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Distribution and disposal of radioactivity in mice infected with Fe59-labeled cercariae of Schistosoma mansoni
EXPERIMENTAL
PARASITOLOGY
11,
147-151 (1961)
Distribution and Disposal of Radioactivity Infected with Fe5’-Labeled Cercariae Schistosoma mansoni Francisco U.S.A. Tropical
J. Corretjer
Research Medical
and
Laboratory,
Milton
in Mice of
E. Rubinil
and Department
of Medicine,
University
of Puerto Rico, School of Medicine, San Juan, Puerto Rico (Submitted
for publication,
21 November
1960)
(1) Living cercariae of Schistosoma mansoni may be labeled by incubation with Feso. (2) Such cercariae were infective for newborn mice and carried significant radioactivity into the host. (3) The biologic half-life of this radioactivity was shorter than that of Fe59 fed or injected. (4) Significant radioactivity was found in the blood, liver, spleen, and lungs.
Radioisotopically labeled living cercariae offer many obvious opportunities of examining certain parameters of schistosome infection.2 Ideally such a label should be intimately attached to the cercariae, should be of a reasonably long half-life, and should be a strong energy emitter to minimize tissue absorption and permit simple counting techniques. This report examines the behavior of Fe50 as an in vitro label for the cercariae of Schistosoma mansoni. The data indicate that such radioactivity may be carried into the host in experimental infections.
ported to the radioisotope laboratory as soon as possible, and were not used unless they were visibly motile. Usually studies were initiated within 3 hours of shedding. Radioactivity was monitored with a well or flat surface shielded scintillation detector. All counts were at least 10 times background. Appropriate standards were employed to correct for geometry, physical decay, and minor fluctuations in counting efficiency. Albino mice used in these experiments were of the Bagg strain previously inbred at Walter Reed Army Medical Center. Preparation of FeSS-Labeled Cercariae
METHODS
Living cercariae of S. mansoni were obtained from infected snails (Australorbis glabratus). Shedding took place in dechlorinated water and was induced by exposure to light. The organisms were concentrated by heliotropism and sedimentation (Olivier and Stirewalt, 1952; Gumble et al., 1957). Extensive experience in this laboratory with similarly handled cercariae has shown that they maintained their infectivity for experimental animals. The cercariae were trans-
Live or dead cercariae were incubated with gentle agitation in dechlorinated tap water with radioactive iron without carrier. After varying incubation times, 20-cc samples were removed, assayed, centrifuged at 2000 rpm for 30 minutes, and the top 18-19 cc of water removed. Residual water and cercariae were reassayed; several washings were performed with 10 cc of water or normal saline and recentrifuging, taking care to minimize damage to the organisms. Several selected experiments are shown in Table I. Iron-59 appeared to adhere to both live and dead cercariae. The living cercariae absorbed two to three times as much radioactivity as dead cercariae. Radioactive
1 Present address: Division of Medicine, Walter Reed Army Institute of Research, Washington, D.C. 2 A recent example of radioisotope tagging of helminth larvae parasites reviews similar applications (Dissanaike, 1958). 147
148
CORRET JER
Incubation Isotope 1. Fesscitrate
2.
Fe59
PC
Cercariae, cca
Incubation, min.
Period
% Adherent 2 washingsb
% Adherent 4 washing9
15 30 45
29.5 31.7 29.9
27.9 30.1 28.0
60 90 120 150
33.0 25.4 33.5 33.6
30.6 23.9 32.5 32.1
180
20.6
19.0
15 30 60 120
17.2 21.9 16.1 18.0
170 Alive
15 30 90 60
31.7 22.8 22.5 23.0
170
15 30 90 60
9.5 8.8 6.8 7.7
1.050
908
Alive
0.056
0.038
RUBINI
TABLE I of Cercariae with Fe $9 Efiect of Varying Incubation
500
Heatkilled
3. Fe59
AND
Heatkilled IJ Cercariae concentration was determined b Expressed relative to total radioactivity
by direct counting after iodine killing initially present in counting tube.
iron was firmly adherent to the live cercariae and not removed by serial washings (the slight loss of radioactivity evident after increasing the number of washings from two to four is compatible with the unavoidable loss of cercariae). Increasing incubation time to longer than 15 minutes did not appreciably increase the amount of tagging. Concentration of iron in the water was approximately 10 yg ‘j6. Raising iron concentration lo-fold by the addition of “cold” ferric chloride did not interfere with tagging, and increasing iron concentration SOO- to lOOOfold prevented tagging completely. After the organism was already tagged, adding iron as either ferrous sulfate or ferric chloride did not displace the radioactivity adherent to the living cercariae. RESULTS
Infection with Labeled Cercariae Mice were experimentally infected with cercariae which had been incubated with Fe59-citrate. One-day-old mice were selected because of their susceptibility to infection.
and staining.
Each mouse was exposed to a known number of cercariae by dipping them in the upright position in 1 to 3 cc of incubate. Care was taken to avoid imbibition of the radioactive solution. Control experiments in which the cercariae were killed after incubation by heating were performed with littermates (Table II). In other experiments the dipping was performed in identical manner in a solution containing identical amounts of radioactivity but devoid of cercariae. Approximately four times as much radioactivity adhered to mice exposed to live labeled cercariae as when the labeled cercariae were heat killed. Much of the radioactivity adherent to skin was lost rapidly after dipping and the 12-hour counts are the more pertinent. From these and similar experiments it appeared that the transfer of radioactivity from the incubate containing tagged cercariae to the mouse appeared similar over a threefold range of radioactivity of incubating medium. Infectivity of the cercariae for the mouse was probably little altered by increasing radiation effects on the cercariae. How-
HOST
RADIOACTIVITY
AFTER
INFECTION
WITH
LABELED
CERCARIAE
149
TABLE II Adherence of Fe59 to Newborn Mice Each mouse was dipped for 30 minutes in l-3 cc of water containing Fess and live or dead cercariae, blotted dry, dropped in a test tube, counted in a well counter, and then returned to the dam. Adherent radioactivity was monitored again after 12 hours. No. of mice
State of cercariae
Cercariae mouse
Av. at 12 hr adherent x lo-3
9 5
Alive Heat-killed
3,oooa 3,000
28.1 2 3.5 FCb 8.0 2 1.4
11 6
Alive Heat-killed
750 750
7.1 -+ 0.3 pcb 1.7 + 0.5
5 This is probably a lethal dose for a newborn mouse. However, only one death occurred in 12 hours of observation. b Differences between live and killed are highly significant (t test; P < .OOl).
ever, some newborn mice survived after contact with over 2000 cercariae, which is unusual in our experience and suggests that infectivity of cercariae exposed to Fe5” was reduced even at lowest radiation dosage. Survival studies in mice exposed to labeled and unlabeled cercariae (circa 700 cercariae per mouse) did not reveal an obvious difference in mortality, and worm recovery on liver perfusion was similar at 6 to 7 weeks after infection. Biologic Half-Life of Fe5g Infected and noninfected radioactive mice were counted daily to evaluate biological halflife. The dam was observed to lick repeatedly the young, and she and her feces contained appreciable radioactivity. Furthermore, the young huddled together constantly and presumably cross-contaminated their skins. Accordingly control experiments were performed in which Fe5g was force fed or injected intraperitoneally. The results are reported in Table III. Retention of Fe5g in mice infected with labeled live cercariae differs from those injected, fed, or exposed to killed labeled cercariae. Distribution of Fe59 The distribution of the radioactivity carried by the labeled living cercariae into the infected mouse was examined (Table IV). Anesthetized mice were killed by exsanguination 24 and 48 hours after exposure; blood, skin, and the various organs were weighed and counted at constant volume. At 24 hours after exposure, the skin was the main site of radioactivity; by 48 hours a
12-fold reduction in relative skin radioactivity occurred. Activity in other organs relative to blood also decreased but much less than in the skin. Internal radioactivity increased more than that in the organs examined, the ratio (counts per gram of organ/counts per gram of blood) dropped. If 55% of each organ were assumed to be blood, then a significant proportion of the monitored radioactivity could be accounted for. All spleen radioactivity could be attributed to its blood content; the gut, liver, and lungs would have definite radioactivity but less than 5% of that in the entire body; the skin would be little affected and still contain more than 25% of Fe5” contained in the entire body. Much of the activity in internal organs could not be accounted for by any reasonable estimate of blood content, which suggests that some iron5” was introduced into the body with the intact cercariae. DISCUSSION
It would appear that Fe”!) tags living cercariae and to a lesser extent dead cercariae. When the living infective cercariae previously labeled with Fesg are brought in contact with a suitable host the radioactivity is carried into the body of the host, presumably by the head as the tail is probably left at the site of penetration (Standen, 1953). The behavior of such radioactive iron is different from fed or injected iron in that it localizes in lungs and intestine, as well as liver, and a large proportion is not incorporated into the red cell mass. Its rapid half-life suggests the tag does not remain with a viable grow-
150
CORRET JER
Biological
AND
TABLE III Half-Life
RUBINI
of Fe.59
Exposure
‘Hr
Number of mice
Live labeled cercariae
0 12 36 60 84
17
177.4
27 27 27 7
100.0a
3 9 9 9
198.7 100.0a 77.0 62.5
1 7 7 7
119.4 100.0~ 82.6 68.9
9 9 8 7 7
107.3 100.0~ 99.1 93.7 90.0
Dead labeled cercariae
0 12 36 60 84
Fed Fess solution
Av. half-life hrb
61.2 42.3 33.6
in
40.8
67.2
-
0 12 36 60 84
Injected intraperitoneally with Fe59 solution
Av. body activitya (% of 12 hr)
88.8
-
0 12 36 60 84
283.2
a Body activity 12 hr after exposure was selected as point of reference as much of the skin radioactivity of mice exposed to either live or dead cercariae was rapidly lost. Plots of log body activity vs time became linear after 12 hr. b Average of individually determined half-lives.
Distribution
TABLE IV of Radioactivity (‘jk of Total Body Counts) in Newborn Exposed to Live Cercariae (Counts/min/gram
organ relative
to blood) 48 hr. (5 mice)
24 hr. (5 mice)
Organ Blood Skin Liver Spleen Lungs GI Tract Carcass
Av. activity relative to blood5 1
12.310 0.482 0.097 0.122 0.654
Mice
Av. % total activity in mouseb
Av. activity relative to blooda
3.3 58.3 1.2 0.2 0.3 3.0 31.5
0.888 0.087 0.049 0.098 0.102
Q Average of individual determinations on each mouse. b Obtained by dividing amount of radioactivity in pooled organs from activity present in group of mice at time of sacrifice X 100.
Av. % total activity in mouseb
1
each mouse by amount
27.5 24.4 2.4 1 .o
2.4 4.7 37.8 of radio-
HOST
RADIOACTIVITY
AFTER
INFECTION
ing worm, but rather remains bound to some component of the cercariae in a form not readily utilizable by the host. Whether living worms shed the iron by exchange with serum iron of the host, or whether the radiation effects on the cercariae cause an alteration in their survival, and hence loss of radioactivity after death, cannot be answered by these experiments. Exchange for unlabeled iron stores of the host seems unlikely because most of the retained Febg is not used to manufacture red cells, especially in the newborn mouse whose iron stores are probably minimal. The selective residual radioactivity in the lungs is especially interesting as iron released into the lungs differs from iron released elsewhere in the body, and in man remains in situ for protracted periods (Soergel, 1957). The radioactivity in lung tissue might be interpreted as suggesting that the tag remains with the cercariae through its passage into the lungs and if the cercariae are entrapped, it remains in situ (Filho, 1959). This interpretation of our data would be contrary to the opinion of Olivier and Schneidermann (1953) that the migrating worms do not arrive in the lungs until the fourth day after infection. We have tried to find radioactivity in the maturing organism, and recovered the growing adult worms after 5 to 7 weeks by perfusing the liver. Although detectable radioactivity remains in the mouse at this time, recovered worms contained no discernible activity, and most of the residual radioactivity in mice older than one month could be accounted for by that in the red cells. Radioautographic studies of similarly infected mice at short intervals may be a
WITH
LABELED
151
CERCARIAE
more fruitful approach to this problem, to see whether any appreciable radioactivity coincides with the site of the parasite. ACKNOWLEDGMENT
The authors are indebted to Drs. Seymour and Lawrence S. Ritchie, Division of Zoology, U.S. Army Tropical Research Laboratory, for advice and criticism, and Genoveva Montalvo for technical assistance.
Garson Medical Medical to Miss
REFERENCES
G. A., JEFFRY, G. M., AND BARTON, B. P. 1958. Radioactive tagging of hookworm larvae (Necator americanus) with P32. Experimental Parasitology 7, 249.253. FILHO, A. M. 1959. Pulmonary lesions in mice experimentally infected with Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene 8, 527-536. GUMBLE, A., OTORE, Y., RITCKIE, L. S., AND HUNTER, G. W., III. 1957. The effect of light, temperature and pH on the emergence of Schistosoma japonicum cercariae from Oncomelania nosophora. Transactions of the American Microscopical Society 76, 87-92. DISSANAIKE,
OLIVIER,
L.,
AND
SCHNEIDERMANN,
M.
1953.
Ac-
quired resistance to Schistosoma mansoni infections in laboratory animals. American Journal of Tropical Medicine and Hygiene 2, 298-306. OLIVIER, L., AND STIREWALT, M. A. 1952. An efficient method for exposure of mice to cercariae of Schistosoma mansoni. Journal of Parasitology 38, 19-23. SOERGEL,K. H. 1957. Ideopathic pulmonary hemosiderosis. Review and report of two cases. Pediatrics 19, 1101-1108. STANDEN, 0. D. 1953. The penetration of the cercariae of Schistosoma mansoni into the skin and lymphatics of the mouse. Transactions of the Royal Society of Tropical Medicine and Hygiene 47, 292.298.
PARASITOLOGY
11,
147-151 (1961)
Distribution and Disposal of Radioactivity Infected with Fe5’-Labeled Cercariae Schistosoma mansoni Francisco U.S.A. Tropical
J. Corretjer
Research Medical
and
Laboratory,
Milton
in Mice of
E. Rubinil
and Department
of Medicine,
University
of Puerto Rico, School of Medicine, San Juan, Puerto Rico (Submitted
for publication,
21 November
1960)
(1) Living cercariae of Schistosoma mansoni may be labeled by incubation with Feso. (2) Such cercariae were infective for newborn mice and carried significant radioactivity into the host. (3) The biologic half-life of this radioactivity was shorter than that of Fe59 fed or injected. (4) Significant radioactivity was found in the blood, liver, spleen, and lungs.
Radioisotopically labeled living cercariae offer many obvious opportunities of examining certain parameters of schistosome infection.2 Ideally such a label should be intimately attached to the cercariae, should be of a reasonably long half-life, and should be a strong energy emitter to minimize tissue absorption and permit simple counting techniques. This report examines the behavior of Fe50 as an in vitro label for the cercariae of Schistosoma mansoni. The data indicate that such radioactivity may be carried into the host in experimental infections.
ported to the radioisotope laboratory as soon as possible, and were not used unless they were visibly motile. Usually studies were initiated within 3 hours of shedding. Radioactivity was monitored with a well or flat surface shielded scintillation detector. All counts were at least 10 times background. Appropriate standards were employed to correct for geometry, physical decay, and minor fluctuations in counting efficiency. Albino mice used in these experiments were of the Bagg strain previously inbred at Walter Reed Army Medical Center. Preparation of FeSS-Labeled Cercariae
METHODS
Living cercariae of S. mansoni were obtained from infected snails (Australorbis glabratus). Shedding took place in dechlorinated water and was induced by exposure to light. The organisms were concentrated by heliotropism and sedimentation (Olivier and Stirewalt, 1952; Gumble et al., 1957). Extensive experience in this laboratory with similarly handled cercariae has shown that they maintained their infectivity for experimental animals. The cercariae were trans-
Live or dead cercariae were incubated with gentle agitation in dechlorinated tap water with radioactive iron without carrier. After varying incubation times, 20-cc samples were removed, assayed, centrifuged at 2000 rpm for 30 minutes, and the top 18-19 cc of water removed. Residual water and cercariae were reassayed; several washings were performed with 10 cc of water or normal saline and recentrifuging, taking care to minimize damage to the organisms. Several selected experiments are shown in Table I. Iron-59 appeared to adhere to both live and dead cercariae. The living cercariae absorbed two to three times as much radioactivity as dead cercariae. Radioactive
1 Present address: Division of Medicine, Walter Reed Army Institute of Research, Washington, D.C. 2 A recent example of radioisotope tagging of helminth larvae parasites reviews similar applications (Dissanaike, 1958). 147
148
CORRET JER
Incubation Isotope 1. Fesscitrate
2.
Fe59
PC
Cercariae, cca
Incubation, min.
Period
% Adherent 2 washingsb
% Adherent 4 washing9
15 30 45
29.5 31.7 29.9
27.9 30.1 28.0
60 90 120 150
33.0 25.4 33.5 33.6
30.6 23.9 32.5 32.1
180
20.6
19.0
15 30 60 120
17.2 21.9 16.1 18.0
170 Alive
15 30 90 60
31.7 22.8 22.5 23.0
170
15 30 90 60
9.5 8.8 6.8 7.7
1.050
908
Alive
0.056
0.038
RUBINI
TABLE I of Cercariae with Fe $9 Efiect of Varying Incubation
500
Heatkilled
3. Fe59
AND
Heatkilled IJ Cercariae concentration was determined b Expressed relative to total radioactivity
by direct counting after iodine killing initially present in counting tube.
iron was firmly adherent to the live cercariae and not removed by serial washings (the slight loss of radioactivity evident after increasing the number of washings from two to four is compatible with the unavoidable loss of cercariae). Increasing incubation time to longer than 15 minutes did not appreciably increase the amount of tagging. Concentration of iron in the water was approximately 10 yg ‘j6. Raising iron concentration lo-fold by the addition of “cold” ferric chloride did not interfere with tagging, and increasing iron concentration SOO- to lOOOfold prevented tagging completely. After the organism was already tagged, adding iron as either ferrous sulfate or ferric chloride did not displace the radioactivity adherent to the living cercariae. RESULTS
Infection with Labeled Cercariae Mice were experimentally infected with cercariae which had been incubated with Fe59-citrate. One-day-old mice were selected because of their susceptibility to infection.
and staining.
Each mouse was exposed to a known number of cercariae by dipping them in the upright position in 1 to 3 cc of incubate. Care was taken to avoid imbibition of the radioactive solution. Control experiments in which the cercariae were killed after incubation by heating were performed with littermates (Table II). In other experiments the dipping was performed in identical manner in a solution containing identical amounts of radioactivity but devoid of cercariae. Approximately four times as much radioactivity adhered to mice exposed to live labeled cercariae as when the labeled cercariae were heat killed. Much of the radioactivity adherent to skin was lost rapidly after dipping and the 12-hour counts are the more pertinent. From these and similar experiments it appeared that the transfer of radioactivity from the incubate containing tagged cercariae to the mouse appeared similar over a threefold range of radioactivity of incubating medium. Infectivity of the cercariae for the mouse was probably little altered by increasing radiation effects on the cercariae. How-
HOST
RADIOACTIVITY
AFTER
INFECTION
WITH
LABELED
CERCARIAE
149
TABLE II Adherence of Fe59 to Newborn Mice Each mouse was dipped for 30 minutes in l-3 cc of water containing Fess and live or dead cercariae, blotted dry, dropped in a test tube, counted in a well counter, and then returned to the dam. Adherent radioactivity was monitored again after 12 hours. No. of mice
State of cercariae
Cercariae mouse
Av. at 12 hr adherent x lo-3
9 5
Alive Heat-killed
3,oooa 3,000
28.1 2 3.5 FCb 8.0 2 1.4
11 6
Alive Heat-killed
750 750
7.1 -+ 0.3 pcb 1.7 + 0.5
5 This is probably a lethal dose for a newborn mouse. However, only one death occurred in 12 hours of observation. b Differences between live and killed are highly significant (t test; P < .OOl).
ever, some newborn mice survived after contact with over 2000 cercariae, which is unusual in our experience and suggests that infectivity of cercariae exposed to Fe5” was reduced even at lowest radiation dosage. Survival studies in mice exposed to labeled and unlabeled cercariae (circa 700 cercariae per mouse) did not reveal an obvious difference in mortality, and worm recovery on liver perfusion was similar at 6 to 7 weeks after infection. Biologic Half-Life of Fe5g Infected and noninfected radioactive mice were counted daily to evaluate biological halflife. The dam was observed to lick repeatedly the young, and she and her feces contained appreciable radioactivity. Furthermore, the young huddled together constantly and presumably cross-contaminated their skins. Accordingly control experiments were performed in which Fe5g was force fed or injected intraperitoneally. The results are reported in Table III. Retention of Fe5g in mice infected with labeled live cercariae differs from those injected, fed, or exposed to killed labeled cercariae. Distribution of Fe59 The distribution of the radioactivity carried by the labeled living cercariae into the infected mouse was examined (Table IV). Anesthetized mice were killed by exsanguination 24 and 48 hours after exposure; blood, skin, and the various organs were weighed and counted at constant volume. At 24 hours after exposure, the skin was the main site of radioactivity; by 48 hours a
12-fold reduction in relative skin radioactivity occurred. Activity in other organs relative to blood also decreased but much less than in the skin. Internal radioactivity increased more than that in the organs examined, the ratio (counts per gram of organ/counts per gram of blood) dropped. If 55% of each organ were assumed to be blood, then a significant proportion of the monitored radioactivity could be accounted for. All spleen radioactivity could be attributed to its blood content; the gut, liver, and lungs would have definite radioactivity but less than 5% of that in the entire body; the skin would be little affected and still contain more than 25% of Fe5” contained in the entire body. Much of the activity in internal organs could not be accounted for by any reasonable estimate of blood content, which suggests that some iron5” was introduced into the body with the intact cercariae. DISCUSSION
It would appear that Fe”!) tags living cercariae and to a lesser extent dead cercariae. When the living infective cercariae previously labeled with Fesg are brought in contact with a suitable host the radioactivity is carried into the body of the host, presumably by the head as the tail is probably left at the site of penetration (Standen, 1953). The behavior of such radioactive iron is different from fed or injected iron in that it localizes in lungs and intestine, as well as liver, and a large proportion is not incorporated into the red cell mass. Its rapid half-life suggests the tag does not remain with a viable grow-
150
CORRET JER
Biological
AND
TABLE III Half-Life
RUBINI
of Fe.59
Exposure
‘Hr
Number of mice
Live labeled cercariae
0 12 36 60 84
17
177.4
27 27 27 7
100.0a
3 9 9 9
198.7 100.0a 77.0 62.5
1 7 7 7
119.4 100.0~ 82.6 68.9
9 9 8 7 7
107.3 100.0~ 99.1 93.7 90.0
Dead labeled cercariae
0 12 36 60 84
Fed Fess solution
Av. half-life hrb
61.2 42.3 33.6
in
40.8
67.2
-
0 12 36 60 84
Injected intraperitoneally with Fe59 solution
Av. body activitya (% of 12 hr)
88.8
-
0 12 36 60 84
283.2
a Body activity 12 hr after exposure was selected as point of reference as much of the skin radioactivity of mice exposed to either live or dead cercariae was rapidly lost. Plots of log body activity vs time became linear after 12 hr. b Average of individually determined half-lives.
Distribution
TABLE IV of Radioactivity (‘jk of Total Body Counts) in Newborn Exposed to Live Cercariae (Counts/min/gram
organ relative
to blood) 48 hr. (5 mice)
24 hr. (5 mice)
Organ Blood Skin Liver Spleen Lungs GI Tract Carcass
Av. activity relative to blood5 1
12.310 0.482 0.097 0.122 0.654
Mice
Av. % total activity in mouseb
Av. activity relative to blooda
3.3 58.3 1.2 0.2 0.3 3.0 31.5
0.888 0.087 0.049 0.098 0.102
Q Average of individual determinations on each mouse. b Obtained by dividing amount of radioactivity in pooled organs from activity present in group of mice at time of sacrifice X 100.
Av. % total activity in mouseb
1
each mouse by amount
27.5 24.4 2.4 1 .o
2.4 4.7 37.8 of radio-
HOST
RADIOACTIVITY
AFTER
INFECTION
ing worm, but rather remains bound to some component of the cercariae in a form not readily utilizable by the host. Whether living worms shed the iron by exchange with serum iron of the host, or whether the radiation effects on the cercariae cause an alteration in their survival, and hence loss of radioactivity after death, cannot be answered by these experiments. Exchange for unlabeled iron stores of the host seems unlikely because most of the retained Febg is not used to manufacture red cells, especially in the newborn mouse whose iron stores are probably minimal. The selective residual radioactivity in the lungs is especially interesting as iron released into the lungs differs from iron released elsewhere in the body, and in man remains in situ for protracted periods (Soergel, 1957). The radioactivity in lung tissue might be interpreted as suggesting that the tag remains with the cercariae through its passage into the lungs and if the cercariae are entrapped, it remains in situ (Filho, 1959). This interpretation of our data would be contrary to the opinion of Olivier and Schneidermann (1953) that the migrating worms do not arrive in the lungs until the fourth day after infection. We have tried to find radioactivity in the maturing organism, and recovered the growing adult worms after 5 to 7 weeks by perfusing the liver. Although detectable radioactivity remains in the mouse at this time, recovered worms contained no discernible activity, and most of the residual radioactivity in mice older than one month could be accounted for by that in the red cells. Radioautographic studies of similarly infected mice at short intervals may be a
WITH
LABELED
151
CERCARIAE
more fruitful approach to this problem, to see whether any appreciable radioactivity coincides with the site of the parasite. ACKNOWLEDGMENT
The authors are indebted to Drs. Seymour and Lawrence S. Ritchie, Division of Zoology, U.S. Army Tropical Research Laboratory, for advice and criticism, and Genoveva Montalvo for technical assistance.
Garson Medical Medical to Miss
REFERENCES
G. A., JEFFRY, G. M., AND BARTON, B. P. 1958. Radioactive tagging of hookworm larvae (Necator americanus) with P32. Experimental Parasitology 7, 249.253. FILHO, A. M. 1959. Pulmonary lesions in mice experimentally infected with Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene 8, 527-536. GUMBLE, A., OTORE, Y., RITCKIE, L. S., AND HUNTER, G. W., III. 1957. The effect of light, temperature and pH on the emergence of Schistosoma japonicum cercariae from Oncomelania nosophora. Transactions of the American Microscopical Society 76, 87-92. DISSANAIKE,
OLIVIER,
L.,
AND
SCHNEIDERMANN,
M.
1953.
Ac-
quired resistance to Schistosoma mansoni infections in laboratory animals. American Journal of Tropical Medicine and Hygiene 2, 298-306. OLIVIER, L., AND STIREWALT, M. A. 1952. An efficient method for exposure of mice to cercariae of Schistosoma mansoni. Journal of Parasitology 38, 19-23. SOERGEL,K. H. 1957. Ideopathic pulmonary hemosiderosis. Review and report of two cases. Pediatrics 19, 1101-1108. STANDEN, 0. D. 1953. The penetration of the cercariae of Schistosoma mansoni into the skin and lymphatics of the mouse. Transactions of the Royal Society of Tropical Medicine and Hygiene 47, 292.298.