INFECTION AND IMMUNITY, May 1979, p. 337-345 0019-9567/79/05-0337/09$02.00/0
Vol. 24, No. 2
Comparative Behavior of Virulent Strains of Treponema pallidum and Treponema pertenue in Gradient Cultures of Various Mammalian Cells A. HOWARD FIELDSTEEL,* JAMES G. STOUT, AND FRANCES A. BECKERt Life Sciences Division, SRI International, Menlo Park, California 94025 Received for publication 21 February 1979
Two strains of virulent Treponema pallidum and two of virulent T. pertenue were investigated for their ability to attach to and survive in gradient cultures of five different mammalian cells under aerobic conditions. The strains of T. pallidum studied were the high-rabbit-passage Nichols and the low-rabbit-passage KKJ. The former was known to readily attach to cottontail rabbit epithelial cells (SflEp) and to survive in the virulent state for up to 21 days. We therefore compared attachment of the other virulent treponemes with that of T. pallidum (Nichols). The KKJ strain of T. pallidum behaved in a fashion similar to T. pallidum (Nichols) in all of the cultures. Both strains exhibited preferential attachment to cells of Sf1Ep and those derived from the ear of a nude athymic (nu/nu) mouse. In these cultures, we observed a consistent three- to fivefold increase in attached treponemes up to 12 days after initial inoculation. The strains of T. pertenue were the human-derived Gauthier and cynocephalus-derived FB. These two strains of T. pertenue also attached to cells of all five types of cultures, but in smaller numbers than were seen with T. pallidum and equally to all of the cultures. Neither preferential attachment to SflEp and nude mouse ear cells nor increased attachment with time was seen.
Since 1906 there have been many reports of cultivation of Treponemapallidum in vitro (12). The isolated organisms were uniformly avirulent and could not be identified immunologically as T. pallidum. It is probable that the isolated treponemes were saprophytic organisms that were part of the normal genital flora. Recently, Jones et al. (8) reported the successful cultivation of virulent T. pallidum through nine passages in tissue cultures of baby hamster kidney. Foster et al. (4), despite intensive efforts, failed to confirm the results of Jones and his colleagues. Sandok et al. (9) have demonstrated two- to fivefold increases of virulent T. pallidum over a maximum period of 5 days in either cell-free medium or the supernatant fluids of tissue culture containing prepuce cells. They have called this phenomenon "unsustained multiplication." In attempts at cultivation of T. pallidum in vitro, these investigators and others (2, 3) have utilized the Nichols strain of virulent T. pallidum. In our initial efforts to cultivate T. pallidum in tissue culture we also used this strain and were not able to demonstrate replication. However, we were able to show, under aerobic t Present address: Syntex Research, Palo Alto, CA 94043.
conditions, that these treponemes had a 50% survival time (ST5o) of about 14 days and remained virulent in gradient cultures of SflEp cells for up to 21 days (1). We then directed our efforts towards the study of virulent treponemes other than the Nichols strain of T. pallidum. This was done for several reasons. First, we felt that since T. pallidum (Nichols) had been passaged in rabbit testes many hundreds of times for more than 65 years, its behavior in tissue culture might be different from that of other strains with a low-passage history in rabbits. Second, because a close relationship is known to exist between T. pallidum and other virulent treponemes of human origin (10), it was worth determining how these other treponemes behaved in tissue culture. It was our hope that they might be more amenable to in vitro cultivation. In the work reported herein, we reinvestigated T. pallidum (Nichols) and compared it with a low-rabbit-pasage strain of T. pallidum (KKJ) and two strains of T. pertenue in five different cultures of mammalian
cells.
MATERIALS AND METHODS Animals. Six- to 8-month-old New Zealand white male rabbits weighing 3 to 4 kg and free of treponemal 337
338
FIELDSTEEL, STOUT, AND BECKER
infection, as determined by nonreactivity to the VDRL test, were used for passage of all treponemes. The animals were housed at a temperature of 16 to 18'C. Treponemes. T. pallidum (Nichols) was propagated in rabbit testes as described previously (1). T. pallidum (KKJ) was obtained from P. Hardy of the Johns Hopkins University, Baltimore, Md. It was isolated there in 1973 from the cerebrospinal fluid of a newborn infant with congenital syphilis. When received in our laborabory, it was in rabbit testis passage 3. The experiments reported here utilized animal passages 4 through 6. These organisms were propagated in cortisone-treated rabbits by procedures identical to those utilized for propagation of T. pallidum (Nichols). T. pertenue (Gauthier) (7) was obtained from J. W. Clark of the Center for Disease Control, Atlanta, Ga., after an unknown number of passages in rabbit testis after original isolation in hamsters. The organisms were propagated by intratesticular inoculation of 1.3 X 107 to 1.7 x 107 T. pertenue suspended in 0.5 ml of basal reduced medium (BRM). All animals were treated daily with cortisone acetate (6 mg/kg) beginning 3 days after inoculation. Harvest of the organisms was approximately 3 weeks postinfection. Treponema FB was isolated from a lymph node of a cynocephalus baboon by A. Fribourg-Blanc et al. in 1966 (5). It is reported to be a strain of T. pertenue and has also been called T. pertenue, variant cynocephalus (11). A frozen suspension of Treponema FB was obtained from P. Hardy and propagated as described for T. pertenue. All treponemes were extracted from infected testes as described previously (1). For use in tissue culture studies, treponemal suspensions were diluted to a concentration of 3.0 x 107 per ml either in infected rabbit testis extract that had been cleared of treponemes by centrifugation at 12,000 x g for 10 min or in normal testis extract. Treponemes for all experiments were derived from freshly harvested material. However, all inocula were derived from large pools of organisms that had been suspended in BRM containing 15% glycerol, sealed in individual ampoules containing 1.2 ml, and stored in liquid N2. Tissue culture medium components. Earle balanced salt solution was prepared from reagent-grade chemicals, sterilized by membrane filtration (Millipore Corp.), and stored at room temperature. Essential and nonessential amino acids, vitamins, sodium pyruvate, and fetal bovine serum were obtained from Flow Laboratories. HEPES (N-2-hydroxyethyl piperazine-N'-2ethanesulfonic acid), reduced glutathione, DL-cysteine HC1, and dithiothreitol were supplied by Sigma Chemical Co. Resazurin was obtained from Difco Laboratories. Heparin, sodium salt (100 U/mg), was obtained from Nutritional Biochemicals Corp. All other chemicals used in these experiments were also reagent grade. Six lots of fetal bovine serum were screened for their ability to support survival of T. pallidum, and considerable variation was observed. One lot in which maximum survival occurred was used throughout this study. Fetal bovine serum was inactivated at 56°C for 30 min and stored in small aliquots at -20°C until
INFECT. IMMUN.
needed. Amino acid concentrates, vitamins, and glutamine in small aliquots were also stored at -20°C. HEPES was prepared as a 1 M stock (pH 7.3), heparin as a 1-mg/ml stock, resazurin as a 0.20-mg/ml stock, and NaHCO3 as a 7.5% stock. All were sterilized by membrane filtration (Millipore Corp.) and stored at 40C. BRM. BRM and BRM-50 were prepared and preequilibrated from the original formulation (1), with some modifications. Because several lots of commercially prepared 1Ox Eagle minimal essential medium (MEM) were found to be toxic to T. pallidum (Nichols), the 1Ox MEM was replaced with 1Ox Earle balanced salt solution, a 5Ox essential amino acid solution, and a 100x vitamin solution. BRM stock was thus prepared by combining the following compounds in distilled water to a final volume of 100 ml: 10 ml of lOx Earle balanced salt solution without phenol red and NaHCO3, 2 ml of 5Ox MEM amino acids, 1 ml of 100x MEM vitamins, 1 ml of 200 mM L-glutamine, 1 ml of 100x MEM nonessential amino acids, 3.75 ml of 7.5% NaHCO3, 10 mg of sodium pyruvate, 40 mg of reduced glutathione, 20 mg of DL-cysteine HCl, 10 mg of dithiothreitol, and 1 ml of sodium heparin solution containing 100 U/ml. The heparin was found to be a necessary component of the medium because we found that it prevented formation of fibrin clots that caused clumping of the treponemes and obscured the monolayer with particulate debris. Tissue cultures. Cultures of cottontail rabbit epithelium (SflEp), human adult foreskin (HAF), rabbit testis, nude mouse ear (NME), and fetal rabbit skin were grown as described previously (1). The SflEp culture was obtained from the Naval Biomedical Research Laboratory, Oakland, Calif. All other cultures were initiated in our laboratory. Passage levels utilized were: SflEp, 78 to 95; HAF, 18 to 22; rabbit testis, 2 to 5; NME, 9 to 11; and fetal rabbit skin, 9 to 11. For use in experiments, cells suspended in 1 ml of Eagle MEM plus 10% fetal bovine serum were seeded in Leighton tubes. The tubes were sealed with silicone rubber stoppers and incubated aerobically in a horizontal position for 2 or 3 days at 33°C. At the time the treponemes were added, the cell monolayer was approximately 20% confluent. Gradient cultures (1) used throughout these studies were initiated by placing the Leighton tube cultures in a vertical position and adding approximately 6 ml of BRM-50 that contained 106 treponemes per ml and a 1:30 dilution of testis extract. All experiments were also performed under aerobic conditions at 33°C. Enumeration of treponemes. Counts were performed as described previously (1), but with the modification that two fields were counted on the gradient cover slip at each indicated depth. The data from two to ten replicate experiments were averaged and expressed both as average percent motile and average number per field at each level, as well as totals for all five levels observed.
RESULTS Survival of T. pallidum in gradient tissue cultures. These experiments were designed primarily to determine whether T. pallidum sur-
VOL. 24, 1979
T. PALLIDUM AND T. PERTENUE IN TISSUE CULTURE
vives as well in gradient cultures of other mammalian cells as it does in those of SflEp. However, equally important, we wanted to determine whether the Nichols strain of T. pallidum was unique among pathogenic treponemes in its ability to attach to tissue culture cells and survive for relatively long periods of time. From the data presented in Table 1, it is readily apparent that the ability of T. pallidum (Nichols) to attach and survive varied greatly among the cell lines. The SflEp cells gave the best results and the HAF cells gave the poorest. To determine whether the long rabbit passage history had influenced the ability of T. pallidum to replicate or to attach to and survive in various tissue cultures, a series of experiments was carried out with the low-rabbit-passage KKJ strain of T. pallidum (Table 2). The tissue cultures were the same as those used in the previous set of experiments. Simultaneous controls consisting of the Nichols strain of T. pallidum were
339
utilized in all experiments. Because the procedures and results for these controls were the same as for the T. pallidum (Nichols) experiments that were carried out above (Table 1), the two sets of data have been averaged together. Therefore, Table 1 represents the controls for the experiments in Table 2. The ranking of the tissue cultures with respect to the ability of T. pallidum to attach and survive was the same for the KKJ strain as for the Nichols strain. If anything, the KKJ strain appeared to attach to a slightly greater degree and to have a longer ST5o than did the Nichols strain in both the SflEp cultures and the NME. Survival of KKJ was also poorest in the HAF cultures. Survival of T. pertenue in gradient tissue
cultures. Other investigators have shown that nonvirulent, cultivable treponemes did not attach to tissue cultures (2, 3, 6), and it had been assumed that the phenomenon of attachment was characteristic of pathogenic, noncultivable
TABLE 1. Survival of T. pallidum (Nichols) in gradient cultures of various mammalian tissues Avg % motile treponemesh (avg no. of treponemes counted) Depth in Cell strain or line gradient Day 14 Day 9 Day 12 Day 7 Day 5 Day 2 (mm) 55 (53) 5 (10) 54 (30) 22 (19) 25 (16) 54 (8) 5 SflEp 88 (101) 53 (46) 88 (93) 79 (53) 76 (63) 94 (23) 11 95 (95) 75 (35) 94 (111) 97 (153) 93 (76) 96 (33) 17 56 (37) 89 (73) 95 (95) 95 (137) 92 (66) 96 (28) 23 15 (8) 29 (14) 78 (42) 73 (26) 70 (30) 93 (22) 29 53 (136) 83 (336) 96 (455) 86 (301) 80 (254) 92 (114) Avg (total) 0 (1) 42 (5) 42 (5) 74 (5) 85 (4) 5 HAF 44 (4) 14 (2) 90 (10) 79 (14) 11 97 (6) 22 (2) 0 (0) 54 (4) 90 (9) 100 (14) 17 0 (3) 0 (2) 0 (1) 33 (2) 100 (4) 23 0 (1) 0 (3) 6 (2) 50 (1) 85 (2) 29 5 (7) 26 (16) 62 (22) 78 (31) 96 (30) Avg (total) 0 (1) 54 (2) 89 (14) 87 (4) 33 (1) 27 (9) 5 RT 61 (8) 65 (19) 88 (23) 97 (90) 92 (28) 88 (21) 11 0 ( N XAvt > 3n 0 0 X o 0 N t o > >Q~~~~~~~~~~~~~~I C .0Oeteeo°>b°te°bn
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Vol. 24, No. 2
Comparative Behavior of Virulent Strains of Treponema pallidum and Treponema pertenue in Gradient Cultures of Various Mammalian Cells A. HOWARD FIELDSTEEL,* JAMES G. STOUT, AND FRANCES A. BECKERt Life Sciences Division, SRI International, Menlo Park, California 94025 Received for publication 21 February 1979
Two strains of virulent Treponema pallidum and two of virulent T. pertenue were investigated for their ability to attach to and survive in gradient cultures of five different mammalian cells under aerobic conditions. The strains of T. pallidum studied were the high-rabbit-passage Nichols and the low-rabbit-passage KKJ. The former was known to readily attach to cottontail rabbit epithelial cells (SflEp) and to survive in the virulent state for up to 21 days. We therefore compared attachment of the other virulent treponemes with that of T. pallidum (Nichols). The KKJ strain of T. pallidum behaved in a fashion similar to T. pallidum (Nichols) in all of the cultures. Both strains exhibited preferential attachment to cells of Sf1Ep and those derived from the ear of a nude athymic (nu/nu) mouse. In these cultures, we observed a consistent three- to fivefold increase in attached treponemes up to 12 days after initial inoculation. The strains of T. pertenue were the human-derived Gauthier and cynocephalus-derived FB. These two strains of T. pertenue also attached to cells of all five types of cultures, but in smaller numbers than were seen with T. pallidum and equally to all of the cultures. Neither preferential attachment to SflEp and nude mouse ear cells nor increased attachment with time was seen.
Since 1906 there have been many reports of cultivation of Treponemapallidum in vitro (12). The isolated organisms were uniformly avirulent and could not be identified immunologically as T. pallidum. It is probable that the isolated treponemes were saprophytic organisms that were part of the normal genital flora. Recently, Jones et al. (8) reported the successful cultivation of virulent T. pallidum through nine passages in tissue cultures of baby hamster kidney. Foster et al. (4), despite intensive efforts, failed to confirm the results of Jones and his colleagues. Sandok et al. (9) have demonstrated two- to fivefold increases of virulent T. pallidum over a maximum period of 5 days in either cell-free medium or the supernatant fluids of tissue culture containing prepuce cells. They have called this phenomenon "unsustained multiplication." In attempts at cultivation of T. pallidum in vitro, these investigators and others (2, 3) have utilized the Nichols strain of virulent T. pallidum. In our initial efforts to cultivate T. pallidum in tissue culture we also used this strain and were not able to demonstrate replication. However, we were able to show, under aerobic t Present address: Syntex Research, Palo Alto, CA 94043.
conditions, that these treponemes had a 50% survival time (ST5o) of about 14 days and remained virulent in gradient cultures of SflEp cells for up to 21 days (1). We then directed our efforts towards the study of virulent treponemes other than the Nichols strain of T. pallidum. This was done for several reasons. First, we felt that since T. pallidum (Nichols) had been passaged in rabbit testes many hundreds of times for more than 65 years, its behavior in tissue culture might be different from that of other strains with a low-passage history in rabbits. Second, because a close relationship is known to exist between T. pallidum and other virulent treponemes of human origin (10), it was worth determining how these other treponemes behaved in tissue culture. It was our hope that they might be more amenable to in vitro cultivation. In the work reported herein, we reinvestigated T. pallidum (Nichols) and compared it with a low-rabbit-pasage strain of T. pallidum (KKJ) and two strains of T. pertenue in five different cultures of mammalian
cells.
MATERIALS AND METHODS Animals. Six- to 8-month-old New Zealand white male rabbits weighing 3 to 4 kg and free of treponemal 337
338
FIELDSTEEL, STOUT, AND BECKER
infection, as determined by nonreactivity to the VDRL test, were used for passage of all treponemes. The animals were housed at a temperature of 16 to 18'C. Treponemes. T. pallidum (Nichols) was propagated in rabbit testes as described previously (1). T. pallidum (KKJ) was obtained from P. Hardy of the Johns Hopkins University, Baltimore, Md. It was isolated there in 1973 from the cerebrospinal fluid of a newborn infant with congenital syphilis. When received in our laborabory, it was in rabbit testis passage 3. The experiments reported here utilized animal passages 4 through 6. These organisms were propagated in cortisone-treated rabbits by procedures identical to those utilized for propagation of T. pallidum (Nichols). T. pertenue (Gauthier) (7) was obtained from J. W. Clark of the Center for Disease Control, Atlanta, Ga., after an unknown number of passages in rabbit testis after original isolation in hamsters. The organisms were propagated by intratesticular inoculation of 1.3 X 107 to 1.7 x 107 T. pertenue suspended in 0.5 ml of basal reduced medium (BRM). All animals were treated daily with cortisone acetate (6 mg/kg) beginning 3 days after inoculation. Harvest of the organisms was approximately 3 weeks postinfection. Treponema FB was isolated from a lymph node of a cynocephalus baboon by A. Fribourg-Blanc et al. in 1966 (5). It is reported to be a strain of T. pertenue and has also been called T. pertenue, variant cynocephalus (11). A frozen suspension of Treponema FB was obtained from P. Hardy and propagated as described for T. pertenue. All treponemes were extracted from infected testes as described previously (1). For use in tissue culture studies, treponemal suspensions were diluted to a concentration of 3.0 x 107 per ml either in infected rabbit testis extract that had been cleared of treponemes by centrifugation at 12,000 x g for 10 min or in normal testis extract. Treponemes for all experiments were derived from freshly harvested material. However, all inocula were derived from large pools of organisms that had been suspended in BRM containing 15% glycerol, sealed in individual ampoules containing 1.2 ml, and stored in liquid N2. Tissue culture medium components. Earle balanced salt solution was prepared from reagent-grade chemicals, sterilized by membrane filtration (Millipore Corp.), and stored at room temperature. Essential and nonessential amino acids, vitamins, sodium pyruvate, and fetal bovine serum were obtained from Flow Laboratories. HEPES (N-2-hydroxyethyl piperazine-N'-2ethanesulfonic acid), reduced glutathione, DL-cysteine HC1, and dithiothreitol were supplied by Sigma Chemical Co. Resazurin was obtained from Difco Laboratories. Heparin, sodium salt (100 U/mg), was obtained from Nutritional Biochemicals Corp. All other chemicals used in these experiments were also reagent grade. Six lots of fetal bovine serum were screened for their ability to support survival of T. pallidum, and considerable variation was observed. One lot in which maximum survival occurred was used throughout this study. Fetal bovine serum was inactivated at 56°C for 30 min and stored in small aliquots at -20°C until
INFECT. IMMUN.
needed. Amino acid concentrates, vitamins, and glutamine in small aliquots were also stored at -20°C. HEPES was prepared as a 1 M stock (pH 7.3), heparin as a 1-mg/ml stock, resazurin as a 0.20-mg/ml stock, and NaHCO3 as a 7.5% stock. All were sterilized by membrane filtration (Millipore Corp.) and stored at 40C. BRM. BRM and BRM-50 were prepared and preequilibrated from the original formulation (1), with some modifications. Because several lots of commercially prepared 1Ox Eagle minimal essential medium (MEM) were found to be toxic to T. pallidum (Nichols), the 1Ox MEM was replaced with 1Ox Earle balanced salt solution, a 5Ox essential amino acid solution, and a 100x vitamin solution. BRM stock was thus prepared by combining the following compounds in distilled water to a final volume of 100 ml: 10 ml of lOx Earle balanced salt solution without phenol red and NaHCO3, 2 ml of 5Ox MEM amino acids, 1 ml of 100x MEM vitamins, 1 ml of 200 mM L-glutamine, 1 ml of 100x MEM nonessential amino acids, 3.75 ml of 7.5% NaHCO3, 10 mg of sodium pyruvate, 40 mg of reduced glutathione, 20 mg of DL-cysteine HCl, 10 mg of dithiothreitol, and 1 ml of sodium heparin solution containing 100 U/ml. The heparin was found to be a necessary component of the medium because we found that it prevented formation of fibrin clots that caused clumping of the treponemes and obscured the monolayer with particulate debris. Tissue cultures. Cultures of cottontail rabbit epithelium (SflEp), human adult foreskin (HAF), rabbit testis, nude mouse ear (NME), and fetal rabbit skin were grown as described previously (1). The SflEp culture was obtained from the Naval Biomedical Research Laboratory, Oakland, Calif. All other cultures were initiated in our laboratory. Passage levels utilized were: SflEp, 78 to 95; HAF, 18 to 22; rabbit testis, 2 to 5; NME, 9 to 11; and fetal rabbit skin, 9 to 11. For use in experiments, cells suspended in 1 ml of Eagle MEM plus 10% fetal bovine serum were seeded in Leighton tubes. The tubes were sealed with silicone rubber stoppers and incubated aerobically in a horizontal position for 2 or 3 days at 33°C. At the time the treponemes were added, the cell monolayer was approximately 20% confluent. Gradient cultures (1) used throughout these studies were initiated by placing the Leighton tube cultures in a vertical position and adding approximately 6 ml of BRM-50 that contained 106 treponemes per ml and a 1:30 dilution of testis extract. All experiments were also performed under aerobic conditions at 33°C. Enumeration of treponemes. Counts were performed as described previously (1), but with the modification that two fields were counted on the gradient cover slip at each indicated depth. The data from two to ten replicate experiments were averaged and expressed both as average percent motile and average number per field at each level, as well as totals for all five levels observed.
RESULTS Survival of T. pallidum in gradient tissue cultures. These experiments were designed primarily to determine whether T. pallidum sur-
VOL. 24, 1979
T. PALLIDUM AND T. PERTENUE IN TISSUE CULTURE
vives as well in gradient cultures of other mammalian cells as it does in those of SflEp. However, equally important, we wanted to determine whether the Nichols strain of T. pallidum was unique among pathogenic treponemes in its ability to attach to tissue culture cells and survive for relatively long periods of time. From the data presented in Table 1, it is readily apparent that the ability of T. pallidum (Nichols) to attach and survive varied greatly among the cell lines. The SflEp cells gave the best results and the HAF cells gave the poorest. To determine whether the long rabbit passage history had influenced the ability of T. pallidum to replicate or to attach to and survive in various tissue cultures, a series of experiments was carried out with the low-rabbit-passage KKJ strain of T. pallidum (Table 2). The tissue cultures were the same as those used in the previous set of experiments. Simultaneous controls consisting of the Nichols strain of T. pallidum were
339
utilized in all experiments. Because the procedures and results for these controls were the same as for the T. pallidum (Nichols) experiments that were carried out above (Table 1), the two sets of data have been averaged together. Therefore, Table 1 represents the controls for the experiments in Table 2. The ranking of the tissue cultures with respect to the ability of T. pallidum to attach and survive was the same for the KKJ strain as for the Nichols strain. If anything, the KKJ strain appeared to attach to a slightly greater degree and to have a longer ST5o than did the Nichols strain in both the SflEp cultures and the NME. Survival of KKJ was also poorest in the HAF cultures. Survival of T. pertenue in gradient tissue
cultures. Other investigators have shown that nonvirulent, cultivable treponemes did not attach to tissue cultures (2, 3, 6), and it had been assumed that the phenomenon of attachment was characteristic of pathogenic, noncultivable
TABLE 1. Survival of T. pallidum (Nichols) in gradient cultures of various mammalian tissues Avg % motile treponemesh (avg no. of treponemes counted) Depth in Cell strain or line gradient Day 14 Day 9 Day 12 Day 7 Day 5 Day 2 (mm) 55 (53) 5 (10) 54 (30) 22 (19) 25 (16) 54 (8) 5 SflEp 88 (101) 53 (46) 88 (93) 79 (53) 76 (63) 94 (23) 11 95 (95) 75 (35) 94 (111) 97 (153) 93 (76) 96 (33) 17 56 (37) 89 (73) 95 (95) 95 (137) 92 (66) 96 (28) 23 15 (8) 29 (14) 78 (42) 73 (26) 70 (30) 93 (22) 29 53 (136) 83 (336) 96 (455) 86 (301) 80 (254) 92 (114) Avg (total) 0 (1) 42 (5) 42 (5) 74 (5) 85 (4) 5 HAF 44 (4) 14 (2) 90 (10) 79 (14) 11 97 (6) 22 (2) 0 (0) 54 (4) 90 (9) 100 (14) 17 0 (3) 0 (2) 0 (1) 33 (2) 100 (4) 23 0 (1) 0 (3) 6 (2) 50 (1) 85 (2) 29 5 (7) 26 (16) 62 (22) 78 (31) 96 (30) Avg (total) 0 (1) 54 (2) 89 (14) 87 (4) 33 (1) 27 (9) 5 RT 61 (8) 65 (19) 88 (23) 97 (90) 92 (28) 88 (21) 11 0 ( N XAvt > 3n 0 0 X o 0 N t o > >Q~~~~~~~~~~~~~~I C .0Oeteeo°>b°te°bn
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