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Correlation of virulence, susceptibility to leptospiricidal activity test mediated by antiserum plus complement and colonial morphology of five lines of a strain of Leptospira interrogans serovar copenhageni
Z bl. Bakt. Hyg., I. Abt. Orig. A 251, 230-236 (1981)
Department of Hygiene and Microbiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Jap an (Head : Prof. Dr. R. Yanagawa)
Correlation of Virulence, Susceptibility to Leptospiricidal Activity Test Mediated by Antiserum plus Complement and Colonial Morphology of Five Lines of a Strain of Leptospira interrogans Serovar copenhageni Die Beziehungen zwischen der Virulenz, der Empfindlichkeit im Leptospirozidie-Aktivitatstest und der Koloniemorphologie bei fiinf verschiedenen Linien eines Stammes der Leptospira interrogans Serovar copenhageni WASHINGTON YEPEZ PLASCENCIO, RYO YANAGAWA, and KOUKO UENO With 2 Figures ' Received August 24, 1981
Abstract Correlation of virulence, susceptibility to lepto spiricidal activity test mediated by anti serum plus complement (LAT ) and colonial morphology was investigated using five lines of Lept ospira interrogans serova r copenbageni Shibaura. Line No . 2, a moder ately virulent strai n, passaged in guinea pigs for 24 successive times caused occasional death of guinea pigs. Lept ospiras recovered from d eceas~d animals were characterized by decreased susceptibility to LAT and pred ominance of Iiazy colonies upon culture. Lepto spiras recovered from surviving guinea pigs were of increased susceptibility to LAT and grew predominantly in non-h azy colonies. Virulent line No .1 subjected to passage through liquid medium for 20 successive times resulted in a slight decrease in virulence and slight increase in susceptibility to LAT, but the predominance of hazy colonies upon culture was invariable. The other three lines, Nos. 3, 4 and 5 which have been maintained for more than 10 years by passage through different media respectively, were avirulent, susceptible to LAT and produced only non-hazy colonies.
Zusammenfassung Bei fiinf verschiedenen Linien cines Stammes der Leptospira interrogans Serovar copenhageni wurd en die Beziehun gen zwischen Virulenz, der Empfindlichkeit im LeptospirozidieAktivitarsrest mit Antiserum und Komplement (LAT) und der Koloniemorphologie untersucht,
Virulence, Susceptibility to LAT and Colony Form of Leptospira
231
Der Stamm Nr. 2, ein miigig virulenter Stamm, wurde nacheinander 21mal einer Meerschweinchenpassage unterzogen. Nur einige dieser Tiere verendeten an der Infektion. Leptospiren, die aus diesen verstorbenen Tieren angeziichtet wurden, zeigten eine verminderte Empfindlichkeit im LAT und vorherrschend verschwommene Kolonieformen. Leptospiren, die aus iiberlebenden Meerschweinchen isoliert wurden, hatten dagegen eine hohere Empfindlichkeit im LAT und nicht verschwommene Kolonien. Der virulente Stamm Nr. 1, der 20mal in fliissigem Medium passagiert wurde, hatte einen leichten Viruienzabfall und ein leichtes Ansteigen der Empfindlichkeit im LAT. Die verschwommenen Kolonieformen bleiben bei ihm unverandert erhalten. Die Stamme Nr. 3, 4 und 5, die mehr als zehn Jahre durch die verschiedensten Niihrmedien passagiert wurden, waren avirulent, im LAT empfindlich und bildeten nur nichtverschwommene Kolonien.
Introduction Avirulent leptospiras have been found to be susceptible to leptospiricidal activity test mediated by antiserum plus complement (LAT), while virulent leptospiras were found to be resistant to these serum substances (3). The change of a virulent culture of the avirulent state as a result of growth in culture media was found to be accompanied by the loss of resistance to antibody plus complement (3). Although no association of leptospiral virulence with colonial morphology of leptospiras has been reported (4, 6), virulence was reported to be linked with colonial morphology and shape of leptospiras in some strains (2, 4). Paine and Van der Hoeden (2) described a large colony of typically hooked icterohaemorrhagiae virulent for hamsters and guinea pigs that became gradually replaced on culture by a serologically identical small-colony avirulent straight mutant. The purpose of the present study is to correlate virulence, susceptibility to LAT and colonial morphology of the stock culture of 5 lines of strain Shibaura of copenhageni, divergent in passage history and virulence, during limited range of passage through animals and medium.
Materials and Methods Strains: Five lines of Leptospira interrogans serovar copenhageni strain Shibaura with different history of passage and/or cloning were used in this study (Table 1). Line No.1, the virulent line isolated from a rat in Tokyo (1964) was received from the Japanese National Institute of Health. Thenceforth, it has been passaged through guinea pigs for 315 times. Line No.2 (clone 1 of Shibaura) (12),a moderately virulent line was cloned from line No.1 by plating (1973) and since then passaged once or twice a year through guinea pigs. Line No.3, a streptomycin resistant mutant derivedfrom line No.1 (7),50 serial passages through media containing 100 fig streptomycin/ml (January, 1975) then passaged through serum medium every 2 months. Line No.4 was derived by 199 serial passages of line No.1 through guinea pigs then cultivation in boiled serum medium (9), thereafter 190 times passage through Shenberg's medium (5), then growth in boiled serum medium (36th passage; December, 1980). Line No.5 was derived by 192 passages of line No.1 through guinea pigs, then 67 times passage through serum medium for more than 10 years (December, 1980). Serum medium: 0.2% tryptose phosphate broth (TPB, Difco), containing 10% normal rabbit serum and a small amount of phenol red as pH indicator was used as the serum medium. Normal serum medium with 1% agar (Special agar noble, Difco) (1) was used as the solid normal serum medium.
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W. Y.Plascencio, R. Yanagawa, and K.Ueno
Table 1. Five lines of copenbageni Shibaura 1 Line No.
Origin
Virulence
Passages
1 2
Rat Line No.1 Line No.1
Virulent Moderately virulent Avirulent
4
Line No.1
Avirulent
5
Line No.1
Avirulent
Guinea pigs Guinea pigs/ serum medium Serum medium with and without streptomycin Shenberg's synthetic medium/boiled serum medium Serum medium
3
1
Other remarks
Clonized from line No.1 Streptomycin resistant
Further information is given in the text.
Immune sera: Rabbits were hyperimmunized against the five lines of copenhageni Shibaura, following the method described previously (10). The fifty percent microscopic agglutination end titers of these immune sera were 1:25600 (anti-line No.2 and anti-line No.4), 1:12800 (anti-line No.1) and 1:6400 (anti-line No.3 and anti-line No.5). Complement: Pooled normal guinea pig serum used as the complement source was obtained from adult guinea pigs. Guinea pigs: Guinea pigs of the Hartley strain weighing approximately 250 g were purchased from Shizuoka Agricultural Cooperative Association for Laboratory Animals, Shizuoka, Japan. Hamsters: Female Syrian hamsters, approximately 3 weeks old were used in the virulence studies. Microscopic agglutination test and precipitin-absorption test: These tests were done as previously described (11). Leptospiricidal activity test (LAT) : This test was done as described previously (8). Homologous antiserum was used for the test. Titers were expressed as more than 90% of lysis. Serial passage through guinea pigs: First passage was done by intraperitoneal inoculation of guinea pig with 2 ml (1 x 108 leptospiras per ml) of line No.2 cultured in serum medium. Subsequently up to the 6th passage, blood cultures obtained during the febrile stage of inoculated guinea pig was similarly inoculated into the succeeding guinea pig. Guinea pig 7th was inoculated directly with 1 ml of blood from guinea pig 6th. The same procedure was used in guinea pig 8th to 15th. Guinea pig 16th was injected intraperitoneally with 2 ml of 10 times concentrated culture from passage 11th plus passage 16th. Guinea pig 17th was inoculated with 1 ml of blood, 1 ml of renal suspension and 1 ml of hepatic suspension from guinea pig 16th. Guinea pig 18th was inoculated with 2 ml of 10 times concentrated culture from passage 17th. Guinea pig 19th, 20th and 21st were inoculated according to the procedure used in guinea pig 17th. Guinea pig 22nd was injected with 2 ml of 10 times concentrated cultures from passage 17th plus passage 21st. Guinea pig 23rd was inoculated in the same manner as guinea pig 17th (The body weight of the guinea pigs used for the 22nd and 23rd passages were exceptionally small, 150 g). Finally guinea pig 24th was inoculated with 2 ml of blood culture from guinea pig 23rd. Serial passage through serum medium: Line No.1 was successively passed through serum medium, 20 times every 4 or 5 days.
Virulence, Suscept ibility to LAT and Colony Form of Lept ospir a
233
Cultures on solid medium : Fresh cultures (1 x 108 leptospiras per ml) in liquid medium or th e blood of guinea pigs in th e period of leptospiremia wer e diluted and inoculated onto the solid seru m med ium. Colonies usually grew 6 to 8 days after inoculation. Colonial morphology wa s recorded one da y after the appearance of colonies. At least 100 colonies were ex amined ; haz y colonies were th ose with haz y margin wh erea s non-hazy colonies were those with smo oth margin , either opalescent or translucent and either small or large.
Results Th e five lines of copenhagen; Shibaura were found to vary in virulence and susceptibility to LAT (Table 2). Line No.1 was virulent, resistant to LAT and T able 2. Susceptibility to LAT , viru lence and colonial morphology of five lines of copenhagen; Shiba ura Line T iter of No. LAT with homologous antiserum 1 1 2 3 4
5
< 100
Resistance or sensitivity to LAT
Virulence in ham sters LD"o
ID"o
2
64000
Resistant Sensitive
> 10"
< 10 4 > 108
12800 0 160 00 32 000
Sensitive Sensitive Sensitive
> 108 > 10" > 10"
> 10" > 10" > 108
104
Virulence in guinea pigs 3 Death Leprospir emia
Colonial morphology
+
Hazy Non-hazy with few hazy Non -hazy Non -hazy Non-h azy
+ +
1 Recip rocal of the highest dilut ion of th e antisera show ing 90% or more lept ospiricidal activity. 3 Infection was dete rm ined by the recover y of leptospira s from the kidn eys, 21 days afte r incub ation. 3 Determ ined by inoculat ion of 10" cells.
Fig. 1
Fig. 2
Fig. 1 (Left). Colonies of Line No . 1, wit h hazy margin (hazy colonies). Scale : 3 mm . Fig. 2 (Righr). Colo nies of Line No . 2, with smooth margin (non-hazy colonies). Scale : 3 mm.
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W. Y. Plascencio, R. Yanagawa, and K. Ueno
produced hazy colonies (Fig. 1). Line No.2 was moderately virulent causing leptospiremia that is not always lethal, susceptible to LAT and formed colonies consisting mostly of non-haz y (Fig. 2) and few hazy type. Lines Nos. 3, 4 and 5 were avirulent, susceptible to LAT and formed only non-hazy colonies. Line No.2 was serially passaged for 24 times through guinea pigs and examined during the course of passage for changes in susceptibility to LAT and colonial morphology (Table 3). Guinea pigs inoculated died at intervals during the passage. Leptospiras recovered from dying guinea pigs were formed to be rather resistant to LAT and majority of the colonies had hazy margin. In contrast, leptospiras recovered from surviving guinea pigs retained the susceptibility to LAT and grew Table 3. Susceptibility to LAT and colonial morphology of line No.2 recovered from guinea pigs used in the passage Passages thro ugh guinea pigs
o 5th 11th 12th 14th 15th 17th 21st 24th
LAT of recovered lept ospira with ant iline No. 2 serum
Fate of ino culated gui nea pigs
Colonial morphology
64000 1 64000
Survi ved Survived Deceased Sur vived Sur vived Survived Decea sed Sur vived Decea sed
Hazy
Non-hazy
%
%
8
92 98 37 80
2
-ooo
63 20 23
16000 64000 32000 4000 16000 4000
77
22 69 NP 95
78 31 NT
5
1 Reciprocal of the highe st dilution of the antiserum showing 90 % or more leptospiricidal activity. 2 Not tested.
Table 4. Virulence, susceptibility to LAT and colonial morphology of line No.1 in relation to passages in medium Leptospiric ida l activity test 2
Pa ssages th rough medium
Virulence in hamsters 1 LD so 10 5 0
1 : 100
1:1000
1:2000
1:4000
Before passage 10th pas sage 20th pas sage
10' < 104 10' < 10' 10" 10"
65% 75% 95 %
65% 75% 95%
75%
75%
Colonial morphology Hazy Non-hazy % % 100 NT3 100
0 NT 0
1 Infect ion was determined by the recovery of leptospiras from kidneys, 21 days after incubation. 2 Da shes represent less th an 50% of lysis. 3 Not tested.
Virulence, Susceptibility to LAT and Colony Form of Leptospira
235
predominantly in non-hazy colonies. The use of exceptionally small guinea pigs for passage 22nd and 23rd resulted in premature death of these guinea pigs before any change in these characters could be observed. Line No.1 was subjected to continuous passage through serum medium 20 times to decrease its virulence. Table 4 shows that a slight reduction of virulence of line No.1 was accompanied by a slight increase of susceptibility to LAT, but the predominance of hazy colonies was invariable. Dark field microscopy showed that leptospiras of all the lines possessed similar hooked shape. Microscopic agglutination test and precipitin-absorption test in gel indicated that the lines used were antigenically similar to each other with the exception of line No.4, which was antigenically different from the others.
Discussion Correlation was found between virulence in animals and resistance to LATin the experiment using virulent and moderately virulent lines of copenhageni Shibaura. These findings generally agree with those reported by Johnson and Harris (3). In the present experiment using a moderately virulent strain, however, leptospiras recovered from the blood of dying guinea pigs showed relative resistance to the leptospiricidal activity test, whereas leptospiras recovered from surviving guinea pigs showed susceptibility to LAT. The close relationship between virulence and susceptibility to LAT was thus demonstrated in the present study, using the moderately virulent strain. Correlation between virulence and colonial morphology was also observed. This is consistent with those reported by Faine and Van der Hoeden (2) and Petrov and Chernukha (4). Using moderately virulent strain, we found that hazy colonies were predominant in the leptospiras recovered from dying guinea pigs and non-hazy colonies were predominant in the leptospiras recovered from surviving guinea pigs. Thus the relation of virulence, colonial morphology and resistance to LATwas shown. We were unable to demonstrate differences in leptospiral shape and motility, since all colonial types were composed of hooked leptospiras of similar motility. Thus, we differ from Faine and Van der Hoeden (2) in thinking that the basis of virulence could lie in the hooked shape of virulent leptospiras. Acknowledgement. Dr. W. Yepe z Plascencia was the visiting scientist from Instituto Nacional de Higiene "Leopold Izquieta Perez" Julian Coronel 905 and Esmeraldas, P.O. Box 3961, Guayaquil, Equador, under the support of Japan International Cooperation Agency.
References 1. Cox, C.D. and A.D.Larson : Colonial growth of leptospirae.J. Bact. 73 (1957) 587-589 2. Faine, S. and J.Van der Hoeden : Virulence linked colonialand morphologicalvariation in Leptospira. J. Bact. 88 (1964) 1493-1496 3. Johnson, R. C. and V.G. Harris: Antileptospiralactivityof serum.IILeptospiralvirulence factor. J. Bact. 93 (1967) 513-519
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4. Petrou, E. M. and Y.G. Chernukha: Leptospires colonial variations. Zbl. Bakt. Hyg., LAbt. Orig. A 243 (1979) 511-521 5. Shenberg, E.: Growth of pathogenic leptospira in chemically defined media. J. Bact. 93 (1967) 1598-1606 6. Stalheim, O. H. V. and]. B.Wilson: Leptospiral colonial morphology. J. Bact. 86 (1963) 482-489 7. Takashima, I. and R. Yanagawa: Isolation and characterization of streptomycin-resistant mutant of leptospiras. Jap. J. Microbiol. 16 (1972) 535-537 8. Torres, j., K. Vena, and R. Yanagauia: Specificityof leptospiricidal activity test mediated by antiserum and complement. Zbl. Bakt. Hyg., LAbt. Orig. A 249 (1981) 124-132 9. Yanagauia, R. and]. B.Wilson: Two types of leptospirae distinguishable by their growth in boiled serum medium. J. infect. Dis. 110 (1962) 70-74 10. Yanagaioa, R., M.Shinagawa, and I. Takashima: Serological studies of leptospiras by immunodiffusion. Zbl. Bakt. Hyg., I.Abt. Orig. A 238 (1974) 369-377 11. Yanagatoa, R. and Y.Adachi: Identification of some japanese leptospiral strains as serotypes copenhageni and icterohaemorrhagiae by precipitin-absorption test in gel. Zbl. Bakt, Hyg., LAbt. Orig. A 237 (1977) 96-103 12. Yanagaioa, R., E.Shimono, and E. M. Shinjo: Isolation of antigenic variants from leptospiras grown in vitro and from heart blood of guinea pigs inoculated with a clonized strain of leptospira. Zbl. Bakt. Hyg., LAbt. Orig. A 245 (1979) 345-355 Prof. Dr. Ryo Yanagawa, Department of Hygiene and Microbiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, 060, Japan
Department of Hygiene and Microbiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, Jap an (Head : Prof. Dr. R. Yanagawa)
Correlation of Virulence, Susceptibility to Leptospiricidal Activity Test Mediated by Antiserum plus Complement and Colonial Morphology of Five Lines of a Strain of Leptospira interrogans Serovar copenhageni Die Beziehungen zwischen der Virulenz, der Empfindlichkeit im Leptospirozidie-Aktivitatstest und der Koloniemorphologie bei fiinf verschiedenen Linien eines Stammes der Leptospira interrogans Serovar copenhageni WASHINGTON YEPEZ PLASCENCIO, RYO YANAGAWA, and KOUKO UENO With 2 Figures ' Received August 24, 1981
Abstract Correlation of virulence, susceptibility to lepto spiricidal activity test mediated by anti serum plus complement (LAT ) and colonial morphology was investigated using five lines of Lept ospira interrogans serova r copenbageni Shibaura. Line No . 2, a moder ately virulent strai n, passaged in guinea pigs for 24 successive times caused occasional death of guinea pigs. Lept ospiras recovered from d eceas~d animals were characterized by decreased susceptibility to LAT and pred ominance of Iiazy colonies upon culture. Lepto spiras recovered from surviving guinea pigs were of increased susceptibility to LAT and grew predominantly in non-h azy colonies. Virulent line No .1 subjected to passage through liquid medium for 20 successive times resulted in a slight decrease in virulence and slight increase in susceptibility to LAT, but the predominance of hazy colonies upon culture was invariable. The other three lines, Nos. 3, 4 and 5 which have been maintained for more than 10 years by passage through different media respectively, were avirulent, susceptible to LAT and produced only non-hazy colonies.
Zusammenfassung Bei fiinf verschiedenen Linien cines Stammes der Leptospira interrogans Serovar copenhageni wurd en die Beziehun gen zwischen Virulenz, der Empfindlichkeit im LeptospirozidieAktivitarsrest mit Antiserum und Komplement (LAT) und der Koloniemorphologie untersucht,
Virulence, Susceptibility to LAT and Colony Form of Leptospira
231
Der Stamm Nr. 2, ein miigig virulenter Stamm, wurde nacheinander 21mal einer Meerschweinchenpassage unterzogen. Nur einige dieser Tiere verendeten an der Infektion. Leptospiren, die aus diesen verstorbenen Tieren angeziichtet wurden, zeigten eine verminderte Empfindlichkeit im LAT und vorherrschend verschwommene Kolonieformen. Leptospiren, die aus iiberlebenden Meerschweinchen isoliert wurden, hatten dagegen eine hohere Empfindlichkeit im LAT und nicht verschwommene Kolonien. Der virulente Stamm Nr. 1, der 20mal in fliissigem Medium passagiert wurde, hatte einen leichten Viruienzabfall und ein leichtes Ansteigen der Empfindlichkeit im LAT. Die verschwommenen Kolonieformen bleiben bei ihm unverandert erhalten. Die Stamme Nr. 3, 4 und 5, die mehr als zehn Jahre durch die verschiedensten Niihrmedien passagiert wurden, waren avirulent, im LAT empfindlich und bildeten nur nichtverschwommene Kolonien.
Introduction Avirulent leptospiras have been found to be susceptible to leptospiricidal activity test mediated by antiserum plus complement (LAT), while virulent leptospiras were found to be resistant to these serum substances (3). The change of a virulent culture of the avirulent state as a result of growth in culture media was found to be accompanied by the loss of resistance to antibody plus complement (3). Although no association of leptospiral virulence with colonial morphology of leptospiras has been reported (4, 6), virulence was reported to be linked with colonial morphology and shape of leptospiras in some strains (2, 4). Paine and Van der Hoeden (2) described a large colony of typically hooked icterohaemorrhagiae virulent for hamsters and guinea pigs that became gradually replaced on culture by a serologically identical small-colony avirulent straight mutant. The purpose of the present study is to correlate virulence, susceptibility to LAT and colonial morphology of the stock culture of 5 lines of strain Shibaura of copenhageni, divergent in passage history and virulence, during limited range of passage through animals and medium.
Materials and Methods Strains: Five lines of Leptospira interrogans serovar copenhageni strain Shibaura with different history of passage and/or cloning were used in this study (Table 1). Line No.1, the virulent line isolated from a rat in Tokyo (1964) was received from the Japanese National Institute of Health. Thenceforth, it has been passaged through guinea pigs for 315 times. Line No.2 (clone 1 of Shibaura) (12),a moderately virulent line was cloned from line No.1 by plating (1973) and since then passaged once or twice a year through guinea pigs. Line No.3, a streptomycin resistant mutant derivedfrom line No.1 (7),50 serial passages through media containing 100 fig streptomycin/ml (January, 1975) then passaged through serum medium every 2 months. Line No.4 was derived by 199 serial passages of line No.1 through guinea pigs then cultivation in boiled serum medium (9), thereafter 190 times passage through Shenberg's medium (5), then growth in boiled serum medium (36th passage; December, 1980). Line No.5 was derived by 192 passages of line No.1 through guinea pigs, then 67 times passage through serum medium for more than 10 years (December, 1980). Serum medium: 0.2% tryptose phosphate broth (TPB, Difco), containing 10% normal rabbit serum and a small amount of phenol red as pH indicator was used as the serum medium. Normal serum medium with 1% agar (Special agar noble, Difco) (1) was used as the solid normal serum medium.
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Table 1. Five lines of copenbageni Shibaura 1 Line No.
Origin
Virulence
Passages
1 2
Rat Line No.1 Line No.1
Virulent Moderately virulent Avirulent
4
Line No.1
Avirulent
5
Line No.1
Avirulent
Guinea pigs Guinea pigs/ serum medium Serum medium with and without streptomycin Shenberg's synthetic medium/boiled serum medium Serum medium
3
1
Other remarks
Clonized from line No.1 Streptomycin resistant
Further information is given in the text.
Immune sera: Rabbits were hyperimmunized against the five lines of copenhageni Shibaura, following the method described previously (10). The fifty percent microscopic agglutination end titers of these immune sera were 1:25600 (anti-line No.2 and anti-line No.4), 1:12800 (anti-line No.1) and 1:6400 (anti-line No.3 and anti-line No.5). Complement: Pooled normal guinea pig serum used as the complement source was obtained from adult guinea pigs. Guinea pigs: Guinea pigs of the Hartley strain weighing approximately 250 g were purchased from Shizuoka Agricultural Cooperative Association for Laboratory Animals, Shizuoka, Japan. Hamsters: Female Syrian hamsters, approximately 3 weeks old were used in the virulence studies. Microscopic agglutination test and precipitin-absorption test: These tests were done as previously described (11). Leptospiricidal activity test (LAT) : This test was done as described previously (8). Homologous antiserum was used for the test. Titers were expressed as more than 90% of lysis. Serial passage through guinea pigs: First passage was done by intraperitoneal inoculation of guinea pig with 2 ml (1 x 108 leptospiras per ml) of line No.2 cultured in serum medium. Subsequently up to the 6th passage, blood cultures obtained during the febrile stage of inoculated guinea pig was similarly inoculated into the succeeding guinea pig. Guinea pig 7th was inoculated directly with 1 ml of blood from guinea pig 6th. The same procedure was used in guinea pig 8th to 15th. Guinea pig 16th was injected intraperitoneally with 2 ml of 10 times concentrated culture from passage 11th plus passage 16th. Guinea pig 17th was inoculated with 1 ml of blood, 1 ml of renal suspension and 1 ml of hepatic suspension from guinea pig 16th. Guinea pig 18th was inoculated with 2 ml of 10 times concentrated culture from passage 17th. Guinea pig 19th, 20th and 21st were inoculated according to the procedure used in guinea pig 17th. Guinea pig 22nd was injected with 2 ml of 10 times concentrated cultures from passage 17th plus passage 21st. Guinea pig 23rd was inoculated in the same manner as guinea pig 17th (The body weight of the guinea pigs used for the 22nd and 23rd passages were exceptionally small, 150 g). Finally guinea pig 24th was inoculated with 2 ml of blood culture from guinea pig 23rd. Serial passage through serum medium: Line No.1 was successively passed through serum medium, 20 times every 4 or 5 days.
Virulence, Suscept ibility to LAT and Colony Form of Lept ospir a
233
Cultures on solid medium : Fresh cultures (1 x 108 leptospiras per ml) in liquid medium or th e blood of guinea pigs in th e period of leptospiremia wer e diluted and inoculated onto the solid seru m med ium. Colonies usually grew 6 to 8 days after inoculation. Colonial morphology wa s recorded one da y after the appearance of colonies. At least 100 colonies were ex amined ; haz y colonies were th ose with haz y margin wh erea s non-hazy colonies were those with smo oth margin , either opalescent or translucent and either small or large.
Results Th e five lines of copenhagen; Shibaura were found to vary in virulence and susceptibility to LAT (Table 2). Line No.1 was virulent, resistant to LAT and T able 2. Susceptibility to LAT , viru lence and colonial morphology of five lines of copenhagen; Shiba ura Line T iter of No. LAT with homologous antiserum 1 1 2 3 4
5
< 100
Resistance or sensitivity to LAT
Virulence in ham sters LD"o
ID"o
2
64000
Resistant Sensitive
> 10"
< 10 4 > 108
12800 0 160 00 32 000
Sensitive Sensitive Sensitive
> 108 > 10" > 10"
> 10" > 10" > 108
104
Virulence in guinea pigs 3 Death Leprospir emia
Colonial morphology
+
Hazy Non-hazy with few hazy Non -hazy Non -hazy Non-h azy
+ +
1 Recip rocal of the highest dilut ion of th e antisera show ing 90% or more lept ospiricidal activity. 3 Infection was dete rm ined by the recover y of leptospira s from the kidn eys, 21 days afte r incub ation. 3 Determ ined by inoculat ion of 10" cells.
Fig. 1
Fig. 2
Fig. 1 (Left). Colonies of Line No . 1, wit h hazy margin (hazy colonies). Scale : 3 mm . Fig. 2 (Righr). Colo nies of Line No . 2, with smooth margin (non-hazy colonies). Scale : 3 mm.
234
W. Y. Plascencio, R. Yanagawa, and K. Ueno
produced hazy colonies (Fig. 1). Line No.2 was moderately virulent causing leptospiremia that is not always lethal, susceptible to LAT and formed colonies consisting mostly of non-haz y (Fig. 2) and few hazy type. Lines Nos. 3, 4 and 5 were avirulent, susceptible to LAT and formed only non-hazy colonies. Line No.2 was serially passaged for 24 times through guinea pigs and examined during the course of passage for changes in susceptibility to LAT and colonial morphology (Table 3). Guinea pigs inoculated died at intervals during the passage. Leptospiras recovered from dying guinea pigs were formed to be rather resistant to LAT and majority of the colonies had hazy margin. In contrast, leptospiras recovered from surviving guinea pigs retained the susceptibility to LAT and grew Table 3. Susceptibility to LAT and colonial morphology of line No.2 recovered from guinea pigs used in the passage Passages thro ugh guinea pigs
o 5th 11th 12th 14th 15th 17th 21st 24th
LAT of recovered lept ospira with ant iline No. 2 serum
Fate of ino culated gui nea pigs
Colonial morphology
64000 1 64000
Survi ved Survived Deceased Sur vived Sur vived Survived Decea sed Sur vived Decea sed
Hazy
Non-hazy
%
%
8
92 98 37 80
2
-ooo
63 20 23
16000 64000 32000 4000 16000 4000
77
22 69 NP 95
78 31 NT
5
1 Reciprocal of the highe st dilution of the antiserum showing 90 % or more leptospiricidal activity. 2 Not tested.
Table 4. Virulence, susceptibility to LAT and colonial morphology of line No.1 in relation to passages in medium Leptospiric ida l activity test 2
Pa ssages th rough medium
Virulence in hamsters 1 LD so 10 5 0
1 : 100
1:1000
1:2000
1:4000
Before passage 10th pas sage 20th pas sage
10' < 104 10' < 10' 10" 10"
65% 75% 95 %
65% 75% 95%
75%
75%
Colonial morphology Hazy Non-hazy % % 100 NT3 100
0 NT 0
1 Infect ion was determined by the recovery of leptospiras from kidneys, 21 days after incubation. 2 Da shes represent less th an 50% of lysis. 3 Not tested.
Virulence, Susceptibility to LAT and Colony Form of Leptospira
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predominantly in non-hazy colonies. The use of exceptionally small guinea pigs for passage 22nd and 23rd resulted in premature death of these guinea pigs before any change in these characters could be observed. Line No.1 was subjected to continuous passage through serum medium 20 times to decrease its virulence. Table 4 shows that a slight reduction of virulence of line No.1 was accompanied by a slight increase of susceptibility to LAT, but the predominance of hazy colonies was invariable. Dark field microscopy showed that leptospiras of all the lines possessed similar hooked shape. Microscopic agglutination test and precipitin-absorption test in gel indicated that the lines used were antigenically similar to each other with the exception of line No.4, which was antigenically different from the others.
Discussion Correlation was found between virulence in animals and resistance to LATin the experiment using virulent and moderately virulent lines of copenhageni Shibaura. These findings generally agree with those reported by Johnson and Harris (3). In the present experiment using a moderately virulent strain, however, leptospiras recovered from the blood of dying guinea pigs showed relative resistance to the leptospiricidal activity test, whereas leptospiras recovered from surviving guinea pigs showed susceptibility to LAT. The close relationship between virulence and susceptibility to LAT was thus demonstrated in the present study, using the moderately virulent strain. Correlation between virulence and colonial morphology was also observed. This is consistent with those reported by Faine and Van der Hoeden (2) and Petrov and Chernukha (4). Using moderately virulent strain, we found that hazy colonies were predominant in the leptospiras recovered from dying guinea pigs and non-hazy colonies were predominant in the leptospiras recovered from surviving guinea pigs. Thus the relation of virulence, colonial morphology and resistance to LATwas shown. We were unable to demonstrate differences in leptospiral shape and motility, since all colonial types were composed of hooked leptospiras of similar motility. Thus, we differ from Faine and Van der Hoeden (2) in thinking that the basis of virulence could lie in the hooked shape of virulent leptospiras. Acknowledgement. Dr. W. Yepe z Plascencia was the visiting scientist from Instituto Nacional de Higiene "Leopold Izquieta Perez" Julian Coronel 905 and Esmeraldas, P.O. Box 3961, Guayaquil, Equador, under the support of Japan International Cooperation Agency.
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