Bill Chobotar

COMPARISON OF FOUR MURINE EIMERIA SPECIES IN IMMUNOCOMPETENT AND IMMUNODEFICIENT MICE

Factors associated with immune-mediated protection against coccidial parasites were examined in a series of experiments utilizing immunocompromised scid/scid(SCID) and scid/scid.beige/beige(SCID/Bg) mice, as well as immunocompetent BALB/c mice. Number of oocysts produced per g feces each day and prepatent and patent periods were assessed for 4 eimerian parasites (Eimeria papillata, Eimeria vermiformis, Eimeria falciformis, and Eimeria ferrisi) using the 3 murine strains. The number of infections required to elicit a protective immune response was also determined for each coccidial species in BALB/c mice. We report the first description of patent infections in inbred immunocompetent and immunodeficient mice infected with E. papillata. Results indicate that during primary infections, parasite replication is under partial immunological control for all Eimeria species. However, the control is mechanistically different for E. papillata because the adaptive immune response does not contribute to the control of primary infections. Both coccidial species infecting intestinal villar epithelial cells (E. papillata and E. ferrisi) were affected by the beige mutation using parasite output as an indicator, whereas E. falciformis, which infects intestinal crypt cells, is not. BALB/c mice were more resistant to challenge infections with upper intestinal parasites (E. papillata and E. vermiformis) in comparison to challenge infections with lower intestinal and cecal parasites (E. falciformis and E. ferrisi).

Fine structural study of the microgametogenesis of Eimeria auburnensis.

SummaryThe microgametogenesis of Eimeria auburnensis from experimentally infected calves killed 16 to 18 days after inoculation was investigated with the electron microscope. Early microgametocytes had nuclei with compact nucleoli and relatively few electron-dense masses. In this stage, deep invaginations from the surface evidently served for the intake of nutrients from the parasitophorous vacuole. In older stages, the nuclei had no compact nucleoli and had more elctrondense material. An intranuclear fiber apparatus was present in some nuclei, apparently in an early stage of division. Nuclear division appeared in electron micrographs to occur by a kind of fission, in which the intranuclear fiber apparatus may participate. Numerous fissures appeared in the interior of the microgametocyte and the nuclei were arranged in irregular rows in association with these. Centrioles, either single or double, were observed between the nuclei and the membrane lining the fissures. Such nuclei often had an intranuclear fiber apparatus; one of the osmiophilic poles of this apparatus protruded outward from the nucleus in the vicinity of the centriole. Directly over this pole, electron-dense material, probably representing the anlage of the perforatorium, occurred immediately beneath the surface membrane of the fissure. As many as 9 closely arranged micropores were observed in this membrane in some specimens. In the nearly mature microgamete, the basal bodies of the flagella lay at the anterior end; the mitochondrion, with numerous, regularly arranged tubules began slightly posterior to this. The strongly osmiophilic, condensed nucleus was a little farther posterior; this remained connected by a narrow stalk with the uncondensed portion of the nucleus in the residual cytoplasm until the microgametes were almost completely mature. Usually, 2 flagella were present. Rarely, 3 flagella were observed, and evidence of a rudimentary middle flagellum was found in a number of specimens.ZusammenfassungDie Mikrogamogonie des Rindercoccids Eimeria auburnensis wurde mit Hilfe des EM untersucht. Junge Mikrogamonten haben Nuklei mit kompaktem Nukleolus und relativ wenig elektronendichten, unregelmäßig im Kernplasma verteilten Partikeln. Schon in jungen Mikrogamonten zeigen sich von der Oberfläche her tiefe, kanalartige Einstülpungen, die tief bis in das Zentrum des Gamonten führen und mit amorphem Material angefüllt sind, wie es in der parasitophoren Vakuole enthalten ist. In älteren Stadien ist der kompakte Nukleolus nicht mehr vorhanden. Dafür sind große Anteile des Kernplasmas stark elektronendicht. Innerhalb der Kerne werden intranukleäre Fibrillen beobachtet. Die Kernteilung erscheint im elektronenoptischen Bild als Zweiteilung, an der sich vermutlich die intranukleären Fibrillen beteiligen. Im Cytoplasma der Gamonten entstehen zahlreiche Spalträume, um die sich die Kerne anordnen. Centriolen in Ein- oder Zweizahl sind zwischen den einzelnen Kernen und den Membranen der Spalträume sichtbar. In diesem Stadium zeigen sich oft ebenfalls intranukleäre Fibrillen, die aus einem osmiophilen Pol in der Nähe der Centriolen entspringen. Oberhalb dieses spindelartigen Pols entwickelt sich eine osmiophile Platte unter der Membran des Spaltraumes. In dieser Membran können sich bis zu 9 Mikroporen dicht nebeneinander befinden. In der Spitze des langgestreckten Mikrogameten liegen die Basalkörper der Geißeln, weiter unterhalb ein besonders strukturiertes Mitochondrium und schließlich der langgestreckte, stark osmiophile Kern. Meistens werden an den Mikrogameten 2 Geißeln beobachtet, in wenigen Fällen sind aber 3 vorhanden und manchmal ergeben sich nur Hinweise auf eine dritte rudimentäre Geißel.

Ultrastructural observations of host-cell invasion by sporozoites ofEimeria papillata in vivo

Scanning and transmission electron microscopy were used to study the invasion of mouse small-intestinal epithelium by sporozoites ofEimeria papillata. Some mice received oocysts by gavage and others received either sporocysts or sporozoites by direct injection into the small intestine. The highest concentration of invaded cells were found in ligated intestinal tissues studied at 5–45 min after the inoculation of sporozoites. Sporozoites actively invaded anterior end first, which resulted in extensive damage to the host cell. Such cells showed disrupted microvilli; protuberances of cytoplasm into the lumen, apparently the result of a disrupted plasma membrane; vacuolization of the cytoplasm; and damage to the mitochondria. These damaged cells were rapidly vacated as the sporozoite moved laterally into one or more adjacent intact host cells without entering the lumen. It is suggested that the host cell initially entered from the lumen becomes so severely traumatized that the parasite of necessity enters an adjacent cell as a prelude to further development. Various aspects of host-cell invasion by coccidia and malarial parasites are reviewed.

Scanning and transmission electron microscopy of host cell pathology associated with penetration byEimeria papillata sporozoites

Scanning and electron microscopy was used to study the pathogenesis that occurred in mouse epithelial cells that had been penetrated byEimeria papillata sporozoites. Optimal penetration of parasites injected into nonligated and ligated mouse intestine was found to occur at 4–15 min post-inoculation. During initial penetration, the parasite caused disruption of the microvilli of the intestinal cells, which led to detachment of the microvilli from the plasma membrane of the penetrated cell. Host cells penetrated by the parasite showed extensive destruction of the internal cellular organization together with blebbing of host-cell cytoplasm and release of internal organelles such as mitochondria. Ultimately, the penetrated cells completely broke down, leaving vacuolated areas next to ultrastructurally normal epithelial cells.

Development of the first-generation schizonts of Elmeria auburnensis.

Thirty-one Holstein-Friesian calves were inoculated with Eimeria auburnensis oocysts; one or more were killed at daily intervals from 4 to 17 days after inoculation. Also, eight calves were infected by the introduction of excysted sporozoites of E. auburnensis into intestinal fistulas, and two calves by placing sporozoites into the intestine via laparotomy. The tissues from calves were prepared by several histological techniques for microscopic examination. First-generation asexual stages were located in epithelial cells at or near the base of the crypts in the jejunum and ileum. Numerous intracellular sporozoites were observed in fistulas of calves killed 4 and 5 days after inoculation. No typically rounded, uninucleate trophozoites were found. In 5-day fistulas, many of the parasites had an appearance similar to that of sporozoites, except that they were larger and had two to six nuclei. Often, such sporozoite-shaped schizonts had lateral bulges with one or more nuclei. This probably represents a stage in the formation of spheroidal schizonts. Early schizonts had nuclei which were randomly arranged; in later schizonts the nuclei assumed a peripheral location. Beginning on the 8th day after inoculation, numerous invaginations of the peripheral nuclear layer of the developing schizont occurred. Each invagination had a lightly staining lumen, which was continuous with the parasitophorous vacuole; each wall bordering the lumen had a single layer of closely spaced nuclei. In schizonts approaching maturity, merozoites were formed by budding from the walls of the invaginations. In mature schizonts, first observed at 10 days after inoculation, the merozoites were randomly distributed throughout the space occupied by the schizont, including the parasitophorous vacuole. Such schizonts contained many thousands of merozoites and occupied the whole width of the crypt, with few or no epithelial cells remaining along the sides. A small amount of glycogen was observed in intracellular sporozoites; more occurred in schizonts, and first-generation merozoites had many granules of glycogen in the middle two-thirds of their bodies. Nuclei of asexual stages were Feulgen-positive. Crescent-shaped bodies of unknown function were associated with 5-day sporozoites and 5and 6-day schizonts. In response to the presence of the parasite, host cell nuclei increased in size; the chromatin became less prominent, and the nucleoli became greatly enlarged. There was no apparent increase in volume of the host cell cytoplasm. Some of the epithelial cells adjacent to the parasitized cells underwent degeneration. Eimeria auburnensis Christensen and Porter, 1939 is a coccidium of cattle having a low degree of pathogenicity as compared with E. bovis and E. zuernii. E. auburnensis is of special interest because the sexual stages are located more deeply in the host tissues than usual, parasitizing cells of the lamina propria, rather than epithelial cells (Hammond, Clark, and Miner, 1961). Little is known about the location and development of the asexual stages in the life cycle of this species. Davis and Bowman (1962) found large schizonts and microgametocytes in the lamina propria near the muscularis mucosae in the small intestine Received for publication 25 October 1968. * Supported in part by NSF Research grant GB5338, and by Public Health Service Fellowship 5-F1-GM-31,285 from the Institute of General Medical Sciences. Published as Journal Paper No. 831, Utah Agricultural Experiment Station. tPresent address: Department of Biology, Andrews University, Berrien Springs, Michigan 49104. of two calves. The relatively large size of the sporozoites (Nyberg and Hammond, 1965; Hammond, Chobotar, and Ernst, 1968) provides a favorable situation for the study of early postinvasion changes in the developing parasites and in host cells. The study reported herein was undertaken to determine the development of the first-generation schizont of E. auburnensis and its relationship to the host cells and tissues. METHODS OF PROCEDURE All calves were purchased locally when less than 1 week old, and housed in individual 5by 10-ft pens constructed of plywood panels. The rear half of each pen was covered with corrugated sheet metal. The floor consisted of a 6-inch layer of coarse gravel, covered with straw, which was changed weekly. At the time of purchase, each calf was given intramuscularly 500,000 international units of vitamin A, 75,000 of vitamin D2, and 50 of vitamin E. Each calf was fed twice daily 2 to 3 pints of raw whole milk mixed with 1 pint of warm water; also, hay and mixed grain were provided. At the beginning of the 4th or 5th week the animals were placed on a diet of

Light and electron microscope study ofSarcocystis sp. from the fallow deer (Cervus dama)

By means of light and electron microscopy a study was made ofSarcocystis sp. from 11 fallow deer (Cervus dama). Cysts ofSarcocystis sp. were found in the tongue and abdominal muscle of 3 of 11 deer from forests near Bonn (FRG). These measured 212–560 μm in length and 54–120 μm in width and contained metrocytes and merozoites. The cyst wall, which had narrow band-like protrusions, is compared with otherSarcocystis sp. from Cervidae.

A fine structural study of asexual stages of the murine coccidium Eimeria ferrisi levine and ivens 1965

SummaryThe schizogony of Eimeria ferrisi was studied in experimentally infected Mus musculus. Developmental stages occurred in epithelial cells of the cecum and colon. During transformation of invasive stages into schizonts the inner membrane complex of the pellicle, the conoid, subpellicular microtubules and micronemes gradually disappeared. The micropore, however, seemed to persist. Dividing nuclei had eccentric intranuclear spindles consisting of microtubules which extended between 2 centrocones, in close relationship with centrioles. During the last nuclear division anlagen of merozoites appeared as extensions on the surface of schizonts. The outer single membrane of the schizont became the outer membrane of the merozoite pellicle. Cytoplasmic organelles, typical of eimerian merozoites were incorporated into the developing merozoites. Finally the merozoite became detached leaving behind a residual cytoplasm. Fully developed merozoites had a 3-layered pellicle, the outer single unit membrane was continuous around the merozoite with the inner complex having interruptions at the anterior and posterior poles and at the micropores. Thirty-two subpellicular microtubules, originating at the anterior polar ring extended to the posterior region of each merozoite. The apical complex consisted of a conoid, preceded by 2 rings and surrounded by a polar ring. Two rhoptries were present having club-shaped terminal ends and slender ductules in the conoid region. Some merozoites had enlarged rhoptries, with the distal vesical appearing dense and osmiophilic. The Golgi complex, endoplasmic reticulum, mitochondria, polysaccharide granules were similar to those seen in other eimerian merozoites.

Electron microscope observations concerning the penetration of a host cell by Eimeria ferrisi in vivo

Invas ive s tages of Sporozoan paras i tes m a y enter host cells by produeing an invag ina t ion in the hos t eell (e ry throcyte) membrane as in Plasmoclium (Ladda, Aikawa, Sprinz, 1969), b y phagocytos i s of m a m m a l i a n maerophages as deser ibed for Toxoplasma gondii (Jones, ¥ e h , Hirsch, 1972) or b y an in t e r rup t ion of the host eell m e m b r a n e as r epor t ed for several species of Eimeria. In Plasmodium species the merozoite makes contaet with the host cell, forming a depression in the host eell (erythrocyte) membrane whieh becomes a cavity, the parasitophorous vacuole, enelosing the parasite (Ladda et al., 1959). In this process the invaginated plasma membrane of the host cell becomes the lining of the parasitophorous vacuole. The different meehanisms of cell entry of these parasites may be due to the type of host cells. Observations of sporozoites of Eimeria species have shown that such sporozoites enter and leave bovine kidney eells readily and quickly after inoculation into eell eultures. In both, light and electron mieroscope studies sporozoites of several Eimeria species were eonstricted at the point of entrance, in some cases resulting in longitudinal folding of the sporozoite pelliele (Roberts, Speer, I-Iammond, 1971). These workers indieated that during penetration by sporozoites of E. larimerensis the host eell membrane may have been interrupted either at the initial site of entry or after beeoming invaginated as penetration was proeeeding. In a recent study Jensen and Hammond (1974) saw no evidence of host cell membrane disruption by sporozoites of E. magna while penetrating cultured bovine kidney cells. Previous observa t ions of pene t r a t i on in e imer ian speeies were done using eell eultures, bu t reeen t ly dur ing a s t n d y oB the deve lopment and u l t r a s t rue tu re of the t issue s tages of E. ]errisi in M u s musculus we ob ta ined serial seetions of a merozoi te pene t r a t ing an in tes t ina l epi thel ia l cell. Mater ia l for s tudy was ob ta ined and proeessed as p rev ious ly deser ibed (Chobotar, Seholtyseek, Sénaud, Erns t , 1975). I n a seet ion th rough near the longi tud ina l p lane of the merozoi te the body was m a r k e d l y eons t r ie ted a t the po in t of en t rance into the epi thel ia l cell (Fig. 1). Pene t r a t i on of the merozoi te had proceeded more t h a n hMLway, to the level of the nueleus whieh had s t re tched into a dumb-be] l shape a t the cons t r ic ted por t ion of the merozoi te (Fig. 1). I n one seet ion a por t ion of the host eell m e m b r a n e tn rned inward for a shor t d is tanee and a b r u p t l y t e rmina ted , ind ica t ing a b reak in the cell membrane (Fig. 2). I n deeper pa r t s of the eell, por t ions of the paras i te appea red to be in con tac t wi th the hos t eell cy toplasm, whereas in o ther por t ions

The ultrastructure of macrogametes of Eimeria ferrisi Levine and Ivens 1965 in Mus musculus

SummaryMacrogametes of Eimeria ferrisi occurred in epithelial cells of the cecum and colon of Mus musculus and were studied by electron microscopy. Young stages were identified as macrogamonts by the presence of wall-forming bodies. At first an ou terlimiting membrane and remnants of the inner membrane complex of the former merozoite pellicle were present; the latter was later lost but in mature macrogametes 3 limiting membranes were observed. Type II wall-forming bodies appeared before type I; the former developed in expanded cisternae of the endoplasmic reticulum whereas the latter were smaller in size and appeared in the ground substance of the cytoplasm. After formation of the oocyst wall the bodies of the 2 types were no longer visible. The persistence of micronemes in mature macrogametes and the presence of numerous layers of rough endoplasmic reticulum during wall formation have not been previously reported.

CELLULAR DYNAMICS AND CYTOKINE RESPONSES IN BALB/C MICE INFECTED WITH EIMERIA PAPILLATA DURING PRIMARY AND SECONDARY INFECTIONS

BALB/c mice were infected with the intestinal intracellular parasite Eimeria papillata to characterize lymphocyte responses and cytokine profiles throughout primary and secondary infections. Lymphocytes from the mesenteric lymph node (MLN) and the gastrointestinal tract (GIT) of infected mice were phenotypically analyzed using flow cytometry and immunofluorescence microscopy, respectively. Lymphocytes isolated from the MLN during primary infections of BALB/c mice with E. papillata do not proliferate, compared to day 0 uninfected controls, when stimulated in vitro with conconavalin A and express TH2-type cytokines (interleukin [IL]-4 and IL-10) on day 3 PI followed by the release of TH1-type cytokines (IL-2 and interferon-gamma) during patency. In the small intestine, significantly more T cells and their subsets were observed during primary infection. During secondary infections, IL-2 was the only 1 of the 4 cytokines that was expressed earlier and at higher levels in the MLN when compared to primary infections. In the small intestine, significantly more alphabeta+ and CD8+ T lymphocytes were observed in mice during secondary infection. Oocyst antigens did not induce cellular proliferation at any time point during primary or secondary infections. We conclude that primary oral infection of BALB/c mice with E. papillata is associated with localized immunosuppression that may be mediated, in part, by early TH2-type cytokines. Immunity to secondary infection may be mediated by intestinal alphabeta+ CD8+ T lymphocytes through an IL-2-dependent mechanism.