Host Cell Vacuoles Research Articles

Microtubules are associated with intracellular movement and spread of the periodontopathogen Actinobacillus actinomycetemcomitans.

Actinobacillus actinomycetemcomitans SUNY 465, the invasion prototype strain, enters epithelial cells by an actin-dependent mechanism, escapes from the host cell vacuole, and spreads intracellularly and to adjacent epithelial cells via intercellular protrusions. Internalized organisms also egress from host cells into the assay medium via protrusions that are associated with just a single epithelial cell. Here we demonstrate that agents which inhibit microtubule polymerization (e.g., colchicine) and those which stabilize polymerized microtubules (e.g., taxol) both increase markedly the number of intracellular A. actinomycetemcomitans organisms. Furthermore, both colchicine and taxol prevented the egression of A. actinomycetemcomitans from host cells into the assay medium. Immunofluorescence microscopy revealed that protrusions that mediate the bacterial spread contain microtubules. A. actinomycetemcomitans SUNY 465 and 652, strains that are both invasive and egressive, interacted specifically with the plus ends (growing ends) of the filaments of microtubule asters in a KB cell extract. By contrast, neither A. actinomycetemcomitans 523, a strain that is invasive but not egressive, nor Haemophilus aphrophilus, a noninvasive oral bacterium with characteristics similar to those of A. actinomycetemcomitans, bound to microtubules. Together these data suggest that microtubules function in the spread and movement of A. actinomycetemcomitans and provide the first evidence that host cell dispersion of an invasive bacterium may involve the usurption of host cell microtubules.

Examination of Listeria monocytogenes intracellular gene expression by using the green fluorescent protein of Aequorea victoria.

The ActA protein of Listeria monocytogenes is an essential virulence factor and is required for intracellular bacterial motility and cell-to-cell spread. plcB, cotranscribed with actA, encodes a broad-specificity phospholipase C that contributes to lysis of host cell vacuoles and cell-to-cell spread. Construction of a transcriptional fusion between actA-plcB and the green fluorescent protein gene of Aequorea victoria has facilitated the detailed examination of patterns of actA/plcB expression within infected tissue culture cells. actA/plcB expression began approximately 30 min postinfection and was dependent upon entry of L. monocytogenes into the host cytosol. L. monocytogenes Deltahly mutants, which are unable to escape from host cell vacuoles, did not express actA/plcB at detectable levels within infected tissue culture cells; however, complementation of the hly defect allowed entry of the bacteria into the host cytoplasm and subsequent actA/plcB expression. These results emphasize the ability of L. monocytogenes to sense the different host cell compartment environments encountered during the course of infection and to regulate virulence gene expression in response.

Ultrastructural variation of cultured Ehrlichia chaffeensis.

The ultrastructure of Ehrlichia chaffeensis (Arkansas strain) was studied in non-irradiated and irradiated monolayers of mouse embryo, Vero, BGM and L929 cells, and in non-irradiated DH82 cells. Within the intracellular parasitophorous vacuoles (morulae), two types of ehrlichial cells were found regularly--those with uniformly dispersed nucleoid filaments and ribosomes (dense-cored cells), which represent the normal life cycle of ehrlichiae. In addition, large reticulate cells were observed, forming long projections of the cell wall, protrusions of cytoplasmic membrane into the periplasmic space, or budding of protoplast fragments (minute forms) into the periplasmic space. Ehrlichiae with abnormalities of protoplast fission were found, apparently leading to formation of giant, multilobular or elongated rod-like ehrlichiae. Morulae were usually surrounded by cisterns of granular endoplasmic reticulum and mitochondria and often contained vesicles, long tubules 25nm in diameter, probably originating from the ehrlichial cell wall, and fibrillar ehrlichial antigen apparently shed from the surface of the cell wall. Some cells contained, in addition to normal morulae, a whole morula that had become dense and contained degenerating ehrlichiae. These results indicate that as well as normal growth and reproduction, ehrlichiae exhibit pathological events: they can be remarkably damaged inside the host cell vacuoles, presumably phagolysosomes, or enter a process morphologically similar to bacterial L-transformation.

Calcium and hydrogen ion concentrations in the parasitophorous vacuoles of epithelial cells infected with the microsporidian Encephalitozoon hellem.

Microsporidia of the genus Encephalitozoon undergo merogony and sporogony in a parasitophorous vacuole within the host cell. Cultured green monkey kidney cells infected with Encephalitozoon hellem were loaded with the fluorescent dyes fura-2 or BCECF in order to measure intracellular concentrations of calcium and hydrogen ions respectively. Both the parasitophorous vacuole calcium concentration and pH values resembled those of the host cell cytoplasm in infected cells. Calcein entered the parasitophorous vacuole but not other host cell vacuoles or parasite stages within the parasitophorous vacuole. The lack of a pH or calcium concentration gradient across the parasitophorous vacuole membrane and the permeability of this membrane to a large anion such as calcein suggest that the vacuole membrane surrounding E. hellem resembles that surrounding some other intracellular parasites such as Toxoplasma gondii. A potential role is discussed for the parasitophorous vacuole calcium concentration in germination in situ.

Biochemical and Molecular Characterization of Nucleoside Triphosphate Hydrolase Isozymes from the Parasitic Protozoan Toxoplasma gondii

We have previously reported the presence of a novel nucleoside triphosphate hydrolase (NTPase) in the rapidly multiplying tachyzoite form of a virulent strain (RH) of Toxoplasma gondii. On further examination, it was found that the purified enzyme was not a single enzyme but was a mixture of two isozymes termed NTPase-I and NTPase-II. The two isozymes had similar molecular masses of approximately 240-270 kDa by gel filtration and contained four identical subunits of molecular mass 66-67 kDa by SDS-polyacrylamide gel electrophoresis. Both forms of the NTPase were activated by dithiothreitol, and NTPase-I had a specific activity 4.5-fold higher than NTPase-II in hydrolysis of ATP. The primary difference between these isozymes lies in their ability to hydrolyze nucleoside triphosphate versus diphosphate substrates. While NTPase-II hydrolyzed ATP to ADP and ADP to AMP at almost the same rate, NTPase-I hydrolyzed ADP to AMP at a much slower rate (0.7% of the rate for ATP). The complete cDNAs for NTPase-I and NTPase-II were sequenced and found to encode the same size predicted open reading frame of which only 16 of 628 amino acids differed between the two isozymes. Both forms of the NTPase contained an NH2-terminal hydrophobic signal peptide, consistent with our previous findings that these enzymes are secreted into the host cell vacuole occupied by the parasite. The gene encoding NTPase-II was found in all strains of Toxoplasma, while the NTPase-I was confined only to virulent strains. Expression of this highly active ATPase (NTPase-I) may contribute to intracellular survival and virulence of T. gondii.

Characterization of Listeria monocytogenes pathogenesis in a strain expressing perfringolysin O in place of listeriolysin O.

Listeriolysin O (LLO) is a pore-forming cytolysin that enables Listeria monocytogenes to escape from a host cell vacuole. The structural gene for the related cytolysin perfringolysin O (pfo) was cloned downstream from the promoter for hly, the gene encoding LLO, both on a plasmid and on the L. monocytogenes chromosome. Both strains secreted active PFO, although regulation was not identical to that of LLO. The chromosomal PFO-expressing strain was characterized for intracellular growth and cell-to-cell spread. It escaped from a host cell vacuole with 64% efficiency compared with the wild type as determined by immunofluorescent staining of bacteria for F-actin, a marker for entry into the cytoplasm. In addition, it replicated intracellularly with a doubling time similar to that of the wild type for 5 h, after which growth was aborted because of a cytotoxic effect on the host cell and influx of extracellular gentamicin. The chromosomal PFO strain was able to plaque in mouse L2 fibroblasts, but it did so at 20% efficiency compared with the wild type and the plaques were significantly smaller. Both strains expressing PFO were completely avirulent in mice. These results indicate that PFO can mediate escape from a host cell vacuole but cannot complement an hly deletion strain for virulence.

Regulation of the prfA transcriptional activator of Listeria monocytogenes: multiple promoter elements contribute to intracellular growth and cell-to-cell spread

The prfA gene product is a transcriptional activator of Listeria monocytogenes determinants of pathogenicity. In this study, we provide direct evidence that the PrfA protein is a site-specific DNA-binding protein. Additionally, we describe the characterization of two classes of L. monocytogenes mutants which contain transposon insertions either in the prfA structural gene (exemplified by strain DP-L1075) or within the prfA promoter region (exemplified by strain DP-L973). Both mutants are completely avirulent and secrete greatly reduced levels of listeriolysin O and phosphatidylinositol-specific phospholipase C, and both are fully complemented by the introduction of prfA on a multicopy plasmid. The behaviors of the two mutants differ markedly within cultured macrophages. Following infection, no cytoplasmic growth was observed for DP-L1075 whereas DP-L973 escaped from the phagosome and grew in the cell cytoplasm. However, DP-L973 was defective in nucleation of actin filaments and spread to adjacent cells. Transcription of prfA in DP-L973 was directed from a single, previously unidentified promoter (prfAp2) located close to the prfA initiation codon. This promoter is therefore capable of providing sufficient prfA expression for escape from the host cell vacuole but is insufficient for wild-type levels of bacterially induced actin polymerization and cell-to-cell spread. Transcription directed from both prfAp1 and prfAp2 promoters was increased in the absence of a functional prfA gene product, suggesting that PrfA protein contributes to down-regulating its own expression.

Localization of a Low Molecular Weight Antigen of Eimeria tenella by Use of Hybridoma Antibodies

Three monoclonal antibodies (Mabs), found by western blot analysis to recognize 10-kDa bands of Eimeria tenella sporozoite preparations, were used with immunoelectron (IE) microscopy, immunogold-silver staining (IGSS), and indirect immunofluorescent antibody (IFA) light microscopy to determine the location and distribution of the antigens in or on extra- and intracellular parasites. All 3 of the Mabs (designated C3, E5, and 1231) were found by IE microscopy to label amylopectin granules of extracellular sporozoites. Additionally, these Mabs extensively gold-labeled the sporocyst wall. In cultured primary chicken kidney cells inoculated with sporozoites of E. tenella, IGSS showed surface labeling of the parasite and intense labeling of the infected host cells by 6 hr postinoculation (PI). At 24 hr PI, host cell vacuoles in infected and uninfected cells were labeled by the 3 Mabs by IFA. The E5 and C3 Mabs also were seen to label the host cell membrane of newly infected cells. The C3 and 1231 Mabs showed little label of the host cells by 48 hr PI, but the parasites still were labeled up to 96 hr PI. The E5 Mab had intense IFA labeling of infected host cells at 48 hr PI. The results of this study indicate that parasites apparently release antigenic material during the early stages of parasite development and that this material is found internally and/or on the surface of the infected host cells.

Piscirickettsia salmonis gen. nov., sp. nov., the causative agent of an epizootic disease in salmonid fishes.

A novel intracellular pathogen morphologically similar to the ehrlichiae has been isolated in cell culture and identified as the cause of an epizootic disease of salmonid fish. Like the ehrlichiae, the salmonid pathogen, designated strain LF-89, replicates within membrane-bound cytoplasmic vacuoles in host cells. This agent is the first with characteristics of this type to be isolated from a fish. Analysis of the LF-89 16S rRNA indicated that, unlike the ehrlichiae, LF-89 is a gamma proteobacterium distantly related to Coxiella burnetii and perhaps Wolbachia persica. A new genus and species (Piscirickettsia salmonis gen. nov., sp. nov.) are proposed for this organism, with ATCC(R) VR 1361 as the type strain.

Rickettsia-like microorganisms in the ovarian primordial of moltingIxodes ricinus(acari : ixodidae) larvae and nymphs

A large number of coccoid, or oval to spindle-shaped rickettsialike microorganisms are detected intracellularly within the primordial ovaries of all examined molting larvae and nymphs, descendants of a female Ixodes ricinus collected in a forest near Neuchâtel, Switzerland, and within all molting nymphal I. ricinus captured as starved ticks in the same area. The morphology of the microorganisms is identical in all ticks. They measure 0.3-0.4 µm in diameter and 0.6-1.3 µm, occasionally up to about 2.5 µm in length. The microorganisms possess a highly rippled membranous outer cell wall and an inner plasma membrane. Both cellular boundaries show a thickness of approximately 6-10 nm. The microorganisms are always enclosed, singly or in groups, within membrane limited vacuoles of host cells, within vacuolated mitochondria, or within vacuoles with their wall consisting partially of a deformed mitochondrion and partially of a plasma membrane of host cells. With the advance of the development of the nymphal ovaries, the groups of microorganisms associated with mitochondria become larger and increase in number, and they are dominant within the posterior part of the organs in the nymphs examined at day 21 after repletion. The relationship between the microorganisms and the mitochondria is discussed. The morphology of the gonads of the examined immature ticks is also described.

Endocytobionts inDermacentor reticulatus ticks (Ixodidae): An electron microscope study

Endocytobionts (ECBs) were detected in the ovaries of Dermacentor reticulatus. Their developmental cycle is directly related to the developmental stages of tick oocytes. Two basic forms of ECBs occur in tick cells, i.e. dense forms, occurring singly or in aggregates situated free in the cytoplasm of host cells, and the light forms which are larger, pleomorphic and always situated inside vacuoles of host cells. The light forms occur together with dense forms in all oocytes. The dense forms occur freely and independently in funicular cells of the oocyte, the epithelial cells of oviducts, and additionally in the cells of the Malpighian tubules. A probable function of ECBs in the tick host is discussed.

Immuno‐histochemical and ultrastructural characteristics of a cyst‐forming sporozoon associated with encephalomyelitis and myositis in dogs

Light-microscopy, including immuno-histochemical staining, and transmission and scanning electron microscopy were applied for the characterization of a Toxoplasma-like parasite associated with encephalomyelitis and myositis in dogs of the boxer breed. Most parasites were tachyzoites, occurring in clusters, up to 60 micron in diameter, in parasitophorous vacuoles of host cells. Cyst stages with a definite cyst wall were few, but could be demonstrated in all cases. Immunostaining with antiserum against Toxoplasma gondii was negative, while both tachyzoites and bradyzoites were stained with serum from affected dogs. Ultrastructurally, the parasite was characterized by a great number of rhoptries, few amylopectin granules, lack of micropores in the tachyzoites, and the occurrence of numerous dense bodies in the bradyzoites. Numerous intravacuolar tubules were present in the space of the parasitophorous vacuole. The cyst wall resembled that of T. gondii. Until a definite host is found, the parasite may be regarded as an "orphan", cyst-forming sporozoon that is ultrastructurally different from other known protozoan parasites. It has a light-microscopical similarity to T. gondii but is antigenically different.

The importance of methane and thiosulfate in the metabolism of the bacterial symbionts of two deep-sea mussels

Undescribed hydrocarbon-seep mussels were collected from the Louisiana Slope, Gulf of Mexico, during March 1986, and the ultrastructure of their gills was examined and compared to Bathymodiolus thermophilus, a mussel collected from the deep-sea hydrothermal vents on the Galapagos Rift in March 1985. These closely related mytilids both contain abundant symbiotic bacteria in their gills. However, the bacteria from the two species are distinctly different in both morphology and biochemistry, and are housed differently within the gills of the two mussels. The symbionts from the seep mussel are larger than the symbionts from B. thermophilus and, unlike the latter, contain stacked intracytoplasmic membranes. In the seep mussel three or fewer symbionts appear to be contained in each host-cell vacuole, while in B. thermophilus there are often more than twenty bacteria visible in a single section through a vacuole. The methanotrophic nature of the seep-mussel symbionts was confirmed in 14C-methane uptake experiments by the appearance of label in both CO2 and acid-stable, non-volatile, organic compounds after a 3 h incubation of isolated gill tissue. Furthermore, methane consumption was correlated with methanol dehydrogenase activity in isolated gill tissue. Activity of ribulose-1,5-biphosphate (RuBP) carboxylase and 14CO2 assimilation studies indicate the presence of either a second type of symbiont or contaminating bacteria on the gills of freshly captured seep mussels. A reevaluation of the nutrition of the symbionts in B. thermophilus indicates that while the major symbiont is not a methanotroph, its status as a sulfur-oxidizing chemoautotroph, as has been suggested previously, is far from proven.

Phagosome acidification blocked by intracellular Toxoplasma gondii.

Toxoplasma gondii belongs to a group of highly virulent intracellular parasites that reside in host cell vacuoles which resist typical phagosome-lysosome fusion. Live Toxoplasma replicate prodigiously within modified phagocytic vacuoles formed during invagination of the host plasma membrane. In contrast, heat-killed Toxoplasma or specific antibody (heat-inactivated)-coated live Toxoplasma-containing vacuoles readily undergo lysosome fusion and digestion in normal macrophages. Of newly recognized significance to Toxoplasma survival is the microbicidal effect of phagosome acidification, which reportedly can occur independently of fusion with other acidic vesicles. We report here that modified live Toxoplasma-containing vacuoles fail to acidify in normal macrophages, as indicated by the sensitive pH probe fluorescein. In contrast, when live Toxoplasma are coated with specific antibody (heat-inactivated), they trigger phagosome acidification when entering normal macrophages. A similar acidification is observed when normal phagocytes ingest dead Toxoplasma. Extracellular Toxoplasma are highly susceptible to acidic pH conditions, indicating that the acidification block in the modified vacuoles may be important for intracellular survival.

A new rickettsiale of Gambia rats (Cricetomys gambianus) in Senegal : Grahamella kaniae n. sp. (Bartonellacae)

Giant Rat (Cricetomys gambianus Waterh) is a big rodent very common in West Africa. Its burrow is bordering upon huts and lofts in the african villages. Omnivorous, it is more or less a commensal of the man. The blood of 20 adults male and female specimens captured in the Sine Saloum (Fatik) and Presqu'Ille du Cap Vert (Dakar) regions was examined (peripheric blood smears). In 60 p. 100 (12/20) of hemograms, an intraerythrocytic organism of the genus Grahamella Brumpt 1911 was observed. Rod-shaped and carmine-coloured by staining technic of May-Grünwald and Giemsa, it is an unflagellate element. Slightly narrowed in the median part, it is 1 to 1.5 mu long and 0.25 mu wide. Multiplication by bipartition in the red cell plasma that may be occupied by 30 to 40 Grahamella. In chronic infection without morbid signs. 1/50 to 1/60 hematocyte is only infected. Electron micrograph of thin sections of red cells infected by Grahamella shows an element with a cell wall surrounding cytoplasmic masses which are enclosed in a cytoplasmic membrane. Nuclear region (bundles of moniliform chains = nucleic acids) is not surrounded by a nuclear membrane. Intraerythrocytic position. Bipolar densification of the cell plasma. The prokaryote is directly included in the erythrocyte plasma : no host cell vacuole is visible. Stress, polyinfection, polyparasitism or immuno-depressor effects, determine an intense proliferation of Grahamella by micrococcus elements (diameter : 0.1 to 0.3 mu) invading the systemic circulation. In that cases : septicaemia, anaemia; haematopoiesis with respiratory and nervous disorders; hyperthermia and weakness precede the death. This prokaryote is the first Baratonellacae found in the blood of the Giant Rat. It is namded Grahamella knaiae n. sp ("Kania" is the name of C. gambianus in "serere" language of Fatik region of Senegal). The fleas seem to be the vectors or this prokaryotic element.

Aspects of the relation betweenCyanophora paradoxa(Korschikoff) and its endosymbiotic cyanellesCyanocyta korschikoffiana(Hall & Claus) - I. Growth, ultrastructure, photosynthesis and the obligate nature of the association

Ultrastructural studies of the symbiotic relation between the apochlorotic cryptomonadCyanophora paradoxaand the cyanobacteriumCyanocyta korschikoffianahave demonstrated that the endosymbiotic cyano­bacteria are within host cell vacuoles and possess a highly reduced prokaryotic cell wall which becomes particularly evident during binary fission. Division by the endosymbiotic cyanobacteria was found not to be in synchrony with division by the host cryptomonad. The cyano­bacteria divided most rapidly when the host population had reached its asymptote, such that, in cultures in extended stationary phase, it is possible to observe as many as eight cyanobacteria in one host cell. Nutritionally,C. paradoxawas found to be an obligate phototroph; its auxotropic capabilities are confined to acetate and cyanocobalamin. Analysis of the rate of photosynthesis by the intact association demon­strated that saturation occurred at the 140 μE cm-2s-1(where E is einstein), and at saturating light intensities, the rate of CO2fixation was 30 μmol CO2(mg chla)-1h-1. The best rate of CO2fixation obtained by the isolated cyanobacteria represented 12% of the rate found in the intact association. Two independent methods of analysis showed that at best, only 15% of the carbon fixed by the cyanobacteria was translocated to the host, mostly as glucose and probably sucrose. Studies with metabolic inhibitors directed against the prokaryotic cyanobacteria demonstrated the close interdependence of the host with the endosymbionts, in that disruption of the endosymbionts always resulted in death of the host. Our inability to grow the cyanobacteria away from the host, combined with the apparent inability of the host to survive in the absence of the photosynthetic endosymbiotic cyanobacteria provide strong support for the concept that the association represents a well integrated mutually obligate relation, but the concept that the cyanobacteria are chloroplasts cannot be supported.

Symbiosis of Algae and Invertebrates: Aspects of the Symbiont Surface and the Host-Symbiont Interface

MUSCATINE, L., POOL, R. R. & TRENCH, R. K. 1975. Symbiosis of algae and invertebrates: aspects of the symbiont surface and the host-symbiont interface. Trans. Amer. Micros. Soc., 94: 450-469. The surface and interfacial morphology and function of selected algal and chloroplast symbionts and their host cells are reviewed. Symbionts may be intercellular or intracellular with respect to host digestive cells. Intracellular symbionts are generally within host cell vacuoles, but some chloroplast symbionts seem to lie free in the host cytoplasm. Cell walls may be modified, unmodified, or lacking in symbionts compared to the condition in free-living counterparts. While wall modification may be viewed as an adaptation for a symbiotic existence, it is not a prerequisite for metabolic interactions such as translocation of reduced organic carbon from algae to host cells. The function of symbiont and host surfaces is described in connection with symbiont acquisition and resistance to host digestion. Evidence is presented for cellular recognition of symbiont surface antigens by host cells. Hypothetical mechanisms for resistance of symbionts to host digestion are described and compared to known mechanisms employed by symbiotic bacteria.

The fine structure of pea root nodules. 2. Senescence and disintegration of the bacteroid tissue

The nitrogen-fixing cells of pea root nodules ultimately disintegrate. Ultrastructural observations were made on the bacteroid tissue of nodules on the proximal 3 cm of the main root of Pisum sativum cv. Rondo induced by Rhizobium leguminosarum A 171. Host cells and bacteroids simultaneously undergo ultrastructural changes considered to the features of senescence. These changes can be observed in bacteroid cells at the base of nodules harvested 14 days after culture starts. When the host cell vacuole disappears senescent bacteroid cells dis-integrate completely. A minority of the bacteroid population shows an earlier degeneration.

Fine Structure of the Schizonts and Merozoites of Eimeria acervulina in the Chicken

Fine structural changes observed during development of merozoites were similar in the 1st and later asexual generations of Eimeria acervulina in the chicken. Merozoites were formed at the surface of multinucleated schizonts. The anlage of the merozoite first appeared as a cone-shaped elevation of the surface, in which the inner membrane complex and the organelles of the apical complex developed. The nucleus, Golgi complex, and other organelles were incorporated into the developing merozoite while it was attached by the posterior end to the residual cytoplasm. The inner membrane of the pellicle terminated anteriorly at a polar ring and posteriorly at a thickening suggestive of a posterior polar ring. Twenty-two subpellicular microtubules extended along the merozoite from the anterior polar ring to the posterior end. The nucleus and its Golgi complex were situated in the posterior half of the merozoite and mitochondria, endoplasmic reticulum, refractile bodies, numerous micronemes, and 2 rhoptries in the anterior half. Mitochondria and endoplasmic reticulum were found posterior to the nucleus. The conoid had a spiral structure and 2 anterior preconoidal rings. A micropore just anterior to the nucleus was well developed in the lst-generation merozoites. Polysaccharide granules were found only in the 2ndand 3rd-generation merozoites. Entry of merozoites into villus epithelial cells caused deformity of microvilli. Secondand 3rd-generation schizonts lying close to the brush border deformed microvillar filaments in the superficial web zone. Developing schizonts were within parasitophorous vacuoles in host cells whose endoplasmic reticulum and numbers of mitochondria had increased. Mature schizonts were in cells whose organelles, including the nucleus, had degenerated. Tyzzer (1929) described the life cycle and the morphology of the various stages of Eimeria acervulina in the chicken. E. acervulina has since been frequently used to study the effect of intestinal coccidiosis on the chicken host (Preston-Mafham and Sykes, 1970; Turk and Stephens, 1967; Hein, 1968), but as yet these effects have not been examined at the fine structural level. Lee and Millard (1971a) described the fine structure and development of the macrogamete and oocysts of E. acervulina. The ultrastructure of the schizonts and merozoites and of the changes in host cells parasitized by schizonts are described in this paper. MATERIALS AND METHODS Two-week-old, male White Leghorn chicks, raised coccidia-free, were inoculated with sporulated oocysts of Eimeria acervulina as previously described (Fernando and McCraw, 1973). They were killed at 12, 24, 36, 48, and 56 hr, and 3, 4, and 5 days postinfection, and tissues were processed for electron microscopy. The birds killed up to 56 hr postinfection received 4 million sporulated oocysts and those killed at later periods Received for publication 2 March 1973. * Supported by Ontario Health Research Grant No. PR.297 and by the Ontario Ministry of Agriculture and Food. received 500,000 sporulated oocysts each. A section of the duodenum, about 2 inches proximal to the entry of the bile duct, was cut into pieces, 1 to 2 mm square, and fixed in 3% glutaraldehyde in Millonig's buffer at 4 C. The tissues were fixed from 4 to 48 hr, the best results often being obtained with longer fixation periods. They were washed for 24 hr or longer, first in Millonig's buffer and then in 0.2 mi sucrose solution, buffered at pH 7.4 with 0.1 M phosphate, postfixed in 1% osmium tetroxide in Millonig's buffer for 1 hr, washed in the same buffer, and dehydrated through a graded series of acetone, all at 4 C. The tissues were embedded in Epon and sectioned on a Reichert OM ultramicrotome. Ultrathin sections were mounted on 200-mesh copper grids and stained with lead citrate (Venable and Coggeshall, 1965), followed by a saturated solution of uranyl acetate in 50% ethanol. They were examined and photographed at 60 kv in a Phillips EM 300 electron microscope.

The life cycle of Isospora felis Wenyon, 1923, a coccidium of the cat.

SYNOPSIS. A pure strain of Isospora felis derived from a single oocyst was used to study the endogenous cycle. One and a half to two‐month‐old laboratory‐reared, coccidia‐free kittens were used thruout the study. The endogenous stages occurred in the epithelial cells of the distal parts of the villi in the ileum and occasionally duodenum and jejunum. All stages lay above the host cell nucleus. There were 3 asexual generations. The 1st generation schizonts were 11–30 by 10–23 μ when mature and contained 16–17 banana‐shaped merozoites 11–15 by 3–5 μ. They became mature in 96 or sometimes in 120 hours. The 1st generation merozoites entered new host cells, rounded up and formed 2nd generation schizonts. These formed within themselves 2–10 or more spindle‐shaped bodies resembling 1st generation merozoites in shape and size. These were 2nd generation merozoites. They were uninucleate 120 hours after inoculation, but by 144 hours they became larger, multinucleate and some lost their elongate shape and became ovoid. They were then 3rd generation schizonts. They were 12–16 by 4–5 μ. Each formed up to 6 or more banana‐shaped merozoites 6–8 by 1–2 μ. The 3rd generation schizonts and merozoites developed within the same host cell and parasitophorous vacuole as the 2nd generation schizonts and merozoites. Mature schizonts containing only 3rd generation merozoites appeared 144 hours after inoculation, were most abundant 168 hours after inoculation, and might be present as late as 216 hours after inoculation. They were 14–36 by 13–22 μ and contained 36 to more than 70 merozoites.The 3rd generation merozoites entered the sexual cycle. The mature microgametocytes were 24–72 by 18–32 μ and contained a central residuum and a large number of microgametes 5–7 by 0.8 μ with 2 posteriorly‐directed flagella. The mature macrogametes were 16–22 by 8–13 μ. Gametogony occurred 144–216 hours after inoculation.The prepatent period was 168–192 hours and the patent period 10–11 days. Peak oocyst production occurred on the 6th day of the patent period.