Pathogenesis of Sarcocystis falcatula (Apicomplexa: Sarcocystidae) in the budgerigar (Melopsittacus undulatus). III. Pathologic and quantitative parasitologic analysis of extrapulmonary disease

Forty-four budgerigars (Melopsittacus undulatus) were administered sporocysts of Sarcocystis falcatula orally and were examined at necropsy intervals from less than 12 hr to 168 days. Tissue were examined by touch preparations (of organ cut surfaces), light microscopy, and electron microscopy. Meront and cyst burdens were determined in various organs and correlated with duration of infection, inoculum, and the meront or cyst burdens of other organs. Host inflammatory tissue reactions were quantitated and correlated with meront and cyst burdens. Quantitation of meronts was more accurate in tissue sections than in touch preparations, but quantitation of merozoites was better in touch preparations. More than 97% of meronts were found in capillary, venular, and venous endothelial cells. Cysts were found only in cardiac and skeletal myocytes. Merogony began in the lamina propria of the small intestine less than 12 hr postinoculation (PI). Meronts were in liver and lung by the second day PI and in other organs by 3-7 days PI. Mean meront burdens were highest in lung (33 meronts/mm2), lower in liver and kidney (1-3 meronts/mm2), and infrequent in other organs (less than 0.9 meronts/mm2). Cysts were first seen in cardiac myocytes 7 days PI. They developed through the metrocyte stage and then degenerated, rarely reaching maturity. Cysts were first noted in skeletal muscle at 8 days PI. In leg, upper esophagus, and tongue, cysts matured between 44 and 77 days PI. In pectoral muscles, the majority of cysts degenerated during the late metrocyte and early intermediate stages (28-42 days PI). In addition to a previously reported and often fatal acute interstitial pneumonitis, S. falcatula-infected budgerigars also sustained a chronic active hepatitis, interstitial myocarditis, myositis, nephritis, splenitis, and encephalitis. These lesions weakly correlated with meront burdens in most sites during early infection (up to 50 days PI).     

The intermediate host spectrum in a Sarcocystis species of birds

The life cycle of an avian Sarcocystis has been completed in the laboratory, originating with naturally infected icterids and passing alternately between opossums (Didelphis virginiana) and experimentally infected birds. To determine the intermediate host range, six avian species, including canaries (Serinus canarius), zebra finches (Poephila guttata), budgerigars (Melopsittacus undulatus), pigeons (Columba livia), chickens (Gallus gallus), and guinea fowl (Numida meleagris), were inoculated orally with Sarcocystis sporocysts derived from experimentally infected opossums. All birds but the Galliformes were susceptible to merogony. Pigeons (Columbiformes) were susceptible to early merogony but apparently not to muscle stages. Passeriformes and Psittaciformes were completely susceptible and the parasite developed into muscle cysts in them.     

Pathogenesis of Sarcocystis falcatula (Apicomplexa: Sarcocystidae) in the budgerigar (Melopsittacus undulatus). IV. Ultrastructure of developing, mature and degenerating sarcocysts

Sarcocysts in cardiac and skeletal muscles of budgerigars (Melopsittacus undulatus) were examined transmission electron microscopically 5 to 168 days after experimental infection with Sarcocystis falcatula. The ultrastructure of the primary cyst wall, amorphous substance, metrocytes and bradyzoites in developing, degenerating and mature sarcocysts is described and compared with precystic merozoites studied previously. Sufficient morphologic differences between precystic merozoites, metrocytes and bradyzoites (cystozoites) were found which seem to justify their semantic differentiation. Significant differences in immature and mature primary cyst wall morphology were encountered. If primary cyst wall morphology is to be used in determination and differentiation of species of Sarcocystis, then caution must be used to employ only mature sarcocysts.     

Pathogenesis of Sarcocystis falcatula (Apicomplexa: Sarcocystidae) in the Budgerigar (Melopsittacus undulatus). IV. infrastructure of Developing, Mature and Degenerating Sarcocysts

ABSTRACT. Sarcocysts in cardiac and skeletal muscles of budgerigars (Melopsittacus undulatus) were examined transmission electron microscopically 5 to 168 days after experimental infection with Sarcocystis falcatula. The ultrastnicture of the primary cyst wall, amorphous substance, metrocytes and bradyzoites in developing, degenerating and mature sarcocysts is described and compared with precystic merozoites studied previously. Sufficient morphologic differences between precystic rnerozoites, metrocytes and bradyzoites (cystozoites) were found which seem to justify their semantic differentiation. Significant differences in immature and mature primary cyst wall morphology were encountered. If primary cyst wall morphology is to be used in determination and differentiation of species of Sarcocystis , then caution must be used to employ only mature sarcocysts.     

Sarcocystis lindsayi n. sp. (Protozoa: Sarcocystidae) from the South American opossum, Didelphis albiventris from Brazil

A new species, Sarcocystis lindsayi n. sp., is proposed for a parasite resembling Sarcocystis falcatula. It was obtained from the lungs and muscles of budgerigars (Melopsittacus undulatus) fed sporocysts from a naturally-infected South American opossum, Didelphis albiventris, from Jaboticabal, Brazil. Sarcocysts of S. lindsayi n. sp. in budgerigars are microscopic, up to 600 microm long and up to 50 microm wide. The cyst wall is up to 2 microm thick. Ultrastructurally, the sarcocyst wall consists of numerous slender villar protrusions (up to 2.0 microm long and up to 0.3 microm wide), each with a stylet at its tip. Schizonts in cell culture divide by endopolygeny leaving a residual body. Sporocysts are approximately 12 x 7 microm. The parasite is genetically distinct from other organisms that also cycle between opossums and avian species and resemble S. falcatula. Diagnostic genetic variation has been observed in the nuclear large subunit ribosomal RNA gene, the internal transcribed spacer (ITS-1), and each of two other genetic loci. Although the structure of the sarcocyst wall may not provide sufficient grounds for differential diagnosis, several other attributes including schizont morphology and genetic variation at each of these genetic loci permit identification of S. lindsayi n. sp.. Natural intermediate hosts for S. lindsayi n. sp. are not known, and fuller characterization of these and other Sarcocystis species would benefit from experimental avian hosts that are more permissive to the maturation of sarcocysts.     

Naturally occurring apicomplexan acute interstitial pneumonitis in thick-billed parrots (Rhynchopsitta pachyrhyncha)

This report describes acute interstitial pneumonitis due to an apicomplexan parasite with schizogony in endothelial cells of pulmonary vessels accompanied by early and metrocyte stages of sarcocysts in the heart of a thick-billed parrot (Rhynchopsitta pachyrhyncha). The pattern of this disease is similar to that of the acute phase (approximately 10-15 days postinoculation) of experimental infections of budgerigars, Melopsittacus undulatus, with high doses of sporocysts of Sarcocystis falcatula.     

Characterization of an unidentified Sarcocystis falcatula-like parasite from the South American opossum, Didelphis albiventris from Brazil

An unidentified isolate of a Sarcocystis falcatula-like parasite was obtained from the lungs of budgerigars (Melopsittacus undulatus) fed sporocysts from a naturally-infected South American opossum, Didelphis albiventris from Brazil. Four captive budgerigars fed sporocysts from the opossum intestine died of acute sarcocystosis 8, 10, and 12 days after oral inoculation (DAI); one budgerigar was killed 12 DAI when it was lethargic. Schizonts and merozoites found in the lungs of the budgerigars reacted mildly with polyclonal S. falcatula antibody. The parasite was isolated in equine kidney cell cultures inoculated with lung tissue from a budgerigar that was killed 12 DAI. Two budgerigars inoculated subcutaneously with 100,000 culture-derived S. falcatula merozoites developed acute sarcocystosis and S. falcatula-like schizonts were found in their lungs 15 and 16 DAI. Four budgerigars kept as unfed controls in the same environment remained free of Sarcocystis infection. The parasite underwent schizogony in African green monkey kidney cells and bovine turbinate cells. Merozoites divided by endopolygeny, often leaving a residual body. Polymerase chain reaction studies using primers JNB33/JNB54 and Hinf I and Dra I digestion indicated that the isolate was not S. falcatula. Results of this study indicated that the South American opossum, D. albiventris, is a definitive host for yet another S. falcatula-like parasite.     

Prevalence of sarcocystis species sporocysts in wild-caught opossums (Didelphis virginiana)

Sarcocystis sporocysts were found in intestinal scrapings from 24 (54.5%) of 44 opossums (Didelphis virginiana). The number of sporocysts varied from a few (< 10,000) to 245 million. Sporocysts from 23 of 24 opossums were fed to captive budgerigars (Melopsittacus undulatas), and the inocula from 21 opossums were infective, indicating the presence of Sarcocystis falcatula. Sporocysts from 24 opossums were fed to gamma-interferon-knockout (KO) or nude mice; inocula from 14 opossums were infective to mice. Sarcocystis neurona was detected in tissues of KO mice by specific staining with anti-S. neurona antibodies, and the parasite was cultured in vitro from the brains of KO mice fed sporocysts from 8 opossums. Sarcocystis speeri was identified by specific staining with anti-S. speeri antibodies in tissues of KO mice fed inocula from 8 opossums; 3 opossums had mixed S. neurona and S. speeri infections. Thus, the prevalences of sporocysts of different species of Sarcocystis in opossums were: S. falcatula 21 of 44 (47.7%), S. neurona 8 of 44 (18.1%), and S. speeri 8 of 44 (18.1%) opossums. Sarcocystis neurona alone was found in 1 opossum, and S. speeri alone was found in 1 opossum. Mixed Sarcocystis infections were present in 21 opossums.     

Characterization of Sarcocystis falcatula isolates from the Argentinian opossum, Didelphis albiventris

Two isolates of Sarcocystis falcatula were obtained from the lungs of budgerigars (Melopsittacus undulatus) fed sporocysts from two naturally-infected South American opossums (Didelphis albiventris). The two isolates were designated SF-1A and SF-2A. Both isolates induced fatal infections in budgerigars. Both isolates underwent schizogony in African green monkey kidney cells. The structure of schizonts in the lungs of budgerigars was more variable than that observed in cell culture. The two isolates were identified as S. falcatula by the two species-specific Hinf 1 restriction fragments dervied from digestion of a PCR amplification using primers JNB33/JNB54. Thus, the South American opossum, D. albiventris, is a definitive host for S. falcatula.     

Establishment of the new genus Paranosema based on the ultrastructure and molecular phylogeny of the type species Paranosema grylli Gen. Nov., Comb. Nov. (Sokolova, Selezniov, Dolgikh, Issi 1994), from the cricket Gryllus bimaculatus Deg

The ultrastructure of the microsporidian parasite Nosema grylli, which parasitizes primarily fat body cells and haemocytes of the cricket Gryllus bimaculatus (Orthoptera, Gryllidae) is described. All observed stages (meront, meront/sporont transitional stage ("second meront"), sporont, sporoblast, and spore) are found in direct contact with the host cell cytoplasm. Nuclei are diplokaryotic during almost all stages of the life cycle, but a brief stage with one nucleus containing an abundance of electron-dense material is observed during a "second merogony." Sporogony is disporous. Mature spores are ovocylindrical in shape and measure 4.5+/-0.16micromx2.2+/-0.07 microm (n=10) on fresh smears and 3.3+/-0.06 micromx1.4+/-0.07 microm (n=10) on ultrathin sections. Spores contain 15-18 coils of an isofilar polar filament arranged in one or two layers. Comparative phylogenetic analysis using rDNA shows N. grylli to be closely related to another orthopteran microsporidian, Nosema locustae, and to Nosema whitei from the confused flour beetle, Tribolium confusum. Antonospora scoticae, a parasite of the communal bee Andrena scotica, is a sister taxon to these three Nosema species. The sequence divergence and morphological traits clearly separate this group of "Nosema" parasites from the "true" Nosema clade containing Nosema bombycis. We therefore propose to change the generic name of N. grylli and its close relative N. locustae to Paranosema n. comb. We leave N. whitei in former status until more data on fine morphology of the species are obtained.     

Structural and functional characterization of cyst cells in Sarcocystis sp. (Sporozoa, Apicomplexa)

By means of light and electron microscopy, the structural pattern of muscle cysts (sarcocysts) was examined for the four species of the genus Sarcocystis: S. muris (from murine skeletal muscles), Sarcocystis sp. and S. fusiformis (from, respectively, heart and skeletal muscles of buffalo), and S. ovifelis (from ovine tong muscles). The orderly fashion of the interior of the cyst is attained by partitition of its space into numerous compartments with the involvement of the intermediate filaments. These, in their turn, are bound to each other by thin filaments to make eventually a common filamentous net. The net limits separate groups of cells referred to as cyst zoites. The common net of filaments and microtubules (when present) may be regarded not only as the organizer of the cyst interior cytoskeleton, but also as the main mechanism of substance transportation in various directions: from the host cell to the sarcocyst, and within or outside the cyst. The role of dedifferentiation, proliferation and differentiation processes is suggested in the establishment of the fixed sequence of events throughout the unidirectional development of cyst cells and their interaction, from precystic meronts to cyst merozoites (gamonts). Special attention is paid to metrocyte morphogenesis and functioning. In the present work, metrocytes subjected to apoptosis were recognized. It is suggested that phenomenon of programmed cell death in metrocytes may be associated with the control of cell number in mature and ageing sarcocysts.     

Structure of Cyst Wall Precursors and Kinetics of Their Appearance During the Encystment of Laurentiella acuminata (Hypotrichida,Oxytrichidae)1

The encystment of Laurenliella acuminata was divided into five stages: stage A (precystic semitransparent cell with dark-globules), stage B (precystic transparent cell), stage C (precystic pigmented cell), stage D (spherical shape without cyst wall) and stage E (young resting cyst), on the basis of observations of changes in morphology and pigmentation during encystment. The duration of these stages was also established. Observations by electron microscopy confirmed that the cyst wall, composed of four layers, is derived from different kinds of precursors which are synthesized “de novo.” The ectocyst precursors are composed of stacks of between 5 and 12 small thin plates or discs; these stacks are about 0.9 μm in length and 0.06 μm in height. The mesocyst precursors are fibrillar bodies of variable shapes, about 2.4 μm in maximum length and 0.12–0.16 μm in diameter. These precursors appear in the cytoplasm of the precystic cell during the first precystic stage (stage A). The endocyst precursors are rounded bodies surrounded by a fine membrane, and their contents appeared similar to the endocyst. The granular layer precursors are spherical bodies about 0.1–0.2 μm in diameter, surrounded by a double membrane presenting ribosomes adhering to its outer membrane. Both endocyst and granular layer precursors are observed in the precystic cytoplasm from stage B. On the basis of ultrastructural studies, a formation and growth model of the cyst wall of the hypotrichous ciliate Laurentiella acuminata is proposed.     

Isolates of Sarcocystis falcatula-like organisms from South American opossums Didelphis marsupialis and Didelphis albiventris from S?o Paulo, Brazil.

Isolates of Sarcocystis falcatula-like organisms from South American opossums were characterized based on biological and morphological criteria. Sporocysts from intestinal scrapings of 1 Didelphis marsupialis and 8 Didelphis albiventris from S?o Paulo, Brazil, were fed to captive budgerigars (Melopsittacus undulatus). Budgerigars fed sporocysts from all 9 isolates became ill and S. falcatula-like schizonts were identified in sections of their lungs by immunohistochemical staining. Sarcocystis falcatula-like organisms were cultured from lungs of budgerigars fed sporocysts from D. marsupialis and from lungs of budgerigars fed sporocysts from 3 of 8 D. albiventris. The 33/54 locus amplified by polymerase chain reaction from culture-derived merozoites contained both a HinfI endonuclease recognition site previously suggested to diagnose S. falcatula and a DraI site thought to diagnosed S. neurona. Development of the isolate from D. marsupialis was studied in cell culture; its schizonts divided by endopolygeny, leaving a residual body. Morphological and genetic variation differentiated this Sarcocystis isolate originating in D. marsupialis from the Cornell I isolate of S. falcatula. This is the first report of a S. falcatula infection in the South American opossum, D. marsupialis.     

Encephalitozoon hellem in Two Eclectus Parrots (Eclectus roratus): Identification from Archival Tissues

ABSTRACT Members of the phylum Microspora are obligate, intracellular, single-celled parasites identified in a wide range of vertebrate and invertebrate hosts. Only a few cases of microsporidial infections have been documented in psittacine birds including peach-faced, masked, and Fischer's lovebirds ( Agapornis roseicollis, A. personata , and A. fischeri. respectively), budgerigars ( Melopsittacus undulatus ), and a double yellow-headed Amazon parrot ( Amazona ochrocephala ). Parasite identification has typically been limited to phylum or genus, and no avian species of microsporidia has clearly been described. In this report, microsporidia were identified in the kidney and intestine of a new host, the eclectus parrot ( Eclectus roratus ). Parasites were identified as Encephalitozoon hellem using morphologic, ultrastructural, and small subunit ribosomal RNA gene sequence data obtained from archived tissues. This parasite species was first identified in immunocompromised humans and may be a potential zoonotic pathogen. The epidemiology and prevalence of this parasite in humans and birds should be further explored.     

Neural sarcocystosis in a straw-necked ibis (Carphibis spinicollis) associated with a Sarcocystis neurona-like organism and description of muscular sarcocysts of an unidentified Sarcocystis species.

A Sarcocystis neurona-like parasite was associated with acute sarcocystosis in the brain of an ibis (Carphibis spinicollis). Numerous schizonts and merozoites were found extravascularly in encephalitic lesions. These schizonts reacted positively with anti-S. neurona and anti-S. falcatula polyclonal antibodies in an immunohistochemical test. Sarcocysts of an unidentified Sarcocystis species were present in the brain, heart, and skeletal muscles. Sarcocysts in skeletal muscles were microscopic, and the sarcocyst wall was up to 3 microm thick. The villar protrusions on the sarcocyst wall were up to 4.5 microm long, constricted at the base, and expanded laterally. Schizonts and sarcocysts distinct from those of S. falcatula.     

Intracellular Parasitism and the Problem of Sarcocystosis

The obligatory heterogenous tissue cyst-forming coccidia of the genus Sarcystosis are regarded as an excellent example of the specific coexistence of two organisms, i.e., the host and parasite. These parasitic protozoans are known as causative agents of the chronic, often life-threatening disease, sarcocystosis, which still cannot be effectively controlled. In Sarcocystis, the entire phase of asexual multiplication was transferred to the intermediate host. Of special interest is the parasite's ability to persist in this host at the stage of tissue cyst or sarcocyst. This is a giant meront, in which unidirectional development proceeds starting from a little differentiated metrocyte, through intermediate cells, and towards highly differentiated cyst merozoites (gamonts) unable to further divide. The life span of the sarcocyst depends, to a great extent, on self-regulation within the cyst itself and on relations between the cyst and its immediate environment. A totally new field of research into Sarcocystis was initiated by the discovery that the intracellular parasite damages both cyst harboring and intact muscle cells, apart from the adjacent connective and nervous tissue. The previously unknown cytopathological effects of sarcocysts have been described and characterized. The changes observed within and outside the sarcocysts have been analyzed in terms of general biological processes: proliferation, differentiation, and programmed cell death.     

Ultrastructure of shizonts and merozoites of Sarcocystis falcatula in the lungs of budgerigars (Melopsittacus undulatus).

Transmission electron microscopy was used to study the ultrastructure of schizogony of Sarcocystis falcatula in the lungs of budgerigars (Melopsittacus undulatus). Schizogony occurred exclusively by endopolygeny within endothelial cells of pulmonary capillaries, venules, and small veins. Early schizonts were elongate with a large nucleus and nucleolus, surrounded by a pellicle consisting of a plasmalemma and an inner single membrane, and contained most of the organelles and inclusion bodies found in merozoites of Sarcocystis species. As development proceeded, schizonts increased in size and conformed to the shapes of the pulmonary blood vessels. As micronemes, dense granules, the conoid, and subpellicular microtubules disappeared, there was an increase in the size and number of mitochondria, Golgi complexes, and Golgi adjuncts (apicoplasts). As the nucleus elongated, there was a progressive increase in the number of spindles located at various intervals along the nuclear envelope. Eventually, 2 merozoites formed internally immediately above each spindle. During endopolygeny, a portion of the nucleus was incorporated into each merozoite bud along with 1 or 2 Golgi adjuncts, a Golgi complex, mitochondria, endoplasmic reticulum, and ribosomes. During merozoite formation, micronemes appeared in close association with the Golgi complex and gradually increased in number. The pellicle invaginated around the merozoites so they budded at the schizont surface leaving behind a small, central residual body. Dense granules appeared after merozoites were completely formed. Schizonts were 24 x 6.8 microm and contained 24-96 merozoites. Merozoites were 5.1 x 1.8 microm and were found free in the pulmonary air passages and pulmonary capillaries and within nearly all cells of the lung except red blood cells.     

Experimental transmission of Sarcocystis speeri Dubey and Lindsay, 1999 from the South American opossum (Didelphis albiventris) to the North American opossum (Didelphis virginiana)

Sarcocystis speeri Dubey and Lindsay, 1999 from the South American opossum Didelphis albiventris was successfully transmitted to the North American opossum Didelphis virginiana. Sporocysts from a naturally infected D. albiventris from Argentina were fed to 2 gamma-interferon knockout (KO) mice. The mice were killed 64 and 71 days after sporocyst feeding (DAF). Muscles containing sarcocysts from the KO mouse killed 71 DAF were fed to a captive D. virginiana; this opossum shed sporocysts 11 days after ingesting sarcocysts. Sporocysts from D. virginiana were fed to 9 KO mice and 4 budgerigars (Melopsittacus undulatus). Schizonts, sarcocysts, or both of S. speeri were found in tissues of all 7 KO mice killed 29-85 DAF; 2 mice died 39 and 48 DAF were not necropsied. Sarcocystis stages were not found in tissues of the 4 budgerigars fed S. speeri sporocysts and killed 35 DAE These results indicate that S. speeri is distinct from Sarcocystis falcatula and Sarcocystis neurona, and that S. speeri is present in both D. albiventris and D. virginiana.     

Pathogenesis of infection with Sarcocystis rauschorum (Apicomplexa) in experimentally infected varying lemmings (Dicrostonyx richardsoni)

This study describes the sequential formation of lesions associated with the endogenous development of Sarcocystis rauschorum (Apicomplexa: Sarcocystidae) in varying lemmings, Dicrostonyx richardsoni. Lethal doses of sporocysts (greater than 500) were orally administered to lemmings examined 1-6 days postinoculation (DPI) whereas sublethal doses were administered to lemmings examined subsequently. Transient necrosis and purulent inflammation, in association with precystic merogony, occurred in the liver by 4.5 DPI, peaked at 6 DPI and subsided beginning at 11 DPI with the liver returning to normal by 15 DPI. Cyst formation in skeletal and cardiac muscle was associated with purulent inflammation and sarcolemmal proliferation beginning at 9 DPI. These lesions persisted to 42 DPI. In addition, multifocal nonsuppurative meningoencephalitis was present in six of 11 infected lemmings examined between 11 and 15 DPI.     

Identification of Sarcocystis hominis-like (Protozoa: Sarcocystidae) cyst in water buffalo (Bubalus bubalis) based on 18S rRNA gene sequences

DNA templates were extracted from isolates of Sarcocystis hominis-like cysts collected from cattle and water buffalo, as well as from Sarcocystis fusiformis cysts and Sarcocystis suihominis cysts. The 18S rRNA genes were amplified using DNA from a single cyst as the templates. Approximately 1,367-1,440 bp sequences were obtained. The sequence difference in isolates of Sarcocystis hominis-like cysts from water buffaloes, and isolates of S. hominis cysts from cattle were very low, only about 0.1%, much lower than the lowest value (1.7%) among different species. Combined with their morphological structure, these sequence data indicate that the 4 isolates from cattle and water buffalo might be the same species, i.e., S. hominis, suggesting that both cattle and water buffalo may serve as the intermediate hosts for this parasite. Apparently, this is the first report using a single cyst to do such work and is a useful way to distinguish the Sarcocystis cyst in an intermediate host that may be simultaneously infected by several different Sarcocystis species.