JOURNAL OF CLINICAL MICROBIOLOGY, May 1976, p. 513-518 Copyright X 1976 American Society for Microbiology
Vol. 3, No. 5 Printed in U.SA.
Antibody Response to Rocky Mountain Spotted Fever R. H. KENYON,* P. G. CANONICO, L. S. SAMMONS, L. R. BAGLEY, AND C. E. PEDERSEN, JR. U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21701
Received for publication 17 February 1976
Various techniques were compared to determine the most sensitive method for detection of Rocky Mountain spotted fever antibody. A radiometabolic technique for detection of Rocky Mountain spotted fever antibody is also described. In infected monkeys, the fluorescent antibody technique yielded the earliest evidence of seroconversion; with some monkeys the microagglutination procedure was equally effective. The fluorescent antibody and microagglutination measurements showed higher titers than those for complement fixation, Weil-Felix, or the radiometabolic techniques. A variety of immunological techniques for the detection of humoral antibody in Rocky Mountain spotted fever (RMSF) has been described. These include complement fixation (CF) (10), Weil-Felix (16), microagglutination (MA) (5), and fluorescent (3) antibody (FA) reactions. The present studies were designed to determine the most sensitive and reliable technique for the detection of RMSF antibody. Standard methods for antibody detection are compared and the temporal course of antibody response is described after experimental RMSF infection of Macaca mulatta and accidental, laboratory-acquired infections in two humans. The median lethal dose of RMSF rickettsial preparations has been difficult to determine for rhesus monkeys in this laboratory. Monkey deaths have occurred using inocula with 101 through 107 plaque-forming units (PFU)/ml. Generally, the greater the infection dose, the shorter the incubation period and the greater the percentage of deaths. Monkeys that had survived RMSF infection were selected to study antibody response over a period of time. Antibody response was also measured in two monkeys with fatal infections. In addition, a radiometabolic technique for rapid detection of RMSF opsonizing antibody is described and evaluated. MATERIALS AND METHODS CF test. CF tests were performed with the microtiter method of Casey (2). A standard rickettsial CF antigen was supplied by the Center for Disease Control, Atlanta, Ga. MA test. MA tests for RMSF antibody were performed as described by Fiset et al. (5). Antigen for this test was prepared from the Sheila Smith strain of Rickettsia rickettsii grown in duck embryo cell (DEC) cultures: rickettsial suspensions collected after differential centrifugation were inactivated
with 0.1% formalin and extracted twice with ethyl ether to remove egg lipids. The antigen suspensions were standardized to contain 1 mg of rickettsiae/ml. Antigen-antiserum suspensions were incubated at 20 C for 18 h, at which time 25 u1 of 0.02% acridine orange was added; results were recorded at 24 h. Weil-Felix antibody tests. Proteus OX2 and OX19 slide antigen, tube antigen, and positive antiserum were purchased from Difco Laboratories, Detroit, Mich. Serum titrations were performed in microtiter plates with 200 Al of diluted serum and 25 A1l of 1:8 saline dilution of slide antigen in each well. Sealed plates were incubated in a 37 C water bath, and agglutination was recorded after 4 h. The tube test was performed as described by Weil and Felix (16). Indirect FA test. WI-38 cells grown on cover slips in Leighton tubes were infected with R. rickettsii at a multiplicity of infection of 5 rickettsiae/cell. At 72 h postinfection, cells were fixed and stored in acetone at -70 C. Human or monkey test sera were diluted serially in 0.015 M phosphate-buffered saline, pH 7.3, and applied to infected cells for 25 min in a moist staining chamber. Preparations were washed with phosphate-buffered saline, and conjugated rabbit anti-human globulin (Microbiological Associates, Bethesda, Md.) or rabbit anti-monkey globulin (prepared in our laboratory) was applied for 30 min. Slides were washed with three changes of phosphate-buffered saline and mounted with phosphate-buffered glycerol. The lowest dilution of the immune sera showed no positive staining on uninfected WI-38 cells. The conjugate itself showed no positive staining on infected WI-38 cells at the working dilution for both conjugates. Stained cells were observed with a Zeiss microscope fitted with BG-38 and BG-12 exciter filters, 10 x oculars, and a 40 x Planapochromat objective. A titration end point was considered to be the highest dilution of test serum with which specific rickettsial fluorescence was observed. Opsonizing antibody test. Serum opsonizing activity was evaluated with a radiometabolic assay as described by Canonico et al. for Francisella tularensis (1). Rhesus monkey peripheral neutrophils (PMN) were harvested from heparinized venous 513
514 blood
J. CLIN. MICROBIOL.
KENYON ET AL.
described by Skoog and Beck (12). Leuko-
tions and at 2-week intervals thereafter for 3 months. pended in glucose-free Hanks balanced salt solution Two human patients developed laboratory-acat 107 PMN/ml. quired infections with RMSF. Patient histories indiTest antigen was prepared from DEC-grown R. cated that both individuals were involved in separickettsii; rickettsiae collected after differential cen- rate unreported laboratory accidents with yolk sactrifugation were resuspended in Hanks balanced grown R. rickettsii 6 to 7 days prior to early sympsalt solution and inactivated by ultraviolet light (45 toms of illness. In both cases the suspected infection min, 4 cm from two 15-W germicidal lamps). The was by aerosols, which were accidentally created antigen suspension was standardized by direct count while working with a needle and syringe. Patient 2 (11) at approximately 2 x 109 rickettsiae/ml. For the had received immunization with a commercially radiometabolic assays, 0.025 ml of test serum, 0.1 ml available RMSF vaccine 4 years previously. Within of antigen, and 0.2 ml of Hanks balanced salt solu- 72 h after the first symptoms appeared, each patient tion supplemented with 4 mM KCN and 0.5 mCi of was seen by a physician and tetracycline chemother[ 1-'4C]glucose were mixed in a nonsiliconized plastic apy was initiated. The presumptive diagnosis for test tube with a rubber cap from which a plastic both was confirmed by isolation of R. rickettsii from center well (Kontes, Vineland, N.J.) containing guinea pigs inoculated with blood samples obtained filter paper saturated with 0.1 ml of hyamine hy- prior to chemotherapy; rickettsemia, however, droxide was suspended. After a 15-min opsonization proved too low for successful recovery by plaque period, 0.1 ml of leukocyte suspension was added techniques. Serum samples collected at intervals and the reaction was incubated for 30 min at 37 C during and after hospitalization were stored at with vigorous shaking (120 strokes/min). The reac- -70 C. Preinfection serum samples for both individtion was stopped by injection of 0.5 ml of trichloroa- uals were available from a previous immunization cetic acid. Released '4CO2 was collected during an surveillance program. additional 45-min incubation period before the plasRESULTS tic center well was transferred to 10 ml of Scintolute (Isolab, Akron, Ohio) in a scintillation vial. RadioMicrotiter technique for Weil-Felix reacactivity was counted with a liquid scintillation counter (Nuclear-Chicago Corp., Austin, Tex.). Op- tion. A comparison of the microtiter, slide, and sonizing activity was equated to hexose monophos- tube techniques for the Weil-Felix reaction is phate shunt activation and reported as counts of shown in Table 1. Although titers were compa'4CO2 released per minute from the oxidation of [1- rable with the three methods, end point reac'4C]glucose by 106 PMN/30 min. tions were achieved more rapidly and were easRickettsiae. The Sheila Smith strain ofR. rickett- iest to read with the microtiter technique. Consii was used. Seed stock was propagated in embryosequently, in this study, all Weil-Felix determinated chicken eggs (15), and all dilutions were made nations were performed by microtitration. using sucrose phosphate glutamate buffer, pH 7.2 Temporal course of antibody responses. An(7). Viable yolk sac-grown rickettsiae were titrated tibody titers and rickettsemia levels were deby the plaquing method described by Weinberg et al. (17), and rickettsemia levels were determined by the termined in sequential blood samples from individual monkeys infected by the i.v. route with methods of Wike and Burgdorfer (18). Serum donors. Six well-conditioned, healthy rhe- 50 or 106 PFU of R. rickettsii (Fig. 1). The sus monkeys, weighing 3 to 4 kg, were purchased monkey inoculated with the 106 dose was rickfrom Prime Laboratories (Farmingdale, N.J.), ettsemic from days 1 to 7, with maximum rickhoused in individual cages, and fed a commercial ettsemia on day 4, and the monkey inoculated ration (Wayne Feed Supply Co., Gaithersburg, with the 50 PFU dose was rickettsemic from Md.). Monkeys selected for use had the following serological profile: MA s 1:2, CF - 1:16, OX2 and days 3 to 11, with the maximum on day 9. OX19 - 1:4, and FA c 1:4. A fourfold or greater Antibody titers were detected earlier and increase in titer was considered significant. Mon- peaked more rapidly after the 106 dose; by day keys were infected by intravenous (i.v.) or subcuta- 4, when rickettsemia was maximum, CF antineous (s.c.) inoculation with the indicated dose of body, OX19 antibody, FA, and MA responses viable yolk sac-grown rickettsiae. Prior to inocula- were detectable. Opsonizing activity appeared tion and on indicated days thereafter, monkeys were on day 7 and achieved maximum values on days bled from the femoral vein. Samples of serum for antibody determinations and of whole blood for rick- TABLE 1. Comparison of slide, tube, and microtiter ettsiae levels were stored at -70 C. techniques for detection of Weil-Felix antibody' Adult rhesus monkeys were vaccinated with inactivated DEC-grown or commercial yolk sac-grown Reciprocal Antibody Technique RMSF vaccine (8). Group I (four monkeys) was im(0X2) (0X19)titer munized with three inoculations at 2-week intervals Slide 320 320 with 0.5 ml of DEC vaccine. Group II (four monkeys) Tube 160 320 was similarly immunized with commercial vaccine. Microtiter 256 256 Monkeys were bled for serum to test for opsonic " activity just prior to the second and third inoculaKnown positive sera. as
cytes separated by dextran sedimentation
were sus-
ANTIBODY RESPONSE TO RMSF
VOL. 3, 1976
o000 E
515
A
500:
CL&
0
w
Ni~
0c
a. DAYS AFTER INOCULATION
FIG. 1. Rickettsemia levels and antibody activity in monkeys that survived i.v. infection with (A) 50 R. rickettsii or (B) 106 R. rickettsii. Opsonic activity is reported as arithmetic mean '4C counts per minute per 106 PMN; each horizontal band represents the mean value ± 2 standard deviations for preinoculation sera. Other antibody measurements are presented as reciprocal log2 titers.
11 to 15. In contrast, after the 50 PFU dose, FA, CF, and MA antibodies were detectable before maximum rickettsemia, i.e., on day 4 for FA and on day 8 for CF and MA; therefore, FA and MA titers increased concomitantly and more rapidly than did CF activity until all achieved peak values on days 15 to 17. As was observed after the 10" dose, opsonizing activity was not detected during rickettsemia; it appeared on day 11 and increased rapidly to achieve a maximum value on day 18. Minimal amounts of OX19 and OX2 agglutinins were present from days 8 to 28. Despite the difference in injected dose, maximum CF, FA, MA, and opsonic activities were essentially identical for both monkeys. Responses of monkeys after s.c. inoculation with 10:1 or 104 PFU of R. rickettsii are presented in Fig. 2 and 3, respectively. Both monkeys injected with 101 rickettsiae had rickettsial levels .10:1 PFU/ml of blood on day 3 and continued to increase until both died of RMSF infection. One monkey that died by day 9 failed to develop detectable antibody; the second monkey died on day 12, soon after an antibody response, detected by FA, MA, CF, and WeilFelix techniques, was initiated. Serum opsonic activity, however, failed to increase in either monkey. Two monkeys that received 104 PFU s.c. survived infection and had elevated FA and MA titers by day 6, when maximum rickettsemia was observed (Fig. 3). A moderate CF antibody response was detected somewhat later (days 9
and 12), and antibody against OX19, inapparent in one monkey, nearly coincided with CF antibody in the other. Increased opsonic activity developed more slowly, appearing on days 12 and 17; in the first monkey the opsonic antibody level was at a maximum on day 21 and thereafter decreased rapidly, but in the second it continued to increase through day 90. Monkeys vaccinated with commercial or DEC-grown vaccine failed to show enhanced opsonic activity at any time after vaccination. Although these monkeys were not challenged, two other groups similarly immunized resisted subsequent s.c. challenge with 104 R. rickettsii 30 days after the last inoculation. Antibody response patterns for the two humans with RMSF were somewhat different (Fig. 4). At no time did sera from these individuals have CF or OX2 antibody titers or detectable opsonic activity. Only sera from the individual who had no prior RMSF vaccination (patient 1) exhibited MA activity; the titer increased rapidly and remained elevated through day 90. However, at 2 to 3 weeks after initial signs of illness, both individuals developed gradually rising FA titers that continued at maximum levels through day 90. A weak OX19 antibody response was initiated somewhat earlier and remained at maximum levels from days 21 through 90. DISCUSSION The primary purpose of this investigation was to determine the most sensitive method for
516
J. CLIN. MICROBIOL.
KENYON ET AL. B
A
-J
LL.
oJ Z
IOX19 .-
r
FA
3O ww
CF, MA
20- OPSONIC ACTIVITY 15-
N
0 x
10d
a-
53
6
9 0 6 9 3 12 DAYS AFTER INOCULATION FIG. 2. Rickettsemia levels and antibody activity in monkeys that succumbed to infection after 0
s.c.
inoculation with 10OR. rickettsii. Opsonic activity is reported as arithmetic mean '4C counts per minute per 106 PMN; each horizontal band represents the mean value + 2 standard deviations for preinoculation sera. Other antibody measurements are presented as reciprocal log2 titers.
-B
500 4
1
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512-
0
FA
> MA --O
FA
_
MA
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Vol. 3, No. 5 Printed in U.SA.
Antibody Response to Rocky Mountain Spotted Fever R. H. KENYON,* P. G. CANONICO, L. S. SAMMONS, L. R. BAGLEY, AND C. E. PEDERSEN, JR. U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21701
Received for publication 17 February 1976
Various techniques were compared to determine the most sensitive method for detection of Rocky Mountain spotted fever antibody. A radiometabolic technique for detection of Rocky Mountain spotted fever antibody is also described. In infected monkeys, the fluorescent antibody technique yielded the earliest evidence of seroconversion; with some monkeys the microagglutination procedure was equally effective. The fluorescent antibody and microagglutination measurements showed higher titers than those for complement fixation, Weil-Felix, or the radiometabolic techniques. A variety of immunological techniques for the detection of humoral antibody in Rocky Mountain spotted fever (RMSF) has been described. These include complement fixation (CF) (10), Weil-Felix (16), microagglutination (MA) (5), and fluorescent (3) antibody (FA) reactions. The present studies were designed to determine the most sensitive and reliable technique for the detection of RMSF antibody. Standard methods for antibody detection are compared and the temporal course of antibody response is described after experimental RMSF infection of Macaca mulatta and accidental, laboratory-acquired infections in two humans. The median lethal dose of RMSF rickettsial preparations has been difficult to determine for rhesus monkeys in this laboratory. Monkey deaths have occurred using inocula with 101 through 107 plaque-forming units (PFU)/ml. Generally, the greater the infection dose, the shorter the incubation period and the greater the percentage of deaths. Monkeys that had survived RMSF infection were selected to study antibody response over a period of time. Antibody response was also measured in two monkeys with fatal infections. In addition, a radiometabolic technique for rapid detection of RMSF opsonizing antibody is described and evaluated. MATERIALS AND METHODS CF test. CF tests were performed with the microtiter method of Casey (2). A standard rickettsial CF antigen was supplied by the Center for Disease Control, Atlanta, Ga. MA test. MA tests for RMSF antibody were performed as described by Fiset et al. (5). Antigen for this test was prepared from the Sheila Smith strain of Rickettsia rickettsii grown in duck embryo cell (DEC) cultures: rickettsial suspensions collected after differential centrifugation were inactivated
with 0.1% formalin and extracted twice with ethyl ether to remove egg lipids. The antigen suspensions were standardized to contain 1 mg of rickettsiae/ml. Antigen-antiserum suspensions were incubated at 20 C for 18 h, at which time 25 u1 of 0.02% acridine orange was added; results were recorded at 24 h. Weil-Felix antibody tests. Proteus OX2 and OX19 slide antigen, tube antigen, and positive antiserum were purchased from Difco Laboratories, Detroit, Mich. Serum titrations were performed in microtiter plates with 200 Al of diluted serum and 25 A1l of 1:8 saline dilution of slide antigen in each well. Sealed plates were incubated in a 37 C water bath, and agglutination was recorded after 4 h. The tube test was performed as described by Weil and Felix (16). Indirect FA test. WI-38 cells grown on cover slips in Leighton tubes were infected with R. rickettsii at a multiplicity of infection of 5 rickettsiae/cell. At 72 h postinfection, cells were fixed and stored in acetone at -70 C. Human or monkey test sera were diluted serially in 0.015 M phosphate-buffered saline, pH 7.3, and applied to infected cells for 25 min in a moist staining chamber. Preparations were washed with phosphate-buffered saline, and conjugated rabbit anti-human globulin (Microbiological Associates, Bethesda, Md.) or rabbit anti-monkey globulin (prepared in our laboratory) was applied for 30 min. Slides were washed with three changes of phosphate-buffered saline and mounted with phosphate-buffered glycerol. The lowest dilution of the immune sera showed no positive staining on uninfected WI-38 cells. The conjugate itself showed no positive staining on infected WI-38 cells at the working dilution for both conjugates. Stained cells were observed with a Zeiss microscope fitted with BG-38 and BG-12 exciter filters, 10 x oculars, and a 40 x Planapochromat objective. A titration end point was considered to be the highest dilution of test serum with which specific rickettsial fluorescence was observed. Opsonizing antibody test. Serum opsonizing activity was evaluated with a radiometabolic assay as described by Canonico et al. for Francisella tularensis (1). Rhesus monkey peripheral neutrophils (PMN) were harvested from heparinized venous 513
514 blood
J. CLIN. MICROBIOL.
KENYON ET AL.
described by Skoog and Beck (12). Leuko-
tions and at 2-week intervals thereafter for 3 months. pended in glucose-free Hanks balanced salt solution Two human patients developed laboratory-acat 107 PMN/ml. quired infections with RMSF. Patient histories indiTest antigen was prepared from DEC-grown R. cated that both individuals were involved in separickettsii; rickettsiae collected after differential cen- rate unreported laboratory accidents with yolk sactrifugation were resuspended in Hanks balanced grown R. rickettsii 6 to 7 days prior to early sympsalt solution and inactivated by ultraviolet light (45 toms of illness. In both cases the suspected infection min, 4 cm from two 15-W germicidal lamps). The was by aerosols, which were accidentally created antigen suspension was standardized by direct count while working with a needle and syringe. Patient 2 (11) at approximately 2 x 109 rickettsiae/ml. For the had received immunization with a commercially radiometabolic assays, 0.025 ml of test serum, 0.1 ml available RMSF vaccine 4 years previously. Within of antigen, and 0.2 ml of Hanks balanced salt solu- 72 h after the first symptoms appeared, each patient tion supplemented with 4 mM KCN and 0.5 mCi of was seen by a physician and tetracycline chemother[ 1-'4C]glucose were mixed in a nonsiliconized plastic apy was initiated. The presumptive diagnosis for test tube with a rubber cap from which a plastic both was confirmed by isolation of R. rickettsii from center well (Kontes, Vineland, N.J.) containing guinea pigs inoculated with blood samples obtained filter paper saturated with 0.1 ml of hyamine hy- prior to chemotherapy; rickettsemia, however, droxide was suspended. After a 15-min opsonization proved too low for successful recovery by plaque period, 0.1 ml of leukocyte suspension was added techniques. Serum samples collected at intervals and the reaction was incubated for 30 min at 37 C during and after hospitalization were stored at with vigorous shaking (120 strokes/min). The reac- -70 C. Preinfection serum samples for both individtion was stopped by injection of 0.5 ml of trichloroa- uals were available from a previous immunization cetic acid. Released '4CO2 was collected during an surveillance program. additional 45-min incubation period before the plasRESULTS tic center well was transferred to 10 ml of Scintolute (Isolab, Akron, Ohio) in a scintillation vial. RadioMicrotiter technique for Weil-Felix reacactivity was counted with a liquid scintillation counter (Nuclear-Chicago Corp., Austin, Tex.). Op- tion. A comparison of the microtiter, slide, and sonizing activity was equated to hexose monophos- tube techniques for the Weil-Felix reaction is phate shunt activation and reported as counts of shown in Table 1. Although titers were compa'4CO2 released per minute from the oxidation of [1- rable with the three methods, end point reac'4C]glucose by 106 PMN/30 min. tions were achieved more rapidly and were easRickettsiae. The Sheila Smith strain ofR. rickett- iest to read with the microtiter technique. Consii was used. Seed stock was propagated in embryosequently, in this study, all Weil-Felix determinated chicken eggs (15), and all dilutions were made nations were performed by microtitration. using sucrose phosphate glutamate buffer, pH 7.2 Temporal course of antibody responses. An(7). Viable yolk sac-grown rickettsiae were titrated tibody titers and rickettsemia levels were deby the plaquing method described by Weinberg et al. (17), and rickettsemia levels were determined by the termined in sequential blood samples from individual monkeys infected by the i.v. route with methods of Wike and Burgdorfer (18). Serum donors. Six well-conditioned, healthy rhe- 50 or 106 PFU of R. rickettsii (Fig. 1). The sus monkeys, weighing 3 to 4 kg, were purchased monkey inoculated with the 106 dose was rickfrom Prime Laboratories (Farmingdale, N.J.), ettsemic from days 1 to 7, with maximum rickhoused in individual cages, and fed a commercial ettsemia on day 4, and the monkey inoculated ration (Wayne Feed Supply Co., Gaithersburg, with the 50 PFU dose was rickettsemic from Md.). Monkeys selected for use had the following serological profile: MA s 1:2, CF - 1:16, OX2 and days 3 to 11, with the maximum on day 9. OX19 - 1:4, and FA c 1:4. A fourfold or greater Antibody titers were detected earlier and increase in titer was considered significant. Mon- peaked more rapidly after the 106 dose; by day keys were infected by intravenous (i.v.) or subcuta- 4, when rickettsemia was maximum, CF antineous (s.c.) inoculation with the indicated dose of body, OX19 antibody, FA, and MA responses viable yolk sac-grown rickettsiae. Prior to inocula- were detectable. Opsonizing activity appeared tion and on indicated days thereafter, monkeys were on day 7 and achieved maximum values on days bled from the femoral vein. Samples of serum for antibody determinations and of whole blood for rick- TABLE 1. Comparison of slide, tube, and microtiter ettsiae levels were stored at -70 C. techniques for detection of Weil-Felix antibody' Adult rhesus monkeys were vaccinated with inactivated DEC-grown or commercial yolk sac-grown Reciprocal Antibody Technique RMSF vaccine (8). Group I (four monkeys) was im(0X2) (0X19)titer munized with three inoculations at 2-week intervals Slide 320 320 with 0.5 ml of DEC vaccine. Group II (four monkeys) Tube 160 320 was similarly immunized with commercial vaccine. Microtiter 256 256 Monkeys were bled for serum to test for opsonic " activity just prior to the second and third inoculaKnown positive sera. as
cytes separated by dextran sedimentation
were sus-
ANTIBODY RESPONSE TO RMSF
VOL. 3, 1976
o000 E
515
A
500:
CL&
0
w
Ni~
0c
a. DAYS AFTER INOCULATION
FIG. 1. Rickettsemia levels and antibody activity in monkeys that survived i.v. infection with (A) 50 R. rickettsii or (B) 106 R. rickettsii. Opsonic activity is reported as arithmetic mean '4C counts per minute per 106 PMN; each horizontal band represents the mean value ± 2 standard deviations for preinoculation sera. Other antibody measurements are presented as reciprocal log2 titers.
11 to 15. In contrast, after the 50 PFU dose, FA, CF, and MA antibodies were detectable before maximum rickettsemia, i.e., on day 4 for FA and on day 8 for CF and MA; therefore, FA and MA titers increased concomitantly and more rapidly than did CF activity until all achieved peak values on days 15 to 17. As was observed after the 10" dose, opsonizing activity was not detected during rickettsemia; it appeared on day 11 and increased rapidly to achieve a maximum value on day 18. Minimal amounts of OX19 and OX2 agglutinins were present from days 8 to 28. Despite the difference in injected dose, maximum CF, FA, MA, and opsonic activities were essentially identical for both monkeys. Responses of monkeys after s.c. inoculation with 10:1 or 104 PFU of R. rickettsii are presented in Fig. 2 and 3, respectively. Both monkeys injected with 101 rickettsiae had rickettsial levels .10:1 PFU/ml of blood on day 3 and continued to increase until both died of RMSF infection. One monkey that died by day 9 failed to develop detectable antibody; the second monkey died on day 12, soon after an antibody response, detected by FA, MA, CF, and WeilFelix techniques, was initiated. Serum opsonic activity, however, failed to increase in either monkey. Two monkeys that received 104 PFU s.c. survived infection and had elevated FA and MA titers by day 6, when maximum rickettsemia was observed (Fig. 3). A moderate CF antibody response was detected somewhat later (days 9
and 12), and antibody against OX19, inapparent in one monkey, nearly coincided with CF antibody in the other. Increased opsonic activity developed more slowly, appearing on days 12 and 17; in the first monkey the opsonic antibody level was at a maximum on day 21 and thereafter decreased rapidly, but in the second it continued to increase through day 90. Monkeys vaccinated with commercial or DEC-grown vaccine failed to show enhanced opsonic activity at any time after vaccination. Although these monkeys were not challenged, two other groups similarly immunized resisted subsequent s.c. challenge with 104 R. rickettsii 30 days after the last inoculation. Antibody response patterns for the two humans with RMSF were somewhat different (Fig. 4). At no time did sera from these individuals have CF or OX2 antibody titers or detectable opsonic activity. Only sera from the individual who had no prior RMSF vaccination (patient 1) exhibited MA activity; the titer increased rapidly and remained elevated through day 90. However, at 2 to 3 weeks after initial signs of illness, both individuals developed gradually rising FA titers that continued at maximum levels through day 90. A weak OX19 antibody response was initiated somewhat earlier and remained at maximum levels from days 21 through 90. DISCUSSION The primary purpose of this investigation was to determine the most sensitive method for
516
J. CLIN. MICROBIOL.
KENYON ET AL. B
A
-J
LL.
oJ Z
IOX19 .-
r
FA
3O ww
CF, MA
20- OPSONIC ACTIVITY 15-
N
0 x
10d
a-
53
6
9 0 6 9 3 12 DAYS AFTER INOCULATION FIG. 2. Rickettsemia levels and antibody activity in monkeys that succumbed to infection after 0
s.c.
inoculation with 10OR. rickettsii. Opsonic activity is reported as arithmetic mean '4C counts per minute per 106 PMN; each horizontal band represents the mean value + 2 standard deviations for preinoculation sera. Other antibody measurements are presented as reciprocal log2 titers.
-B
500 4
1
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512-
0
FA
> MA --O
FA
_
MA
64C-)
