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Radioactive tagging of hookworm larvae (Necator americanus) with P32
EXPERIMENTAL
PARASITOLOGY
Radioactive
7,
249-253 (1958)
Tagging of Hookworm americanus) with
G. A. Dissanaike,’
G. M. Jeffery,2 Columbia,
(Submitted
Larvae Pa2
(Necator
and B. P. Barton’
South Carolina
for publication,
10 July
1957)
Tagging of organisms with radioactive isotopes has been reported frequently in recent years. Extension of this field to some of the problems encountered in the life histories of helminth parasites has been of considerable interest. Application of this technique has been reported for certain nematodes as Onchocerca voZvulus (Barter, et al., 1948), Trichinella (McCoy, et al., 1941; Stoner and Hankes, 1955), Wuchereria buncrofti and Setaria digit& (Dissanaike, et al., 1957). So far as we can determine, no such tagging has been attempted with human hookworm. Since such radioactive tagging might have wide usefulness in the study of life cycles of hookworm and similar parasitic nematodes, tagging of the freeliving stages of hookworm larvae has been attempted. METHODS AND MATERIALS
Fecal samples known to contain heavy concentrations of Necator americanus eggs were used as a source. Eggs were usually further concentrated by flotation in saturated sodium chloride (sp. gravity 1.20). Eggs were washed twice in tap water and were used in tap water suspensions. Charcoal cultures were prepared in small petri dishes (9 cm) and made up to the P32 concentrations indicated in Table I. The P32was obtained from a stock solution of HQOd in HCl (P-32. P-l, Oak Ridge National Laboratory). Hookworm material was used as follows: A. Whole fecal material containing eggs. 1 Department of Physics, University of South Carolina, Columbia, South Carolina. * Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Laboratory of Tropical Diseases, Post Office Box 717, Columbia, South Carolina. 249
250
DISSANAIKE,
JEFFERY, AND BARTON
B. Concentrated eggs (about 2000) with about one gram of fecal material, made up to 10 cc with the medium indicated in Table I. C. Same as (B), with addition of bacteria previously immersed for 2% days in 250 ~lc/cc of P32 in nutrient broth. D. Same as (C) except isotonic saline substituted for nutrient broth. After 14 days larvae were recovered from the charcoal cultures using a Baermann apparatus and were centrifuged and washed in tap water three times. The final supernatant was tested for beta activity. Larvae were held in the final suspension for 8 to 15 days, at which time they were isolated individually under the dissecting microscope and placed in clean depression slides. When a sufficient number was collected from each sample the larvae were washed again in situ, finally enumerated and allowed to dry. The beta activity of each batch of larvae was measured using a thin end-window Geiger-Miiller counter connected to a conventional rate meter. The solid angle of the counter was xo of a sphere, and the background counting rate was 16 per minute. In order to detect the possibility and extent of surface contamination of the larvae, a set of controls, cultured and recovered by the technique described above but without addition of P32to the medium, was immersed for 30 minutes in 40 PC/CCof P32solution. These larvae were then washed, isolated, enumerated and tested for beta activity in the same manner as described previously. RESULTS
The larvae apparently developed normally in all experimental and control cultures. Even in the culture with 90 PC/CC of P32, where the radiation dosage received is estimated at 30,000 rads (3,000,OOOergs/g) for the 14-day period, there was no indication of retarded growth; quite to the contrary this culture apparently produced the largest number of larvae. The experimental results are presented in Table I. The beta activity tabulated is corrected to a value corresponding to the date of removal of the larvae from the cultures, i.e., 14 days after introduction of Ps2 in the cultures, and is also corrected for the background count. Confirmatory tests of cultures 3, 4, and 6 gave consistent values. No beta activity was detected by tests of the final supernatant collected during larval washing. The control larvae, subjected to P32 immersion for 30 minutes, showed no significant beta activity.
TAGGING
HOOKWORM
TABLE
LARVAE
WITH
251
P3’
I
Abso &ion of Pa2in hookworm larvae (Necator americanus) Group
A B
C
D Controls
Activity0 (@mnts per min.)
ulture NO.
Tap water Feces c. 2000 eggs Tap water c. 2000 eggs Nutrient broth c. 2000 eggs Tap water
50 40 90
c. 2000 eggs Nutrient broth c. 2000 eggs Isotonic saline c. 2000 eggs Nutrient broth Feces Tap water
50
14 15 9 9 10 11 8
76 165 613 468 935 835 1095
4.0 4.3 16.1 15.6 31.2 28.8 45.6
nil
(4 2% 8 (b) 30 8 25 12
1780 1681 7
61.4 56.0 0.3
nil
25
7
0.3
50
50
(a) (b) (a) (b)
19 38 38 30 30 XI 24
12
a Activity corrected to value corresponding to date of isolation of larvae from cultures, i.e., 14 days after the introduction of Pa*. b Cultures 4 and 6 each contained over 109 bacteria preimmersed in Pal; culture 5 contained twice this number. Bacteria were from cultures of unidentified non-pathogens isolated from fecal material and other SXXYXS.
The degree of absorption of the P32 by the larvae appeared to be influenced by (a) the addition of bacteria previously immersed in 250 PC/CC of P32, (b) the medium used for rearing the larvae, and (c) the strength of P32. Apparently the greatest amount of absorption resulted from cultivation of the larvae in an isotonic saline (NaCl) medium in the presence of added bacteria which had been previously immersed in isotonic saline containing P32. DISCUSSION
Of interest is the greater absorption of P32 in the cultures where activated bacteria were added. Tagging of bacteria by immersion in P32 solutions is a well known technique, and it is probable that these, bacteria were utilized as food by the developing larvae. These larvae thereby absorbed more P32 than did those in cultures exposed to P32 in solution only. The higher activity of the larvae reared in isotonic saline (with added bacteria
pre-exposed
to P32 in saline)
is also of much
interest.
Earlier
work (e.g. Shearer, 1919) has shown that when bacteria (Bacillus coli)
252
DISSANAIKE,
JEFFERY,
AND
BARTON
are suspended in isotonic saline the ohmic resistance across the bacterial cell falls off with time to a fixed value presumably because the NaCl decreases the resistance of the cell membrane, and, in the absence of divalent cations, lowers the effective “barrier” for the ion transport across the membrane. Such an effect would promote the absorption of (PO& ions and, therefore, possibly account for the higher activity of the larvae in the saline group. As an alternative explanation, the immersion of bacteria in P32in nutrient broth would probably result in enormous multiplication of bacteria during the period of exposure (234 days), while similar immersion only in isotonic saline would result in little multiplication. Thus, one would expect each individual bacterium produced in saline to have a greater amount of activity than a similar individual produced in the nutrient solution. Ingestion of similar numbers of bacteria by larvae in the two groups would in turn result in more activity in the saline group. This effect might, however, be minimized by other conditions. The larval culture enriched with bacteria immersed in nutrient broth (and P”“) would contain an enormously higher proportion of “activated” to “non-activated” bacteria than would the larval culture enriched with the smaller number of bacteria pre-immersed in isotonic saline. Thus, the larvae in the former would be more apt to engulf “activated” bacteria, even though each individual bacterium might possessa smaller amount of radioactivity. It is possible that the activity per larva might be further increased by an increased concentration of P32in the culture (since the larvae were not seemingly affected by the radiation dosage), and also by previously immersing in P32even the fecal matter used as food in the cultures of groups B, C, and D. The results must therefore be considered preliminary but may be of value in indicating possible techniques for investigation of little-known life cycles of various nematodes. Adaptation to use in following the worms through the vertebrate hosts would require use of isotopes which are gamma active and have much longer lifetimes. SUMMARY
Larvae of Necator americanus were successfully tagged with P32by cultivation of the larvae through the free-living stages in the presence of the isotope. The degree of absorption of P32by the larvae was influenced by the strength of P32in the cultures, the addition of bacteria previously activated, and by the nature of the medium for larval or bacterial cultivation.
253
TAGGING HOOKWORM LARVAE WITH P3’ REFERENCES
BARTER, F. C., BURCH, T. A., COWIE, D. B., ASHBURN, L. L., AND BRADY, F. J.
1948. Experimental
therapy
of onchocerciasis with trivalent
antimonials.
Ann. N. Y. Acad. Sci. 60, 89-96. DISSANAIBE, A. S., DISSANAIKE, G. A., NILES, W. J., AND SURENDRANATHAN,
R. 1957. Further studies on radioactive mosquitoes and filarial larvae using autoradiographic technique. Ezptl. Parasitol. 6, 261-270. MCCOY, 0. R., DOWNING, V. F., AND VAN VOORHIS, S. N. 1941. The penetration of radioactive phosphorus into encysted Trichinella larvae. J. Parasitol. 27, 53-58. SHEARER, C. 1919. Studies on the action of electrolytes on bacteria. Part I. The action of monovalent and divalent salts on the conductivity of bacterial emulsions. J. Hyg. 18, 337-360. STONER, R. D., AND HANKES, L. V. 1955. Incorporation of Q-labeled amino acids by Trichinella spiralis larvae. Exptl. Parasitol. 4. 435-444.
PARASITOLOGY
Radioactive
7,
249-253 (1958)
Tagging of Hookworm americanus) with
G. A. Dissanaike,’
G. M. Jeffery,2 Columbia,
(Submitted
Larvae Pa2
(Necator
and B. P. Barton’
South Carolina
for publication,
10 July
1957)
Tagging of organisms with radioactive isotopes has been reported frequently in recent years. Extension of this field to some of the problems encountered in the life histories of helminth parasites has been of considerable interest. Application of this technique has been reported for certain nematodes as Onchocerca voZvulus (Barter, et al., 1948), Trichinella (McCoy, et al., 1941; Stoner and Hankes, 1955), Wuchereria buncrofti and Setaria digit& (Dissanaike, et al., 1957). So far as we can determine, no such tagging has been attempted with human hookworm. Since such radioactive tagging might have wide usefulness in the study of life cycles of hookworm and similar parasitic nematodes, tagging of the freeliving stages of hookworm larvae has been attempted. METHODS AND MATERIALS
Fecal samples known to contain heavy concentrations of Necator americanus eggs were used as a source. Eggs were usually further concentrated by flotation in saturated sodium chloride (sp. gravity 1.20). Eggs were washed twice in tap water and were used in tap water suspensions. Charcoal cultures were prepared in small petri dishes (9 cm) and made up to the P32 concentrations indicated in Table I. The P32was obtained from a stock solution of HQOd in HCl (P-32. P-l, Oak Ridge National Laboratory). Hookworm material was used as follows: A. Whole fecal material containing eggs. 1 Department of Physics, University of South Carolina, Columbia, South Carolina. * Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Laboratory of Tropical Diseases, Post Office Box 717, Columbia, South Carolina. 249
250
DISSANAIKE,
JEFFERY, AND BARTON
B. Concentrated eggs (about 2000) with about one gram of fecal material, made up to 10 cc with the medium indicated in Table I. C. Same as (B), with addition of bacteria previously immersed for 2% days in 250 ~lc/cc of P32 in nutrient broth. D. Same as (C) except isotonic saline substituted for nutrient broth. After 14 days larvae were recovered from the charcoal cultures using a Baermann apparatus and were centrifuged and washed in tap water three times. The final supernatant was tested for beta activity. Larvae were held in the final suspension for 8 to 15 days, at which time they were isolated individually under the dissecting microscope and placed in clean depression slides. When a sufficient number was collected from each sample the larvae were washed again in situ, finally enumerated and allowed to dry. The beta activity of each batch of larvae was measured using a thin end-window Geiger-Miiller counter connected to a conventional rate meter. The solid angle of the counter was xo of a sphere, and the background counting rate was 16 per minute. In order to detect the possibility and extent of surface contamination of the larvae, a set of controls, cultured and recovered by the technique described above but without addition of P32to the medium, was immersed for 30 minutes in 40 PC/CCof P32solution. These larvae were then washed, isolated, enumerated and tested for beta activity in the same manner as described previously. RESULTS
The larvae apparently developed normally in all experimental and control cultures. Even in the culture with 90 PC/CC of P32, where the radiation dosage received is estimated at 30,000 rads (3,000,OOOergs/g) for the 14-day period, there was no indication of retarded growth; quite to the contrary this culture apparently produced the largest number of larvae. The experimental results are presented in Table I. The beta activity tabulated is corrected to a value corresponding to the date of removal of the larvae from the cultures, i.e., 14 days after introduction of Ps2 in the cultures, and is also corrected for the background count. Confirmatory tests of cultures 3, 4, and 6 gave consistent values. No beta activity was detected by tests of the final supernatant collected during larval washing. The control larvae, subjected to P32 immersion for 30 minutes, showed no significant beta activity.
TAGGING
HOOKWORM
TABLE
LARVAE
WITH
251
P3’
I
Abso &ion of Pa2in hookworm larvae (Necator americanus) Group
A B
C
D Controls
Activity0 (@mnts per min.)
ulture NO.
Tap water Feces c. 2000 eggs Tap water c. 2000 eggs Nutrient broth c. 2000 eggs Tap water
50 40 90
c. 2000 eggs Nutrient broth c. 2000 eggs Isotonic saline c. 2000 eggs Nutrient broth Feces Tap water
50
14 15 9 9 10 11 8
76 165 613 468 935 835 1095
4.0 4.3 16.1 15.6 31.2 28.8 45.6
nil
(4 2% 8 (b) 30 8 25 12
1780 1681 7
61.4 56.0 0.3
nil
25
7
0.3
50
50
(a) (b) (a) (b)
19 38 38 30 30 XI 24
12
a Activity corrected to value corresponding to date of isolation of larvae from cultures, i.e., 14 days after the introduction of Pa*. b Cultures 4 and 6 each contained over 109 bacteria preimmersed in Pal; culture 5 contained twice this number. Bacteria were from cultures of unidentified non-pathogens isolated from fecal material and other SXXYXS.
The degree of absorption of the P32 by the larvae appeared to be influenced by (a) the addition of bacteria previously immersed in 250 PC/CC of P32, (b) the medium used for rearing the larvae, and (c) the strength of P32. Apparently the greatest amount of absorption resulted from cultivation of the larvae in an isotonic saline (NaCl) medium in the presence of added bacteria which had been previously immersed in isotonic saline containing P32. DISCUSSION
Of interest is the greater absorption of P32 in the cultures where activated bacteria were added. Tagging of bacteria by immersion in P32 solutions is a well known technique, and it is probable that these, bacteria were utilized as food by the developing larvae. These larvae thereby absorbed more P32 than did those in cultures exposed to P32 in solution only. The higher activity of the larvae reared in isotonic saline (with added bacteria
pre-exposed
to P32 in saline)
is also of much
interest.
Earlier
work (e.g. Shearer, 1919) has shown that when bacteria (Bacillus coli)
252
DISSANAIKE,
JEFFERY,
AND
BARTON
are suspended in isotonic saline the ohmic resistance across the bacterial cell falls off with time to a fixed value presumably because the NaCl decreases the resistance of the cell membrane, and, in the absence of divalent cations, lowers the effective “barrier” for the ion transport across the membrane. Such an effect would promote the absorption of (PO& ions and, therefore, possibly account for the higher activity of the larvae in the saline group. As an alternative explanation, the immersion of bacteria in P32in nutrient broth would probably result in enormous multiplication of bacteria during the period of exposure (234 days), while similar immersion only in isotonic saline would result in little multiplication. Thus, one would expect each individual bacterium produced in saline to have a greater amount of activity than a similar individual produced in the nutrient solution. Ingestion of similar numbers of bacteria by larvae in the two groups would in turn result in more activity in the saline group. This effect might, however, be minimized by other conditions. The larval culture enriched with bacteria immersed in nutrient broth (and P”“) would contain an enormously higher proportion of “activated” to “non-activated” bacteria than would the larval culture enriched with the smaller number of bacteria pre-immersed in isotonic saline. Thus, the larvae in the former would be more apt to engulf “activated” bacteria, even though each individual bacterium might possessa smaller amount of radioactivity. It is possible that the activity per larva might be further increased by an increased concentration of P32in the culture (since the larvae were not seemingly affected by the radiation dosage), and also by previously immersing in P32even the fecal matter used as food in the cultures of groups B, C, and D. The results must therefore be considered preliminary but may be of value in indicating possible techniques for investigation of little-known life cycles of various nematodes. Adaptation to use in following the worms through the vertebrate hosts would require use of isotopes which are gamma active and have much longer lifetimes. SUMMARY
Larvae of Necator americanus were successfully tagged with P32by cultivation of the larvae through the free-living stages in the presence of the isotope. The degree of absorption of P32by the larvae was influenced by the strength of P32in the cultures, the addition of bacteria previously activated, and by the nature of the medium for larval or bacterial cultivation.
253
TAGGING HOOKWORM LARVAE WITH P3’ REFERENCES
BARTER, F. C., BURCH, T. A., COWIE, D. B., ASHBURN, L. L., AND BRADY, F. J.
1948. Experimental
therapy
of onchocerciasis with trivalent
antimonials.
Ann. N. Y. Acad. Sci. 60, 89-96. DISSANAIBE, A. S., DISSANAIKE, G. A., NILES, W. J., AND SURENDRANATHAN,
R. 1957. Further studies on radioactive mosquitoes and filarial larvae using autoradiographic technique. Ezptl. Parasitol. 6, 261-270. MCCOY, 0. R., DOWNING, V. F., AND VAN VOORHIS, S. N. 1941. The penetration of radioactive phosphorus into encysted Trichinella larvae. J. Parasitol. 27, 53-58. SHEARER, C. 1919. Studies on the action of electrolytes on bacteria. Part I. The action of monovalent and divalent salts on the conductivity of bacterial emulsions. J. Hyg. 18, 337-360. STONER, R. D., AND HANKES, L. V. 1955. Incorporation of Q-labeled amino acids by Trichinella spiralis larvae. Exptl. Parasitol. 4. 435-444.