John M. Vetterling

CONTINUOUS-FLOW DIFFERENTIAL DENSITY FLOTATION OF COCCIDIAL OOCYSTS AND A COMPARISON WITH OTHER METHODS

A continuous-flow differential density flotation (CFDDF) method is described for recover- ing coccidial oocysts free of fecal debris. Using decreasing molar concentrations of sucrose and a chem- ical centrifuge with an unperforated basket, oocysts are recovered from large quantities (> 10 liters) of unsieved fecal material. This method is compared with other commonly used flotation methods (coverslip flotation, gravity pan flotation, zonal gradients, and linear gradients). With the CFDDF method 85.4% of the total oocysts are recovered, the percentage sporulation in the harvested frac- tion is increased from 85.2 to 97.2%, and 1 liter of fecal suspension per man-hour can be processed. Other techniques require 13 to 41 man-hours per liter of fecal suspension (including sieving time) and recover only 40.5 to 65.7% of the total oocysts without increasing the percentage of sporulation.

Ultrastructure of dormant, activated, and intracellular sporozoites of Eimeria adenoeides and E. tenella.

Electron microscopy of dormant sporozoites of the poultry coccidia, Eimeria adenoeides and E. tenella, those activated by excystation, and those subsequently inoculated into monolayer cultures of chick kidney and bovine embryonic kidney revealed changes related to increased metabolic activity of the parasite and its association with the host cell. In activated sporozoites, the anterior refractile body was frequently fragmented or found posterior to the nucleus, apparently coalescing with the posterior refractile body. The conoid occasionally was found protruded anterior to the polar ring, with a membranous swirl where it touched the host cell. The perinuclear cisterna was dilated and connected directly with the endoplasmic reticulum. No nucleolus was observed. Mitochondria were swollen and the tubular cristae were enlarged and distinct. The enlarged Golgi apparatus usually indented the nucleus. Amylopectin granules were fewer in number and smaller than in dormant sporozoites. Micropores were seen in the pellicle of many sporozoites. Intracellular sporozoites were similar to the activated sporozoites except that (1) amylopectin granules were further reduced in size and numbers, indicating that they were utilized during the excystation-penetration process, (2) the nucleus had enlarged, and (3) a nucleolus had formed. Other features which were considered to be interactions between the parasite and the host cell were observed. Occasionally, the outer membrane of the pellicle was broken where it touched the membrane of the parasitophorous vacuole. Membranous swirls were formed within the parasitophorous vacuole. The ultrastructure of activated E. tenella sporozoites has been reported by Ryley (1969), and that of intracellular E. tenella sporozoites by Scholtyseck and Strout (1968) and Strout and Scholtyseck (1970). In addition, we have studied the surface structure of E. tenella and E. adenoeides sporozoites using scanning electron microscopy (Vetterling, Madden, and Dittemore, 1971). In the present report, we describe differences in ultrastructure of dormant, activated, and intracellular sporozoites of Eimeria adenoeides Moore and Brown, 1951 and Eimeria tenella (Railliet and Lucet, 1891) Fantham, 1909. MATERIALS AND METHODS Oocysts were collected, sporulated, and cleaned of fecal debris as previously described (Vetterling, 1969). Sporocysts were released from oocysts by grinding (Doran and Vetterling, 1968), and treated with excystation fluid at 43 C (Doran and Vetterling, 1967a) until sporozoites excysted. Sporozoite suspensions were cleaned of oocyst and sporocyst hulls by passage through 2 columns of packed glass beads by the method of Wagenbach (1969) as modified by Doran (1970a). Bovine embryonic kidney (cell line) and chicken kidney (primary) cells were cultured on 10by Received for publication 5 May 1972. 15 35-mm Mylar cover slips in Leighton tubes using the culture techniques previously reported by Doran (1970a, b, c; 1971). Sporozoites and infected monolayer cell cultures used in this report were portions of the same material used by Doran (loc. cit.). Dormant sporozoites, within oocysts and in sporocysts released by grinding, sporozoites activated by in vitro excystation, and intracellular sporozoites in monolayer cell cultures 10 min, 30 min, 1 hr, 5 hr, and 30 hr after inoculation (PI) with sporozoites were fixed using a variety of fixatives and buffers: 1 to 3% glutaraldehyde in 0.05 to 0.02 M phosphate or cacodylate buffer with or without 3 m:M calcium. They were postfixed in 1% OsO4 in the same buffer. The samples were then dehydrated through ethanol, transferred to propylene oxide, and embedded in epon or eponaraldite. Sections were stained with uranyl magnesium acetate (Frasca and Parks, 1965) and lead citrate (Venable and Coggeshall, 1965).

Klossiella equi Baumann, 1946 (Sporozoa: Eucoccidia: Adeleina) from equids.

Kidneys from 5 of 40 ponies (Equus caballus) and from 3 of 14 burrows (Equus asinus) were found infected with Klossiella equi. In addition to previously reported sporogonous stages in epithelial cells of Henles loop, schizogonic stages in endothelial cells of Bowmans capsule and epithelial cells of the proximal convoluted tubules are described. The association of macroand microgametocytes in syzygy is discounted, and a microgametocyte with 8 to 10 microgametes is characterized. Microgametes in the process of migrating to macrogametes are reported. A life cycle for this parasite is proposed. Baumann (1946) first described Klossiella equi from the kidney of a horse in Hungary. He suggested that (1) macrogametes and microgametes develop in syzygy with the formation of four microgametes and (2) after syngamy, the zygote grows and produces, by multiple nuclear fission followed by budding from a large central residual mass, a large number of sporoblasts that develop into sporocysts. Akcay and Urman (1954) found the parasite in kidneys of 72 donkeys in Turkey incidental to experimental work on equine infectious anemia. The species was also described from a burro (Equus asinus) in the United States by Seibold and Thorson (1955a). These workers observed four distinct developmental stages in kidney tubule cells and proposed the following descriptive terms: (1) uninuclear, (2) multinuclear, (3) budding, and (4) thin-walled sac with sporocysts enclosed. In the fourth stage, they counted as many as 12 sporozoites in the sporocysts. They later (1955b) concluded that their parasite was identical to Baumanns K. equi after examining his slides. Newberne, Robinson, and Bowen (1958) found K. equi in the kidney of a zebra from Africa that died in a Florida zoo. Four different sporogonic stages were found in the tubule cells of the medulla. One to four small eosinophilic masses, which were present within the vacuoles of the uninuclear form, were thought to be the microgametocytes suggested by Baumann (1946). Sasmore (1959) found four morphological stages of the parasite Received for publication 28 September 1971. 589 in 28 burros (E. asinus) and one horse (E. caballus) in Tennessee. He proposed the following terms: (1) trophozoite, (2) preschizont, (3) schizont, and (4) sporocyst. He also speculated upon a possible interrelationship of K. equi with skin and intestinal globidial cysts. Hartman (1961) also found K. equi in eight burros. He followed the terminology proposed by Seibold and Thorson (1955a), described the sporogonic phases of the life cycle, and mentioned that the small bodies in the uninuclear phase may be macroand microgametocytes. Klossiella equi has been found in eight equids used in piroplasmosis research during the past 3 years at this laboratory. This report describes the endogenous stages that were found and presents our version of the life cycle of the parasite. MATERIALS AND METHODS Kidney tissues, taken from ponies (Equus caballus) and burros (Equus asinus) that died or were killed during studies of piroplasmosis, were either quick-frozen and sectioned in a cryostat or fixed with Hellys fluid, infiltrated with paraffin, and sectioned. Sections were stained with either Heidenhains iron hematoxlyin with no counterstain (Lillie, 1965), Himes and Moribers trichrome (Himes and Moriber, 1956), or Whipfs polychrome stain (Vetterling and Thompson, in press). Sections were examined at either 400 or 800 X with light-field or dark-field fluorescent micros-

Scanning electron microscopy of Eimeria tenella microgametogenesis and fertilization.

Microgametes of Eimeria tenella were observed in cecal tissue from infected chicks killed six days after inoculation with oocysts. Development from the microgamont stage to maturity was followed. We observed three commonly found, but distinctly different, stages in developing microgametes while they were still associated with microgamonts. In the first, only the flagella could be seen protruding through the enveloping microgamont layer. In the second, both the bodies and flagella could be seen; the flagella had increased in size, and the bodies appeared bulbous. In the third, nearly mature microgametes were randomly arranged around the microgamont, and the manner of flagellar attachment to the bodies near the apical conjunction of the perforatorium was seen. A free microgamete and a macrogamete with one penetrating and two adhering microgametes were found. On flagellum and most of the body of the penetrating microgamete had entered the macrogamete. All micorgametes seen had two flagella.

EIMERIA CASCABELI SP. N. (EIMERIIDAE, SPOROZOA) FROM RATTLESNAKES, WITH A REVIEW OF THE SPECIES OF EIMERIA FROM SNAKES*

The species of Eimeria previously reported from snakes are reviewed and the information tabulated. A new species of Eimeria is described from the rattlesnakes, Crotalus viridis (Rafinesque) of northern Colorado and Crotalus viridis helleri Meek of southern California. Two schizogonous gen- erations and gametocytes were found in the epithelial cells lining the gall bladder and extrahepatic bile ducts. Sporulated oocysts were abundant in the bile. The sporulated oocysts and endogenous stages are described and illustrated. Attempts to transfer this parasite to sidewinders, Crotalus cerastes laterorepens (Hallowell), and garter snakes, Thamnophis sauritis (L.), were unsuccessful. Two new species incor- rectly identified by their authors are named.

Scanning electron microscopy of poultry coccidia after in vitro excystation and penetration of cultured cells

SummaryThe surface structure of Eimeria tenella and E. adenoeides oocysts, sporocysts, and sporozoites were studied by scanning electron microscopy to determine if changes in these surfaces occurred during in vitro excystation and following penetration into cells cultured in monolayers. Oocysts have a rough textured proteinaceous coat. Surface sterilization with NaOCl removed the outer covering of the oocysts, leaving a smooth surface. Sporocysts released from ruptured oocysts had a smooth textured surface. The sporocyst hull covered all of the Stieda body except the tip which was located partly outside the narrow end of the sporocyst. The opening of the sporocyst was slightly ruffled. Excysted sporozoites had a rough surface with small pits and grooves suggestive of micropores and other pellicular invaginations and constrictions. The anterior end of the sporozoites possessed a well defined truncated protrusion of the conoidal complex, the rostrum. We attribute this phenomenon to the activation of the sporozoite by the trypsin-bile solution.

Ultrastructure of Cytoplasmic and Nuclear Changes in Eimeria tenella during First-Generation Schizogony in Cell Culture

Eimeria tenella sporozoites were inoculated into primary cultures of chick kidney cells. Cells fixed from 1 1/2 to 54 hr later were examined with the electron microscope. At 1 1/2 and 24 hr, most intracellular sporozoites were fusiform and retained organelles typical of extracellular sporozoites. However, at 35 hr, rounded trophozoites were present without these structures; only a refractile body, nucleus, mitochondria, and endoplasmic reticulum remained. Binucleate parasites were also present at that time, but at 48 hr many multinucleate schizonts were present. Nuclei, with adjacent conoids, were at the periphery of these schizonts. Partly developed merozoites, each containing a conoid and a nucleus, protruded into the parasitophorous vacuole. At 54 hr, fully developed merozoites were separated from the residual body. Merozoites resembled sporozoites but lacked the large refractile bodies seen in sporozoites. Linear inclusions were present near the merozoite nucleus and in the residual body. Round vacuoles and ribosomes were also found in the residuum. Nucleoli were first seen in sporozoite nuclei at 1 1/2 hr. They were also present in merozoites but were more prominent in trophozoites and schizonts. Peripheral and scattered nuclear heterochromatins were prominent in intracellular sporozoites and diminished in trophozoites, but increased after several nuclear divisions and were again prominent in the merozoite. Small, distinct interchromatin granules were found in all stages. Intranuclear spindles, centrocones, and centrioles were found in connection with nuclear divisions. Ultrastructure of first-generation schizogony in cell culture was similar to that described for second-generation E. tenella in the chicken and to schizogony of other species of Eimeria.