J. PROTOZOOL. 23(4), 530-534 (1976).
Comparative I n Vitro Development of Precocious and Normal Strains of Eimeria tenella (Coccidia) L R. McDOUGALD and T.K. JEFFERS Lilly Research Laboratories, Greenfield, Indiana 46140 SYNOPSIS. Eimeria tenella strain Wis-F is known to develop in chickens with a significantly shortened prepatent period and its pathogenicity is virtually completely attenuated. I n nitro development of this strain paralleled development of the control (Wisconsin) strain through the f i s t asexual generation. Instead of entering 2nd generation schizogony, however, most of the Wis-F merozoites developed into microgamonts or macrogamonts. Wall-forming bodies were prominent in developing macrogametes at 80-88 hr and began coalescing into the oocyst wall by 88 hr. Microgamete development paralleled that of macrogametes, with the appearance of multinucleate, immature forms at 72-80 hr and with recognizable, spermlike microgametes being prominent at 88-96 hr. Pathogenicity attenuation and reduction of the length of the prepatent period clearly resulted from omission of a portion of the life cycle (2nd generation schizogony). Index Key Words: Eimeria tenella; Wis-F strain; Wisconsin strain; chick kidney cell cultures.
J
EFFERS ( 1 ) reported pathogenicity attenuation and a significant reduction in the length of the prepatent period of Eimeria tenella subjected to genetic selection. I n contrast to the normal development time of 138 hr, the specially selected strain (Wis-F) formed oocysts in 102 hr. Further intensive selection for precociousness resulted in additional reduction of the length of the prepatent period and in a shortened life cycle characterized by loss of the 2nd asexual generation (4). The present study was conducted to compare development in vitro of the Wis-F and Wisconsin (control) strains of E. tenella during the first 96 hr postinoculation. MATERIALS AND METHODS
Cell Cultures.-Primary cultures of kidney cells were prepared from 2-week-old chicks by conventional cell culture technic. The culture medium contained 0.17% (w/v) lactalbumin hydrolysate and 5% (v/v) fetal calf serum in modified Earle's balanced salt solution (3). Cells were planted at a concentration of 200,00O/ml in Leighton tubes and incubated 72 hr prior to infection. Cultures were incubated in an atmosphere of 2.5% (v/v) C02 and air, at 40 C. The Parasites.-Freshly excysted sporozoites of the selected Wis-F and Wisconsin (control) strains of E. tenella were prepared as previously described ( 2 ) . Inocula of 200,000 organisms/ ml of culture medium were used to infect the cell cultures. Obseruation Methods.-Living cultures were observed periodically by phase-contrast microscopy. Representative duplicate cultures were fixed in Bouin's fluid at 8 hr intervals from 16 to 96 hr postinoculation and stained with Ehrlich's hematoxylin.
RESULTS Because of the relative asynchrony of nuclear division common among developing schizonts and even within individual schizonts, cultures fixed at each time interval contained a variety of developing forms. The selected photomicrographs represented the average stage of development rather than the most advanced forms. Wisconsin (Control) Struin.-Uninucleate trophozoites were observed by 24 h r (Fig. 1). At 32 hr, numerous schizonts with 2-8 nuclei were present (Fig. 2). Some schizonts were multinucleate by 40 hr (Fig. 3), but nuclear division continued through 48 hr with large, immature (Fig. 4 ) and mature (Fig. 9) schizonts present at 48 hr. Merozoites liberated from firstgeneration schizonts were numerous at 56 h r (Fig. l o ) , and by 64 hr had formed clusters of intracellular trophozoites (Fig. 11 ). At 72 hr, there were numerous clusters of trophozoites (Fig. 12) and small schizonts (Fig. 17). Nuclei were progressively more numerous a t 80 and 88 h r as schizonts increased in size (Figs. 18, 19). Numerous clusters of mature schizonts which contained merozoites typical of the 2nd generation of E. tenella (Fig. 20) were found at 96 hr. Wis-F Strain.-Development of the Wis-F closely paralleled that of the Wisconsin strain through the first generation (Figs. 5-8). Merozoites of the first generation were typical in size (-6 p m long), and clusters of intracellular trophozoites were numerous at 56-64 hr (Figs. 13, 14). By 64 hr, some of the trophozoites were larger, but still uninucleate, and a few forms were recognizable as macrogamonts. Some intracellular forms had also become binucleate. At 72 hr, immature macrogametes
+ All figures are photomicrographs of developmental stages of the specially selected Wis-F, and the nonselected Wisconsin (control, C) strains of Eimeria tenella in primary chick kidney cell cultures. As indicated by the bars in the first figures of each plate, the magnification of all figures is x 1,100. Figs. 1-8.[Development of the parasites at 24-48 hr postinoculation.] 1. C, 24 hr. Uninucleate trophozoite. 2. C, 32 hr. Small schizont. 3. C, 40 hr. Schizont. 4. C, 48 hr. Schizont. 5. Wis-F, 24 hr. Trophozoite. 6. Wis-F, 32 hr. Small schizont. 7. Wis-F, 40 hr. Schizont. 8. Wis-F, 48 hr. Mature 1st-generation schizont. Figs. 9-16.[Development of the parasites at 48-72 hr postinoculation.] 9. C,48 hr. Mature 1st-generation schizont. 10. C, 56 hr. Merozoites leaving the 1st-generation schizont. 11. C, 64 hr. Second-generation trophozoites. 12. C, 72 hr. Second-generation schizonts ( binucleate) and trophozoites undergoing nuclear division. 13. Wis-F, 56 hr. First-generation merozoites and 2nd-generation trophozoite. 14. Wis-F, 64 hr. Larger 2nd-generation trophozoites. 15. Wis-F, 72 hr. Immature macrogametes. 16. Wis-F, 72 hr. Immature microgarnetes. Figs. 17-24.[Development of the parasites at 80-96 hr postinoculation.] 17. C, 80 hr. Binucleate and tetranucleate 2nd-generation schizonts. 18. C, 80 hr. Multinucleate 2nd-generation schizonts. 19. C, 88 hr. Larger multinucleate 2nd-generation schizonts. 20. C, 96 hr. Mature 2nd-generation schizonts. 21. Wis-F, 80 hr. Macrogamete with prominent wall-forming bodies. 22. Wis-F, 88 hr. Macrogamete with wall formation and mature microgametocyte. 23. Wis-F, 96 hr. Oocyst, impervious to stain. 24. Wis-F, 88 hr. Mature microgametes.
530
In Vitro DEVELOPMENT OF PRECOCIOUS E. tenella
53 1
532
In Vitro DEVELOPMENT OF PRECOCIOUS E. tenella
In Vitro DEVELOPMENT OF PRECOCIOUS E. tenella
533
534
In Vitro DEVELOPMENT IF PRECOCIOUS E. tenella
were recognizable by their large size and distinct karyosome (Fig. 15). Wall-forming bodies were prominent in macrogametes by 80 hr (Fig. 21 ), and wall formation was in progress at 80-88 hr (Fig. 22). Oocysts with completely formed walls were recognized as they became impervious to stain; they were numerous at 88-96 hr (Fig. 24). Development of microgametocytes paralleled development of macrogametocytes, with multinucleate gamonts found at 72-80 hr (Fig. 16) and flagellated microgametes common at 80-96 hr (Figs. 22, 23). Mature 2nd-generation schizonts were occasionally seen at 96 hr. In live cultures the vigorous activity of microgametes was apparent at 80-96 hr; free-swimming, extracellular microgametes were present at that time. DISCUSSION Development rate of the Wis-F strain in vitro approximated its prepatent period in chickens, with oocyst formation in less than 96 hr. Maturation of the first-generation schizonts in the Wis-F strain closely paralleled the same process in the Wisconsin (control) strain. At 64 hr, however, divergent development of the Wis-F strain was apparent, with the emergence of recognizable sexual stages, particularly macrogamonts. The control strain continued to develop at a rate considered normal for the species ( 5 ) , with mature 2nd generation schizonts becoming prominent at 96 hr. Maturation of macrogametes and microgametes in cultures at 80-88 h r postinfection with the Wis-F strain, with no evidence of normal 2nd-generation schizogony prior to gametogony, left no doubt that a part of the life cycle had been omitted in most of the population. This explains the prominent shortening of the prepatent period and pathogenicity attenuation. Mature 2nd-generation schizonts were occasionally seen in Wis-F cultures. Development of the Wis-F strain to the oocyst in less than 90 hr has now been observed in over 12 separate experiments with an equal number of passages in the chick. Transmission of the 2nd-schizont-lacking (SSL) property through the life cycle is therefore a stable genetic trait and appears to be the result
of mutation. Development of a portion of the merozoites into 2nd-generation schizonts probably indicates that the SSL property arose from a single oocyst, but that the population still contains other, non-SSL variants. No attempt has been made to “purify” the Wis-F strain other than by imposition of selection pressure by development time selection. Conceivably, 2 separate mechanisms could exist whereby the prepatency could be shortened: (a) early maturation of schizonts with fewer merozoites, and ( b ) the SSL property. I t now seems apparent that both phenomena could have accounted for the results observed by Jeffers (1) during genetic selection of the Wis-F strain. Wis-F oocysts used in the present study were derived from the original selected strain ( 1 ) by continued selection for early oocyst production through 46 generations in chickens, thereby reducing the percentage of the population capable of 2nd generation schizogony and further shortening the prepatent period. I n one instance, passage of oocysts was observed in 72-hr fecal collections (unpublished results). Further studies are planned to isolate parasites carrying the “early maturation” property, in addition to the “SSL” property. ACKNOWLEDGMENT Technical assistance of D. Lee and R. Galloway is gratefully acknowledged. REFERENCES 1. Jeffers TK. 1975. Attenuation of Eimeria tenella through selection for precociousness. J. Parasitol. 61, 1083-90. 2. McDougald LR, Galloway RB. 1973. Eimeria tenella: anticoccidial drug activity in cell cultures. Exp. Parasitol. 34, 18996. 3. -- 1976. Anticoccidial drugs: effects on infectivity and ’survival intracellularly of Eimeria tenella sporozoites. Exp. Parasitol., in press. 4. , Jeffers TK. 1976. Eimeria tenella (Sporozoa: Coccidia ) : gametogony following a single asexual generation. Science 192, 258-9. 5 . Strout RG, Ouellette CA. 1970. Schizogony and gametogony of Eimeria tenella in cell culture. A m . J . V e t . Res. 31, 91 1-8.
J. PROTOZOOL. 23 ( 4 ) , 534-537 (1976).
The Attraction of Trypanosow muxi to Vertebrate Cells I n Vitro LaOornlory
of
JAMES A. DVORAK and CHRISTINE L. HOWE Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20014, USA
SYNOPSIS. A video technic is described that permits a quantification of the degree of attraction of Trypanosoma cruri trypomastigotes to vertebrate cells in uitro. Bovine embryo skeletal muscle cells (BESM), HeLa cells and Vero cells all attract a myotropic strain of T . cruzi trypomastigotes. BESM cells, however, are 2-fold more attractive to trypomastigotes than HeLa cells and 10-fold more attractive than Vero cells. Heat-inactivation of BESM cells abolishes their ability to respire and also to attract T . cruri trypomastigotes. As there is no difference in the endogenous oxygen consumption between BESM, HeLa, and Vero cells, it is unlikely that differences in the attraction of trypomastigotes to the 3 cell types are due to variations in the magnitude of PO, or pC02 gradients in the milieu around the cells. Index Key Words: Trypanosoma cruri; bovine embryo skeletal muscle cell cultures; HeLa cell cultures; Vero cell cultures; attraction; video analysis.
0
N E of the hallmarks of Chagas’ disease is the predilection of the causative agent, Trypanosoma cruzi, for specific tissues or organ systems. Although parasities can be found in virtually all tissues (1, 11, 19) and the parasites are presumably carried through all organs in proportion to blood flow, there are major foci of parasites only in certain tissues. Strains of T . cruzi are classified as myotropic, neurotropic, or reticulotropic de-
pending upon the host tissues in which they are found. Paradoxically, it has been shown that T . cruzi can infect and reproduce in virtually all cell types in uitro. I t becomes relevant, therefore, to determine if there is any relationship between vertebrate cell type and the ability of T. cruzi trypomastigotes to find the cell. I n this report we describe a newly developed analytical
Comparative I n Vitro Development of Precocious and Normal Strains of Eimeria tenella (Coccidia) L R. McDOUGALD and T.K. JEFFERS Lilly Research Laboratories, Greenfield, Indiana 46140 SYNOPSIS. Eimeria tenella strain Wis-F is known to develop in chickens with a significantly shortened prepatent period and its pathogenicity is virtually completely attenuated. I n nitro development of this strain paralleled development of the control (Wisconsin) strain through the f i s t asexual generation. Instead of entering 2nd generation schizogony, however, most of the Wis-F merozoites developed into microgamonts or macrogamonts. Wall-forming bodies were prominent in developing macrogametes at 80-88 hr and began coalescing into the oocyst wall by 88 hr. Microgamete development paralleled that of macrogametes, with the appearance of multinucleate, immature forms at 72-80 hr and with recognizable, spermlike microgametes being prominent at 88-96 hr. Pathogenicity attenuation and reduction of the length of the prepatent period clearly resulted from omission of a portion of the life cycle (2nd generation schizogony). Index Key Words: Eimeria tenella; Wis-F strain; Wisconsin strain; chick kidney cell cultures.
J
EFFERS ( 1 ) reported pathogenicity attenuation and a significant reduction in the length of the prepatent period of Eimeria tenella subjected to genetic selection. I n contrast to the normal development time of 138 hr, the specially selected strain (Wis-F) formed oocysts in 102 hr. Further intensive selection for precociousness resulted in additional reduction of the length of the prepatent period and in a shortened life cycle characterized by loss of the 2nd asexual generation (4). The present study was conducted to compare development in vitro of the Wis-F and Wisconsin (control) strains of E. tenella during the first 96 hr postinoculation. MATERIALS AND METHODS
Cell Cultures.-Primary cultures of kidney cells were prepared from 2-week-old chicks by conventional cell culture technic. The culture medium contained 0.17% (w/v) lactalbumin hydrolysate and 5% (v/v) fetal calf serum in modified Earle's balanced salt solution (3). Cells were planted at a concentration of 200,00O/ml in Leighton tubes and incubated 72 hr prior to infection. Cultures were incubated in an atmosphere of 2.5% (v/v) C02 and air, at 40 C. The Parasites.-Freshly excysted sporozoites of the selected Wis-F and Wisconsin (control) strains of E. tenella were prepared as previously described ( 2 ) . Inocula of 200,000 organisms/ ml of culture medium were used to infect the cell cultures. Obseruation Methods.-Living cultures were observed periodically by phase-contrast microscopy. Representative duplicate cultures were fixed in Bouin's fluid at 8 hr intervals from 16 to 96 hr postinoculation and stained with Ehrlich's hematoxylin.
RESULTS Because of the relative asynchrony of nuclear division common among developing schizonts and even within individual schizonts, cultures fixed at each time interval contained a variety of developing forms. The selected photomicrographs represented the average stage of development rather than the most advanced forms. Wisconsin (Control) Struin.-Uninucleate trophozoites were observed by 24 h r (Fig. 1). At 32 hr, numerous schizonts with 2-8 nuclei were present (Fig. 2). Some schizonts were multinucleate by 40 hr (Fig. 3), but nuclear division continued through 48 hr with large, immature (Fig. 4 ) and mature (Fig. 9) schizonts present at 48 hr. Merozoites liberated from firstgeneration schizonts were numerous at 56 h r (Fig. l o ) , and by 64 hr had formed clusters of intracellular trophozoites (Fig. 11 ). At 72 hr, there were numerous clusters of trophozoites (Fig. 12) and small schizonts (Fig. 17). Nuclei were progressively more numerous a t 80 and 88 h r as schizonts increased in size (Figs. 18, 19). Numerous clusters of mature schizonts which contained merozoites typical of the 2nd generation of E. tenella (Fig. 20) were found at 96 hr. Wis-F Strain.-Development of the Wis-F closely paralleled that of the Wisconsin strain through the first generation (Figs. 5-8). Merozoites of the first generation were typical in size (-6 p m long), and clusters of intracellular trophozoites were numerous at 56-64 hr (Figs. 13, 14). By 64 hr, some of the trophozoites were larger, but still uninucleate, and a few forms were recognizable as macrogamonts. Some intracellular forms had also become binucleate. At 72 hr, immature macrogametes
+ All figures are photomicrographs of developmental stages of the specially selected Wis-F, and the nonselected Wisconsin (control, C) strains of Eimeria tenella in primary chick kidney cell cultures. As indicated by the bars in the first figures of each plate, the magnification of all figures is x 1,100. Figs. 1-8.[Development of the parasites at 24-48 hr postinoculation.] 1. C, 24 hr. Uninucleate trophozoite. 2. C, 32 hr. Small schizont. 3. C, 40 hr. Schizont. 4. C, 48 hr. Schizont. 5. Wis-F, 24 hr. Trophozoite. 6. Wis-F, 32 hr. Small schizont. 7. Wis-F, 40 hr. Schizont. 8. Wis-F, 48 hr. Mature 1st-generation schizont. Figs. 9-16.[Development of the parasites at 48-72 hr postinoculation.] 9. C,48 hr. Mature 1st-generation schizont. 10. C, 56 hr. Merozoites leaving the 1st-generation schizont. 11. C, 64 hr. Second-generation trophozoites. 12. C, 72 hr. Second-generation schizonts ( binucleate) and trophozoites undergoing nuclear division. 13. Wis-F, 56 hr. First-generation merozoites and 2nd-generation trophozoite. 14. Wis-F, 64 hr. Larger 2nd-generation trophozoites. 15. Wis-F, 72 hr. Immature macrogametes. 16. Wis-F, 72 hr. Immature microgarnetes. Figs. 17-24.[Development of the parasites at 80-96 hr postinoculation.] 17. C, 80 hr. Binucleate and tetranucleate 2nd-generation schizonts. 18. C, 80 hr. Multinucleate 2nd-generation schizonts. 19. C, 88 hr. Larger multinucleate 2nd-generation schizonts. 20. C, 96 hr. Mature 2nd-generation schizonts. 21. Wis-F, 80 hr. Macrogamete with prominent wall-forming bodies. 22. Wis-F, 88 hr. Macrogamete with wall formation and mature microgametocyte. 23. Wis-F, 96 hr. Oocyst, impervious to stain. 24. Wis-F, 88 hr. Mature microgametes.
530
In Vitro DEVELOPMENT OF PRECOCIOUS E. tenella
53 1
532
In Vitro DEVELOPMENT OF PRECOCIOUS E. tenella
In Vitro DEVELOPMENT OF PRECOCIOUS E. tenella
533
534
In Vitro DEVELOPMENT IF PRECOCIOUS E. tenella
were recognizable by their large size and distinct karyosome (Fig. 15). Wall-forming bodies were prominent in macrogametes by 80 hr (Fig. 21 ), and wall formation was in progress at 80-88 hr (Fig. 22). Oocysts with completely formed walls were recognized as they became impervious to stain; they were numerous at 88-96 hr (Fig. 24). Development of microgametocytes paralleled development of macrogametocytes, with multinucleate gamonts found at 72-80 hr (Fig. 16) and flagellated microgametes common at 80-96 hr (Figs. 22, 23). Mature 2nd-generation schizonts were occasionally seen at 96 hr. In live cultures the vigorous activity of microgametes was apparent at 80-96 hr; free-swimming, extracellular microgametes were present at that time. DISCUSSION Development rate of the Wis-F strain in vitro approximated its prepatent period in chickens, with oocyst formation in less than 96 hr. Maturation of the first-generation schizonts in the Wis-F strain closely paralleled the same process in the Wisconsin (control) strain. At 64 hr, however, divergent development of the Wis-F strain was apparent, with the emergence of recognizable sexual stages, particularly macrogamonts. The control strain continued to develop at a rate considered normal for the species ( 5 ) , with mature 2nd generation schizonts becoming prominent at 96 hr. Maturation of macrogametes and microgametes in cultures at 80-88 h r postinfection with the Wis-F strain, with no evidence of normal 2nd-generation schizogony prior to gametogony, left no doubt that a part of the life cycle had been omitted in most of the population. This explains the prominent shortening of the prepatent period and pathogenicity attenuation. Mature 2nd-generation schizonts were occasionally seen in Wis-F cultures. Development of the Wis-F strain to the oocyst in less than 90 hr has now been observed in over 12 separate experiments with an equal number of passages in the chick. Transmission of the 2nd-schizont-lacking (SSL) property through the life cycle is therefore a stable genetic trait and appears to be the result
of mutation. Development of a portion of the merozoites into 2nd-generation schizonts probably indicates that the SSL property arose from a single oocyst, but that the population still contains other, non-SSL variants. No attempt has been made to “purify” the Wis-F strain other than by imposition of selection pressure by development time selection. Conceivably, 2 separate mechanisms could exist whereby the prepatency could be shortened: (a) early maturation of schizonts with fewer merozoites, and ( b ) the SSL property. I t now seems apparent that both phenomena could have accounted for the results observed by Jeffers (1) during genetic selection of the Wis-F strain. Wis-F oocysts used in the present study were derived from the original selected strain ( 1 ) by continued selection for early oocyst production through 46 generations in chickens, thereby reducing the percentage of the population capable of 2nd generation schizogony and further shortening the prepatent period. I n one instance, passage of oocysts was observed in 72-hr fecal collections (unpublished results). Further studies are planned to isolate parasites carrying the “early maturation” property, in addition to the “SSL” property. ACKNOWLEDGMENT Technical assistance of D. Lee and R. Galloway is gratefully acknowledged. REFERENCES 1. Jeffers TK. 1975. Attenuation of Eimeria tenella through selection for precociousness. J. Parasitol. 61, 1083-90. 2. McDougald LR, Galloway RB. 1973. Eimeria tenella: anticoccidial drug activity in cell cultures. Exp. Parasitol. 34, 18996. 3. -- 1976. Anticoccidial drugs: effects on infectivity and ’survival intracellularly of Eimeria tenella sporozoites. Exp. Parasitol., in press. 4. , Jeffers TK. 1976. Eimeria tenella (Sporozoa: Coccidia ) : gametogony following a single asexual generation. Science 192, 258-9. 5 . Strout RG, Ouellette CA. 1970. Schizogony and gametogony of Eimeria tenella in cell culture. A m . J . V e t . Res. 31, 91 1-8.
J. PROTOZOOL. 23 ( 4 ) , 534-537 (1976).
The Attraction of Trypanosow muxi to Vertebrate Cells I n Vitro LaOornlory
of
JAMES A. DVORAK and CHRISTINE L. HOWE Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20014, USA
SYNOPSIS. A video technic is described that permits a quantification of the degree of attraction of Trypanosoma cruri trypomastigotes to vertebrate cells in uitro. Bovine embryo skeletal muscle cells (BESM), HeLa cells and Vero cells all attract a myotropic strain of T . cruzi trypomastigotes. BESM cells, however, are 2-fold more attractive to trypomastigotes than HeLa cells and 10-fold more attractive than Vero cells. Heat-inactivation of BESM cells abolishes their ability to respire and also to attract T . cruri trypomastigotes. As there is no difference in the endogenous oxygen consumption between BESM, HeLa, and Vero cells, it is unlikely that differences in the attraction of trypomastigotes to the 3 cell types are due to variations in the magnitude of PO, or pC02 gradients in the milieu around the cells. Index Key Words: Trypanosoma cruri; bovine embryo skeletal muscle cell cultures; HeLa cell cultures; Vero cell cultures; attraction; video analysis.
0
N E of the hallmarks of Chagas’ disease is the predilection of the causative agent, Trypanosoma cruzi, for specific tissues or organ systems. Although parasities can be found in virtually all tissues (1, 11, 19) and the parasites are presumably carried through all organs in proportion to blood flow, there are major foci of parasites only in certain tissues. Strains of T . cruzi are classified as myotropic, neurotropic, or reticulotropic de-
pending upon the host tissues in which they are found. Paradoxically, it has been shown that T . cruzi can infect and reproduce in virtually all cell types in uitro. I t becomes relevant, therefore, to determine if there is any relationship between vertebrate cell type and the ability of T. cruzi trypomastigotes to find the cell. I n this report we describe a newly developed analytical
