well-defined zone of immuno-
z cytes, i.e., lymphocytes and plasma cells. Those
0 hamsters that died typically had a diffuse gran-
z30 D X ulomatous interstitial pneumonia.
E /\ The cellularity or reactivity of the lesions in
LO 20 / \hamsters given the lower doses of BCG was less
20 g \ than in hamsters given the larger doses. This
was especially true in animals killed near the
IO - / \;/ w \ , end of the experiment. Figures 5B and C illus-
trate the extent and morphological characteris-
tics of the scattered pulmonary granulomas in
0( 5 l hamsters given low doses of BCG. These indi-
TIME AFTER INFECTION (weeks) vidual lesions were circumscribed and poorly
cellular, and contained primarily macrophages.
FIG. 3. Response to 25 pg of PPD of splenic lym- These lesions were primarily located in clear-
phocytes obtained from hamsters infected with M.
bovis (strain BCG). Infection of animals injecteda
with: (A) 107 CFU; (B) 106 CFU; (C) 10 CFU; (D) subpleural regions. Little or no diffuse intersti-
104 CFU. tial pneumonia was noted. The hepatic and274 ZWILLING AND DAVIS INFECT. IMMUN.
'V
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P
'.- . s\c%4 te
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_' 't
-b 7 * .VOL. 14, 1976 BCG INFECTION IN HAMSTERS 275
minimal loss of function. These animals sur- animals. This observation has been made in
vived well, and secondary lesions were not sig- several laboratories (1, 2, 3, 6); however, there
nificant. is no agreement as to the cause of death. Hau-
duroy and Rosset (1, 2) first reported that the
DISCUSSION Syrian golden hamster was sensitive to BCG
Systemic infection of Syrian golden hamsters and concluded that death was caused by a pro-
with large doses of BCG leads to death of the gressive tuberculosis infection. Van Deinse and
4-
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.r
.4V
,
p.
- "'at-
Pr.s*
L
. .^ B,
I.
. N W+.
V.
:~~~~~~~~~~~i
",'"','ff'''D-'',A'v''i D.
44$
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FIG. 6. The microscopic appearance of the livers from hamsters 7 weeks after exposure to 1 0' (A), 106 (B),
10:4(C), or 104 (D) BCG organisms. Decreasing the dose of organisms decreased the number and size of hepatic
granulomas (G). There was also a decrease in the number of lymphocytes associated with the hypersensitivity
granulomas (arrows). The dark black foci within the lesions represent phagocytized organisms. Hematoxylin
and eosin stain. x50.276 ZWILLING AND DAVIS INFECT. IMMUN.
Sen6chal (6) demonstrated that large doses of The lymphoproliferative response of hamster
heat-killed BCG or tuberculin caused fatal dis- splenic lymphocytes immune to BCG was deter-
ease in hamsters and concluded that animals mined. Cells from animals receiving 107 and 10"
injected with living BCG died as a result of CFU exhibited little stimulation when incu-
intoxication from bacterial metabolic products. bated with PPD. This was probably due to the
Our results indicate that animals infected sys- generalized stimulation of the immune'system
temically with large numbers of BCG die as a by BCG, which resulted in a high degree of
result of a diffuse granulomatous interstitial splenomegaly in these animals. Histologically,
pneumonia, whereas animals infected with the splenomegaly was due to reticuloendothe-
lower doses localize the granuloma and survive. lial and parafollicular cell hyperplasia. Similar
The lesions we observed were granulomas of observations have been reported by Mackaness
the delayed hypersensitivity type as previously et al. (4) in mice.
reported (3). The lesions were markedly cellu- Hamsters are particularly susceptible to
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lar with large numbers of viable macrophages BCG infection. This offers the opportunity to
and lymphocytes. Neither caseous necrosis nor study both acute and chronic disease in the
any other indication of intoxication was pres- same animal model system. Van Deinse and
ent. There was a partial resolution of hepatic Sen6chal (6) demonstrated that large doses of
lesions in animals receiving the high doses of BCG or tuberculin resulted in toxicity with
BCG. We also have not observed any deaths in acute lesions of local edema, necrosis, and exu-
animals inoculated with nonviable BCG cell dative suppurative inflammation. By lowering
walls, although these animals became sensi- the dose of organisms we can modify survival,
tized to tuberculin (unpublished observations). mortality, and the morphological lesions. Ham-
The survival of the animals was inversely sters do not usually develop significant sponta-
proportional to the dose of BCG. Jespersen and neous pulmonary infections and therefore lend
Bentzon (3) reported similar findings using a themselves to studies designed to elucidate al-
strain of BCG designated Dubos Philadelphia veolar inflammatory processes.
by the authors. This strain is also referred to as ACKNOWLEDGMENTS
the Phipps strain. The authors reported that This investigation was supported by Public Health Serv-
the median survival time of hamsters receiving ice grant CA-16342 from the National Cancer Institute.
an intraperitoneal injection of 6 x 107 units was We wish to thank Laura Campolito for her excellent
141 days. In our studies, animals receiving 107 technical assistance.
CFU had a median survival time of 115 days. LITERATURE CITED
Jespersen and Bentzon reported that animals 1. Hauduroy, P., and W. Rosset. 1951. Sensibilite du ham-
which received 6 x 106 units survived longer ster au bacille BCG. Presse Med. 59:121.
than 365 days. In contrast, we found that ani- 2. Hauduroy, P., and W. Rosset. 1963. Dauze aus
mals receiving 10" CFU had a median survival d'experimentation sur l'infection des hamsters par le
BCG. Ann Inst. Pasteur Paris 104:131-132.
time of 140 days. No other comparable data are 3. Jespersen, A., and M. W. Bentzon. 1964. The virulence
available. In our study, all hamsters receiving of various strains of BCG determined on the golden
104 CFU of BCG survived. hamster. Acta Tuberc. Pneumol. Scand. 44:222-249.
Hamsters infected with the several doses of 4. Mackaness, G. B., D. J. Duclair, and P. H. Lagrange.
1973. Immunopotentiation with BCG. I. Immune re-
BCG exhibited positive reactions to PPD in- sponse to different strains and preparations. J. Natl.
jected into the footpad within 8 weeks after Cancer Inst. 51:165-1667.
infection. Van Deinse and Senechal (6) stated 5. Morton, D. L., F. R. Eilber, R. A. Malmgren, and W.
that Hussel found that hamsters inoculated C. Wood. 1970. Immunological factors which influ-
ence response to immunotherapy of malignant mela-
with 2 x 106 tubercle bacilli reacted positively noma. Surgery 68:158-164.
to tuberculin after 24 days of infection. In con- 6. Van Deinse, F., and R. Senechal. 1954. Le BCG est-el
trast they also reported that Murohashi and verulent pour le hamster dore de syrie? Ann Inst.
Yaganisawa (see reference 6) were not able to Pasteur Paris 87:117-130.
7. Zbar, B., and T. Tanaka. 1971. Immunotherapy of can-
detect tuberculin reactivity in hamsters receiv- cer: regression of tumors after intralesional injection
ing 10 mg of BCG 6 and 12 weeks previously. of living Mycobacterium bovis. Science 172:271-273.You can also read
