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Hatchability, viability, and infectivity of Hydatigera taeniaeformis eggs
EXPERIMENTAL PARASITOLOGY12, 120-124 (1962)
Hatchability,
Viability,
and
Infectivity taeniae]ormis E g g s 1
o f Hydatigera
Joan L. Huffman and Arthur W. Jones Department of Zoology and Entomology, University of Tennessee, Knoxville (Submitted for publication, 26 September 1961) Eggs of Hydatigera taeniaeformis were tested in vitro for hatchability in an effort to predict in vivo results in laboratory animals. Hatching was accomplished in solutions containing cholesterol, trypsin, pancreatin, bile and sodium tauroglycolate. Twelve hatching solutions were used. Each differed from the others in either bile concentration, pancreatin concentration or hydrogen ion concentration. The hatching medium containing five ~ bile concentration gave the largest percentage of hatched eggs. There were no appreciable effects of changes in panereatin concentrations and hydrogen ion concentrations. Viability was determined by observing motility in the hatched oncospheres and in the ability of the oncospheres to take up vital stains. In vivo tests were made using the best hatching medium. The per cent eggs hatched was compared to the per cent infectivity in the rat. The correlation coefficient was 0.73 for the results obtained. Although this was not a high correlation, it is thought that this method of determining infectivities should be further investigated.
The infectivity of cestode eggs is difficult to estimate in advance. The eggs of the cat tapeworm, Hydatigera taeniae]ormis Batsch, 1786, were used to determine accurate ways of predicting infectivity in the laboratory animal. The present paper describes attempts to determine a suitable hatching medium for H. taeniaeJormis eggs, to observe motility, to study viability based on the ability of the living oncosphere to resist vital stains, and to test these results in the laboratory animal. The term "hatched" is used in this study to indicate the complete separation and freedom of an oncosphere from an embryophore. The term "viable" is used to mean a living form, determined (insofar as is possible) either by observation of motility or by vital staining. Several workers (Bullock, Dunning, and Curtis, 1934; and Wantland, 1953) have succeeded in hatching H. taeniaeJormis eggs. Silverman (1954a) hatched and activated T. :aginata and T. pisiJormis eggs in a solution 1 Supported in part by the U.S. Atomic Energy
Commission Contract No. AT(40-1) 1749.
containing pancreatin, bile, cholesterol and trypsin. The hatching media used in the present study were modifications of Silverman's. MATERIALS AND METHODS
Eggs Eggs were obtained from gravid proglottids. The number of gravid proglottids in tapeworms varies considerably among members of the family Taeniidae. Segarra (1958) found that the last six proglottids of H. taeniaeformis were usually gravid. All gravid segments contain not only mature and immature eggs, but also stages of developing intermediate forms. According to Silverman (1954a) the hatching process of taeniid eggs occurs in two stages, the passive digestion of the striated embryophore, and the activation of the hexacanth embryo to rupture its enclosing oncospheral membrane. The tapeworms used in the hatching experiments were obtained from the intestines of cats in Knox County, Tennessee. One hundred thirty-two cats were examined for H. taenicLeJormis of which 8.7~o harbored from 120
HATCHABILITY, VIABILITY, INFECTIVITY OF CESTODE EGGS
one to four worms each. One cat was infected with two mature and fourteen immature worms. Worms were washed in physiological saline, placed in a closed container, and stored at 4~ The worms were designated by letters in their order of recovery. Each was examined to be sure that at least six gravid proglottids were present. Only the last six proglottids, designated one through six from the end of the strobila, were used in each test. The work here was designed for use of six suitable tapeworms and twelve hatching solutions. Random distribution of the proglottids among the hatching solutions was achieved by selection of numbered tags from a container. Immediately before each hatching test was performed, the last proglottid was removed from the worm, placed in a few drops of physiological saline and teased apart to release the eggs. The eggs were washed three times in physiological saline and then drained free of all excess saline after centrifugation. The eggs were then placed in a water bath at 38 ~ C. for 5 minutes before the hatching medium was added.
Media The media used in the hatching experiments were modifications of Silverman's (1954a) medium for T. saginata and T. pisi]ormis. The same components used by Silverman were used in these tests, but concentrations were altered. The chemicals used in the hatching experiments and their concentrations appear below:
solutions were all brought to this temperature before the hatching solutions were added to the eggs. Each test ran for 3 hours. During each test a sample of eggs was removed from the water bath every 30 minutes to a slide and 100 eggs were counted at random. Each count included the number of motile oncospheres released from the embryophores, the number of non-motile oncospheres released from the embryophores and the number of oncospheres not hatched. For every hatch done, samples of 600 eggs were counted for each hatching medium. Each solution was tested three times with a different batch of eggs used each time. Care was taken to avoid pressure on the eggs while they were being counted to eliminate the possibility of mechanically forcing the oncospheres out of the embryophores. Each hatching medium was prepared immediately before the experiment was begun and fresh solutions were used in each of the three trials. An analysis of variance was performed on the data collected. By means of a three-way classification it was attempted to determine whether any single medium was capable of yielding a higher percentage hatch and whether there was any significant interaction among the three variables.
Vital staining After the embryos were hatched, they were tested with several vital stains to see if living oncospheres were more resistant to vital stains than dead oncospheres. Five water soluble vital stains were used at a concentration
Bile (Ox) . . . . . . . . . . . . Cholesterol . . . . . . . . . . . . Trypsin . . . . . . . . . . . . . Pancreatin . . . . . . . . . . . . Sodium tauroglycolate (bile salts) . . . Phosphate buffer (K2HPO4 9 NaH2PO~) The two pancreatin concentrations (0.5 and 1.5%) were tested with each of three bile concentrations (0.2, 1.0 and 5.0%) at two pH concentrations (7.0 and 7.6).
Hatch All testing was done at a constant temperature of 38~ The eggs and the hatching
121
. . . . . .
. . . . .
. . . . . . . . . .
0.2%, 1.0%, 5.0% 0.2% 0.2% 0.5%, 1.5% 0.5% 0.5%
of 0.1%. These were Nile blue sulfate, Janus green B, methylene blue, neutral red and a combination of bromphenol blue and nigrosin. One drop of hatched oncospheres was gently mixed with an equal amount of dye. One hundred oncospheres were examined and the percentage of viable embryos was deter-
122
HUFFMAN AND JONES
mined by counting those oncospheres which resisted the vital stains. Each stain was tested in triplicate with eggs giving high hatchability. In order to compare the dyes used, all dyes were tested against the same hatch of oncospheres in order to avoid the effect of differences that might be in the eggs themselves.
In vivo In}ections In vivo tests were performed to determine infectivities following the in vitro tests, using media and vital stains described above. The best hatching medium was tested for its value in predicting infectivities in laboratory rats. These tests were made using eight groups with five rats in each group. Hydatigera eggs from one proglottid were placed in a test tube, centrifuged, washed three times in physiological saline and divided into two equal parts. One half of the eggs was hatched in the hatching medium selected. At the end of 3 hours, a sample of the eggs was removed and counted. The percentage of hatched oncospheres was determined and recorded. The other half of the eggs was suspended in saline to give from one hundred to one thousand eggs per cc. This concentration was determined by standard hemacytometer techniques for white blood cells. A known number of eggs was given to each of five, 40-day-old, Rockland strain, male rats by way of a stomach tube after light etheranesthetization. At the end of 21 days the rats were killed, their livers were removed and the cysts were counted. Percentages were calculated from the number of cysts compared with the number of eggs administered. RESULTS
There was a great amount of variability in the results of the hatching experiments among the eggs from different worms. There was also great variability in the hatching among eggs from different segments of the same worm (Table I). The percentage of eggs hatched from each proglottid is independent of the percentage of eggs hatched from any other. The hatching media selected for this study were all effective to some extent. The single medium which appeared to give the best overall hatch contained, in addition to the
unaltered Silverman and 1.5% pancreatin hatching medium was effective than several
ingredients, 5~, bile, at a pH of 7.0. This not significantly more of the other solutions.
TABLE I
Percentage Hatch oJ Eggs ]rom Different Proglottids of the Same Worm
Proglottid numbera Worm
I
II
III
IV
V
VI
A B C D E F
7% 35% 34% 98% 8% 98%
45% 12% 44% 67% 83% 99%
3% 40% 54% 1% 78% 2%
0 50% 4% 26% 99% 94%
lS% 65% 77% 6% 98% 65%
72% 97% 94% 20% 89% 77%
a Proglottid I is the terminal proglottid of the worm. Prog!ottid VI is the sixth proglottid from the end.
The length of time required to hatch H.
taeniaeformis eggs varied among different batches of eggs and even among eggs from the same proglottid. The shortest time required in this series of hatching was 10 minutes and the longest was 2 ~ hours. The hatching media did not seem to affect the length of time required for hatching the eggs (Segarra, 1958). Oncosphere motility, ranging from 0 to 14%, showed no significant difference among the twelve hatching media. The hatch increased as the bile concentrations increased. No differences resulted from varying the pancreatin concentrations or the hydrogen ion concentrations. An increase in the percentage of hatched eggs occurred each time the bile concentration was increased. An analysis of variance test was performed on the three variables, bile, pancreatin, and hydrogen ion concentration. There was a significant difference only among the bile concentrations, at the five % level of significance. There was no apparent correlation between the hatching results and the percentage of eggs showing motility or resisting the vital stains. Motility ranged from 1 to 3% in each of the hatching media. High percentages of hatching yielded high resistance to the vital stains in some instances and low resistance in others regardless of the media being tested (Table II).
I-IATCHABILITY~ VIABILITY, INFECTIVITY OF CESTODE EGGS TABLE I I
Comparison o/ Eggs Hatched, Motility and Vital Stain Resistance
% Vital stain
% Eggs hatched
% Motileoncospheres
resistant oncospheres
2 0 1 1 1 2 1 4 1 2 4 1
32 6 14 2 29 20 5 11 2 22 15 3
51 48 19 43 66 64 61 62 77 77 82 77
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infectivity and hatchability, the relationship between the two variables is not close enough to consider either variable as a predicter for the other. It is possible, however, to say that for any in vitro hatch the in vivo infectivity for a group of animals would fall between 10% and 70% of the in vitro estimate. While this relationship is not a close one, it seems to be closer than previously reported comparisons (Shaw, 1957). DISCUSSION
There was a linear relationship between hatchability and infectivity, as shown in Fig. 1. The standard error of estimate had a relatively high value of 16.13. The significant coefficient of correlation was 0.73. Although there is a significant correlation between the
90 -
123
7b x
Per cent hatchability
FIG. 1. The relationship of the in vitro hatchability to the in vivo infectivity, s E is the standard error of estimate with a value of 16.13. Ninety-five per cent of the points fall between the two parallel lines 2s E distance from the line of regression.
As the hatching medium begins to act on the embryophore the egg becomes dark brown in color and no structure within the egg can be distinguished. The hatching medium penetrates the embryophore in a manner that is not fully known. It is thought that the digestive juices soften the cement substance which binds the blocks of the shell together (Silverman, 1954a and b; Lee, 1959). When the embryophore disintegrates, these blocks separate and float out into the surrounding medium, permitting the oncosphere, in its oncospheral membrane, to move freely into the medium. The embryos are often as active within the embryophore as they are after they have hatched. All investigators who have studied taeniid egg hatching agree that the activity of the oncospheres is stimulated by the addition of bile or bile salts to the hatching solutions. The movements of the embryo are responsible for the rupture of the oncospheral membrane. The hooks tear the membrane by extending and retracting. The separation of the oncosphere from the oncospheral membrane cannot always be demonstrated since the delicate membrane closely adheres to the oncosphere. No attempts were made in this study to distinguish between oncospheres released from the oncospheral membrane and those still contained within the membrane. I t does not seem possible to predict hatchability for the eggs of all gravid segments of any one worm by merely hatching eggs from one segment of that worm. All segments are independent units and the eggs from individual segments vary in per cent infectivity. This fact makes infecting laboratory animals difficult and complex since every proglottid
124
HUFFMAN AND JONES
must be treated independently and a sample of eggs from every proglottid must be hatched in order to predict infectivity. E v e r y proglottid, even from the same worm, gives a different per cent hatch. I t is not possible to estimate infectivity of eggs for an entire worm from one segment of the worm. F o r all the solutions tested bile concentration seemed to be the only critical factor in hatching the tapeworm eggs. A p p a r e n t l y the concentrations of pancreatin and hydrogen ion were sufficiently high at their low levels to produce whatever effect they had in the hatching process. Increased concentrations did not yield higher hatches. M o t i l i t y is independent of the media used and of the concentrations in each. Silverman (1954a) and others have shown that bile is essential for the activation of the hexacanth embryo. A p p a r e n t l y only a small percentage of bile is necessary and a n y increase in that percentage does not alter the motility. Oncosphere motility is p r o b a b l y a variable within each individual proglottid and the hatching process can stimulate only those oncospheres which have sufficiently m a t u r e d in the proglottid. Low motility percentages were obtained in these experiments. T h e viability which was tested here is p r o b a b l y time limited or temporary. This concept of viability as determined by motility is valid only for a particular stage in the development of a group of stored eggs. Various stages of the developing egg are released from the proglottid in nature. This variability is advantageous to the propagation of tapeworms since the time involved in transmission can be long in m a n y instances (Silverman, 1954b; Jones et al., 1960). The eggs tested in this experiment were hatched a t a specific time in their development. Only a few displayed motility, or viability, but this fact does not indicate that the proportion of potentially viable embryos is small, since development can occur over a long period of time. There was no direct correlation between the number of hatched oncospheres and the
number of dye resistant oncospheres. T h e concept of viability, presented above, might also serve to explain the great variability which was obtained from the vital stains. W i t h the information obtained from these experiments, it seems possible to make predictions regarding animal infectivity since a correlation exists between the in vitro and the in vivo results provided one allows for the large standard error which also is evident. Within the standard error of estimate it is possible to estimate in advance the infectivity of cestode eggs. This information can be valuable in determining the number of eggs to be given an animal experimentally. REFERENCES BULLOCK, F. D., DUNNING, W. F., AND CURTIS, M. R. 1934. Observations of the digestion of the shells of the eggs of Taenia taeniaejormis. American Journal o] Cancer 20, 390-397. JONES, A. W., SEGARRA,J. M., AND WYANT, K. D. 1960. Growth and hatching of taeniid eggs. Journal o/ Parasitology 46, 170-174. LEE, H. 1959. Development of the Taeniid Embryophore. Association o/ Southeastern Biologists Bulletin 6, 28. SEGARRA,J. M. 1958. A study on the eggs of Hydatigera taeniae/ormis. Unpublished Master's Thesis, Department of Zoology and Entomology, University of Tennessee. SHAW, G. L. 1957. An in vitro method for predicting the number of infective eggs in a given sample of Taenia taeniae/ormis eggs. Unpublished Master's Thesis, Department of Zoology and Entomology, University of Tennessee. SILVERMAN, P. H. 1954a. Studies on the biology of some tapeworms of the genus Taenia. I. Factors affecting hatching and activation of taeniid ova, and some criteria of their viability. Annals o/ Tropical Medicine and Parasitology 48, 207215. SILVERMAN, P. H. 1954b. Studies on the biology of some tapeworms of the genus Taenia. II. The morphology and development of the taeniid hexacanth embryo and its enclosing membranes, with some notes on the state of development and propagation of gravid segments. Annals of Tropical Medicine and Parasitology 48, 356-366. WANTLAND, W. W. 1953. Cysticercus ]asciolaris in the Syrian hamster. Journal o] Parasitology 39, 667-668.
Hatchability,
Viability,
and
Infectivity taeniae]ormis E g g s 1
o f Hydatigera
Joan L. Huffman and Arthur W. Jones Department of Zoology and Entomology, University of Tennessee, Knoxville (Submitted for publication, 26 September 1961) Eggs of Hydatigera taeniaeformis were tested in vitro for hatchability in an effort to predict in vivo results in laboratory animals. Hatching was accomplished in solutions containing cholesterol, trypsin, pancreatin, bile and sodium tauroglycolate. Twelve hatching solutions were used. Each differed from the others in either bile concentration, pancreatin concentration or hydrogen ion concentration. The hatching medium containing five ~ bile concentration gave the largest percentage of hatched eggs. There were no appreciable effects of changes in panereatin concentrations and hydrogen ion concentrations. Viability was determined by observing motility in the hatched oncospheres and in the ability of the oncospheres to take up vital stains. In vivo tests were made using the best hatching medium. The per cent eggs hatched was compared to the per cent infectivity in the rat. The correlation coefficient was 0.73 for the results obtained. Although this was not a high correlation, it is thought that this method of determining infectivities should be further investigated.
The infectivity of cestode eggs is difficult to estimate in advance. The eggs of the cat tapeworm, Hydatigera taeniae]ormis Batsch, 1786, were used to determine accurate ways of predicting infectivity in the laboratory animal. The present paper describes attempts to determine a suitable hatching medium for H. taeniaeJormis eggs, to observe motility, to study viability based on the ability of the living oncosphere to resist vital stains, and to test these results in the laboratory animal. The term "hatched" is used in this study to indicate the complete separation and freedom of an oncosphere from an embryophore. The term "viable" is used to mean a living form, determined (insofar as is possible) either by observation of motility or by vital staining. Several workers (Bullock, Dunning, and Curtis, 1934; and Wantland, 1953) have succeeded in hatching H. taeniaeJormis eggs. Silverman (1954a) hatched and activated T. :aginata and T. pisiJormis eggs in a solution 1 Supported in part by the U.S. Atomic Energy
Commission Contract No. AT(40-1) 1749.
containing pancreatin, bile, cholesterol and trypsin. The hatching media used in the present study were modifications of Silverman's. MATERIALS AND METHODS
Eggs Eggs were obtained from gravid proglottids. The number of gravid proglottids in tapeworms varies considerably among members of the family Taeniidae. Segarra (1958) found that the last six proglottids of H. taeniaeformis were usually gravid. All gravid segments contain not only mature and immature eggs, but also stages of developing intermediate forms. According to Silverman (1954a) the hatching process of taeniid eggs occurs in two stages, the passive digestion of the striated embryophore, and the activation of the hexacanth embryo to rupture its enclosing oncospheral membrane. The tapeworms used in the hatching experiments were obtained from the intestines of cats in Knox County, Tennessee. One hundred thirty-two cats were examined for H. taenicLeJormis of which 8.7~o harbored from 120
HATCHABILITY, VIABILITY, INFECTIVITY OF CESTODE EGGS
one to four worms each. One cat was infected with two mature and fourteen immature worms. Worms were washed in physiological saline, placed in a closed container, and stored at 4~ The worms were designated by letters in their order of recovery. Each was examined to be sure that at least six gravid proglottids were present. Only the last six proglottids, designated one through six from the end of the strobila, were used in each test. The work here was designed for use of six suitable tapeworms and twelve hatching solutions. Random distribution of the proglottids among the hatching solutions was achieved by selection of numbered tags from a container. Immediately before each hatching test was performed, the last proglottid was removed from the worm, placed in a few drops of physiological saline and teased apart to release the eggs. The eggs were washed three times in physiological saline and then drained free of all excess saline after centrifugation. The eggs were then placed in a water bath at 38 ~ C. for 5 minutes before the hatching medium was added.
Media The media used in the hatching experiments were modifications of Silverman's (1954a) medium for T. saginata and T. pisi]ormis. The same components used by Silverman were used in these tests, but concentrations were altered. The chemicals used in the hatching experiments and their concentrations appear below:
solutions were all brought to this temperature before the hatching solutions were added to the eggs. Each test ran for 3 hours. During each test a sample of eggs was removed from the water bath every 30 minutes to a slide and 100 eggs were counted at random. Each count included the number of motile oncospheres released from the embryophores, the number of non-motile oncospheres released from the embryophores and the number of oncospheres not hatched. For every hatch done, samples of 600 eggs were counted for each hatching medium. Each solution was tested three times with a different batch of eggs used each time. Care was taken to avoid pressure on the eggs while they were being counted to eliminate the possibility of mechanically forcing the oncospheres out of the embryophores. Each hatching medium was prepared immediately before the experiment was begun and fresh solutions were used in each of the three trials. An analysis of variance was performed on the data collected. By means of a three-way classification it was attempted to determine whether any single medium was capable of yielding a higher percentage hatch and whether there was any significant interaction among the three variables.
Vital staining After the embryos were hatched, they were tested with several vital stains to see if living oncospheres were more resistant to vital stains than dead oncospheres. Five water soluble vital stains were used at a concentration
Bile (Ox) . . . . . . . . . . . . Cholesterol . . . . . . . . . . . . Trypsin . . . . . . . . . . . . . Pancreatin . . . . . . . . . . . . Sodium tauroglycolate (bile salts) . . . Phosphate buffer (K2HPO4 9 NaH2PO~) The two pancreatin concentrations (0.5 and 1.5%) were tested with each of three bile concentrations (0.2, 1.0 and 5.0%) at two pH concentrations (7.0 and 7.6).
Hatch All testing was done at a constant temperature of 38~ The eggs and the hatching
121
. . . . . .
. . . . .
. . . . . . . . . .
0.2%, 1.0%, 5.0% 0.2% 0.2% 0.5%, 1.5% 0.5% 0.5%
of 0.1%. These were Nile blue sulfate, Janus green B, methylene blue, neutral red and a combination of bromphenol blue and nigrosin. One drop of hatched oncospheres was gently mixed with an equal amount of dye. One hundred oncospheres were examined and the percentage of viable embryos was deter-
122
HUFFMAN AND JONES
mined by counting those oncospheres which resisted the vital stains. Each stain was tested in triplicate with eggs giving high hatchability. In order to compare the dyes used, all dyes were tested against the same hatch of oncospheres in order to avoid the effect of differences that might be in the eggs themselves.
In vivo In}ections In vivo tests were performed to determine infectivities following the in vitro tests, using media and vital stains described above. The best hatching medium was tested for its value in predicting infectivities in laboratory rats. These tests were made using eight groups with five rats in each group. Hydatigera eggs from one proglottid were placed in a test tube, centrifuged, washed three times in physiological saline and divided into two equal parts. One half of the eggs was hatched in the hatching medium selected. At the end of 3 hours, a sample of the eggs was removed and counted. The percentage of hatched oncospheres was determined and recorded. The other half of the eggs was suspended in saline to give from one hundred to one thousand eggs per cc. This concentration was determined by standard hemacytometer techniques for white blood cells. A known number of eggs was given to each of five, 40-day-old, Rockland strain, male rats by way of a stomach tube after light etheranesthetization. At the end of 21 days the rats were killed, their livers were removed and the cysts were counted. Percentages were calculated from the number of cysts compared with the number of eggs administered. RESULTS
There was a great amount of variability in the results of the hatching experiments among the eggs from different worms. There was also great variability in the hatching among eggs from different segments of the same worm (Table I). The percentage of eggs hatched from each proglottid is independent of the percentage of eggs hatched from any other. The hatching media selected for this study were all effective to some extent. The single medium which appeared to give the best overall hatch contained, in addition to the
unaltered Silverman and 1.5% pancreatin hatching medium was effective than several
ingredients, 5~, bile, at a pH of 7.0. This not significantly more of the other solutions.
TABLE I
Percentage Hatch oJ Eggs ]rom Different Proglottids of the Same Worm
Proglottid numbera Worm
I
II
III
IV
V
VI
A B C D E F
7% 35% 34% 98% 8% 98%
45% 12% 44% 67% 83% 99%
3% 40% 54% 1% 78% 2%
0 50% 4% 26% 99% 94%
lS% 65% 77% 6% 98% 65%
72% 97% 94% 20% 89% 77%
a Proglottid I is the terminal proglottid of the worm. Prog!ottid VI is the sixth proglottid from the end.
The length of time required to hatch H.
taeniaeformis eggs varied among different batches of eggs and even among eggs from the same proglottid. The shortest time required in this series of hatching was 10 minutes and the longest was 2 ~ hours. The hatching media did not seem to affect the length of time required for hatching the eggs (Segarra, 1958). Oncosphere motility, ranging from 0 to 14%, showed no significant difference among the twelve hatching media. The hatch increased as the bile concentrations increased. No differences resulted from varying the pancreatin concentrations or the hydrogen ion concentrations. An increase in the percentage of hatched eggs occurred each time the bile concentration was increased. An analysis of variance test was performed on the three variables, bile, pancreatin, and hydrogen ion concentration. There was a significant difference only among the bile concentrations, at the five % level of significance. There was no apparent correlation between the hatching results and the percentage of eggs showing motility or resisting the vital stains. Motility ranged from 1 to 3% in each of the hatching media. High percentages of hatching yielded high resistance to the vital stains in some instances and low resistance in others regardless of the media being tested (Table II).
I-IATCHABILITY~ VIABILITY, INFECTIVITY OF CESTODE EGGS TABLE I I
Comparison o/ Eggs Hatched, Motility and Vital Stain Resistance
% Vital stain
% Eggs hatched
% Motileoncospheres
resistant oncospheres
2 0 1 1 1 2 1 4 1 2 4 1
32 6 14 2 29 20 5 11 2 22 15 3
51 48 19 43 66 64 61 62 77 77 82 77
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infectivity and hatchability, the relationship between the two variables is not close enough to consider either variable as a predicter for the other. It is possible, however, to say that for any in vitro hatch the in vivo infectivity for a group of animals would fall between 10% and 70% of the in vitro estimate. While this relationship is not a close one, it seems to be closer than previously reported comparisons (Shaw, 1957). DISCUSSION
There was a linear relationship between hatchability and infectivity, as shown in Fig. 1. The standard error of estimate had a relatively high value of 16.13. The significant coefficient of correlation was 0.73. Although there is a significant correlation between the
90 -
123
7b x
Per cent hatchability
FIG. 1. The relationship of the in vitro hatchability to the in vivo infectivity, s E is the standard error of estimate with a value of 16.13. Ninety-five per cent of the points fall between the two parallel lines 2s E distance from the line of regression.
As the hatching medium begins to act on the embryophore the egg becomes dark brown in color and no structure within the egg can be distinguished. The hatching medium penetrates the embryophore in a manner that is not fully known. It is thought that the digestive juices soften the cement substance which binds the blocks of the shell together (Silverman, 1954a and b; Lee, 1959). When the embryophore disintegrates, these blocks separate and float out into the surrounding medium, permitting the oncosphere, in its oncospheral membrane, to move freely into the medium. The embryos are often as active within the embryophore as they are after they have hatched. All investigators who have studied taeniid egg hatching agree that the activity of the oncospheres is stimulated by the addition of bile or bile salts to the hatching solutions. The movements of the embryo are responsible for the rupture of the oncospheral membrane. The hooks tear the membrane by extending and retracting. The separation of the oncosphere from the oncospheral membrane cannot always be demonstrated since the delicate membrane closely adheres to the oncosphere. No attempts were made in this study to distinguish between oncospheres released from the oncospheral membrane and those still contained within the membrane. I t does not seem possible to predict hatchability for the eggs of all gravid segments of any one worm by merely hatching eggs from one segment of that worm. All segments are independent units and the eggs from individual segments vary in per cent infectivity. This fact makes infecting laboratory animals difficult and complex since every proglottid
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must be treated independently and a sample of eggs from every proglottid must be hatched in order to predict infectivity. E v e r y proglottid, even from the same worm, gives a different per cent hatch. I t is not possible to estimate infectivity of eggs for an entire worm from one segment of the worm. F o r all the solutions tested bile concentration seemed to be the only critical factor in hatching the tapeworm eggs. A p p a r e n t l y the concentrations of pancreatin and hydrogen ion were sufficiently high at their low levels to produce whatever effect they had in the hatching process. Increased concentrations did not yield higher hatches. M o t i l i t y is independent of the media used and of the concentrations in each. Silverman (1954a) and others have shown that bile is essential for the activation of the hexacanth embryo. A p p a r e n t l y only a small percentage of bile is necessary and a n y increase in that percentage does not alter the motility. Oncosphere motility is p r o b a b l y a variable within each individual proglottid and the hatching process can stimulate only those oncospheres which have sufficiently m a t u r e d in the proglottid. Low motility percentages were obtained in these experiments. T h e viability which was tested here is p r o b a b l y time limited or temporary. This concept of viability as determined by motility is valid only for a particular stage in the development of a group of stored eggs. Various stages of the developing egg are released from the proglottid in nature. This variability is advantageous to the propagation of tapeworms since the time involved in transmission can be long in m a n y instances (Silverman, 1954b; Jones et al., 1960). The eggs tested in this experiment were hatched a t a specific time in their development. Only a few displayed motility, or viability, but this fact does not indicate that the proportion of potentially viable embryos is small, since development can occur over a long period of time. There was no direct correlation between the number of hatched oncospheres and the
number of dye resistant oncospheres. T h e concept of viability, presented above, might also serve to explain the great variability which was obtained from the vital stains. W i t h the information obtained from these experiments, it seems possible to make predictions regarding animal infectivity since a correlation exists between the in vitro and the in vivo results provided one allows for the large standard error which also is evident. Within the standard error of estimate it is possible to estimate in advance the infectivity of cestode eggs. This information can be valuable in determining the number of eggs to be given an animal experimentally. REFERENCES BULLOCK, F. D., DUNNING, W. F., AND CURTIS, M. R. 1934. Observations of the digestion of the shells of the eggs of Taenia taeniaejormis. American Journal o] Cancer 20, 390-397. JONES, A. W., SEGARRA,J. M., AND WYANT, K. D. 1960. Growth and hatching of taeniid eggs. Journal o/ Parasitology 46, 170-174. LEE, H. 1959. Development of the Taeniid Embryophore. Association o/ Southeastern Biologists Bulletin 6, 28. SEGARRA,J. M. 1958. A study on the eggs of Hydatigera taeniae/ormis. Unpublished Master's Thesis, Department of Zoology and Entomology, University of Tennessee. SHAW, G. L. 1957. An in vitro method for predicting the number of infective eggs in a given sample of Taenia taeniae/ormis eggs. Unpublished Master's Thesis, Department of Zoology and Entomology, University of Tennessee. SILVERMAN, P. H. 1954a. Studies on the biology of some tapeworms of the genus Taenia. I. Factors affecting hatching and activation of taeniid ova, and some criteria of their viability. Annals o/ Tropical Medicine and Parasitology 48, 207215. SILVERMAN, P. H. 1954b. Studies on the biology of some tapeworms of the genus Taenia. II. The morphology and development of the taeniid hexacanth embryo and its enclosing membranes, with some notes on the state of development and propagation of gravid segments. Annals of Tropical Medicine and Parasitology 48, 356-366. WANTLAND, W. W. 1953. Cysticercus ]asciolaris in the Syrian hamster. Journal o] Parasitology 39, 667-668.