CURRENT ]~'IICROBIOLOGYVol. 25 (1992), pp. 95-97
Current Microbiology 9 Springer-Verlag New York Inc. 1992
Correlation Between the Virulence of Francisella tularensis in Experimental Mice and Its Acriflavine Reaction Tadashi Sato,l Hiromi Fujita] Yoshiro Ohara, 2 and Morio Homma 3 1The Laboratory of Ohara General Hospital, Omachi, Fukusbima; 2Department of Neurological Sciences, Tohoku University School of Medicine, Seiryo-Machi, Sendai; and 3Department of Microbiology, Kobe University School of Medicine, Chuo-ku, Kobe, Japan
Abstract. Correlation between the virulence of Francisella tularensis in experimental mice and its acriflavine reaction was studied. The cultures derived from all four strains (Ebina, CMB2, Schu, and N9) that had long been subcultured on agar media yielded two types of colonies, i.e., acriflavine reaction-positive (acf +) and acriflavine reaction-negative (acf-) colonies. All acf + colonies, regardless of their parent strains, were shown to be low virulent in mice. Acf- colonies were shown to be either high (Ebina, CMB2) or low (Schu, N9) virulent. The low-virulent acfcolonies gained virulence during several passages in mice, whereas the acf + colonies remained low virulent even after the animal passages.
Changes in the virulence of Francisella tularensis have been reported previously. A considerable reduction of the virulence was frequently observed during the prolonged periods of subcultivation on artificial :media [2, 6]. On the other hand, it was also reported that repeated passages through a given animal species resulted in a marked increase in the virulence in the host animal [5]. The mechanism(s) of such a change of virulence needs to be clarified. Besides, many attempts to correlate certain in vitro markers with in vivo virulence have failed so far, and no correlation has been found. Eigelsbach et al. [1] studied the corre, lation between the virulence of F. tularensis in mice and the colonial morphology as well as its agglutination in acriflavine solution (acriflavine reaction). Moody and Downs [3] found a close correlation of colonial types with acriflavine reaction and saline sensitivity, which, however, did not correlate with virulence in mice. In the present paper, we will show some correlation between the virulence of F. tularensis in mice and its acriflavine reaction, which might help to elucidate the mechanism(s) of virulence of F. tularensis.
Laboratory of Ohara General Hospital. The Schu strain, originally isolated from a human ulcer in 1941 in the United States [1], and the CMB2 strain, originally isolated from a muskrat in 1957 in Canada, were both supplied through the courtesy of Rocky Mountain Laboratory, Montana, in 1958. The N9 strain, originally isolated from a vole in 1948 in USSR, was also supplied from Gamaleya Institute, Moscow, in 1967. All of these strains have been passed every 4 weeks on LGH agar media and stored at 4~ after incubation at 37~ overnight. Virulence in mice. To examine the virulence, pathogen-free 6week-old female d d Y mice (Funabashi Farm, Shizuoka, Japan), housed in a barrier environment, were injected intraperitoneally with 0.1 ml of serial tenfold dilutions of standard suspensions of each strain. Three to five mice were tested in each experiment, and the lethal dose for 50% (LDs0) was determined on the 10th day after injection according to the method of Reed and Muench [7]. As a control, 0.1 ml of 0.01 M phosphate-buffered saline (PBS) was injected. The standard suspension was made of 10 mg (wet weight)/ml of F. tularens& in 0.01 M PBS. A 1-mg wet weight of F. tularensis was equal to 1.5-2.7 • 10 9 CFU. Acriflavine test. Acriflavine test was performed according to the method described by Eigelsbach et al [I]. The colony on LGH agar was emulsified in a drop of physiological saline on a slide to which was added a drop of 1 : 1000 neutral acriflavine (Sigma, Missouri) to detect agglutinations with ease. The results were read visually.
Materials and Methods Strains and culture conditions. Four strains ofF. tularensis, which had been maintained on pig liver glucose hemoglobin (LGH) agar media [4] for 30-40 years, were used throughout. The Ebina strain was isolated from a patient with tularemia in 1950 in the
Results Change of virulence through long-term subcultures. Table 1 shows that the virulence of all four strains
Address reprint requests to: Yoshiro Ohara, M.D., Department of Neurological Sciences, Tohoku University School of Medicine, 1-1 Seiryo-Machi, Sendai 980, Japan.
96
CURREYTMICROBIOLOGYVol. 25 (1992)
Table 1. Reduction of the virulence of Francisella tularensis in mice after subcultures on LGH agar media
Strain
At the beginning of subcultures
Ebina CMB2 Schu N9
10-8.0 a 10 .9.3 10-86 10 8.0
After a long period of subcultures 10-5.5 10 .0.2 10-1.7 10 -0.4
(444)b (372) (372) (264)
into mice. A few days after the inoculation, the organisms were recovered from their spleens or livers on Eugon agar media (Difco, Detroit) supplemented with 8% human whole blood and transferred to L G H agar. The same procedures were repeated, and LD50 was examined at each passage. As shown in Table 3, the acf- colonies of both the Schu and N9 strains gained virulence after a few passages, whereas the acf + colonies remained low-virulent even after those passages.
a LDs0for mice. b Number of subcultures.
Discussion was originally high but decreased after a long period of subcultures. The Ebina strain became moderately virulent, with LDs0 of 10 -55, but the CMB2, Schu, and N9 strains became low-virulent, with LDs0 of 10 -0.2 , 10 1.7, and 10 04, respectively.
Correlation between acriflavine reaction of colony and its virulence in mice. All four strains exclusively gave rise to two types of colonies: acriflavine testpositive (acf +) (agglutinations were easily detected visually) and -negative (acf-) (the suspension remained uniformly turbid). Then the virulence of each type of colony in mice was examined. Each colony was grown on L G H agar at 37~ for 24 h after its reaction to acriflavine was checked. The standard suspension was made of a portion of the growth, and LDs0 was determined as described previously. As shown in Table 2, acf- colonies were either highly virulent or low-virulent; all five colonies with the acf- phenotype from the Ebina and CMB2 strains were highly virulent, whereas those from the Schu and N9 were low-virulent. On the other hand, all five colonies with the acf + phenotype were shown to be low-virulent, irrespective of their parental strains. This was also the case with five acf + colonies of Ebina or CMB2 strains, obtained by recloning of the highly virulent acf- colonies of those strains (data not shown). Change of virulence of colony through passages in mice. Although all of the acf colonies derived from the low-virulent Schu and N9 strains were shown to be low-virulent (Table 2), the change in the virulence of those acf- colonies through passages in mice was examined. Both the acf- and acf + colonies derived from low-virulent Schu and N9 strains were grown on L G H agar. The standard suspensions were made in PBS as described previously, and a 0.1-ml aliquot of 10 .2 dilution of the suspension was inoculated
It has been reported that F. tularensis, as well as other certain bacterial species, decreases virulence during subcultures on artificial media [2, 6]. The present data demonstrated that acf + colonies, irrespective of their parent strains, exclusively showed low virulence (Table 2) and that any of the acf + colonies that emerged from acf- colonies were also low-virulent. In addition, the change from acf + to acf hardly occurred compared with the change from acf- to acf + on L G H agar. A phenomenon similar to the above observation was also described by Moody and Downs [3]. As acf + colonies showed low virulence without exceptions, the appearance and predominance of acf + colonies might be one of the most important factors responsible for the decrease in virulence by subcultures on L G H agar. This phenomenon is not, however, consistent with acf colonies. As shown in Table 2, all acfcolonies showed high virulence in the case of the Ebina and the CMB2 strains, but they did not in the case of the Schu and the N9 strains. In addition, the low-virulent acf colonies gained virulence during several passages in mice. In other words, the acfphenotype does not necessarily correlate with the virulence. The factors that regulate the correlation between the reaction to acriflavine and virulence still remain to be clarified. With regard to the increase or decrease of the virulence, Eigelsbach et al. [1] found that the basic mechanism underlying these changes appears to involve spontaneous occurrence of undirected variants (mutants) in growing populations, and their subsequent establishment under environmental conditions that are more favorable for the variant types than for the parent type. The results shown in Table 3 appear to support their hypothesis. A single, low-virulent colony (acf- colony of Schu or N9 strain), which could be thought to have a homogeneous population, was inoculated into mice at the initial passage, and a few highly virulent colonies
T. Sato et al.: Virulence of F. tularensis in Mice and Its Acriflavine Reaction
97
Table 2. The correlation between ac~flavine reaction and the virulence in mice ~ Strain
acf- colonies
Ebina CMB2 Schu N9
10 8.6b, 10 8.4 10 -0.5 , 10 -0.5 ,
10-8,5 10-8.5, l0 8.5, 10-8.5, 10 -0'5, 10 -05, 10 -~ 10 -t'~
10-8.6, 10-8.6, 10 -1'0, 10 15,
acf § colonies 10 8.5 10-9.0 10 -1"5 10-1.5
10-0.25, 100, 100, 10-0.25,
10-0.5, 10~ 10 -0'25, 10-025,
10-0.7, 10-1.0, 10-1.0 10~ 10 0.5, 10-0.5 10 -~ 10 -~ 10 05 10-05, 10-0.5, 10-1.0
Each colony was grown on LGH agar at 37~ for 24 h. The standard suspension was made of a portion of the confluent growth, and LDs0 was determined. h LDs0 for mice.
Table 3. The change of virulence through passages in mice a
Strain
No. of passages in mice
acfcolony
acf + colony
Schu
0
10 15
10 05h
1
10 -4'5
10 -0'5
2 3 0 1 2 3 4
10 6.5 10 8.5 10 -0.5 10 -1"~ 10 30 10 60 10 8.5
10 0.5 10 0.25 10-1~ 10.0.5 10 0.2 10 ~.2 10-0.7
N9
(~0.1 ml of 10 2dilution of the standard suspension was inoculated into mice. The organisms were recovered on Eugon agar media, and transferred to LGH agar media. After those procedures were repeated, LDs0 was determined at each passage. b LDs0 for mice.
h a v e b e e n o b t a i n e d at t h e t h i r d p a s s a g e o f t h e S c h u s t r a i n or' t h e f o u r t h p a s s a g e o f t h e N 9 s t r a i n . T h i s , therefore, indicates that a mutation and subsequent selection might have occurred during those passages. T h e p r e s e n t s t u d y m i g h t p r o v i d e s o m e c l u e s to elucidate the mechanism(s) of the virulence of F. tularensis. All four strains consist exclusively of two types of colonies. One type of colony represents the l o w v i r u l e n c e in m i c e a n d a b s o l u t e l y c o r r e l a t e s w i t h t h e a c f § p h e n o t y p e as w e l l . O n t h e o t h e r h a n d , t h e other type of colony does not necessarily correlate
w i t h t h e v i r u l e n c e a l t h o u g h it e x p r e s s e s t h e a c f phenotype constantly. Therefore, further studies s u c h as a n a l y s e s at t h e c o l o n y l e v e l w i l l b e a n a i d in delineating the mechanism(s) of the virulence of F. tularensis.
ACKNOWLEDGMENT
We are grateful to Dr. J. F. Bell for his invaluable advice and encouragement.
Literature
Cited
1. Eigelsbach HT, Braun W, Herring RT (1951) Studies on the variation of Bacterium tularense. J Bacteriol 61:557-569 2. Foshay L (1932) Induction of avirulence in Pasteurella tularensis. J Infect Dis 51:280-285 3. Moody MD, Downs CM (1955) Studies on tularemia. I. The relation between certain pathogenic and immunogenic properties of variants of Pasteurella tularensis. J Bacteriol 70:297-304 4. Ohara S, Sato M, Sato T (1953) On pig-liver medium and a modified medium for cultivation of Bacterium tularense. Tohoku J Exp Med 58:185-190 5. Philip CB, Davis GE (1935) Tularemia. Observations on a strain of low initial virulence from rabbit ticks. Public Health Rep 50:909-911 6. Ransmeier JC (1943) The reaction of the chick embryo to virulent and non-virulent strains of Bact. tularense. J Infect Dis 72:86-90 7. Reed LJ, Muench H (1938) A simple method of estimating fifty per cent endpoints. Am J Hyg 27:493-497
Current Microbiology 9 Springer-Verlag New York Inc. 1992
Correlation Between the Virulence of Francisella tularensis in Experimental Mice and Its Acriflavine Reaction Tadashi Sato,l Hiromi Fujita] Yoshiro Ohara, 2 and Morio Homma 3 1The Laboratory of Ohara General Hospital, Omachi, Fukusbima; 2Department of Neurological Sciences, Tohoku University School of Medicine, Seiryo-Machi, Sendai; and 3Department of Microbiology, Kobe University School of Medicine, Chuo-ku, Kobe, Japan
Abstract. Correlation between the virulence of Francisella tularensis in experimental mice and its acriflavine reaction was studied. The cultures derived from all four strains (Ebina, CMB2, Schu, and N9) that had long been subcultured on agar media yielded two types of colonies, i.e., acriflavine reaction-positive (acf +) and acriflavine reaction-negative (acf-) colonies. All acf + colonies, regardless of their parent strains, were shown to be low virulent in mice. Acf- colonies were shown to be either high (Ebina, CMB2) or low (Schu, N9) virulent. The low-virulent acfcolonies gained virulence during several passages in mice, whereas the acf + colonies remained low virulent even after the animal passages.
Changes in the virulence of Francisella tularensis have been reported previously. A considerable reduction of the virulence was frequently observed during the prolonged periods of subcultivation on artificial :media [2, 6]. On the other hand, it was also reported that repeated passages through a given animal species resulted in a marked increase in the virulence in the host animal [5]. The mechanism(s) of such a change of virulence needs to be clarified. Besides, many attempts to correlate certain in vitro markers with in vivo virulence have failed so far, and no correlation has been found. Eigelsbach et al. [1] studied the corre, lation between the virulence of F. tularensis in mice and the colonial morphology as well as its agglutination in acriflavine solution (acriflavine reaction). Moody and Downs [3] found a close correlation of colonial types with acriflavine reaction and saline sensitivity, which, however, did not correlate with virulence in mice. In the present paper, we will show some correlation between the virulence of F. tularensis in mice and its acriflavine reaction, which might help to elucidate the mechanism(s) of virulence of F. tularensis.
Laboratory of Ohara General Hospital. The Schu strain, originally isolated from a human ulcer in 1941 in the United States [1], and the CMB2 strain, originally isolated from a muskrat in 1957 in Canada, were both supplied through the courtesy of Rocky Mountain Laboratory, Montana, in 1958. The N9 strain, originally isolated from a vole in 1948 in USSR, was also supplied from Gamaleya Institute, Moscow, in 1967. All of these strains have been passed every 4 weeks on LGH agar media and stored at 4~ after incubation at 37~ overnight. Virulence in mice. To examine the virulence, pathogen-free 6week-old female d d Y mice (Funabashi Farm, Shizuoka, Japan), housed in a barrier environment, were injected intraperitoneally with 0.1 ml of serial tenfold dilutions of standard suspensions of each strain. Three to five mice were tested in each experiment, and the lethal dose for 50% (LDs0) was determined on the 10th day after injection according to the method of Reed and Muench [7]. As a control, 0.1 ml of 0.01 M phosphate-buffered saline (PBS) was injected. The standard suspension was made of 10 mg (wet weight)/ml of F. tularens& in 0.01 M PBS. A 1-mg wet weight of F. tularensis was equal to 1.5-2.7 • 10 9 CFU. Acriflavine test. Acriflavine test was performed according to the method described by Eigelsbach et al [I]. The colony on LGH agar was emulsified in a drop of physiological saline on a slide to which was added a drop of 1 : 1000 neutral acriflavine (Sigma, Missouri) to detect agglutinations with ease. The results were read visually.
Materials and Methods Strains and culture conditions. Four strains ofF. tularensis, which had been maintained on pig liver glucose hemoglobin (LGH) agar media [4] for 30-40 years, were used throughout. The Ebina strain was isolated from a patient with tularemia in 1950 in the
Results Change of virulence through long-term subcultures. Table 1 shows that the virulence of all four strains
Address reprint requests to: Yoshiro Ohara, M.D., Department of Neurological Sciences, Tohoku University School of Medicine, 1-1 Seiryo-Machi, Sendai 980, Japan.
96
CURREYTMICROBIOLOGYVol. 25 (1992)
Table 1. Reduction of the virulence of Francisella tularensis in mice after subcultures on LGH agar media
Strain
At the beginning of subcultures
Ebina CMB2 Schu N9
10-8.0 a 10 .9.3 10-86 10 8.0
After a long period of subcultures 10-5.5 10 .0.2 10-1.7 10 -0.4
(444)b (372) (372) (264)
into mice. A few days after the inoculation, the organisms were recovered from their spleens or livers on Eugon agar media (Difco, Detroit) supplemented with 8% human whole blood and transferred to L G H agar. The same procedures were repeated, and LD50 was examined at each passage. As shown in Table 3, the acf- colonies of both the Schu and N9 strains gained virulence after a few passages, whereas the acf + colonies remained low-virulent even after those passages.
a LDs0for mice. b Number of subcultures.
Discussion was originally high but decreased after a long period of subcultures. The Ebina strain became moderately virulent, with LDs0 of 10 -55, but the CMB2, Schu, and N9 strains became low-virulent, with LDs0 of 10 -0.2 , 10 1.7, and 10 04, respectively.
Correlation between acriflavine reaction of colony and its virulence in mice. All four strains exclusively gave rise to two types of colonies: acriflavine testpositive (acf +) (agglutinations were easily detected visually) and -negative (acf-) (the suspension remained uniformly turbid). Then the virulence of each type of colony in mice was examined. Each colony was grown on L G H agar at 37~ for 24 h after its reaction to acriflavine was checked. The standard suspension was made of a portion of the growth, and LDs0 was determined as described previously. As shown in Table 2, acf- colonies were either highly virulent or low-virulent; all five colonies with the acf- phenotype from the Ebina and CMB2 strains were highly virulent, whereas those from the Schu and N9 were low-virulent. On the other hand, all five colonies with the acf + phenotype were shown to be low-virulent, irrespective of their parental strains. This was also the case with five acf + colonies of Ebina or CMB2 strains, obtained by recloning of the highly virulent acf- colonies of those strains (data not shown). Change of virulence of colony through passages in mice. Although all of the acf colonies derived from the low-virulent Schu and N9 strains were shown to be low-virulent (Table 2), the change in the virulence of those acf- colonies through passages in mice was examined. Both the acf- and acf + colonies derived from low-virulent Schu and N9 strains were grown on L G H agar. The standard suspensions were made in PBS as described previously, and a 0.1-ml aliquot of 10 .2 dilution of the suspension was inoculated
It has been reported that F. tularensis, as well as other certain bacterial species, decreases virulence during subcultures on artificial media [2, 6]. The present data demonstrated that acf + colonies, irrespective of their parent strains, exclusively showed low virulence (Table 2) and that any of the acf + colonies that emerged from acf- colonies were also low-virulent. In addition, the change from acf + to acf hardly occurred compared with the change from acf- to acf + on L G H agar. A phenomenon similar to the above observation was also described by Moody and Downs [3]. As acf + colonies showed low virulence without exceptions, the appearance and predominance of acf + colonies might be one of the most important factors responsible for the decrease in virulence by subcultures on L G H agar. This phenomenon is not, however, consistent with acf colonies. As shown in Table 2, all acfcolonies showed high virulence in the case of the Ebina and the CMB2 strains, but they did not in the case of the Schu and the N9 strains. In addition, the low-virulent acf colonies gained virulence during several passages in mice. In other words, the acfphenotype does not necessarily correlate with the virulence. The factors that regulate the correlation between the reaction to acriflavine and virulence still remain to be clarified. With regard to the increase or decrease of the virulence, Eigelsbach et al. [1] found that the basic mechanism underlying these changes appears to involve spontaneous occurrence of undirected variants (mutants) in growing populations, and their subsequent establishment under environmental conditions that are more favorable for the variant types than for the parent type. The results shown in Table 3 appear to support their hypothesis. A single, low-virulent colony (acf- colony of Schu or N9 strain), which could be thought to have a homogeneous population, was inoculated into mice at the initial passage, and a few highly virulent colonies
T. Sato et al.: Virulence of F. tularensis in Mice and Its Acriflavine Reaction
97
Table 2. The correlation between ac~flavine reaction and the virulence in mice ~ Strain
acf- colonies
Ebina CMB2 Schu N9
10 8.6b, 10 8.4 10 -0.5 , 10 -0.5 ,
10-8,5 10-8.5, l0 8.5, 10-8.5, 10 -0'5, 10 -05, 10 -~ 10 -t'~
10-8.6, 10-8.6, 10 -1'0, 10 15,
acf § colonies 10 8.5 10-9.0 10 -1"5 10-1.5
10-0.25, 100, 100, 10-0.25,
10-0.5, 10~ 10 -0'25, 10-025,
10-0.7, 10-1.0, 10-1.0 10~ 10 0.5, 10-0.5 10 -~ 10 -~ 10 05 10-05, 10-0.5, 10-1.0
Each colony was grown on LGH agar at 37~ for 24 h. The standard suspension was made of a portion of the confluent growth, and LDs0 was determined. h LDs0 for mice.
Table 3. The change of virulence through passages in mice a
Strain
No. of passages in mice
acfcolony
acf + colony
Schu
0
10 15
10 05h
1
10 -4'5
10 -0'5
2 3 0 1 2 3 4
10 6.5 10 8.5 10 -0.5 10 -1"~ 10 30 10 60 10 8.5
10 0.5 10 0.25 10-1~ 10.0.5 10 0.2 10 ~.2 10-0.7
N9
(~0.1 ml of 10 2dilution of the standard suspension was inoculated into mice. The organisms were recovered on Eugon agar media, and transferred to LGH agar media. After those procedures were repeated, LDs0 was determined at each passage. b LDs0 for mice.
h a v e b e e n o b t a i n e d at t h e t h i r d p a s s a g e o f t h e S c h u s t r a i n or' t h e f o u r t h p a s s a g e o f t h e N 9 s t r a i n . T h i s , therefore, indicates that a mutation and subsequent selection might have occurred during those passages. T h e p r e s e n t s t u d y m i g h t p r o v i d e s o m e c l u e s to elucidate the mechanism(s) of the virulence of F. tularensis. All four strains consist exclusively of two types of colonies. One type of colony represents the l o w v i r u l e n c e in m i c e a n d a b s o l u t e l y c o r r e l a t e s w i t h t h e a c f § p h e n o t y p e as w e l l . O n t h e o t h e r h a n d , t h e other type of colony does not necessarily correlate
w i t h t h e v i r u l e n c e a l t h o u g h it e x p r e s s e s t h e a c f phenotype constantly. Therefore, further studies s u c h as a n a l y s e s at t h e c o l o n y l e v e l w i l l b e a n a i d in delineating the mechanism(s) of the virulence of F. tularensis.
ACKNOWLEDGMENT
We are grateful to Dr. J. F. Bell for his invaluable advice and encouragement.
Literature
Cited
1. Eigelsbach HT, Braun W, Herring RT (1951) Studies on the variation of Bacterium tularense. J Bacteriol 61:557-569 2. Foshay L (1932) Induction of avirulence in Pasteurella tularensis. J Infect Dis 51:280-285 3. Moody MD, Downs CM (1955) Studies on tularemia. I. The relation between certain pathogenic and immunogenic properties of variants of Pasteurella tularensis. J Bacteriol 70:297-304 4. Ohara S, Sato M, Sato T (1953) On pig-liver medium and a modified medium for cultivation of Bacterium tularense. Tohoku J Exp Med 58:185-190 5. Philip CB, Davis GE (1935) Tularemia. Observations on a strain of low initial virulence from rabbit ticks. Public Health Rep 50:909-911 6. Ransmeier JC (1943) The reaction of the chick embryo to virulent and non-virulent strains of Bact. tularense. J Infect Dis 72:86-90 7. Reed LJ, Muench H (1938) A simple method of estimating fifty per cent endpoints. Am J Hyg 27:493-497
