Cultivation of Eimeria Dispersa in Bobwhite Quail Embryos
Hansen, Richard Dean
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The problem. To redescribe the sporulated oocysts of Eimeria dispersa from the Bobwhite quail (Colinus virginianus), to study the endogenous development of E. dispersa in Bobwhite quail embryos, and to examine the cross-transmission properties of this species for turkeys, chickens, and Japanese quail. Procedure. Oocysts, isolated from fecal samples of adult Bobwhites, were sporulated in an aqueous solution containing 2.5% potassium dichromate. Oocysts used for rediscription were isolated by sugar flotation and studied with the aid of a compound microscope equipped with achromatic objectives. Oocysts used as a source of inoculum were isolated by sugar flotation, washed, placed in a tissue grinder, and crushed. Sporocysts, freed by crushing, were isolated by centrifugation and placed in an excystation medium containing 5.0% chicken bile and 0.25% trypsin in Tyrode's solution. Between 1,000 and 44,000 sporozoites were inoculated into the allantoic cavity of 10-to 12-day-old embryos with the aid of a tuberculin syringe. The allantoic fluid and chorioallantoic membranes were removed from inoculated eggs at 12-hour intervals from 1/2 through 10 days after inoculation. The chorioallantoic membranes were fixed in Zenker's fluid, stained with hematoxylin and eosin, and studied with bright-field and phase-contrast microscopy. Oocysts were harvested from the allantoic fluid, sporulated, and inoculated into a 3-month-old Bobwhite in order to demonstrate infectivity. In order to examine the cross-transmission properties of E. dispersa, two 2-month-old domestic turkeys, two 2-week-old White Leghorn chickens, and two 2-week-old Japanese quail were inoculated with oocysts of this species. Findings. Subspherical to ovoid oocysts were found in the feces 4 to 4 1/2 days after inoculation. Sporulated oocysts averaged 22.4 by 18.5 micrometers. Sporocysts were assymmetrically ellipsoidal and averaged 14.1 by 6.7 micrometers. Extended free sporozoites averaged 13.8 by 2.7 micrometers. All tissue stages developed within epithelial cells of the chorioallantoic membrane. Intracellular sporozoites were present from day 2 until day 9 1/2 post-inoculation. Fixed intracellular sporozoites averaged 7.8 by 4.0 micrometers. These developed into sporozoite-shaped schizonts which were observed between 2 and 9 1/2 days after inoculation, and averaged 10.0 by 3.7 micrometers. Mature large schizonts. probably representing those of a first generation, developed from the sporozoite-shaped schizonts and were observed between 2 1/2 and 10 days post-inoculation. These large schizonts averaged 16.7 by 12.4 micrometers and contained an average of 24 merozoites. Smaller schizonts, probably those of a second generation, were observed between 4 and 10 days after inoculation and contained an average of 12 merozoites. These small schizonts averaged 9.9 by 7.9 micrometers. Mature microgametocytes averaging 18.7 by 14.9 micrometers were observed on days 4 through 10. Mature macrogametes averaging 19.2 by 15.0 micrometers were observed on days 8 through 10 post-inoculation. Oocysts developing in epithelial cells of the chorioallantoic membrane were observed between 8 and 9 1/2 days after inoculation. Non-sporulated oocysts were found in the allantoic fluid at 9 and 9t days post-inoculation and averaged 19.8 by 17.0 micrometers. The chorioallantoic membranes showed no macroscopic lesions. Slight hyperemia and hemorrhaging were observed in scattered areas near large blood vessels during schizogony and gametogony. In adult birds, the prepatent period was 4 to 4 1/2 days, and oocysts were found in large numbers until 15 days post-inoculation when only a few oocysts were present in fecal samples. When embryo-derived oocysts were inoculated into a coccidia-free juvenile, the prepatent period was delayed until 6 days post-inoculation. Attempts to transmit E. dispersa to domestic turkeys, White Leghorn chickens, and Coturnix quail were unsuccessful. Conclusions. The sporulated oocysts of E. dispersa from the Bobwhite quail are redescribed, and their endogenous development in quail embryos is reported. In embryos, the life cycle appears to be delayed beyond that observed in mature birds. When embryo-derived oocysts are reinoculated into young bobwhite quail. the prepatent period is also delayed beyond that observed in birds inoculated with oocysts derived from natural sources. As in naturally infected birds, 2 generations of schizonts appear to be present in the life cycle in embryos, and the gametocyte stages appear to be similar to those reported for this species. E. dispersa was found to be relatively non-pathogenic in adult Bobwhites. but markedly pathogenic in young Bobwhites 2 weeks of age and younger. Attempts to infect turkeys, chickens, and Japanese quail with sporulated oocysts of E. dispersa isolated from adult Bobwhite quail were unsuccessful, indicating that E. dispersa may be host specific solely for Bobwhite quail. Recommendations. Future research concerning the cultivation of E. dispersa should provide more detailed light microscope descriptions of the endogenous stages in the adult bobwhite quail and its embryos. Also, attempts should be directed toward the description and cultivation of the endogenous stages of this species in cell cultures. The fine-structure of these stages, both in vivo and in vitro, should also be studied. The phenomenon of a lengthened prepatent period in Bobwhites inoculated with embryo-derived oocysts, as opposed to those given naturally-derived oocysts, should also be investigated.
56 leaves. Advisor: Dr. R. S. Wacha