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.     

The Fine Structure of Microgametogenesis in Haemoproteus columbae Kruse*

SYNOPSIS. The structural changes leading to the formation of motile microgametes from a single immobile intracellular gametocyte have been examiued in the electron microscope. After pigeon blood infected with Haemoproteus columbae was exposed to the air at room temperature for a few minutes axonemes appeared in the parasite's cytoplasm and the cytoplasm itself appeared less dense. The axonemes were connected with bundles of intranuclear microtubules that were perhaps spindle fibers. No conventional kinetosomes or centrioles have been observed. After the microgametocyte left the erythrocyte, it assumed the shape of a polarized slug or a dumb-bell. Half of the organism was surrounded by a single membrane and filled by part of the nucleus. The other half was surrounded by the remains of the multiple membranes of the gametocyte and contained pigment granules, mitochondria, axonemes and nuclear extensions. The axonemes and nuclear extensions were segregated at the periphery of the cell, exterior to the gametocyte's inner membrane, and were assembled in situ into microgametes. The mature microgamete appeared to peel off from the gametocyte, leaving a residual body.     

Studies of growth and development of gametocytes in Haemoproteus columbae Kruse.

As determined by their ability to exflagellate and round up, it took the macrogametocyte and the microgametocyte of Haemoproteus columbae in pigeons 68 and 116 h, respectively, after patency to reach maturity. Pigment granules appeared in the undifferentiated gametocytes 8 h after invasion of blood. Vacuoles were observed in young gametocytes and persisted in the older forms. The growth curve of H. columbae is close to the sigmoidal curve for growth in protozoa. Multiple infection was noticed in pigeons with high levels of parasitemia, but no more than 2 gametocytes reached maturity; such multiple infections were rare in relapses. The sex ratio of the gametocytes was 1:1. Strong lateral displacement of the nuclei of infected erythrocytes was the rule; hypertrophy was negligible.     

The Fine Structure of Differentiating Merozoites of Haemoproteus columbae Kruse*

SYNOPSIS. The first sign of merozoite formation in schizonts of Haemoproteus columbae is the accumulation of dense material at intervals beneath the plasma membrane of the schizont. The schizont's membrane then invaginates in deep furrows cleaving the parasite into pseudo-cytomeres. thereby increasing the area of membrane available for differentiation. Signs of differentiation appear under this membrane as soon as it is formed. Rhoptries and polar rings develop in the region of the dense accumulations, the cytoplasm containing these structures begins to elevate, and each evagination differentiates into a merozoite. When the merozoite is half-formed, the cytostome appears, then dense bodies at the apex of the organism, and finally a spherical body intimately associated with a mitochondrion. These merozoites of Haemoproteus are assumed to be the forms that penetrate erythrocytes and become gametocytes. They contain the same organelles as merozoites of Plasmodium. However, the merozoites of Haemoproteus are oval like the erythrocytic merozoites of Plasmodium rather than elongate like the exoerythrocytic merozoites. This body shape may be a generic characteristic or it may indicate a structural difference between exoerythrocytic merozoites and merozoites that infect erythrocytes. When the merozoites of Plasmodium, Haemoproteus and Leucocytozoon are compared, the first 2 genera appear closely related, but Leucocytozoon seems very different. Perhaps it should not be included within the Haemoproteidae.     

An Electronmicroscopic Study of the Microgamete of Haemoproteus columbae Kruse*

SYNOPSIS. Microgametes of Haemoproteus columbae were studied by light microscopy and electron microscopy, using the single-stage replication technic. The filiform microgamete has a swelling or vesicle near the round end; the opposite end is asymmetrically tapered. The gamete is polarized, for it travels in sinuous movements with the round end (the putative anterior end) directed forward. There is no flagellum or undulating membrane and no mitochondrion. The vesicle may contain energy reserves for locomotion or lytic enzymes for penetration. Tubular material often is seen coming from the vesicle and anterior end and may be a kind of acrosomal filament.     

Fine Structure of Haemoproteus columbae Kruse During Macrogametogenesis and Fertilization*

When blood is withdrawn from a pigeon (Columba livia) infected with gametocytes of Haemoproteus columbae, differentiation of the gametes begins immediately. This study examines the formation of the macrogamete and its fertilization. The first visible signs of differentiation are the elongation of the nucleus along with the appearance of an intranuclear spindle and atypical centrioles. Then maturation bodies, the products of nuclear reduction, form in both erythrocytic macrogametocytes and macrogametocytes free of their host cells. Penetration of the macrogamete by the microgamete occurs rapidly. Their plasma membranes fuse, and the microgamete's nucleus, axonemes and cytoplasm enter the macrogamete. The nucleus of the microgamete expands and migrates to lie at an angle to the macrogamete nucleus. The 2 fuse across a small area. The nuclear envelope and the plasma membrane of the zygote are a mosaic of the membranes of the 2 gametes.     

Early Stages in the Differentiation of Gametocytes of Haemoproteus columbae Kruse*

SYNOPSIS The sexes of mature gametocytes of Haemoproteus columbae Kruse circulating in the blood of the domestic pigeon can be identified in the electron microscope by the same criteria that distinguish them in the light microscope. The microgametocyte has a large nucleus and pigment granules restricted to the 2 extremities of its halter-shaped cells. The macrogametocyte has dense granular cytoplasm with scattered pigment granules and a small central nucleus. The sex of young gametocytes cannot yet be recognized. When blood containing mature gametocytes is cooled outside the body of the host visible signs of gametogenesis appear within 30 seconds. The earliest signs are increasing electron lucidity of the cytoplasm and separation of the outer membrane from the body of the parasite. The membrane may form vesicles or whorls or lie free in the erythrocyte's cytoplasm. The middle membrane of the parasite becomes the plasma membrane. Axonemes and microtubules appear in the cytoplasm and nucleoplasm of the microgametocyte. The macrogametocyte lags slightly behind the microgametocyte in development. With the first signs of differentiation, the host cell cytoplasm begins to disappear. The fate of the outer membrane and the erythrocyte's cytoplasm suggests the release of a lytic substance by the parasite.     

Studies of Growth and Development of Gametocytes in Haemoproteus columbae Kruse*

SYNOPSIS As determined by their ability to exflagellate and round up, it took the macrogametocyte and the microgametocyte of Haemoproteus columbae in pigeons 68 and 116 h, respectively, after patency to reach maturity. Pigment granules appeared in the undifferentiated gametocytes 8 h after invasion of blood. Vacuoles were observed in young gametocytes and persisted in the older forms. The growth curve of H. columbae is close to the sigmoidal curve for growth in protozoa. Multiple infection was noticed in pigeons with high levels of parasitemia, but no more than 2 gametocytes reached maturity; such multiple infections were rare in relapses. The sex ratio of the gametocytes was 1:1. Strong lateral displacement of the nuclei of infected erythrocytes was the rule; hypertrophy was negligible.     

The Fine Structure of the Mature Gametes of Haemoproteus columbae Kruse*

SYNOPSIS. The filiform microgamete of Haemoproteus columbae consists of an elongate double-walled nucleus paralleled by 2 axonemes embedded in a homogeneous matrix. At one end of the gamete, the axonemes are sharply flexed back on themselves, but no conventional kinetosome has been recognized. No mitochondria have been seen. Single-walled vesicles occur in the matrix, and the entire gamete is surrounded by a single membrane. The large round macrogamete has a conspicuous central nucleus with its outer membrane drawn out into anastomosing evaginations which extend to the periphery of the cell. A moderately electron dense material fills the space between the 2 nuclear membranes and the lumina of the evaginations. Nucleolar material may occur in scattered masses within the nucleus. One or 2 axonemes appear to arise endogenously next to the nuclear membrane. The cytoplasm is filled with ribosomes and perhaps glycogen granules. Typical protozoan mitochondria and vesicles containing pigment retained from the erythrocytic stage are found in the peripheral cytoplasm. Accumulations of dense-walled vesicles occur in the cytoplasm in conjunction with evaginations of the nuclear membrane. Amid these vesicles triple-ringed discs resembling the cytostomes of merozoites are frequently seen. Several distinct layers of dense material surround the micro-gamete.     

Fine Structure of Haemoproteus columbae Kruse During Differentiation of the Ookinete*

Ookinete differentiation begins in vitro～1 hr after blood infected with mature gametocytes of Haemoproteus columbae is withdrawn from a pigeon. In the undifferentiated zygote, dense material accumulates at the point under the plasma membrane. The conoid and conoidal rings condense from this material. The nucleus is drawn out to a point with the intranuclear spindle (INS) at the peak. Atypical centrioles lie under the forming conoid in the cytoplasm next to the INS. Fibrous material under the inner membrane forms the polar ring from which subpellicular microtubules originate. One hr later the centrioles have disappeared and the nucleus has returned to the center of the organism. The conoidal complex forms the tip of a growing cytoplasmic projection, the anterior end of the ookinete. During this time an elaborate pellicle is differentiating antero-posteriorly; crystalloid formation begins with an extensive proliferation of rough endoplasmic reticulum (ER) continuous with the outer membrane of the nuclear envelope. Crystalloid particles are formed between the lamellae of the ER and collected in a sphere that is later partially surrounded by a small amount of ER. Ookinetes, differentiated 2 hr longer than the ookinetes in vitro, were obtained from the gut of the pigeon fly, Pseudolynchia maura. The differentiated pellicle of these ookinetes consists of a plasma membrane, an inner membrane layer composed of 2 appressed membranes, and in the anterior end, an electron-opaque lamina immediately under the inner membrane. Anterior to the polar ring, this lamina forms a canopy which, posteriorly, is drawn out into projecting ribs which diminish and disappear in the first third of the organism. Fifty to 60 subpellicular microtubules insert on the polar ring. Ookinetes differentiated in vitro were no more than 4 hr old. They lacked micronemes and retained a pellicular cytostome and “internal cytostomes.” The differentiation of micronemes probably occurs at a later time because they are visible after 6 hr in ookinetes in the fly gut. So many degenerating organisms appeared in vitro after 5 hr that this material was discarded.     

Observations on the Fine Structure of the Schizonts of Haemoproteus columbae Kruse*

SYNOPSIS. The schizonts of Haemoproteus columbae resemble the exoerythrocytic schizonts of avian Plasmodium in their fine structure. Haemoproteus infects endothelial cells and grows several hundredfold in volume, destroying the cytoplasm and nucleus of the host cell. The schizont's plasma membrane is trilamellar with a dense outer lamella. Some schizonts have micropores in their plasma membranes, but there is no evidence for ingestion thru them. Instead, numerous vesicles and channels fill the host cell cytoplasm and give its plasma membrane and periparasitic vacuolar membrane the appearance of active pinocytosis. The parasite's membrane shows no sign of pinocytosis, indicating that it probably feeds by diffusion. The growing schizont has numerous mitochondria, nuclei, and ribosome-rich cytoplasm which contains electron-lucent vacuoles and clefts. The latter appear to be artifacts of fixation.     

The Host Restriction of Haemoproteus columbae

SYNOPSIS. Sporozoites of Haemoproteus columbae failed to produce an erythrocytic infection in 4 Columba palumbus , tho 2 C. livia (the natural host of H. columbae ), inoculated with part of the same material, developed parasitemia. H. columbae formed oocysts in all of 6 Ornithomyia avicularia (the natural vector of the haemoproteid of C. palumbus ), but most of the oocysts appeared degenerate and no sporozoites were seen in the 4 O. avicularia which survived for 10–19 days.     

The Fine Structure of Haemoproteus columbae Sporozoites*

SYNOPSIS. The fine structure of Haemoproteus columbae sporozoites has been studied and compared to sporozoite structure as revealed by the light microscope. The sporozoites are ultrastructurally similar to those of other Haemosporidia in that they possess a 3-layered pellicle, subpellicular microtubules, polar ring, micropore, free ribosome-like particles, micronemes, a structure resembling a Golgi complex, an irregular mitochondrion, and a large nucleus. In the anterior region of the sporozoite there are 21–22 regularly arranged longitudinal subpellicular microtubules located peripherally around the cell. In the apical region the microtubules appear thickened on 1 side. The sporozoite of H. columbae has a microneme system in which 1–3 micronemes are associated with the outer pellicular membrane at the anterior end. Micronemes are found throughout the cytoplasm, but occur in greater concentration in the anterior region of the sporozoite. A clear pellicular cavity, located between the polar ring and the termination of the inner pellicular layer, is present at the anterior end of the sporozoite. Vesicular invaginations of the inner pellicular layer have been observed in the anterior region; their function is unknown. Spherical osmophilic bodies are found throughout the cytoplasm.