Vol. 15, No. 3
INFECTION AND IMMUNITY, Mar. 1977, p. 813-816 Copyright © 1977 American Society for Microbiology
Printed in U.S.A.
Indirect Immunofluorescence Antibodies in Natural and Acquired Rickettsia tsutsugamushi Infections of Philippine Rodents1 P. F. D. VAN PEENEN,2 C. M. HO, AND A. L. BOURGEOIS
U.S. Naval Medical Research Unit No. 2, APO San Francisco, California 96263 Received for publication 12 September 1976
Antibodies against Rickettsia tsutsugamushi detected by the indirect fluorescent-antibody test (IFAT) were present in most rats trapped from a human focus of scrub typhus in the Philippines. Rickettsiae were isolated only from rats with positive IFATs. Naturally acquired antibodies persisted for at least 11 months, and antibodies resulting from experimental infections of rats persisted for at least 7 months. A common Philippine rodent, Rattus mindanensis, tolerated experimental infections with both local and standard Karp strains ofR. tsutsugamushi, and such infections always produced a positive IFAT.
with R. tsutsugamushi (6). In addition, the persistence of antibodies acquired through both natural and experimental infection of wild rats was studied.
The role of wild rodents in the ecology of scrub typhus is controversial. There is a considerable body of evidence suggesting that chiggers alone are involved in transmission of infection and that rodents simply serve as sources of chigger blood meals (5). However, evidence of infection in rodents is uneven, especially in Southeast Asia, indicating that rodent species vary in attractiveness or susceptibility to infection (4). In addition, there is recent experimental evidence that chiggers may, in fact, acquire Rickettsia tsutsugamushi infection from mice and transmit it either to other mice by interrupted feeding or by transovarial transmission to trombiculid progeny (6). Although evidence for infection of rodents from the field -has usually been by actual isolation of rickettsiae, recent studies have relied upon seroepidemiological evidence. The indirect fluorescent-antibody test (IFAT) has been used extensively in Malaysia to detect antibodies against R. tsutsugamushi in wild mammals (3, 4, 7). In our laboratory, the test was applied to sera of rodents collected in a human focus of scrub typhus in the Republic of the Philippines, and antibodies were found in many of the rodent sera examined. The present study was undertaken to learn whether the presence of antibodies in wild rats was associated with harborage of viable scrub typhus rickettsiae, since it is known that rickettsiae will persist for long periods in animals infected experimentally
MATERIALS AND METHODS Rodents. Wild rats were collected from a study area in Central Luzon (15'09'N, 120027'E, 300-m elevation) and transported alive to laboratories in Taipei, Taiwan. Wild Rattus mindanensis were bred in the laboratory, and F1 progeny were used in some experiments. Outbred adult laboratory mice of the Swiss white strain, weighing 18 to 25 g, were used for isolation of rickettsiae. Strains of R. tsutsugamushi. Rickettsiae of the Karp, Gilliam, and Kato strains of R. tsutsugamushi were obtained in the 46th, 147th, and 123rd egg passage, respectively, from the Naval Medical Research Institute, Bethesda, Md. Murine typhus rickettsiae in low egg passage were also obtained from the Naval Medical Research Institute. TSV-798 is a highly lethal Taiwan scrub typhus strain that was isolated originally from the spleen of Rattus losea and has since been passed continuously by intraperitoneal injection of laboratory mice for about 14 generations. Strain PVS 350-54 was isolated from a pool of organs from four R. mindanensis from the Central Luzon focus and had undergone only three mouse passages. Strain PVS-193 was isolated from the spleen and kidney of a single R. mindanensis from the Central Luzon focus and had also undergone only three mouse passages. Antibodies. The indirect fluorescent-antibody test (IFAT) was performed as described by Bozeman and Elisberg (1) and modified by Gan et al. (2). Sera for IFAT were obtained from the ocular sinuses or by heart puncture. Antigens consisted of single spots of Karp, Kato, and Gilliam strains of R. tsutsugamushi in heavily infected yolk sac suspensions. A R. mooseri spot was also included, and known positive and negative control sera were run in conjunction
' Reprint requests to: Publications Office, NAMRU-2, Box 14, APO San Francisco 96263, or 7-1 Kung Yuan Road, Taipei, Taiwan, Republic of China. 2 Present address: Department of Preventive Medicine, Uniformed Services University of Health Sciences, Bethesda, MD 20014.
813
VAN PEENEN, HO, AND BOURGEOIS
INF ECT . IMMUN .
with test sera in the IFAT. Fluorescein-conjugated, high-titered rabbit anti-R. norvegicus gamma globulin was used in the IFAT for wild rats, whereas rabbit anti-Mus musculus gamma globulin was used to test for antibodies in mice. All sera were screened at 1:20 or 1:40 dilutions. Isolation of rickettsiae. Isolation of rickettsiae was attempted by aseptically removing the spleen and a kidney from sacrificed animals, grinding with 5 volumes of brain heart infusion broth, and inoculating 0.2 ml of the 20% suspension intraperitoneally into four to six laboratory mice. Mice were examined daily; if morbidity or mortality occurred, mice were sacrificed, and peritoneal smears were stained with Giemsa and examined for rickettsiae. If mice remained healthy, second passages were made after 14 days. Second-passage mice surviving 14 to 21 days were challenged with 100 mean lethal doses (LD50) of either the Karp strain or strain TVS798, and only if mice survived were they considered infected. Biopsies and sequential bleedings. Biopsies of spleen and kidney were performed surgically using open-drop ether. A spleen snip was removed with no attempt at hemostasis, whereas for kidney biopsy an entire kidney was removed after the renal artery and vein had been tied off. Sequential bleedings were done by puncture of the ocular sinus with a capillary pipette.
least one of the standard R. tsutsugamushi strains except for the R. panglima, which was negative at .1:40 but positive at 1:20 in March 1976, after 8 months. However, that rat had IFAT antibodies at a .1:40 serum dilution in all other months. The remaining 15 rats checked monthly for persistence of antibodies consisted of 12 R. mindanensis, 3 R. everetti, and 2 R. exulans whose rickettsial infection status was unknown. Four, all R. mindanensis, were antibody negative for 11 months. Serum from another was positive against only the Karp antigen at 1:20 when first tested but was negative against all antigens when tested subsequently. The remainder, with one exception, consistently had antibodies against at least one of the three standard strains when tested at .1:40 for 9 months. The exception, R. exulans, had antibodies for 5 consecutive months but was negative thereafter at a 1:40 dilution, although antibodies were detected at a 1:20 serum dilution for 3 more months. Antibodies after experimental infection with local R. tsutsugamushi strains. This experiment was continued for 7 months. Nine wild-caught R. mindanensis, which were twice tested and found to be antibody-free, were in-
814
RESULTS
jected intraperitoneally with 102
Antibodies and rickettsiae in wild rats. Sixty-four rats were examined. Most (84%) had antibodies when checked at the -1:20 serum dilution, but rickettsiae were isolated from only eight (Table 1). All isolations were from antibody-positive rats. The rats in this experiment were sacrificed and were not used for studies reported below. Persistence of antibodies in natural infections. Four rats in this experiment were known to be infected, since rickettsiae had been isolated from splenic biopsies. These were single specimens of R. panglima and R. exulans and two R. mindanensis, from which rickettsiae were isolated in August, October, and November 1975, respectively. Sera from all four were tested monthly for 11 months and consistently had positive IFATs at titers .1:40 against at
mouse
LD50 Of
R. tsutsugamushi PVS 350-4. On subsequent monthly bleedings, for 5 months, sera from all contained immunofluorescence antibodies at a 1:40 dilution when tested against the Karp strain. Sera obtained after 6 months lacked antibodies in the case of three rats and, depending on the antigen, were often present only at a 1:20 serum dilution in the others. However, antibodies were present in the 7-month sera from all rats that had antibodies previously. There were monthly variations in titers of antibodies against the Gilliam strain from the same rats. Antibodies against Kato strain rickettsiae at titers .1:40 were present in all sera, except in one R. mindanensis that was negative at 1:40 on one occasion (February 1976) but was positive at that time at a 1:20 dilution and at 1:40 for the first 5 months.
TABLE 1. IFAT and Rickettsia isolations from wild Philippine rodents Isolation and serology (%) Rat species No. examined Antibody positive (%)
Rattus mindanensis 38 76 R. rattus 12 100 7 R. everetti 100 5 R. exulans 80 R. panglima 2 100 a No animals were isolation positive, serology negative.
Both positive Both negative
10 0 29 40 0
24 0 0 20 0
Sertil pegative0la66 100 71 40 100
R. TSUTSUGAMUSHI IN R. MINDANENSIS
VOL. 15, 1977
Twelve laboratory reared, F1 generation R. mindanensis were injected intraperitoneally or subcutaneously with 103 mouse LD50 of R. tsutsugamushi PVS-193. Animals were bled and sacrificed in pairs, one each of those injected intraperitoneally and subcutaneously, at various periods, with results as shown in Table 2. Rickettsiae were isolated, from one animal, as early as 4 days after injection, but antibodies did not appear in any rats until day 11. There was variation in results; inexplicably, antibodies were lacking in both rats sacrificed 14 days after injection. As shown, isolation of rickettsiae was somewhat more successful from animals injected intraperitoneally. Antibodies after experimental injections with the Karp strain. Six antibody-free F1 generation R. mindanensis were inoculated intraperitoneally with 101 to 104 mouse LD50 of Karp strain rickettsiae. All survived with no signs of illness. When serum was tested 21 days after injection, antibodies were present and, in all cases, highest titers were against the homologous strain. Nevertheless, anti-Gilliam and anti-Kato antibodies were detected (Table 3). TABLE 2. Antibodies and rickettsial isolations from 12 laboratory-reared R. mindanensis sacrificed periodically in pairs after injection with R. tsutsugamushi PVS-193 Days after Antibody (1:20)a
Isolation
injection 0 4 7 11 14 21 36 60 90
All neg. NDb IP pos., SC neg.e Both neg. Both neg. Both neg. Both pos. Both pos. Both neg. Both pos. ND Both pos. Both pos. IP pos., SC neg. Both pos. Both neg. Both pos. Both pos. a Neg., Negative; pos., positive. b ND, Not done. e IP, Rat inoculated intraperitoneally; SC, rat inoculated subcutaneously.
TABLE 3. IFAT titers in F1 generation R. mindanensis inoculated with Karp strain
rickettsiaea Inoculum (mouse LDWo)
101
a
DISCUSSION Although proven rickettsial infection was always associated with the presence of IFAT antibodies in wild Philippine rats, presence of antibodies often did not indicate active infection. In fact, rickettsiae could not be isolated from the majority of rats that were antibody positive at the time of capture. Nevertheless, IFAT antibodies caused by natural infection were remarkably stable and, with few exceptions, persisted for 10 to 11 months, which was the longest period, studied. Antibodies produced by experimental infection persisted for 7 months at serum dilutions of .1:40. The "temporary" loss of antibodies in three animals at the 6-month bleeding may have been due to technical problems with antigens used in the IFAT. Although not detailed, the antigenic character of antibodies, except for the IFAT against Gilliam strain rickettsiae, was quite stable in naturally infected rats; for example, rats with positive IFAT against the Karp strain, when first tested, usually had similar test results throughout. As with Malaysian rats (7), Philippine rats tolerated R. tsutsugamushi infection well and showed no signs of illness even after exposure to heavy inocula ofR. tsutsugamushi, whether of local or standard (Karp) strains. Although rickettsiae could not be isolated from a pair of experimentally infected rats 60 days after infection in our experiments, R. tsutsugamushi were isolated from the 90-day pair (Table 2). In addition to the results presented here, we have also isolated rickettsiae from wild-caught rats held in the laboratory for periods of up to 3 months before examination. Serological results obtained with both naturally and experimentally infected Philippine rats confirm the value of the IFAT in seroepidemiological studies. Antibodies persist for periods of time that encompass a good part of the normal life-span of the animals in the wild. Although there was some variation in results obtained by sequential bleedings, it is significant that rickettsiae were never isolated from antibody-free animals and that known infection was always associated with antibody production.
IFAT titer Karp
Gilliam
Kato
Murine
1:640 1:20 1:20 0 1:640 1:80 1:80 0 103 1:1,280 1:20 0 0 103 1:1,280 1:320 1:320 0 104 1:320 0 0 0 104 1:640 0 1:40 0 All rats were originally antibody negative. 102
815
ACKNOWLEDGMENTS We are grateful to Emilio Weiss and D. L. Huxsoll for valuable comments on the manuscript. This study was supported through funds provided by the Naval Medical Research and Development Command, Navy Department, for Work Unit MF51.524.009-0037. The research described in this report involved animals maintained in animal care facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care.
816
VAN PEENEN, HO, AND BOURGEOIS LITERATURE CITED
1. Bozeman, F. M., and B. L. Elisberg. 1963. Serological diagnosis of scrub typhus by indirect immunofluorescence. Proc. Soc. Exp. Biol. Med. 112:568-573. 2. Gan, E., F. C. Cadigan, Jr., and J. S. Walker. 1972. Filter paper collection of blood for use in a screening and diagnostic test for scrub typhus using the IFAT. Trans. R. Soc. Trop. Med. Hyg. 66:588-593. 3. Muul, I., L. B. Liat, and J. S. Walker. 1975. Mammals and scrub typhus ecology in Peninsular Malaysia. Trans. R. Soc. Trop. Med. Hyg. 69:121-130. 4. Muul, I., B. L. Lim, and E. Gan. 1974. Scrub typhus
INFECT. IMMUN. antibody in mammals in three habitats in Sabah. Southeast Asian J. Trop. Med. Public Health 5:80-84. 5. Traub, R., and C. L. Wisseman, Jr. 1974. The ecology of chigger-borne rickettsiosis (scrub typhus). J. Med. Entomol. 11:237-303. 6. Traub, R., C. L. Wisseman, Jr., M. R. Jones, and J. J. O'Keefe. 1975. The acquisition ofRickettsai tsutsugamushi by chiggers (Trombiculid mites) during the feeding process. Ann. N. Y. Acad. Sci. 266:91-114. 7. Walker, J. S., E. Gan, C. T. Chye, and I. Muul. 1973. Involvement of small mammals in the transmission of scrub typhus in Malaysia: isolation and serological evidence. Trans. R. Soc. Trop. Med. Hyg. 67:838-845.
INFECTION AND IMMUNITY, Mar. 1977, p. 813-816 Copyright © 1977 American Society for Microbiology
Printed in U.S.A.
Indirect Immunofluorescence Antibodies in Natural and Acquired Rickettsia tsutsugamushi Infections of Philippine Rodents1 P. F. D. VAN PEENEN,2 C. M. HO, AND A. L. BOURGEOIS
U.S. Naval Medical Research Unit No. 2, APO San Francisco, California 96263 Received for publication 12 September 1976
Antibodies against Rickettsia tsutsugamushi detected by the indirect fluorescent-antibody test (IFAT) were present in most rats trapped from a human focus of scrub typhus in the Philippines. Rickettsiae were isolated only from rats with positive IFATs. Naturally acquired antibodies persisted for at least 11 months, and antibodies resulting from experimental infections of rats persisted for at least 7 months. A common Philippine rodent, Rattus mindanensis, tolerated experimental infections with both local and standard Karp strains ofR. tsutsugamushi, and such infections always produced a positive IFAT.
with R. tsutsugamushi (6). In addition, the persistence of antibodies acquired through both natural and experimental infection of wild rats was studied.
The role of wild rodents in the ecology of scrub typhus is controversial. There is a considerable body of evidence suggesting that chiggers alone are involved in transmission of infection and that rodents simply serve as sources of chigger blood meals (5). However, evidence of infection in rodents is uneven, especially in Southeast Asia, indicating that rodent species vary in attractiveness or susceptibility to infection (4). In addition, there is recent experimental evidence that chiggers may, in fact, acquire Rickettsia tsutsugamushi infection from mice and transmit it either to other mice by interrupted feeding or by transovarial transmission to trombiculid progeny (6). Although evidence for infection of rodents from the field -has usually been by actual isolation of rickettsiae, recent studies have relied upon seroepidemiological evidence. The indirect fluorescent-antibody test (IFAT) has been used extensively in Malaysia to detect antibodies against R. tsutsugamushi in wild mammals (3, 4, 7). In our laboratory, the test was applied to sera of rodents collected in a human focus of scrub typhus in the Republic of the Philippines, and antibodies were found in many of the rodent sera examined. The present study was undertaken to learn whether the presence of antibodies in wild rats was associated with harborage of viable scrub typhus rickettsiae, since it is known that rickettsiae will persist for long periods in animals infected experimentally
MATERIALS AND METHODS Rodents. Wild rats were collected from a study area in Central Luzon (15'09'N, 120027'E, 300-m elevation) and transported alive to laboratories in Taipei, Taiwan. Wild Rattus mindanensis were bred in the laboratory, and F1 progeny were used in some experiments. Outbred adult laboratory mice of the Swiss white strain, weighing 18 to 25 g, were used for isolation of rickettsiae. Strains of R. tsutsugamushi. Rickettsiae of the Karp, Gilliam, and Kato strains of R. tsutsugamushi were obtained in the 46th, 147th, and 123rd egg passage, respectively, from the Naval Medical Research Institute, Bethesda, Md. Murine typhus rickettsiae in low egg passage were also obtained from the Naval Medical Research Institute. TSV-798 is a highly lethal Taiwan scrub typhus strain that was isolated originally from the spleen of Rattus losea and has since been passed continuously by intraperitoneal injection of laboratory mice for about 14 generations. Strain PVS 350-54 was isolated from a pool of organs from four R. mindanensis from the Central Luzon focus and had undergone only three mouse passages. Strain PVS-193 was isolated from the spleen and kidney of a single R. mindanensis from the Central Luzon focus and had also undergone only three mouse passages. Antibodies. The indirect fluorescent-antibody test (IFAT) was performed as described by Bozeman and Elisberg (1) and modified by Gan et al. (2). Sera for IFAT were obtained from the ocular sinuses or by heart puncture. Antigens consisted of single spots of Karp, Kato, and Gilliam strains of R. tsutsugamushi in heavily infected yolk sac suspensions. A R. mooseri spot was also included, and known positive and negative control sera were run in conjunction
' Reprint requests to: Publications Office, NAMRU-2, Box 14, APO San Francisco 96263, or 7-1 Kung Yuan Road, Taipei, Taiwan, Republic of China. 2 Present address: Department of Preventive Medicine, Uniformed Services University of Health Sciences, Bethesda, MD 20014.
813
VAN PEENEN, HO, AND BOURGEOIS
INF ECT . IMMUN .
with test sera in the IFAT. Fluorescein-conjugated, high-titered rabbit anti-R. norvegicus gamma globulin was used in the IFAT for wild rats, whereas rabbit anti-Mus musculus gamma globulin was used to test for antibodies in mice. All sera were screened at 1:20 or 1:40 dilutions. Isolation of rickettsiae. Isolation of rickettsiae was attempted by aseptically removing the spleen and a kidney from sacrificed animals, grinding with 5 volumes of brain heart infusion broth, and inoculating 0.2 ml of the 20% suspension intraperitoneally into four to six laboratory mice. Mice were examined daily; if morbidity or mortality occurred, mice were sacrificed, and peritoneal smears were stained with Giemsa and examined for rickettsiae. If mice remained healthy, second passages were made after 14 days. Second-passage mice surviving 14 to 21 days were challenged with 100 mean lethal doses (LD50) of either the Karp strain or strain TVS798, and only if mice survived were they considered infected. Biopsies and sequential bleedings. Biopsies of spleen and kidney were performed surgically using open-drop ether. A spleen snip was removed with no attempt at hemostasis, whereas for kidney biopsy an entire kidney was removed after the renal artery and vein had been tied off. Sequential bleedings were done by puncture of the ocular sinus with a capillary pipette.
least one of the standard R. tsutsugamushi strains except for the R. panglima, which was negative at .1:40 but positive at 1:20 in March 1976, after 8 months. However, that rat had IFAT antibodies at a .1:40 serum dilution in all other months. The remaining 15 rats checked monthly for persistence of antibodies consisted of 12 R. mindanensis, 3 R. everetti, and 2 R. exulans whose rickettsial infection status was unknown. Four, all R. mindanensis, were antibody negative for 11 months. Serum from another was positive against only the Karp antigen at 1:20 when first tested but was negative against all antigens when tested subsequently. The remainder, with one exception, consistently had antibodies against at least one of the three standard strains when tested at .1:40 for 9 months. The exception, R. exulans, had antibodies for 5 consecutive months but was negative thereafter at a 1:40 dilution, although antibodies were detected at a 1:20 serum dilution for 3 more months. Antibodies after experimental infection with local R. tsutsugamushi strains. This experiment was continued for 7 months. Nine wild-caught R. mindanensis, which were twice tested and found to be antibody-free, were in-
814
RESULTS
jected intraperitoneally with 102
Antibodies and rickettsiae in wild rats. Sixty-four rats were examined. Most (84%) had antibodies when checked at the -1:20 serum dilution, but rickettsiae were isolated from only eight (Table 1). All isolations were from antibody-positive rats. The rats in this experiment were sacrificed and were not used for studies reported below. Persistence of antibodies in natural infections. Four rats in this experiment were known to be infected, since rickettsiae had been isolated from splenic biopsies. These were single specimens of R. panglima and R. exulans and two R. mindanensis, from which rickettsiae were isolated in August, October, and November 1975, respectively. Sera from all four were tested monthly for 11 months and consistently had positive IFATs at titers .1:40 against at
mouse
LD50 Of
R. tsutsugamushi PVS 350-4. On subsequent monthly bleedings, for 5 months, sera from all contained immunofluorescence antibodies at a 1:40 dilution when tested against the Karp strain. Sera obtained after 6 months lacked antibodies in the case of three rats and, depending on the antigen, were often present only at a 1:20 serum dilution in the others. However, antibodies were present in the 7-month sera from all rats that had antibodies previously. There were monthly variations in titers of antibodies against the Gilliam strain from the same rats. Antibodies against Kato strain rickettsiae at titers .1:40 were present in all sera, except in one R. mindanensis that was negative at 1:40 on one occasion (February 1976) but was positive at that time at a 1:20 dilution and at 1:40 for the first 5 months.
TABLE 1. IFAT and Rickettsia isolations from wild Philippine rodents Isolation and serology (%) Rat species No. examined Antibody positive (%)
Rattus mindanensis 38 76 R. rattus 12 100 7 R. everetti 100 5 R. exulans 80 R. panglima 2 100 a No animals were isolation positive, serology negative.
Both positive Both negative
10 0 29 40 0
24 0 0 20 0
Sertil pegative0la66 100 71 40 100
R. TSUTSUGAMUSHI IN R. MINDANENSIS
VOL. 15, 1977
Twelve laboratory reared, F1 generation R. mindanensis were injected intraperitoneally or subcutaneously with 103 mouse LD50 of R. tsutsugamushi PVS-193. Animals were bled and sacrificed in pairs, one each of those injected intraperitoneally and subcutaneously, at various periods, with results as shown in Table 2. Rickettsiae were isolated, from one animal, as early as 4 days after injection, but antibodies did not appear in any rats until day 11. There was variation in results; inexplicably, antibodies were lacking in both rats sacrificed 14 days after injection. As shown, isolation of rickettsiae was somewhat more successful from animals injected intraperitoneally. Antibodies after experimental injections with the Karp strain. Six antibody-free F1 generation R. mindanensis were inoculated intraperitoneally with 101 to 104 mouse LD50 of Karp strain rickettsiae. All survived with no signs of illness. When serum was tested 21 days after injection, antibodies were present and, in all cases, highest titers were against the homologous strain. Nevertheless, anti-Gilliam and anti-Kato antibodies were detected (Table 3). TABLE 2. Antibodies and rickettsial isolations from 12 laboratory-reared R. mindanensis sacrificed periodically in pairs after injection with R. tsutsugamushi PVS-193 Days after Antibody (1:20)a
Isolation
injection 0 4 7 11 14 21 36 60 90
All neg. NDb IP pos., SC neg.e Both neg. Both neg. Both neg. Both pos. Both pos. Both neg. Both pos. ND Both pos. Both pos. IP pos., SC neg. Both pos. Both neg. Both pos. Both pos. a Neg., Negative; pos., positive. b ND, Not done. e IP, Rat inoculated intraperitoneally; SC, rat inoculated subcutaneously.
TABLE 3. IFAT titers in F1 generation R. mindanensis inoculated with Karp strain
rickettsiaea Inoculum (mouse LDWo)
101
a
DISCUSSION Although proven rickettsial infection was always associated with the presence of IFAT antibodies in wild Philippine rats, presence of antibodies often did not indicate active infection. In fact, rickettsiae could not be isolated from the majority of rats that were antibody positive at the time of capture. Nevertheless, IFAT antibodies caused by natural infection were remarkably stable and, with few exceptions, persisted for 10 to 11 months, which was the longest period, studied. Antibodies produced by experimental infection persisted for 7 months at serum dilutions of .1:40. The "temporary" loss of antibodies in three animals at the 6-month bleeding may have been due to technical problems with antigens used in the IFAT. Although not detailed, the antigenic character of antibodies, except for the IFAT against Gilliam strain rickettsiae, was quite stable in naturally infected rats; for example, rats with positive IFAT against the Karp strain, when first tested, usually had similar test results throughout. As with Malaysian rats (7), Philippine rats tolerated R. tsutsugamushi infection well and showed no signs of illness even after exposure to heavy inocula ofR. tsutsugamushi, whether of local or standard (Karp) strains. Although rickettsiae could not be isolated from a pair of experimentally infected rats 60 days after infection in our experiments, R. tsutsugamushi were isolated from the 90-day pair (Table 2). In addition to the results presented here, we have also isolated rickettsiae from wild-caught rats held in the laboratory for periods of up to 3 months before examination. Serological results obtained with both naturally and experimentally infected Philippine rats confirm the value of the IFAT in seroepidemiological studies. Antibodies persist for periods of time that encompass a good part of the normal life-span of the animals in the wild. Although there was some variation in results obtained by sequential bleedings, it is significant that rickettsiae were never isolated from antibody-free animals and that known infection was always associated with antibody production.
IFAT titer Karp
Gilliam
Kato
Murine
1:640 1:20 1:20 0 1:640 1:80 1:80 0 103 1:1,280 1:20 0 0 103 1:1,280 1:320 1:320 0 104 1:320 0 0 0 104 1:640 0 1:40 0 All rats were originally antibody negative. 102
815
ACKNOWLEDGMENTS We are grateful to Emilio Weiss and D. L. Huxsoll for valuable comments on the manuscript. This study was supported through funds provided by the Naval Medical Research and Development Command, Navy Department, for Work Unit MF51.524.009-0037. The research described in this report involved animals maintained in animal care facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care.
816
VAN PEENEN, HO, AND BOURGEOIS LITERATURE CITED
1. Bozeman, F. M., and B. L. Elisberg. 1963. Serological diagnosis of scrub typhus by indirect immunofluorescence. Proc. Soc. Exp. Biol. Med. 112:568-573. 2. Gan, E., F. C. Cadigan, Jr., and J. S. Walker. 1972. Filter paper collection of blood for use in a screening and diagnostic test for scrub typhus using the IFAT. Trans. R. Soc. Trop. Med. Hyg. 66:588-593. 3. Muul, I., L. B. Liat, and J. S. Walker. 1975. Mammals and scrub typhus ecology in Peninsular Malaysia. Trans. R. Soc. Trop. Med. Hyg. 69:121-130. 4. Muul, I., B. L. Lim, and E. Gan. 1974. Scrub typhus
INFECT. IMMUN. antibody in mammals in three habitats in Sabah. Southeast Asian J. Trop. Med. Public Health 5:80-84. 5. Traub, R., and C. L. Wisseman, Jr. 1974. The ecology of chigger-borne rickettsiosis (scrub typhus). J. Med. Entomol. 11:237-303. 6. Traub, R., C. L. Wisseman, Jr., M. R. Jones, and J. J. O'Keefe. 1975. The acquisition ofRickettsai tsutsugamushi by chiggers (Trombiculid mites) during the feeding process. Ann. N. Y. Acad. Sci. 266:91-114. 7. Walker, J. S., E. Gan, C. T. Chye, and I. Muul. 1973. Involvement of small mammals in the transmission of scrub typhus in Malaysia: isolation and serological evidence. Trans. R. Soc. Trop. Med. Hyg. 67:838-845.
