Schizogony and Gametogony of Leucocytozoon simondi and Associated Reactions in the Avian Host*

SYNOPSIS. Stages of development of Leucocytozoon simondi in White Pekin ducklings and their reactions to the parasite were studied on successive days after infecting them artificially with sporozoites from Simulium rugglesi. The minimum prepatent period was 5 days. The first asexual cycle occurred exclusively in the parenchymal cells of the liver. Progeny of these hepatic schizonts followed one of 3 courses: (a) invaded parenchymal liver cells to give rise to another hepatic cycle, (b) penetrated blood cells to form round gametocytes, and (c) were phagocytized by macrophages and grew into megaloschizonts thruout the body. The appearance of elongating gametocytes coincided with the period of maturation and release of merozoites from the megaloschizonts. Experimental evidence supports the hypothesis that the round gametocytes arise from the hepatic schizonts and the elongate forms from the megaloschizonts. Mature megaloschizonts released millions of merozoites, but a high 2nd peak in parasitemia did not develop because of retention of developing gametocytes in the deep circulation, particularly the liver and spleen, and a pronounced host reaction.     

Development of Eimeria callospermophili and E. bilamellata from the Uinta Ground Squirrel Spermophilus armatus in Cultured Cells*

SYNOPSIS. Cell lines or established cell lines of bovine, ovine or human origin and primary cells from whole embryos of groundsquirrels were used in a study of the in vitro development of Eimeria callospermophili and E. bilamellata from the Uinta ground squirrel, Spermophilus armatus. Monolayers in Leighton tube cultures were inoculated with sporozoites of either of these 2 species and examined with phase-contrast microscopy at various intervals. After such examination, coverslips were fixed in Schaudinn's or Zenker's fluid and variously stained. E. callospermophi sporozoites penetrated cells and underwent development to mature 1st generation schizonts in most cell types. At different times after inoculation, both species formed sporozoite-shaped schizonts, which later became spheroidal. Intracellular movements of sporo zoite-shaped schizonts of E. callospermophili were observed and such schizonts penetrated cells when freed by mechanical disintegration of the host cells. Merozoites were formed at the periphery of the schizont in both species. Mature 1st generation schizonts of E. callospermophili, with 6–14 merozoites, were first seen 15 hr after inoculation; the corresponding values for E. bilamellata were 12–27 merozoites and 4 days. Merozoites of both had anterior and posterior refractile bodies. Exposure to a trypsin-bile solution stimulated motility in merozoites of E. callospermophili. Second generation trophozoites and immature schizonts of E. callospermophili were seen in cultures of primary cells of whole ground-squirrel embryos 20–24 hr and 44–48 hr, respectively, after inoculation of sporozoites.     

Schizogonic Development of Leucocytozoon smithi

ABSTRACT The schizogonic development of Leucocytozoon smithi in the liver of experimentally infected turkey poults was examined by electron microscopy. Following intraperitoneal injection, sporozoites migrated to the liver and entered hepatic cells to become intracellular trophozoites. Three to four days post inoculation (PI), trophozoites underwent asexual multiple fission known as merogony or schizogony. Two generations of schizonts were observed. The primary or first generation schizonts, abundant on day 4 PI, appeared as interconnected cytoplasmic masses (pseudocytomeres). Each pseudocytomere was enclosed by a membranous vacuole and contained varying numbers of nuclei. As nuclear division and growth of the schizonts continued, larger discrete cytoplasmic masses or cytomeres were formed with rhoptries and multiple nuclei in various stages of division. Synchronous multiple cytoplasmic cleavage of the schizont resulted in the formation of numerous uninucleate merozoites. Second generation schizonts, which developed from hepatic merozoites released from primary schizonts, were abundant in hepatocytes on day 6 PI. Although tissue samples from liver, lung, spleen, kidney, intestine, brain, blood vessels and lymph nodes were examined, schizogonous forms were observed in liver only. No megaloschizonts were detected in any host tissue examined. Schizogonic development was completed by day 7 PI as merozoites developed into gametocytes within mononuclear phagocytes.     

Schizogonic development of Leucocytozoon smithi.

The schizogonic development of Leucocytozoon smithi in the liver of experimentally infected turkey poults was examined by electron microscopy. Following intraperitoneal injection, sporozoites migrated to the liver and entered hepatic cells to become intracellular trophozoites. Three to four days post inoculation (PI), trophozoites underwent asexual multiple fission known as merogony or schizogony. Two generations of schizonts were observed. The primary or first generation schizonts, abundant on day 4 PI, appeared as interconnected cytoplasmic masses (pseudocytomeres). Each pseudocytomere was enclosed by a membranous vacuole and contained varying numbers of nuclei. As nuclear division and growth of the schizonts continued, larger discrete cytoplasmic masses or cytomeres were formed with rhoptries and multiple nuclei in various stages of division. Synchronous multiple cytoplasmic cleavage of the schizont resulted in the formation of numerous uninucleate merozoites. Second generation schizonts, which developed from hepatic merozoites released from primary schizonts, were abundant in hepatocytes on day 6 PI. Although tissue samples from liver, lung, spleen, kidney, intestine, brain, blood vessels and lymph nodes were examined, schizogonous forms were observed in liver only. No megaloschizonts were detected in any host tissue examined. Schizogonic development was completed by day 7 PI as merozoites developed into gametocytes within mononuclear phagocytes.     

Development of Eimeria crandallis Honess from Sheep in Cultured Cells*

SYNOPSIS. Cell lines of embryonic lamb trachea (LETr), lamb thyroid (LETh), and bovine liver (BEL) as well as an established cell line of Madin-Darby bovine kidney (MDBK) were used in a study of the in vitro development of Eimeria crandallis from sheep. Excysted sporozoites were inoculated into Leighton tubes containing coverslips with monolayers of the different cell types. Coverslips were examined with phase-contrast and interference-contrast at various intervals up to 20 days after inoculation; thereafter the monolayers were fixed and stained in various ways. Freshly excysted sporozoites, with 2–10 spheroidal refractile bodies, entered all of the cell types in relatively small numbers; intracellular sporozoites were first seen 2 min after inoculation. After 24 hr, most intracellular sporozoites had only 1 or 2 refractile bodies. Before and during transformation of sporozoites, the nucleus and peripheral nucleolus increased markedly in size. Transformation resulted in usually spheroid but sometimes ellipsoid trophozoites. Trophozoites were seen first 3–4 days, and binucleate schizonts at 4–5 days after inoculation. Immature schizonts increased considerably in size and eventually had large numbers of nuclei. Some of the parasites became lobulated and the lobules often separated to form individual schizonts. In BEL, LETr and LETh cells, mature schizonts, up to 150 μm in diameter, were seen first 11–14 days after inoculation. The BEL cells were the most favorable for development. Merozoites were formed by a budding process from the surface of the schizonts as well as from blastophores. Some merozoites were seen leaving mature schizonts, but no further development was observed. Merozoites frequently were motile and had a sharply bent posterior end. Marked nuclear and cytoplasmic changes were observed in parasitized cells.     

Gametocyte formation by the progeny of single Plasmodium falciparum schizonts

Gametocytogenesis of the malaria parasite Plasmodium falciparum was studied in monolayers of erythrocytes attached to tissue culture dishes. Merozoites produced by single schizonts in erythrocytes overlaying the monolayer infected the attached erythrocytes and produced clusters of progeny. Parasites in these readily indentifiable clusters then underwent either asexual growth or sexual differentiation. The progeny of most schizonts yielded no gametocytes. However, the progeny of those schizonts that did yield gametocytes showed a marked tendency to produce multiple gametocytes. Gametocytogenesis, therefore, was not random. Instead, the progeny of certain schizonts were committed to produce gametes. However, even those clusters containing several gametocytes also contained asexual forms. Therefore, not all merozoites of a single schizont were committed to gametocytogenesis. In those cells infected with two or more merozoites the formation of a gametocyte was usually associated with a block in the further development of other parasites.     

Schizogony in Haemoproteus columbae Kruse*

SYNOPSIS To fill in some of the gaps in our knowledge of Schizogony of Haemoproteus columbae Kruse, transmission experiments involving inoculation into pigeons (Columba livia Gmelin) of sporozoites from salivary glands of the hippoboscid fly Pseudolynchia canariensis (Macquart) were carried out. We were unable to detect prepatent schizonts or to observe schizogonic development when infection became chronic. Schizonts were mainly confined to lung tissue. Observations of parapatent schizonts were made in smears and tissue sections. A variety of forms was found. Cytomeres were rarely encountered. Two types of morphologically distinct merozoites were seen. One type was twice as large as the other and was thought to repeat the process of schizogony several times before invading erythrocytes. Schizonts with cytoplasmic clefts were not common in our material due to the fixatives used (Bouin's and Carnoy's). Merozoites were occasionally observed inside monocytes, probably being phagocytosed.     

In vitro Development of First-Generation Schizonts of Eimeria canadensis (Bruce, 1921)*†

SYNOPSIS. In vitro development of Eimeria canadensis from cattle was studied in monolayer cultures of various bovine cell lines grown on coverslips in Leighton tubes. Excysted sporozoites were used for inoculation of the cell cultures. Sporozoites entered the host cells within a few minutes, but apart from a reduction in the number of refractile bodies, changed little in appearance during the first 9 days. Beginning at 91/2 days postinoculation, sporozoites developed into sporozoite-shaped schizonts or, less frequently, transformed into trophozoites. Sporozoite-shaped schizonts with as many as 8 nuclei were observed transforming into spheroid schizonts. At 111/2 days, intermediate schizonts had a characteristic single mass of refractile granules and 60–80 nuclei. Deep invaginations, which resulted in the formation of several blastophores, usually occurred when schizonts had about 100 nuclei. Merozoites were formed as a result of radial outgrowth from the surface of spheroid schizonts as well as of blastophores. Mature merozoites were seen 1st after 13 days.     

Discovery of the pre-erythrocytic stages of a saurian malaria parasite, hypnozoites, and a possible mechanism for the maintenance of chronic infections throughout the life of the host

Re-examination of tissue sections from four Takydromus tachydromoides (Sauria: Lacertidae) naturally infected with Plasmodium sasai found liver parenchymal cells, containing uninucleate parasites which may correspond to the hypnozoite stage of primate malaria parasites, schizonts and segmenters in parenchymal cells, and hepatic macrophages which contained numerous schizonts. Following destaining of the original H&E and prolonged restaining with warm Giemsa stain, encysted schizonts, protected by a hyaline wall, were discovered in the connective tissue or capillary endothelium of lung, liver, brain, heart, pancreas, kidney, intestine wall, testis, and both intra- and intermuscularly in the femoral muscles. Unencysted schizonts in the pulmonary endothelium apparently represent the phanerozoic stages, which, following encystment in the various tissues, are recognized as a new stage in the life cycle of reptilian malarial parasites, the chronozoic schizonts. A hypothesis is presented to describe the life cycle of P. sasai, which may be characteristic of other saurian malaria parasites. It interprets the sequence of pre-erythrocytic stages found as follows: sporozoites enter hepatic parenchymal cells where some may become dormant as hypnozoites, and others form cryptozoic schizonts. The cryptozoites parasitize hepatic macrophages and form metacryptozoic schizonts. Metacryptozoites produce phanerozoic schizonts in the capillary endothelium and connective tissue of the lung and other organs. Phanerozoites and possibly metacryptozoites then invade the erythrocytes to begin the erythrocytic cycle. Some of the phanerozoites in endothelium, connective tissue and skeletal muscle become encysted as chronozoic schizonts, and their progeny, chronozoites, renew the erythrocytic cycle throughout the life of the host and produce seasonal relapses of gametocytemia, in spring, at the end of hibernation by the lizard.     

In Vitro Cultivation of the Vascular Phase of Sarcocystis singaporensis

To establish an in vitro culture system for the precystic phase of Sarcocystis singaporensis, we initially tested various excysting fluids for sporocysts. An excysting fluid containing 2.5% bovine taurocholate and 10% bile of the specific intermediate host, Rattus norvegicus, in RPMI medium was the most suitable resulting in excystation of 80% of the sporozoites. Subsequently, we identified brain endothelial cells and pneumonocytes of the rat to promote growth of sporozoites to schizonts. Hepatoma, fibroblastic, or myoblastic cells were not suitable for the parasite's development. First-generation schizonts were seen at days 3-10 postinoculation (PI); a distinct second peak of schizogonic development only occurred in endothelial cells at days 14-18 PI. First-generation schizonts were 26.0 (± 3.8) μm in diameter and contained 32-50 merozoites, second-generation schizonts measured 34.4 (± 10.6) μm and contained 54-72 merozoites. Merozoite yield at large-scale culture conditions (75 cm² flasks) using pneumonocytes as host cells was relatively low. Ultrastructurally, sporozoites and merozoites were quite similar to corresponding stages of other Sarcocystis species. With regard to host cell specificity and developmental kinetics, in vitro cultivation showed close similarities to the situation in vivo.     

Intralymphocytic schizonts associated with an initial infection of Saurocytozoon tupinambi in Tupinambis teguixin

An initial natural infection of Saurocytozoon tupinambi in a juvenile Tupinambis teguixin from Venezuela was studied for 131 days following capture of the host. Intralymphocytic parasites appeared in this sequence: small uninucleate and binucleate stages (days 1–31 and again on day 41); schizonts with 3–102 nuclei (days 8–14 and 29–35); immature gametocytes (days 29–35) and apparently mature gametocytes of Saurocytozoon tupinambi from day 41. Maximum parasitemia of trophozoites and binucleate schizonts occurred on day 4 when 11% of lymphocytes were infected. Maximum parasitemia by larger schizonts occurred on day 8 at 0.13% of lymphocytes, while maximum gametocytemia was found on day 49 with 16.4% of lymphocytes parasitized. Two types of schizonts were observed: intralymphocytic and the same type free of host cells, and fragments of varying size which may have been torn from capillary endothelium.Due to presence of concurrent infection by a small Plasmodium species, identity of intralymphocytic asexual stages with S. tupinambi cannot be established. Presence of asexual and sexual stages in the same type of host cells (lymphocytes and close derivatives), sequential appearance of trophozoites, schizonts and gametocytes over a period of 40 days, and correlated fluctuations in lymphocyte density suggest they are conspecific, and that Saurocytozoon, which has a plasmodiid type of sporogony may prove to further differ from leucocytozoids by presence of an asexual cycle in circulating blood cells.     

Development of Eimeria meleagrimitis Tyzzer from Sporozoites and Merozoites in Turkey Kidney Cell Cultures*

SYNOPSIS. Sporozoites and 1st-, 2nd-, and 3rd-generation merozoites of Eimeria meleagrimitis were inoculated into primary cultures of turkey kidney cells. In vitro-excysted sporozoites developed into mature macrogamonts in 8 days; in vivo-excysted sporozoites developed into 2nd- or 3rd-generation schizonts within 5 to 7 days. First-generation merozoites obtained from infected turkeys produced mature 2nd-generation schizonts within 24 h. Second-generation merozoites from turkeys produced mature macrogamonts and oocysts within 72 h, whereas 3rd-generation merozoites produced these stages within 48 h. The oocysts that developed from 3rd-generation merozoites sporulated at 25 C and were infective for turkeys. The timing of the early stages and the intervals between schizogonic generations in cultures were comparable with those in turkeys. Morphologic parameters, however, indicated that some differences existed between in vitro and in vivo development. Second- and 3rd-generation schizonts and gamonts that developed after inoculation of cultures with merozoites were similar to stages in turkeys. Oocysts, however, were significantly smaller (P < 0.05) in cultures. All stages that developed after inoculation of cultures with sporozoites were smaller (P < 0.05) than their in vivo counter parts.     

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.     

Comparative Development of Eimeria tenella from Sporozoites to Oocysts in Primary Kidney Cell Cultures from Gallinaceous Birds

Eimeria tenella completed its endogenous life cycle in primary cultures of kidney cells from 2- to 3-week-old-chickens, guinea fowl, partridges, pheasants, quail, and turkeys. Similarity in percentage of infection at 4 hr suggested that sporozoites entered cells from all birds in equal numbers. Development was better, however, in chicken cells in that the percentage of survival and of developmental stages during the first 2 days were greater, developmental stages occurring after 2 days usually were found earlier, mature 2nd-generation schizonts and oocysts were larger, and oocyst production was far greater than in nonhost cells. Multinucleate macrogametes, which sometimes reached sizes 3–4 times greater than normal oocysts, are reported for the first time.     

Observations on Haemogregarina balli sp. n. from the Common Snapping Turtle,Chelydra serpentina*

SYNOPSIS. Haemogregarina balli sp. n. is described from the blood and organs of the common snapping turtle Chelydra serpentina serpentina and from the gastric and intestinal ceca of the presumed invertebrate hosts, the leeches Placobdella parasitica and Placobdella ornata. In the peripheral blood of the turtle, male and female gametocytes and immature erythrocytic schizonts are found within erythrocytes. The maturation of erythrocytic schizonts containing 6–8 merozoites is recorded from liver imprints. Schizonts with 13–25 merozoites are found in various cells of the liver, lung and spleen. In the gastric ceca of the leeches the host erythrocytes are digested, releasing the gametocytes and immature erythrocytic schizonts. Immature erythrocytic schizonts degenerate. Association of the gametocytes occurs in the intestinal ceca. The microgametocyte apparently gives rise to 4 nonmotile microgametes, one of which fertilizes the macrogamete while the other remain as condensed, residual nuclei on the periphery of the developing oocyst. The oocyst increases in size with maturity. A mature oocyst produces 8 sporozoites from a single germinal center. Sporozoites liberated from the oocyst are found in the tissues of the leech. Transovarial transmission of the parasite does not occur in the turtle. Attempts at experimental transmission failed. Previously unfed (control) leeches were negative for the parasite. Haemogregarina balli is compared with other haemogregarines described from C. serpentina. Features of species of Haemogregarina and Hepatozoon as well as the taxonomy of these genera are discussed.     

Dactylosoma hannesi n. sp. (Dactylosomatidae,Piroplasmia) Found in the Blood of Grey Mullets (Mugilidae) from South Africa1

ABSTRACT. A new species of Dactylosoma (Dactylosomatidae, Piroplasmia), for which the name Dactylosoma hannesi n. sp. is proposed, was discovered in blood erythrocytes of Mugil cephalus, Liza richardsoni, and L. dumerili (Mugilidae) from Swartkops estuary, located east of Port Elizabeth, South Africa. The life cycle of this species differs in some respects from that described for all other known species of Dactylosoma and Babesiosoma. Mature schizonts contain eight nuclei but undergo division only to two to four daughter cells. During cytoplasmic cleavage, schizonts assume triad, rosette, or cruciform shapes. Merozoites are finally produced through a series of binary fissions of these daughter cells which may also be involved in additional nuclear divisions.     

Further observations on the life cycle and vectors of the haemosporidian Leucocytozoon tawaki and its transmission to the Fiordland crested penguin

Abstract

Hepatic and renal schizonts are described from Fiordland crested penguin (Eudyptes pachyrhynchus) chicks, and renal and splenic schizonts from mature birds. Ookinetes, oocysts, and sporozoites are described from the primary vector, Austrosimulium ungulatum. Penguin chicks are infected when 4–6 weeks old. Some observations are recorded on the biology of the vector in association with the birds.     

Development of Hepatozoon griseisciuri in Cultured Squirrel Cells*

Sporocysts of Hepatozoon griseisciuri obtained from laboratory-reared spiny rat mites (Echinolaelaps echidninus) and laboratory-reared squirrel mites (Haemogamasus reidi) were made bacteria-free and incubated in trypsin-bile for 30 min at 37 C to release sporozoites. Hepatozoon griseisciuri sporozoites were inoculated into monolayer cultures of primary adult squirrel kidney (PSK) cells and cell line cultures of neonatal squirrel kidney (SK), heart (SH), and spleen (SS) cells. Extracellular sporozoites underwent flexing, gliding, and pivoting movements similar to other coccidian sporozoites. Sporozoites entered cells in all the cultures used and were found intracellularly as early as 1 hr and as late as 10 days after inoculation. In SK, SH, and SS cells, development proceeded only to the trophozoite stage. In PSK cells, immature schizonts and mature schizonts containing 12–40 merozoites were present from 5 through 10 days after inoculation. The finding of pairs of intracellular organisms within a single parasitophorous vacuole in PSK cells suggested that endodyogeny or limited schizogony had occurred.     

Plasmodium hegneri n. sp. from the European Teal Anas c. crecca in Taiwan*

SYNOPSIS. Plasmodium hegneri n. sp. is described from the European teal duck, Anas c. crecca, from Taiwan. The blood stages, so far the only ones seen, are distinguished mainly by the elongate character of the gametocytes, which closely resemble Haemoproteus (though the pigment tends to be finer and less abundant), and the failure of both asexual and sexual forms to displace the nucleus or otherwise alter the host cell. Merozoites average 13.4 ± 2.2 (range 10–19) per segmenter. Trophozoites often adhere to the host cell nucleus, and may have a large vacuole and a remarkable long, slender pseudopodium. The species so far has been seen only in the European teal, although blood films from 194 species and over 1200 birds have been examined.     

Plasmodium berghei: preparation of rat hepatic cell suspensions that include infective exoerythrocytic schizonts.

Employing an enzymatic method to dissociate rat liver, we prepared suspensions of liver cells from rats infected with sporozoites of Plasmodium berghei 3 to 10, 18 to 28, or 29 to 36 hr prior to liver dissociation. These suspensions of liver cells included hepatocytes, Kupffer cells, fibroblasts, and unidentified cells, as well as hepatocytes infected with exoerythrocytic schizonts (HEX) of P. berghei. These HEX were infective for recipient rodents when inoculated intraperitoneally into the recipients. The number of infective HEX present in the liver cell suspensions was quantitated by varying the number of HEX inoculated into recipients. This infectivity assay made it possible to compare the numbers of HEX in suspensions of liver cells from different donor rats. Infective HEX were obtained from donor rats in 35 of 41 experiments. The greatest number of infective HEX was obtained from donors injected with sporozoites 18 to 28 hr prior to liver dissociation. For morphological observation of mature HEX in cell suspensions, hepatic cells were prepared from donors infected with sporozoites 48 hr prior to liver dissociation. For experimental purposes, the preparation of infective HEX in suspensions of liver cells is superior to the preparation of infective HEX in liver fragments, because it is possible to quantitate the number of HEX which are present either visually or by means of the infectivity assay.