Heinz Mehlhorn

The sarcosporidia (Protozoa, Sporozoa): life cycle and fine structure.

Publisher Summary Sarcosporidia have adapted their life cycle to the “predator–prey” relationship existing between their hosts. Some carnivores may be final hosts of several sarcocystis species and omnivores may be involved as intermediate as well as final hosts in the life cycles of different sarcosporidian species. Sporocysts or sporulated oocysts shed in the faeces of the final host must always be ingested by the intermediate host. The oocysts and sporocysts of the sarcosporidian life cycles are morphologically identical with those of the genus isospora, and thus, sarcocystis has been classified as isospora species. However, there are four differences: (1) the Sarcosporidia have an obligatory two-host cycle, (2) schizogony has two phases: an extraintestinal multiplication followed by cyst formation mostly within the muscles of the intermediate host, (3) in Sarcosporidia, no schizogonic multiplication occurs in the gut wall of the final host or in cell culture, and (4) in Sarcosporidia, oocysts are excreted fully sporulated; single sporocysts are often observed in the faeces of the final host because of the rupture of the extremely fine oocyst wall.

Ultrastructural study of characteristic organelles (paired organelles, micronemes, micropores) of sporozoa and related organisms

SummaryBy means of electron microscopy a study has been made of different developmental stages of Eimeria callospermophili, E. falciformis, Toxoplasma gondii, Frenkelia spec. (= M-organism), Babesia bigemina, and B. ovis. Major emphasis was given to the analysis of some characteristic organelles of the motile stages of the sporozoans. These organelles were the paired organelle, the micronemes and the micropores.

Fine structure of macrogametes and oocysts of Coccidia and related organisms.

SummaryThe fine structure of the macrogametes of coccidia was investigated, described and diagrammatically depicted. A comparative analysis was made of representative species belonging to various genera. The following species were investigated: Eucoccidium dinophili, Aggregata eberthi, Eimeria perforans, E. stiedae, E. falciformis, E. bovis, E. auburnensis, E. tenella, E. maxima, Toxoplasma gondii, and Klossia helicina. The macrogametes of most of these Eimeria species are relatively uniform in their fine structure, but E. falciformis shows some differences and the macrogametes of T. gondii are also different in some respects. In the macrogametes of E. dinophili, A. eberthi, and K. helicina are found a number of special structures not seen in the others.ZusammenfassungDie Feinstruktur der Makrogameten von Coccidien und verwandten Gruppen wurde untersucht, beschrieben und schematisch dargestellt. Charakteristische Organelle folgender Arten: Eucoccidium dinophili, Aggregata eberthi, Eimeria perforans, Eimeria stiedae, E. falciformis, E. bovis, E. auburnensis, E. tenella, E. maxima, Toxoplasma gondii und Klossia helicina dienten als Ausgangsbasis zu einer vergleichenden Strukturanalyse. Die Makrogameten der Eimeria-Arten erwiesen sich in ihrem Feinbau als relativ einheitlich. Einige bedeutsame Unterschiede ergaben sich allerdings bei den weiblichen Gameten von E. falciformis und T. gondii im Vergleich zu den Verhältnissen bei den untersuchten Eimeria-Arten. Die Makrogameten von E. dinophili, A. eberthi und K. helicina enthielten spezielle Einschlüsse, die bei den Eimeria-Arten nicht in Erscheinung traten. Trotz aller Unterschiede liegt jedoch den hier untersuchten Makrogameten ein gemeinsamer Bauplan zu Grunde.

The fine structure of the conoid of sporozoa and related organisms

SummaryThe merozoites ofEimeria callospermophili andE. stiedae as well as the zoites ofToxoplasma gondii and the M-organism and the erythrocytic merozoites ofBabesia bigemina were studied by means of electron microscopy. The fine structure of the apical pole was analysed, and compared with the results of other studies of sporozoa and related organisms. The following conclusions were made:1.The organelle called conoid, which is defined as a hollow cone-like structure, shows identical components in all parasites studied.2.Babesians, theilerians and plasmodiums have no such organelle; they have, however, other reinforcements of the apical pole.3.The terms “polar ring” and “preconoidal ring” were proposed.4.The fine structure of the intraerythrocytic forms ofBabesia bigemina was described.5.The similarities of the fine structure led to a discussion of taxonomic questions.

Electron microscope studies of microgametogenesis in coccidia and related groups

SummaryThe fine structure of the microgamonts and microgametes of coccidia and related groups was investigated and is herein described and diagrammatically depicted. Particular attention is given to the developmental processes during the differentiation of the microgametes. The species included in the study are: Eimeria perforans, E. maxima, E. tenella, E. auburnensis, E. falciformis, and Toxoplasma gondii. A comparative analysis of the developmental processes as well as the fine structure of the microgametes of these species and those of other Sporozoa is presented.

Scanning and transmission electron microscope studies on the efficacy of praziquantel on Hymenolepis nana (Cestoda) in vitro.

SummaryThe dwarf tapeworm,Hymenolepis nana, was studied by means of scanning and transmission electron microscopy after in vitro exposure to 1, 10, and 100 μg/ml of the anthelmintic praziquantel (Droncit®) for 5, 15, 30, and 60 min. The resulting vacuolization of the tegument was exclusively confined to the neck region of the tapeworms and was already observed after treatment for 5 min with 1 μg/ml.This vacuolization finally led to the disruption of the syncytial layer in the apical region of the tegument. The tegumental microtriches and the surface coat remained unaffected. Proglottids of the middle or posterior regions of the worms never showed destruction.

Identifikation von Merozoiten der vier cystenbildenden Coccidien (Sarcocystis, Toxoplasma, Besnoitia, Frenkelia) auf Grund feinstruktureller Kriterien

SummaryCyst stages of the speciesSarcocystis tenella, Frenkelia spec.,Besnoitia jellisoni, andToxoplasma gondii are compared on the electron microscope. Ultrastructural differences between these 4 species are found sufficient to serve as a basis for their exact taxonomic identification. Merozoites, which closely resemble merozoites of other coccidia in regard to shape and ultrastructure, are found in cysts of all 4 species. In addition to them, cysts ofS. tenella andFrenkelia spec. contain metrocytes which are located at the periphery. Metrocytes are ovoid and have deep infoldings that give them the appearance of separated cells. The typical organelles of merozoites such as conoids and micropores are always found in metrocytes. Usually, more than 2 micropores are present. In both species, the nucleus shows a spherical area of osmiophilic granules in addition to the nucleoli. Besides metrocytes,Frenkelia spec. has so-called intermediate cells of bipolar organization that have micronemes at both ends. InB. jellisoni andT. gondii only merozoites were found inside the cysts. Those ofB. jellisoni differ distinctly from merozoites of any other species in having membrane-bound, electronlight spindle-shaped bodies with an electrondense core (=enigmatic bodies). Their function is not yet understood. Exclusive criteria for the different species are:S. tenella has metrocytes and merozoites which are twice as large as those of any other genus. Micronemes in merozoites are abundant (about 400), and up to 11 rhoptries can be found.Frenkelia spec. has intermediate cells with bipolar organization and merozoites with a distinct posterior cone. Micronemes in merozoites are limited to 50–70, and 5–8 rhoptries are present.B. jellisoni merozoites have 80–100 micronemes, 3–5 rhoptries, and 20–30 enigmatic bodies.T. gondii merozoites are the smallest of all species examined, and the number of micronemes is rather small, too, and does not exceed 50. All stages of the 4 species multiply by endodyogeny. Except of metrocytes and intermediate cells ofFrenkelia spec., 22 subpellicular microtubules are found in all stages. The meanings of these findings in regard to their species- or genus-specific significance and several implications on the functional morphology of some organelles are given in the discussion.ZusammenfassungDie Cystenstadien vonSarcocystis tenella, Frenkelia spec.,Besnoitia jellisoni undToxoplasma gondii wurden elektronenoptisch untersucht und schematisch in Relation zu ihrer Größe dargestellt. Mikromorphologische Unterschiede erlauben eine eindeutige Identifikation dieser Coccidien. In allen Cysten sind Merozoiten enthalten, stets von bananenförmiger Gestalt. Darüber hinaus weisen die Cysten vonS. tenella undFrenkelia spec. noch ovoide Metrocyten mit tiefen Einfaltungen auf. Sie liegen an der Peripherie der Cyste, dicht unterhalb der begrenzenden Primärhülle. Die Metrocyten beider Arten zeigen typische Merozoitenorganelle und sind außerdem noch durch einen walzenförmigen bzw. kugeligen Bereich osmiophiler Grana im Nukleus ausgezeichnet. BeiFrenkelia spec. wird noch ein dritter Zelltypus ausgebildet, der als „intermediäre Zelle” bezeichnet wird und bipolar aufgebaut ist. Als wichtige Kriterien der untersuchten Arten wurden folgende Merkmale herangezogen:S. tenella besitzt Metrocyten und Merozoiten, die nahezu doppelt so groß sind wie die der übrigen Arten. Die Mikronemen treten in außergewöhnlich großer Anzahl auf (ca. 400), desgleichen die Rhoptrien (ca. 11).Frenkelia spec. bildet bipolare intermediäre Zellen aus. Die Merozoiten zeigen einen besonderen hinteren Conus. Die Anzahl der Mikronemen ist auf etwa 50–70 beschränkt; etwa 5–8 Rhoptrien sind vorhanden.B. jellisoni-Merozoiten sind durch 20–30 sog. enigmatische Körper charakterisiert; etwa 80–100 Mikronemen und ca. 3–5 Rhoptrien sind vorhanden.T. gondii weist die kleinsten Merozoiten aller untersuchten Arten auf. Die Anzahl der Mikronemen übersteigt 50 kaum. Alle hier untersuchten Stadien vermehren sich durch Endodyogenie. Mit Ausnahme der Metrocyten und intermediären Zellen vonFrenkelia spec. konnten bei allen Formen 22 subpellikuläre Mikrotubuli nachgewiesen werden. Die feinstrukturellen Ergebnisse wurden abschließend auf ihren Wert als systematische Kriterien geprüft.

Electron microscopy of stages of Isospora felis of the cat in the mesenteric lymph node of the mouse

SummaryStages of Isospora felis of the cat in the mesenteric lymph node of the mouse 25 days after oral inoculation with oocysts, have been described at the ultrastructural level. The organisms occurred singly within parasitophorous vacuoles in host cell cytoplasm and were sporozoite-like, having a large crystalloid body up to 5.5 μm in length posterior to the nucleus. The size and appearance of the parasitophorous vacuole varied. Some vacuoles contained numerous, small, electron dense granules about 30 nm in diameter. Because of the aggregation of granules and their arrangement within the parasitophorous vacuole, the impression was sometimes gained by light microscopy that parasites were surrounded by a sheath or cyst wall. However, a cyst wall was not present. In host cells, spherical, membrane-bound bodies with a homogeneous, electron dense core and a maximum diameter of 0.25 μm were filed along the limiting membrane of the parasitophorous vacuole.These extra-intestinal parasites were considered to be waiting stages, with a biological function similar to that of the tissue cyst stage of other genera of isosporan coccidia.

Licht- und elektronenmikroskopische Untersuchungen an Entwicklungsstadien von Sarcocystis tenella aus der Darmwand der Hauskatze

SummaryIn several experiments young cats were infected with Sarcocystis tenella cysts from the esophagus of sheep and the stages in the cat intestine were examined by means of light and electron microscopy. Eleven to twelve days after infection untreated cats excreted a very small number of fully sporulated sporocysts, which measured 12.4×8.7 μm (14.0–10.5 μm×9.7–8.0 μm). Oocysts were not observed here. Sporocysts could be found even 14 days after the beginning of the excretion. The sporocysts, which had no stieda-body, contained 4 sporozoites and a residual body, thus corresponding to the Isospora-type. In a further experiment a cat was treated for 4 weeks with a corticosteroid (2.5 mg daily, Prednison) before infection. This cat also was fed approximately 100 large cysts of S. tenella, which had been carefully extracted from the esophagus. On the 9th day p.i. the cat was killed and the intestine was examined. The latter showed macroscopically no hemorrhage, but contained a considerable number of very thin-walled oocysts without a micropyle. A remarkable accumulation of oocysts was seen in the area of the posterior small intestine. Almost 90% of the oocysts already contained two sporocysts, in which large, dark or light granules were found like those in the few unsporulated oocysts. The sporulated oocysts measured about 18.0–14.8 μm×14.0–10.5 μm. The sporocysts in this experiment were, with 10.9×6.8 μm (12.4–9.8 μm×8.0–6.2 μm), slightly smaller than those of the earlier experiments. Light microscopical investigations of cross sections of the intestine showed that the parasites were always situated immediately under the epithelial cells in the villi of the intestine. They were also always seen in large parasitophorous vacuoles. The oocysts were found relatively often to form long rows, thus indicating that the parasitized host cells were possibly sunken epithelial cells. Electron microscopical studies showed that the unsporulated oocysts were surrounded by a single, very electron-pale wall, which had at this time a diameter of about 0.25 μm and which shrank during sporulation to 0.1 μm. The cytoplasm of the oocyst was limited by a typical unit membrane and contained large reserve granules (polysaccharides, lipids) with a diameter of about 1.5 μm. In addition small spherical elements (30–40 nm) were found, which formed a cristalline pattern. 0.4 μm sized osmiophilic bodies are probably responsible for the formation of the later sporocyst walls. The large nucleus was often situated at the periphery of the spherical unsporulated oocyst, which also possessed numerous tubular mitochondria. Occasionally micropores were still present as relics in the cytoplasmic membrane. The sporocysts were limited by a 50–60 nm thick osmiophilic layer and contained the same elements as the unsporulated oocyst. Most of the ovoid sporocysts had two u-shaped nuclei at the poles. Sporulated as well as unsporulated oocysts were always situated in a large electron-pale parasitophorous vacuole. At this time the host cell consisted only of two remaining membranes: the outer cytoplasmic membrane and the limiting membrane of the parasitophorous vacuole. Therefore it was not possible to decide which cell-type had been parasitized. The oocyst wall proved very fragile in the sporulated stage, so that often single free sporocysts were observed. The free sporocysts had, in addition to their sporocyst wall, a thin granular outer layer, which may be considered a relic of the oocyst wall. From the ultrastructure of the oocyst wall and the advanced state of sporulation (in the tissue) on the 9th day p.i., it becomes clear why, in the transmission experiments, only fully sporulated sporocysts were found in the feces on the 11th–12th day. Finally our results were compared with the other Isospora species of cat. Apparently the large form of Isospora bigemina seems responsible for the relatively large cysts in the muscles of sheep, described formerly as S. tenella.ZusammenfassungIn mehreren Versuchen wurden junge Katzen mit Cysten von Sarcocystis tenella aus der Oesophagus-Muskulatur von Schafen infiziert und die Stadien im Katzendarm licht- und clektronenoptisch untersucht. Bei den Übertragungsversuchen ergab sich, daß vom 11.–12. Tag p.i. Sporocysten ausgeschieden wurden, die in ihrem Innern 4 Sporozoiten und einen großen Restkörper enthielten. Ein Stieda-Körper fehlte diesen etwa 12,4 × 8,7 μm großen Sporocysten, die für lange Zeit in sehr geringer Anzahl abgesetzt wurden. Bei einer mit einem Corticosteroid (Prednison) vorbehandelten Katze konnte am 9. Tag p.i. im Darminnern eine starke Anhäufung von Parasiten beobachtet werden. Hier fanden sich zahlreiche dünnwandige Oocysten, die meist schon zwei Sporocysten im Innern aufwiesen. Die Oocysten lagen oft in langen Reihen unmittelbar unterhalb der Epithelzellen der Darmvilli innerhalb von großen, lichten parasitophoren Vakuolen. Der Feinbau der unsporulierten Oocysten und der Sporocysten wurde beschrieben und mit den Verhältnissen bei Eimeria-Arten verglichen. Aus der Ultrastruktur der Oocystenhülle und dem Sporulationsstand am 9. Tag p.i. geht hervor, warum bei anderen und unseren Übertragungsversuchen stets völlig sporulierte Sporocysten am 11.–12. p.i. im Kot angetroffen wurden. Im Vergleich mit den anderen Isospora-Arten der Katze zeigte sich, daß offenbar die große Form von I. bigemina für die ziemlich großen Cysten in der Muskulatur von Schafen verantwortlich ist.

Treatment of fish parasites. 3. Effects of levamisole HCl, metrifonate, fenbendazole, mebendazole, and ivermectin on Anguillicola crassus (nematodes) pathogenic in the air bladder of eels.

The effects of five nematocidal drugs (levamisole HCl, metrifonate, fenbendazole, mebendazole, and ivermectin) on the nematode Anguillicola crassus, pathogenic in eels and recently introduced in Europe, were tested under in vivo conditions. The resulting tissue alterations were studied by means of light and electron microscopy. It was found that levamisole and metrifonate were most effective in a freshwater bath (1 mg/l) for 24 h. The 50% lethal dose of levamisole was 250 mg/l per 24 hours, whereas that of metrifonate was only 10 mg/l per 2 hours. Morphological studies of the nematodes showed that there was no drug-specific reaction. In general, the hypodermis, the cytoplasmic portions of the muscle cells, and the intestinal wall were most intensively damaged, leading to an irreversible vacuolation and finally to death.