INFECTION AND IMMUNITY, June 1977, p. 817-820 Copyright C 1977 American Society for Microbiology
Vol. 16, No. 3 Printed in U.S.A.
Immunization of Mice Against Naegleria fowleri Infection D. T. JOHN,* R. R. WEIK, AND A. C. ADAMS
Department of Microbiology, Virginia Commonwealth University, Richmond, Virginia 23298 Received for publication 21 January 1977
Naegleria fowleri produces fatal meningoencephalitis in humans and in experimentally infected laboratory animals. The course of the disease in mice is dependent upon the infecting dose of amoebae, route of inoculation, and prior exposure to Naegleria antigens. DUB/ICR mice were immunized by various routes and antigen preparations, held for 21 days, and, together with noninfected control mice, challenged intravenously (i.v.) or intranasally (i.n.) with 107 or 10" N. fowleri per mouse, respectively. Mice immunized with live or formalinized N. fowleri or live N. gruberi subcutaneously, intraperitoneally, i.v., or i.n. were significantly protected against a subsequent lethal challenge with N. fowleri i.v. or i.n. In general, i.v. inoculation afforded greated protection than other routes of immunization, intact cells immunized mice better than did cell fragments, and N. gruberi appeared to be a better immunogen than N. fowleri. Naegleria fowleri, an ameboflagellate, produces fatal meningoencephalitis in humans and laboratory animals. Although the mouse is appropriate as an experimental model for studying amoebic meningoencephalitis (1, 9), there is little information on immunization and host resistance to infection with N. fowleri. Adams et al. (1) have demonstrated that mice surviving a primary infection with N. fowleri were substantially resistant to intravenous (i.v.) challenge with a dose of amoebae that produced uniformly fatal disease in untreated control mice. (erva (5) reported a humoral immune response indicated by complement fixation in guinea pigs infected intranasally (i.n.) with N. fowleri. Delayed hypersensitivity was observed in guinea pigs inoculated subcutaneously (s.c.) with N. fowleri (8). In this study we have demonstrated that mice immunized intraperitoneally (i.p.), i.n., i.v., or s.c. with N. fowleri or the nonpathogen N. gruberi are protected against an otherwise lethal N. fowleri challenge. MATERIALS AND METHODS Male DUB/ICR mice weighing 20 to 25 g were used in all experiments. Mice were obtained from Flow Research Animals, Inc., Dublin, Va. They were allowed to adjust to their new environment for at least 5 days prior to experimentation. The mice were given free access to water and feed (Purina Lab Chow, Ralston Purina Corp.). N. fowleri (LEE strain) and N. gruberi (EGB strain) were cultured axenically in Nelson medium (1) at 37°C and in Balamuth medium (3) at 28°C, respectively. Tissue culture flasks (75 cm2; Falcon Plastics) containing 50 ml of medium were inoculated with 104 amoebae per ml. Amoebae were har-
vested by centrifugation (450 x g, 5 min, 20°C) at 96 h. The cells were washed twice with Page amoeba saline (1) and suspended in 0.15 M NaCl. For counts, 0.2 ml of cell suspension was added to 9.8 ml of electrolyte solution consisting of 0.5% (vol/ vol) formalin and 0.4% (wt/vol) NaCl in distilled water and Vortex-shaken to disperse aggregates. Cells were then counted in a Coulter counter (model ZBI, Coulter Electronics, Inc., Hialeah, Fla.) at settings: matching switch 20 K, gain 0, bandwidth selector extended, 1/amplification 2, 1/aperture current 1, lower threshold 10, upper threshold maximum. Mice were immunized by a single dose (0.2 ml) of amoebae using the following routes and dosages: 3.5 x 107 live N. fowleri s.c., 106 live N. fowleri i.p., 105 live N. fowleri i.v., 103 live N. fowleri i.n., 107 formalinized N. fowleri i. p. or i.v., 107 freeze-thawed N. fowleri with or without Freund complete adjuvant (0.1 ml) i.p. or i.v., and 107 live N. gruberi i.p. or i.v. Formalinized cells were prepared by Vortex-mixing amoebae in 0.5% formalin saline and washing twice with 0.15 M NaCl. Precounted freeze-thawed cells were prepared in screw-capped vials, frozen at - 70°C for 60 min, and thawed at room temperature. This procedure was repeated four times, after which microscopic examination showed no intact cells. There were 20 mice in each experimental or control group unless otherwise specified. Mice were held for 21 days after immunization before they were challenged i.n. with 106 N. fowleri per mouse or i.v. with 107 N. fowleri per mouse. Percent protection was calculated using the expression 100 - (x/y) (100), where x is the percentage of immunized mice that died andy is the percentage of control mice that died.
RESULTS Mice surviving i.p., i.v., s.c., or i.n. immunization with live N. fowleri were held for 21 817
INFECT. IMMUN. 818 JOHN, WEIK, AND ADAMS days. The four groups of 20 mice each plus 20 control mice were then challenged i.v. with 1O7 N. fowleri per mouse. Ninety percent of nonimmunized control mice died within 5 to 11 days as expected. Mice that survived the primary immunizing dose of N. fowleri were partially ~40 protected against the secondary challenge dose (Fig. 1). The immunizing dose was 3.5 x 107 N. fowleri per mouse s.c., 106 i.p., 105 i.v., or 103 i.n., with cumulative dead of 32, 60, 65, and g20 75%, respectively, after i.v. challenge. Additional groups of mice that survived i.p., i.v., or i.n. immunizing doses of live N. fowleri were similarly treated but were challenged i.n. 10 5 0 20 25 30 15 with 106 live N. fowleri per mouse. The total DAYS AFTER CHALLENGE that died from i.n. challenge when the immuFIG. 2. Mortality of mice immunized with live N. nizing dose was i.p. was 35%; i.v., 35%; and i.n., fowleri various routes and challenged 21 days 50% (Fig. 2). Thus, partial protection was af- later withby106N. per mouse i.n. There were 20 forded mice immunized i.p., i.v., or i.n. with N. mice per group. fowleri Symbols: (-) control; (0) i.n.; (A) fowleri and then challenged i.n. i.p.; (U) i.v. Other mice were immunized with 107 formalinized N. fowleri i.p. or i.v., with no deaths occurring for 21 days. These mice plus 20 con100 trols were challenged i.v. with 107 live N. fowleri per mouse. Eighty-five percent of the non80 immunized control mice died within 5 to 10 days, whereas only 45% of i.v. immunized mice died within 7 to 18 days. Mice immunized i.p. W 60i were less protected, with 75% dead. However, the majority of these mice died at least 7 days after the control mice, indicating that protec- -J 40tion may be inapparent if one looks only at cumulative percent dead (Fig. 3). 20Mice immunized i.p. or i.v. with freezethawed N. fowleri and/or Freund complete adjuvant were challenged with a dose of live N. 15 20 25 30 o _5 lo fowleri i.v. Freund complete. adjuvant alone or DAYS AFTER CHALLENGE 100
W 800-
W-L 60-J
40Q
DAYS AFTER CHALLENGE FIG. 1. Mortality of mice immunized with live N. fowleri by various routes and challenged 21 days later with 107 N. fowleri per mouse i.v. There were 20 mice per group except s.c., which had 25. Symbols: (a) control; (0) i.n.; (N) i.v.; (A) i.p.; (AX) s.c.
FIG. 3. Mortality of mice immunized with formalinized N. fowleri by various routes and challenged 21 days later with 107 N. fowleri per mouse i.v. There were 20 mice per group. Symbols: (S) control; (A) i.p.; (U) i.v.
freeze-thawed N. fowleri with or without adjuvant, all given i.p., afforded little protection to mice challenged i.v. with live N. fowleri. However, mice immunized i.v. with freeze-thawed N. fowleri were 33% protected when challenged i.v. compared to nonimmunized controls (Fig. 4). All mice immunized either i.p. or i.v. with 107 live N. gruberi survived the primary inoculation as expected. Twenty-one days later, these mice plus nonimmunized controls were challenged i.v. with 107 live N. fowleri. None of the nonimmunized controls survived, whereas 35% of the i.p. immunized mice survived and 65% of the i.v. immunized mice survived (Fig. 5). Thus, i.v. immunization with N. gruberi is
VOL. 16, 1977
IMMUNITY TO N. FOWLERI INFECTION
more effective than i.p. immunization in protecting mice against a subsequent lethal challenge with N. fowleri. Immunizations with 107 formalinized N. fowleri, freeze-thawed N. fowleri, or live N. gruberi were administered by both i.v. and i.p. routes prior to challenge with live N. fowleri i.v. In all instances i.v. immunized mice were more protected than i.p. immunized mice (Table 1). Mice immunized i.p., i.v., or i.n. with live N. fowleri were similarly protected whether challenged i.v. or i.n. An immunizing dose of 106 N. fowleri i.p., 105 N. fowleri i.v., or 103 N. fowleri i.n. protected 33, 28, and 17%,
819
respectively, after i.v. challenge and 36, 36, and 9%, respectively, after i.n. challenge (Table 1). DISCUSSION Immunization i.v. with formalinized N. fowleri, freeze-thawed N. fowleri, or live N. gruberi was more effective in protecting mice against a subsequent lethal i.v. challenge than was i.p. immunization. A primary dose given i.p. or i.v. was also protective when challenge was i.n. The i.n. route is thought to be the route of entry of N. fowleri in amoebic meningoencephalitis in humans (4); hence, this route is commonly used, to inoculate mice and other experimental animals (7, 9). However, i.v. and i.p. 100 w
80
-
60
W
40
20-
0
5
10
15
20
25
30
DAYS AFTER CHALLENGE FIG. 4. Mortality of mice immunized with freezethawed N. fowleri with or without Freund complete adjuvant by various routes and challenged 21 days later with 107 N. fowleri per mouse i.v. There were 20 mice per group. Symbols: (@) control; (0) adjuvant alone i.p.; (A) i.p.; (A) i.p. plus adjuvant; (U) i.v.
10I 15 20 25 30 DAYS AFTER CHALLENGE FIG. 5. Mortality of mice immunized with live N. gruberi by various routes and challenged 21 days later with 107 N. fowleri per mouse i.v. There were 20 mice per group. Symbols: (0) control; (A) i.p.; (O) _5
i.v.
TABLE 1. Protection of mice against N. fowleri after immunization by various routes and parasite treatment Immunization Challenge No. of mice % Protection % Dead (control) i.p., live N. fowleri i.n. 20 35 (55) 36 i.v., live N. fowleri i.n. 20 35 (55) 36 i.n., live N. fowleri i.n. 20 50 (55) 9 s.c., live N. fowleri i.p., live N. fowleri i.v., live N. fowleri i.n., live N. fowleri
i.v. i.v. i.v. i.v.
25 20 20 20
32 60 65 75
(71) (90) (90) (90)
55 33 28 17
i.p., formalinized N. fowleri i.v., formalinized N. fowleri
i.v. i.v.
20 20
75 (90) 45 (90)
17 50
i.p., freeze-thawed N. fowleri i.p., freeze-thawed N. fowleri + adjuvant i.p., Freund complete adjuvant i.v., freeze-thawed N. fowleri
i.v. i.v.
20 20
85 (90) 80 (90)
6 11
i.v. i.v.
20 20
85 (90) 60 (90)
6 33
i.p., live N. gruberi i.v., live N. gruberi
i.v. i.v.
20 20
65 (100) 35 (100)
35 65
820 JOHN, WEIK, AND ADAMS routes are more exact than the i.n. route since it is difficult to quantitate the number of amoebae retained by the host after i.n. instillation. These data show similar results whether the secondary challenge is administered i.v. or i.n. (Table 1). Immunization with live N. gruberi was effective in protecting mice against N. fowleri challenge (Table 1), indicating that cross-reacting antigens are also protective antigens to the host. Visvesvara and Healy (10) showed that N. fowleri shared three to six common antigens with N. gruberi. One or more of these common antigens, and possibly others, in N. fowleri are probably protective antigens. Culbertson (7) immunized mice i.p. with virulent Acanthamoeba (A-1) and obtained resistance to homologous i.n. challenge. However, he was unable to demonstrate resistance in mice immunized i.p. with avirulent Acanthamoeba (H-A) and challenged i.n. or in mice immunized i.p. with N. fowleri (HB-1) and subsequently challenged i.n. with the same strain. An active infection with live N. fowleri is not required to elicit a protective response by the host, since mice immunized i.v. with formalinized N. fowleri were protected 50% against a subsequent i.v. challenge (Table 1). However, immunization with freeze-thawed cells did not protect mice to the extent that immunization with formalinized cells did (Table 1). Hence, it appears that the development of protection may depend upon the injection of intact cells. Symptoms of central nervous system involvement were more evident in mice challenged i.v. than i.n. Two to 3 days prior to death, i.v. challenged mice often displayed neurological symptoms such as unilateral movement and partial or complete posterior paralysis. Acute death, occurring within 30 min of i.v. challenge, was frequently noted for N. fowleriimmunized mice. These deaths appeared to be associated with route of immunization and were 20, 15, and 5% for mice immunized with N. fowleri i.v., i.p., and i.n., respectively. Occasionally, similar deaths were noted for nonimmunized controls (4% overall). Acute death was 5 and 0% for i.v. and i.p. N. gruberi-immunized mice. Histological examination of lung tissue taken from mice after acute death revealed many capillaries and small blood vessels blocked by amoebae. Anderson and Jamieson (2), using formalinized N. fowleri, produced an antiserum in rabbits capable of agglutinating N. fowleri and, to a lesser degree, N. gruberi. We have obtained similar results using the mouse model, except that the N. fowleri antiserum did not agglutinate N. gruberi amoebae
INFECT. IMMUN.
(unpublished data). It is possible that acute death may be caused by immune agglutination of amoebae and subsequent blockage of pulmonary blood vessels. N. gruberi, which appears to be a better immunogen than N. fowleri (Table 1), produced few acute deaths (2% overall) compared to N. fowleri (17% overall). If acute death were immunologically mediated and N. gruberi antigens were not involved in acute death, it is likely that crossreacting host-protective antigens are not involved but rather are antigens specific for N. fowleri strains. Alternatively, acute death may have been caused by a toxin that is reportedly produced by N. fowleri (6). In summary, i.v. inoculation afforded greater protection than other routes of immunization, intact cells immunized mice better than cell fragments, and N. gruberi appeared to be a better immunogen than N. fowleri. Man's unwitting exposure to nonpathogenic N. gruberi, abundant in all fresh water, raises the interesting possibility that he may become actively immunized against fatal amoebic meningoen-
cephalitis. ACKNOWLEDGMENTS This investigation was supported in part by Research Grant 771019 from A. H. Robins Co., Richmond, Va., and by Public Health Service grant AI-00382 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Adams, A. C., D. T. John, and S. G. Bradley. 1976. Modification of resistance of mice to Naegleria fowleri infections. Infect. Immun. 13:1387-1391. 2. Anderson, K., and A. Jamieson. 1972. Agglutination test for the investigation of the genus Naegleria. Pathology 4:273-278. 3. Balamuth, W. 1964. Nutritional studies on axenic cultures of Naegleria gruberi. J. Protozool. 11:19-20. 4. Carter, R. F. 1972. Primary amoebic meningoencephalitis. An appraisal of present knowledge. Trans. R. Soc. Trop. Med. Hyg. 66:193-213. 5. Cerva, L. 1971. Experimental infection of laboratory animals by the pathogenic Naegleria gruberi strain Vitek. Folia Parasitol. (Prague) 18:171-176. 6. Chang, S. L. 1971. Small, free-living amoebas: cultivation, quantitation, identification, classification, pathogenesis, and resistance, p. 201-254. In T. C. Cheng (ed.), Current topics in comparative pathobiology, vol. 1. Academic Press Inc., New York. 7. Culbertson, C. G. 1971. The pathogenicity of soil amebas. Annu. Rev. Microbiol. 25:231-254. 8. Diffley, P., M. R. Skeels, and F. Sogandares-Bernal. 1976. Delayed type hypersentisivity in guinea pigs infected subcutaneously with Naegleria fowleri, Carter. Z. Parasitenkd. 49:133-137. 9. Martinez, A. J., E. C. Nelson, and R. J. Duma. 1973. Animal model: primary amebic (Naegleria) meningoencephalitis in mice. Am. J. Pathol. 73:545-548. 10. Visvesvara, G. S., and G. R. Healy. 1975. Comparative antigenic analysis of pathogenic and free-living Naegleria species by the gel diffusion and immunoelectrophoresis techniques. Infect. Immun. 11:95-108.
Vol. 16, No. 3 Printed in U.S.A.
Immunization of Mice Against Naegleria fowleri Infection D. T. JOHN,* R. R. WEIK, AND A. C. ADAMS
Department of Microbiology, Virginia Commonwealth University, Richmond, Virginia 23298 Received for publication 21 January 1977
Naegleria fowleri produces fatal meningoencephalitis in humans and in experimentally infected laboratory animals. The course of the disease in mice is dependent upon the infecting dose of amoebae, route of inoculation, and prior exposure to Naegleria antigens. DUB/ICR mice were immunized by various routes and antigen preparations, held for 21 days, and, together with noninfected control mice, challenged intravenously (i.v.) or intranasally (i.n.) with 107 or 10" N. fowleri per mouse, respectively. Mice immunized with live or formalinized N. fowleri or live N. gruberi subcutaneously, intraperitoneally, i.v., or i.n. were significantly protected against a subsequent lethal challenge with N. fowleri i.v. or i.n. In general, i.v. inoculation afforded greated protection than other routes of immunization, intact cells immunized mice better than did cell fragments, and N. gruberi appeared to be a better immunogen than N. fowleri. Naegleria fowleri, an ameboflagellate, produces fatal meningoencephalitis in humans and laboratory animals. Although the mouse is appropriate as an experimental model for studying amoebic meningoencephalitis (1, 9), there is little information on immunization and host resistance to infection with N. fowleri. Adams et al. (1) have demonstrated that mice surviving a primary infection with N. fowleri were substantially resistant to intravenous (i.v.) challenge with a dose of amoebae that produced uniformly fatal disease in untreated control mice. (erva (5) reported a humoral immune response indicated by complement fixation in guinea pigs infected intranasally (i.n.) with N. fowleri. Delayed hypersensitivity was observed in guinea pigs inoculated subcutaneously (s.c.) with N. fowleri (8). In this study we have demonstrated that mice immunized intraperitoneally (i.p.), i.n., i.v., or s.c. with N. fowleri or the nonpathogen N. gruberi are protected against an otherwise lethal N. fowleri challenge. MATERIALS AND METHODS Male DUB/ICR mice weighing 20 to 25 g were used in all experiments. Mice were obtained from Flow Research Animals, Inc., Dublin, Va. They were allowed to adjust to their new environment for at least 5 days prior to experimentation. The mice were given free access to water and feed (Purina Lab Chow, Ralston Purina Corp.). N. fowleri (LEE strain) and N. gruberi (EGB strain) were cultured axenically in Nelson medium (1) at 37°C and in Balamuth medium (3) at 28°C, respectively. Tissue culture flasks (75 cm2; Falcon Plastics) containing 50 ml of medium were inoculated with 104 amoebae per ml. Amoebae were har-
vested by centrifugation (450 x g, 5 min, 20°C) at 96 h. The cells were washed twice with Page amoeba saline (1) and suspended in 0.15 M NaCl. For counts, 0.2 ml of cell suspension was added to 9.8 ml of electrolyte solution consisting of 0.5% (vol/ vol) formalin and 0.4% (wt/vol) NaCl in distilled water and Vortex-shaken to disperse aggregates. Cells were then counted in a Coulter counter (model ZBI, Coulter Electronics, Inc., Hialeah, Fla.) at settings: matching switch 20 K, gain 0, bandwidth selector extended, 1/amplification 2, 1/aperture current 1, lower threshold 10, upper threshold maximum. Mice were immunized by a single dose (0.2 ml) of amoebae using the following routes and dosages: 3.5 x 107 live N. fowleri s.c., 106 live N. fowleri i.p., 105 live N. fowleri i.v., 103 live N. fowleri i.n., 107 formalinized N. fowleri i. p. or i.v., 107 freeze-thawed N. fowleri with or without Freund complete adjuvant (0.1 ml) i.p. or i.v., and 107 live N. gruberi i.p. or i.v. Formalinized cells were prepared by Vortex-mixing amoebae in 0.5% formalin saline and washing twice with 0.15 M NaCl. Precounted freeze-thawed cells were prepared in screw-capped vials, frozen at - 70°C for 60 min, and thawed at room temperature. This procedure was repeated four times, after which microscopic examination showed no intact cells. There were 20 mice in each experimental or control group unless otherwise specified. Mice were held for 21 days after immunization before they were challenged i.n. with 106 N. fowleri per mouse or i.v. with 107 N. fowleri per mouse. Percent protection was calculated using the expression 100 - (x/y) (100), where x is the percentage of immunized mice that died andy is the percentage of control mice that died.
RESULTS Mice surviving i.p., i.v., s.c., or i.n. immunization with live N. fowleri were held for 21 817
INFECT. IMMUN. 818 JOHN, WEIK, AND ADAMS days. The four groups of 20 mice each plus 20 control mice were then challenged i.v. with 1O7 N. fowleri per mouse. Ninety percent of nonimmunized control mice died within 5 to 11 days as expected. Mice that survived the primary immunizing dose of N. fowleri were partially ~40 protected against the secondary challenge dose (Fig. 1). The immunizing dose was 3.5 x 107 N. fowleri per mouse s.c., 106 i.p., 105 i.v., or 103 i.n., with cumulative dead of 32, 60, 65, and g20 75%, respectively, after i.v. challenge. Additional groups of mice that survived i.p., i.v., or i.n. immunizing doses of live N. fowleri were similarly treated but were challenged i.n. 10 5 0 20 25 30 15 with 106 live N. fowleri per mouse. The total DAYS AFTER CHALLENGE that died from i.n. challenge when the immuFIG. 2. Mortality of mice immunized with live N. nizing dose was i.p. was 35%; i.v., 35%; and i.n., fowleri various routes and challenged 21 days 50% (Fig. 2). Thus, partial protection was af- later withby106N. per mouse i.n. There were 20 forded mice immunized i.p., i.v., or i.n. with N. mice per group. fowleri Symbols: (-) control; (0) i.n.; (A) fowleri and then challenged i.n. i.p.; (U) i.v. Other mice were immunized with 107 formalinized N. fowleri i.p. or i.v., with no deaths occurring for 21 days. These mice plus 20 con100 trols were challenged i.v. with 107 live N. fowleri per mouse. Eighty-five percent of the non80 immunized control mice died within 5 to 10 days, whereas only 45% of i.v. immunized mice died within 7 to 18 days. Mice immunized i.p. W 60i were less protected, with 75% dead. However, the majority of these mice died at least 7 days after the control mice, indicating that protec- -J 40tion may be inapparent if one looks only at cumulative percent dead (Fig. 3). 20Mice immunized i.p. or i.v. with freezethawed N. fowleri and/or Freund complete adjuvant were challenged with a dose of live N. 15 20 25 30 o _5 lo fowleri i.v. Freund complete. adjuvant alone or DAYS AFTER CHALLENGE 100
W 800-
W-L 60-J
40Q
DAYS AFTER CHALLENGE FIG. 1. Mortality of mice immunized with live N. fowleri by various routes and challenged 21 days later with 107 N. fowleri per mouse i.v. There were 20 mice per group except s.c., which had 25. Symbols: (a) control; (0) i.n.; (N) i.v.; (A) i.p.; (AX) s.c.
FIG. 3. Mortality of mice immunized with formalinized N. fowleri by various routes and challenged 21 days later with 107 N. fowleri per mouse i.v. There were 20 mice per group. Symbols: (S) control; (A) i.p.; (U) i.v.
freeze-thawed N. fowleri with or without adjuvant, all given i.p., afforded little protection to mice challenged i.v. with live N. fowleri. However, mice immunized i.v. with freeze-thawed N. fowleri were 33% protected when challenged i.v. compared to nonimmunized controls (Fig. 4). All mice immunized either i.p. or i.v. with 107 live N. gruberi survived the primary inoculation as expected. Twenty-one days later, these mice plus nonimmunized controls were challenged i.v. with 107 live N. fowleri. None of the nonimmunized controls survived, whereas 35% of the i.p. immunized mice survived and 65% of the i.v. immunized mice survived (Fig. 5). Thus, i.v. immunization with N. gruberi is
VOL. 16, 1977
IMMUNITY TO N. FOWLERI INFECTION
more effective than i.p. immunization in protecting mice against a subsequent lethal challenge with N. fowleri. Immunizations with 107 formalinized N. fowleri, freeze-thawed N. fowleri, or live N. gruberi were administered by both i.v. and i.p. routes prior to challenge with live N. fowleri i.v. In all instances i.v. immunized mice were more protected than i.p. immunized mice (Table 1). Mice immunized i.p., i.v., or i.n. with live N. fowleri were similarly protected whether challenged i.v. or i.n. An immunizing dose of 106 N. fowleri i.p., 105 N. fowleri i.v., or 103 N. fowleri i.n. protected 33, 28, and 17%,
819
respectively, after i.v. challenge and 36, 36, and 9%, respectively, after i.n. challenge (Table 1). DISCUSSION Immunization i.v. with formalinized N. fowleri, freeze-thawed N. fowleri, or live N. gruberi was more effective in protecting mice against a subsequent lethal i.v. challenge than was i.p. immunization. A primary dose given i.p. or i.v. was also protective when challenge was i.n. The i.n. route is thought to be the route of entry of N. fowleri in amoebic meningoencephalitis in humans (4); hence, this route is commonly used, to inoculate mice and other experimental animals (7, 9). However, i.v. and i.p. 100 w
80
-
60
W
40
20-
0
5
10
15
20
25
30
DAYS AFTER CHALLENGE FIG. 4. Mortality of mice immunized with freezethawed N. fowleri with or without Freund complete adjuvant by various routes and challenged 21 days later with 107 N. fowleri per mouse i.v. There were 20 mice per group. Symbols: (@) control; (0) adjuvant alone i.p.; (A) i.p.; (A) i.p. plus adjuvant; (U) i.v.
10I 15 20 25 30 DAYS AFTER CHALLENGE FIG. 5. Mortality of mice immunized with live N. gruberi by various routes and challenged 21 days later with 107 N. fowleri per mouse i.v. There were 20 mice per group. Symbols: (0) control; (A) i.p.; (O) _5
i.v.
TABLE 1. Protection of mice against N. fowleri after immunization by various routes and parasite treatment Immunization Challenge No. of mice % Protection % Dead (control) i.p., live N. fowleri i.n. 20 35 (55) 36 i.v., live N. fowleri i.n. 20 35 (55) 36 i.n., live N. fowleri i.n. 20 50 (55) 9 s.c., live N. fowleri i.p., live N. fowleri i.v., live N. fowleri i.n., live N. fowleri
i.v. i.v. i.v. i.v.
25 20 20 20
32 60 65 75
(71) (90) (90) (90)
55 33 28 17
i.p., formalinized N. fowleri i.v., formalinized N. fowleri
i.v. i.v.
20 20
75 (90) 45 (90)
17 50
i.p., freeze-thawed N. fowleri i.p., freeze-thawed N. fowleri + adjuvant i.p., Freund complete adjuvant i.v., freeze-thawed N. fowleri
i.v. i.v.
20 20
85 (90) 80 (90)
6 11
i.v. i.v.
20 20
85 (90) 60 (90)
6 33
i.p., live N. gruberi i.v., live N. gruberi
i.v. i.v.
20 20
65 (100) 35 (100)
35 65
820 JOHN, WEIK, AND ADAMS routes are more exact than the i.n. route since it is difficult to quantitate the number of amoebae retained by the host after i.n. instillation. These data show similar results whether the secondary challenge is administered i.v. or i.n. (Table 1). Immunization with live N. gruberi was effective in protecting mice against N. fowleri challenge (Table 1), indicating that cross-reacting antigens are also protective antigens to the host. Visvesvara and Healy (10) showed that N. fowleri shared three to six common antigens with N. gruberi. One or more of these common antigens, and possibly others, in N. fowleri are probably protective antigens. Culbertson (7) immunized mice i.p. with virulent Acanthamoeba (A-1) and obtained resistance to homologous i.n. challenge. However, he was unable to demonstrate resistance in mice immunized i.p. with avirulent Acanthamoeba (H-A) and challenged i.n. or in mice immunized i.p. with N. fowleri (HB-1) and subsequently challenged i.n. with the same strain. An active infection with live N. fowleri is not required to elicit a protective response by the host, since mice immunized i.v. with formalinized N. fowleri were protected 50% against a subsequent i.v. challenge (Table 1). However, immunization with freeze-thawed cells did not protect mice to the extent that immunization with formalinized cells did (Table 1). Hence, it appears that the development of protection may depend upon the injection of intact cells. Symptoms of central nervous system involvement were more evident in mice challenged i.v. than i.n. Two to 3 days prior to death, i.v. challenged mice often displayed neurological symptoms such as unilateral movement and partial or complete posterior paralysis. Acute death, occurring within 30 min of i.v. challenge, was frequently noted for N. fowleriimmunized mice. These deaths appeared to be associated with route of immunization and were 20, 15, and 5% for mice immunized with N. fowleri i.v., i.p., and i.n., respectively. Occasionally, similar deaths were noted for nonimmunized controls (4% overall). Acute death was 5 and 0% for i.v. and i.p. N. gruberi-immunized mice. Histological examination of lung tissue taken from mice after acute death revealed many capillaries and small blood vessels blocked by amoebae. Anderson and Jamieson (2), using formalinized N. fowleri, produced an antiserum in rabbits capable of agglutinating N. fowleri and, to a lesser degree, N. gruberi. We have obtained similar results using the mouse model, except that the N. fowleri antiserum did not agglutinate N. gruberi amoebae
INFECT. IMMUN.
(unpublished data). It is possible that acute death may be caused by immune agglutination of amoebae and subsequent blockage of pulmonary blood vessels. N. gruberi, which appears to be a better immunogen than N. fowleri (Table 1), produced few acute deaths (2% overall) compared to N. fowleri (17% overall). If acute death were immunologically mediated and N. gruberi antigens were not involved in acute death, it is likely that crossreacting host-protective antigens are not involved but rather are antigens specific for N. fowleri strains. Alternatively, acute death may have been caused by a toxin that is reportedly produced by N. fowleri (6). In summary, i.v. inoculation afforded greater protection than other routes of immunization, intact cells immunized mice better than cell fragments, and N. gruberi appeared to be a better immunogen than N. fowleri. Man's unwitting exposure to nonpathogenic N. gruberi, abundant in all fresh water, raises the interesting possibility that he may become actively immunized against fatal amoebic meningoen-
cephalitis. ACKNOWLEDGMENTS This investigation was supported in part by Research Grant 771019 from A. H. Robins Co., Richmond, Va., and by Public Health Service grant AI-00382 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Adams, A. C., D. T. John, and S. G. Bradley. 1976. Modification of resistance of mice to Naegleria fowleri infections. Infect. Immun. 13:1387-1391. 2. Anderson, K., and A. Jamieson. 1972. Agglutination test for the investigation of the genus Naegleria. Pathology 4:273-278. 3. Balamuth, W. 1964. Nutritional studies on axenic cultures of Naegleria gruberi. J. Protozool. 11:19-20. 4. Carter, R. F. 1972. Primary amoebic meningoencephalitis. An appraisal of present knowledge. Trans. R. Soc. Trop. Med. Hyg. 66:193-213. 5. Cerva, L. 1971. Experimental infection of laboratory animals by the pathogenic Naegleria gruberi strain Vitek. Folia Parasitol. (Prague) 18:171-176. 6. Chang, S. L. 1971. Small, free-living amoebas: cultivation, quantitation, identification, classification, pathogenesis, and resistance, p. 201-254. In T. C. Cheng (ed.), Current topics in comparative pathobiology, vol. 1. Academic Press Inc., New York. 7. Culbertson, C. G. 1971. The pathogenicity of soil amebas. Annu. Rev. Microbiol. 25:231-254. 8. Diffley, P., M. R. Skeels, and F. Sogandares-Bernal. 1976. Delayed type hypersentisivity in guinea pigs infected subcutaneously with Naegleria fowleri, Carter. Z. Parasitenkd. 49:133-137. 9. Martinez, A. J., E. C. Nelson, and R. J. Duma. 1973. Animal model: primary amebic (Naegleria) meningoencephalitis in mice. Am. J. Pathol. 73:545-548. 10. Visvesvara, G. S., and G. R. Healy. 1975. Comparative antigenic analysis of pathogenic and free-living Naegleria species by the gel diffusion and immunoelectrophoresis techniques. Infect. Immun. 11:95-108.
