J. Prolorool.. 38(1), 1991, pp. 53-54 0 1991 by the Society of Protozoologists
Early Development of Eimeria papillata (Apicomplexa: Eimeriidae) in the Mouse STEPHANIE A. STAFFORD and CHRISTINE A. SUNDERMANN Department of Zoology and Wildrife Science, Auburn University, Alabama 36849
ABSTRACT. Early development of Eimeriapapillata (Apicomplexa)in the mouse was evaluated using Nomarski interference-contrast and brightfield microscopy. Sporozoite-shaped meronts, which were motile and contained a large posterior refractile body and a smaller anterior refractile body, were observed entering and leaving host cells in the jejunum of an experimentally infected mouse at 26 h post inoculation (HPI). However, early developmental stages were not observed in tissue of the duodenum, ileum, cecum and colon. The mean length and width of these meronts (n = 20) were 12.0 pm and 3.7 pm, respectively. Spherical or subspherical meronts containing crescent-shaped merozoites were observed at 36 HPI. Key words. Coccidian, eimerian, meront, Mus musculus, parasite.
E
RNST, Chobotar and Hammond (1971) first reported Eimeria papillata from the mouse, Mus musculus, but the life cycle of this coccidian has not yet been described. Generally, intracellular sporozoites of Eimeria species transform into trophozoites upon entry into a host cell and then begin the asexual process of merogony, in which structural components of the sporozoite do not remain. In some species, spherical meronts are produced [8]; but there are also reports of the formation of another type of meront, the sporozoite-shaped meront, in host enterocytes [ 1, 4, 1 1, 121. Sporozoite-shaped meronts are multinucleate forms that retain the structure and general shape of a sporozoite. Unlike spherical meronts they can be motile and thus able to enter and leave cells. The purpose of the present study was to describe the early development of E . papillata and determine which type of first generation meront is produced and also to determine the site of endogenous development in the mouse. MATERIALS AND METHODS Oocysts. Oocysts of E . papillata used in the present study were from an isolate obtained from Dr. B. L. Blagburn (College
I
Alabama Experiment Station Publication number 15-902752P.
of Veterinary Medicine, Auburn University) that has been maintained in our laboratories by passage in coccidian-free mice. Infected fecal material was aerated for seven days in 2.5% (w/ v) potassium dichromate (K,Cr,O,) solution to allow sporulation of oocysts. Flotation with Sheather's sugar solution was used to separate oocysts from fecal debris. Prior to inoculation, sporulated oocysts were washed several times in distilled water (27" C) to remove K,Cr,O, from the suspension. Sporulated oocysts were then counted using a hemocytometer and resuspended in Hanks' balanced salt solution (HBSS) (GIBCO Laboratories, Grand Island, NY)for inoculation. To obtain freshly excysted sporozoites, oocysts were stored in 5.25% (v/v) sodium hypochlorite solution overnight at 4" C, washed in distilled water and placed in HBSS. Oocysts were then placed in a solution of 0.75% sodium taurocholate (bile salt) 0.25% trypsin (=final concentration in HBSS) and warmed in a 37" C water bath for 3 0 min. Infection of mice. Laboratory-reared, coccidia-free, SwissWebster mice were each inoculated orally with 6 million oocysts. Individual mice were sacrificed at 26, 28 and 36 h post-inoculation (HPI). Segments of the duodenum, jejunum, ileum, cecum and colon were excised, and mucosal smears of each were made for microscopical examination. Microscopic examinations. Mucosal scrapings from each area
+
Fig. 1. Nomarski interference-contrast photomicrograph of E. papillata sporozoite freshly excysted from sporulated oocysts. Bar = 10 pm. Fig. 2. Nomarski interference-contrast photomicrograph of sporozoite-shaped meront of Eimeria papillata in mucosal scraping of jejunum at 26 HPI. Prominent anterior (ARB) and posterior (PRB) refractile bodies are present. Bar = 10 pm. Fig. 3. Stained preparation of E. papillata sporozoite-shaped meront from a mucosal scraping obtained 27 HPI. Two nuclei (N), an anterior refractile body (ARB) and a posterior refractile body (PRB) are evident. Bar = 5 pm.
53
54
J. PROTOZOOL., VOL. 38, NO. 1 . JANUARY-FEBRUARY 1991
of the gut were examined at room temperature by Nomarski interference-contrast microscopy to observe living stages of development. Measurements of living stages (n = 20) were made using a calibrated ocular micrometer. Additional mucosal smears were air dried and stained with Diff-Quick@(Dade Diagnostic Products, Aquada, Puerto Rico) and examined with brightfield microscopy.
mental stage, an animal usually must receive a high dose of infective oocysts since multiplication often does not occur as early as 26 HPI. This high dose can be lethal to the host after one or more generations of merogony occur; thus, the earliest stage of development could be overlooked since many workers tend to use lower doses. Possibly, re-evaluation of some eimerian life cycles might support the notion that the sporozoiteshaped meront is not an obscure but is a common developmental form.
RESULTS AND DISCUSSION Various stages of merogony were seen in mucosal scrapings LITERATURE CITED of the jejunum at 26, 28 and 3 6 HPI. No developmental stages 1. Chobotar, B., Hammond, D. M. & Miner, M. L. 1969. Develwere observed in mucosal scrapings of the duodenum, ileum, cecum or colon. Sporozoite-shaped meronts were first observed opment of first-generation schizonts of Eimeria auburnensis. J. Parat 26 HPI and were observed entering and leaving intestinal asitol., 55:385-39 7. 2. Clarke, W. N. & Hammond, D. M. 1969. Development of Eicells of the jejunum. They displayed forward motility and the meria auburnensis in cell culture. J. Protozool.. 16:646-654. bending and flexing movements often associated with excysted 3. Emst, J. V., Chobotar, B. & Hammond, D. M. 1971. The oocysts sporozoites. In order to compare size of sporozoite-shaped mer- of Eimeria vermiformis sp. n. and E. papillata sp. n. (Protozoa: Eionts with that of sporozoites, 2 0 living sporozoites were mea- meriidae) from the mouse Mus musculus. J. Protozool., 18:221-223. sured and had a mean length of 18.0 pm (16.5-19.5 pm) and a 4. Gargus, E. B., Sundermann, C. A., Lindsay, D. S. & Blagburn, B. mean width of 3.1 p m (2.6-3.7 pm) (Fig. 1). Twenty living L. 1987. New observations on first-generation merogony of Eimeria sporozoite-shaped meronts were measured, and the mean length tuskegeensis in Sigmodon hispidus. J. Protozool., 34256-258. 5 . Kelley, G. L. & Youssef, N. N. 1977. Development in cell culand width were 12.0 pm (10.5-15.0 pm) and 3.7 pm (3.2-6.0 pm), respectively. Each sporozoite-shaped meront contained one tures of Eimeria vermiforrnis Ernst, Chobotar, and Hammond, 1971. 53:23-29. large, posterior refractile body and one small, anterior refractile 2.6.Parasitenkd, Lindsay, D. S., Blagbum, B. L., Current, W. L. & Emst, J. V. body (Fig. 2). Stained slides revealed that the meronts were 1985. Development of the swine coccidium Eimeria debliecki Douwes, indeed multinucleate and each contained two to four nuclei (Fig. 1921 in mammalian cell cultures. J. Protozool., 32:669-67 1. 3). Spherical to subspherical meronts and a few remaining spo7. Lindsay, D. S., Current, W. L. & Upton, S. J. 1984. Development rozoite-shaped meronts were observed at 28 HPI. Mature spher- of Eimeria tuskegeensis (Protozoa: Eimeriidae) from the cotton rat, ical to subspherical first-generation meronts containing cres- Sigmodon hispidus, in cell cultures. J. Al. Acad. Sci., 55248-256. 8. Long, P. L. (ed.). 1982. The Biology of the Coccidia. University cent-shaped merozoites, in which refractile bodies were not Park Press, Baltimore, Maryland. present, were observed at 36 HPI. 9. Mueller, B. E. G., de Vos, A. J. & Hammond, D. M. 1973. In There have been only four reports of eimerian species that vitro development of first-generation schizonts of Eimeria canadensis produce sporozoite-shaped meronts in vivo [ 1 , 4 , 1 1, 121where- (Bruce, 1921). J. Protozool., 20:293-297. as the formation of sporozoite-shaped meronts in vitro has been 10. Sampson, J. R. & Hammond, D. M. 1972. Fine structural asreported for Eimeria species from pigs [6], cattle [ 2 , 9, lo], pects of development of Eimeria alabamensis schizonts in cell cultures. rabbits [ l l ] , Uinta ground squirrels [12], cotton rats [7] and J. Parasitol., 58:3 11-322. mice [5]. The observation of sporozoite-shaped meronts in vitro 11. Speer, C. A. & Hammond, D. M. 1971. Development of first has been thought to be a phenomenon resulting from cultivation and second-generation schizonts ofEimeria rnagna from rabbits in cell ofthe parasite outside the natural host. The results ofthe present cultures. 2. Parasitenkd., 37:336-353. 12. Speer, C. A., Hammond, D. M. & Anderson, L. C. 1970. Destudy indicate that the life cycle of E . papillata in vivo includes sporozoite-shaped meronts during early development, which velopment of Eimeria callospermophili and E. bilamellata from the Uinta ground squirrel Spermophilus armatus in cultured cells. J. Prooccurs in the jejunum of the mouse. The significance of this tozool.. 17:274-284. developmental stage is not known, but it is possible that sporozoite-shaped meronts may be more common than the literReceived 9-21-90; accepted I I - 7-90 ature suggests. In order to quickly find the earliest develop-
Early Development of Eimeria papillata (Apicomplexa: Eimeriidae) in the Mouse STEPHANIE A. STAFFORD and CHRISTINE A. SUNDERMANN Department of Zoology and Wildrife Science, Auburn University, Alabama 36849
ABSTRACT. Early development of Eimeriapapillata (Apicomplexa)in the mouse was evaluated using Nomarski interference-contrast and brightfield microscopy. Sporozoite-shaped meronts, which were motile and contained a large posterior refractile body and a smaller anterior refractile body, were observed entering and leaving host cells in the jejunum of an experimentally infected mouse at 26 h post inoculation (HPI). However, early developmental stages were not observed in tissue of the duodenum, ileum, cecum and colon. The mean length and width of these meronts (n = 20) were 12.0 pm and 3.7 pm, respectively. Spherical or subspherical meronts containing crescent-shaped merozoites were observed at 36 HPI. Key words. Coccidian, eimerian, meront, Mus musculus, parasite.
E
RNST, Chobotar and Hammond (1971) first reported Eimeria papillata from the mouse, Mus musculus, but the life cycle of this coccidian has not yet been described. Generally, intracellular sporozoites of Eimeria species transform into trophozoites upon entry into a host cell and then begin the asexual process of merogony, in which structural components of the sporozoite do not remain. In some species, spherical meronts are produced [8]; but there are also reports of the formation of another type of meront, the sporozoite-shaped meront, in host enterocytes [ 1, 4, 1 1, 121. Sporozoite-shaped meronts are multinucleate forms that retain the structure and general shape of a sporozoite. Unlike spherical meronts they can be motile and thus able to enter and leave cells. The purpose of the present study was to describe the early development of E . papillata and determine which type of first generation meront is produced and also to determine the site of endogenous development in the mouse. MATERIALS AND METHODS Oocysts. Oocysts of E . papillata used in the present study were from an isolate obtained from Dr. B. L. Blagburn (College
I
Alabama Experiment Station Publication number 15-902752P.
of Veterinary Medicine, Auburn University) that has been maintained in our laboratories by passage in coccidian-free mice. Infected fecal material was aerated for seven days in 2.5% (w/ v) potassium dichromate (K,Cr,O,) solution to allow sporulation of oocysts. Flotation with Sheather's sugar solution was used to separate oocysts from fecal debris. Prior to inoculation, sporulated oocysts were washed several times in distilled water (27" C) to remove K,Cr,O, from the suspension. Sporulated oocysts were then counted using a hemocytometer and resuspended in Hanks' balanced salt solution (HBSS) (GIBCO Laboratories, Grand Island, NY)for inoculation. To obtain freshly excysted sporozoites, oocysts were stored in 5.25% (v/v) sodium hypochlorite solution overnight at 4" C, washed in distilled water and placed in HBSS. Oocysts were then placed in a solution of 0.75% sodium taurocholate (bile salt) 0.25% trypsin (=final concentration in HBSS) and warmed in a 37" C water bath for 3 0 min. Infection of mice. Laboratory-reared, coccidia-free, SwissWebster mice were each inoculated orally with 6 million oocysts. Individual mice were sacrificed at 26, 28 and 36 h post-inoculation (HPI). Segments of the duodenum, jejunum, ileum, cecum and colon were excised, and mucosal smears of each were made for microscopical examination. Microscopic examinations. Mucosal scrapings from each area
+
Fig. 1. Nomarski interference-contrast photomicrograph of E. papillata sporozoite freshly excysted from sporulated oocysts. Bar = 10 pm. Fig. 2. Nomarski interference-contrast photomicrograph of sporozoite-shaped meront of Eimeria papillata in mucosal scraping of jejunum at 26 HPI. Prominent anterior (ARB) and posterior (PRB) refractile bodies are present. Bar = 10 pm. Fig. 3. Stained preparation of E. papillata sporozoite-shaped meront from a mucosal scraping obtained 27 HPI. Two nuclei (N), an anterior refractile body (ARB) and a posterior refractile body (PRB) are evident. Bar = 5 pm.
53
54
J. PROTOZOOL., VOL. 38, NO. 1 . JANUARY-FEBRUARY 1991
of the gut were examined at room temperature by Nomarski interference-contrast microscopy to observe living stages of development. Measurements of living stages (n = 20) were made using a calibrated ocular micrometer. Additional mucosal smears were air dried and stained with Diff-Quick@(Dade Diagnostic Products, Aquada, Puerto Rico) and examined with brightfield microscopy.
mental stage, an animal usually must receive a high dose of infective oocysts since multiplication often does not occur as early as 26 HPI. This high dose can be lethal to the host after one or more generations of merogony occur; thus, the earliest stage of development could be overlooked since many workers tend to use lower doses. Possibly, re-evaluation of some eimerian life cycles might support the notion that the sporozoiteshaped meront is not an obscure but is a common developmental form.
RESULTS AND DISCUSSION Various stages of merogony were seen in mucosal scrapings LITERATURE CITED of the jejunum at 26, 28 and 3 6 HPI. No developmental stages 1. Chobotar, B., Hammond, D. M. & Miner, M. L. 1969. Develwere observed in mucosal scrapings of the duodenum, ileum, cecum or colon. Sporozoite-shaped meronts were first observed opment of first-generation schizonts of Eimeria auburnensis. J. Parat 26 HPI and were observed entering and leaving intestinal asitol., 55:385-39 7. 2. Clarke, W. N. & Hammond, D. M. 1969. Development of Eicells of the jejunum. They displayed forward motility and the meria auburnensis in cell culture. J. Protozool.. 16:646-654. bending and flexing movements often associated with excysted 3. Emst, J. V., Chobotar, B. & Hammond, D. M. 1971. The oocysts sporozoites. In order to compare size of sporozoite-shaped mer- of Eimeria vermiformis sp. n. and E. papillata sp. n. (Protozoa: Eionts with that of sporozoites, 2 0 living sporozoites were mea- meriidae) from the mouse Mus musculus. J. Protozool., 18:221-223. sured and had a mean length of 18.0 pm (16.5-19.5 pm) and a 4. Gargus, E. B., Sundermann, C. A., Lindsay, D. S. & Blagburn, B. mean width of 3.1 p m (2.6-3.7 pm) (Fig. 1). Twenty living L. 1987. New observations on first-generation merogony of Eimeria sporozoite-shaped meronts were measured, and the mean length tuskegeensis in Sigmodon hispidus. J. Protozool., 34256-258. 5 . Kelley, G. L. & Youssef, N. N. 1977. Development in cell culand width were 12.0 pm (10.5-15.0 pm) and 3.7 pm (3.2-6.0 pm), respectively. Each sporozoite-shaped meront contained one tures of Eimeria vermiforrnis Ernst, Chobotar, and Hammond, 1971. 53:23-29. large, posterior refractile body and one small, anterior refractile 2.6.Parasitenkd, Lindsay, D. S., Blagbum, B. L., Current, W. L. & Emst, J. V. body (Fig. 2). Stained slides revealed that the meronts were 1985. Development of the swine coccidium Eimeria debliecki Douwes, indeed multinucleate and each contained two to four nuclei (Fig. 1921 in mammalian cell cultures. J. Protozool., 32:669-67 1. 3). Spherical to subspherical meronts and a few remaining spo7. Lindsay, D. S., Current, W. L. & Upton, S. J. 1984. Development rozoite-shaped meronts were observed at 28 HPI. Mature spher- of Eimeria tuskegeensis (Protozoa: Eimeriidae) from the cotton rat, ical to subspherical first-generation meronts containing cres- Sigmodon hispidus, in cell cultures. J. Al. Acad. Sci., 55248-256. 8. Long, P. L. (ed.). 1982. The Biology of the Coccidia. University cent-shaped merozoites, in which refractile bodies were not Park Press, Baltimore, Maryland. present, were observed at 36 HPI. 9. Mueller, B. E. G., de Vos, A. J. & Hammond, D. M. 1973. In There have been only four reports of eimerian species that vitro development of first-generation schizonts of Eimeria canadensis produce sporozoite-shaped meronts in vivo [ 1 , 4 , 1 1, 121where- (Bruce, 1921). J. Protozool., 20:293-297. as the formation of sporozoite-shaped meronts in vitro has been 10. Sampson, J. R. & Hammond, D. M. 1972. Fine structural asreported for Eimeria species from pigs [6], cattle [ 2 , 9, lo], pects of development of Eimeria alabamensis schizonts in cell cultures. rabbits [ l l ] , Uinta ground squirrels [12], cotton rats [7] and J. Parasitol., 58:3 11-322. mice [5]. The observation of sporozoite-shaped meronts in vitro 11. Speer, C. A. & Hammond, D. M. 1971. Development of first has been thought to be a phenomenon resulting from cultivation and second-generation schizonts ofEimeria rnagna from rabbits in cell ofthe parasite outside the natural host. The results ofthe present cultures. 2. Parasitenkd., 37:336-353. 12. Speer, C. A., Hammond, D. M. & Anderson, L. C. 1970. Destudy indicate that the life cycle of E . papillata in vivo includes sporozoite-shaped meronts during early development, which velopment of Eimeria callospermophili and E. bilamellata from the Uinta ground squirrel Spermophilus armatus in cultured cells. J. Prooccurs in the jejunum of the mouse. The significance of this tozool.. 17:274-284. developmental stage is not known, but it is possible that sporozoite-shaped meronts may be more common than the literReceived 9-21-90; accepted I I - 7-90 ature suggests. In order to quickly find the earliest develop-
