Ultrastructure of the asexual multiplication of Besnoitia besnoiti (MAROTEL, 1912) in Vero‐ and CRFK‐cell cultures

Abstract

Summary Besnoitia besnoiti merozoites obtained from cysts in naturally infected bovines and passaged in Vero‐cells were multiplied in cultures of Vero‐ and CRFK‐monolayers. Their development was studied for 7 days p. i. by light and electron microscopy. As early as 24 h p. i. intracellularly situated single parasites were found; 48 h p. i. up to 10 merozoites of Besnoitia were seen in a single parasitophorous vacuole. Irregularly arranged clusters of up to 32 zoites on the 3rd day p. i. changed into regularly formed rosettes of Besnoitia. After the 4th day p. i. the extracellular forms predominated. During asexual multiplication of the parasites plaque formations could be observed 4—5 days p. i. as a cytopathogenic effect, followed 6—7 days p. i. by complete destruction of the monolayers. The predominant type of multiplication with Besnoitia is endodyogeny, which starts by the spherical mother cells developing two merozoite‐anlagen. Forming the daughter cell pellicle, the nucleus of the mother cell shrinks to a large extent and finally divides, the thicker posterior merozoite pole being left open. The young daughter cells remain connected with this pole by a common residual body until the final division takes place. A special form of endodyogeny is the rosette‐formation resulting from several endodyogenies without any separation of the young merozoites from the mother cell. The not yet fully differentiated merozoites of a rosette situated in a parasitophorous vacuole thus induce a mass multiplication of finally well‐developed merozoites of Besnoitia besnoiti. Zusammenfassung Die Ultrastruktur der asexuellen Vermehrung von Besnoitia besnoiti (MAROTEL, 1912) in Vero‐ und CRFK‐Zellkulturen Aus Zysten natürlich infizierter Rinder gewonnene, in Vero‐Zellen passagierte Besnoitia besnoiti‐Merozoiten wurden in Vero‐ und CRFK‐Zellkulturen vermehrt. Ihre Entwicklung konnte 7 Tage lang licht‐ und elektronenmikroskopisch untersucht werden. 24 Std. p. i. traten vereinzelt intrazellulär liegende Parasiten, 48 Std. p.i. bis zu 10 Besnoitia‐Merozoiten in einer gemeinsamen parasitophoren Vakuole auf. Bis zu 32 unregelmäßig zu Haufen angeordnete Zoiten am 3.Tg. p.i. wechselten mit regelmäßig geformten Besnoitia‐Rosetten ab. Nach dem 4.Tg. p.i. traten überwiegend extrazelluläre Parasiten auf. Als zytopathischer Effekt konnte 4—5 Tg. p. i. Plaquebildung, 6—7 Tg. p. i. komplette Zellzerstörung beobachtet werden. Die Endodyo genie stellt den Haupttyp der ungeschlechtlichen Vermehrung von Besnoitia dar, wobei in jeder Mutterzelle zwei Merozoitenanlagen gebildet werden. Von hier aus entwickelt sich die Tochterzellpellikula und gleichzeitig vergrößert und teilt sich der Mutterzellkern. Die jungen Tochterzellen bleiben mit ihrem offenen, verdickten hinteren Pol mit einem gemeinsamen Restkörper bis zur endgültigen Teilung verbunden. Eine besondere Form der Endodyogenie ist die Rosettenbildung, die eine Folge von mehreren Endodyogenien ohne Abtrennung der Merozoiten von der Mutterzelle darstellt. Die noch nicht ausdifferenzierten, als Roset...

Ultrastructure and pathology of Besnoitia caprae in the naturally infected goats of Kerman, East of Iran

“…Heydorn et al (1984) described the histological and ultrastructural appearance of Besnoitia cysts from the eyelid of goats in Kenya and showed that B. caprae bradyzoites had typical apicomplexa features, namely a three-layered pellicle, conoid, rhoptries, microtubules, micronemes, and polar ring. Similar features were reported for the related organisms, B. jellisoni (Sheffield 1966(Sheffield , 1968, B. besnoiti (Neuman 1974;Gobel et al 1985;Shkap et al 1988;Njenga et al 1995), and Toxoplasma gondii (Ogino and Yoneda 1966;Sheffield and Melton 1968;Aikawa et al 1977).…”

First in vitro isolation of Besnoitia besnoiti from chronically infected cattle in Germany

“…Interestingly, B. besnoiti from other sources were able to replicate fast in Vero cells, irrespective of the stage or source used for inoculation, i.e. cystozoites from cattle from South Africa (Gö bel et al, 1985) or tachyzoites isolated from gerbils previously infected with cystozoites from naturally infected cattle from Israel (Shkap et al, 1987). It is not yet clear whether B. besnoiti from different parts of the world exhibit different characteristics regarding in vitro cultivation.…”

“…This cell line was used for the isolation and long-term cultivation of a B. besnoiti isolate from Spain (BbSpain1) (Ferná ndez-García et al, in press). In a few publications various cell lines were compared for in vitro isolation and permanent cultivation of B. besnoiti (Neuman, 1974;Gö bel et al, 1985;Shkap et al, 1987). In our study, we employed cell lines (Vero, BHK 21 , MARC-145) which had successfully been used for the isolation or the long-term cultivation of B. besnoiti by other working groups.…”

“…Such a characterization can only be achieved if many isolates from different countries become available for analysis. There are reports on the in vitro isolation of B. besnoiti in cell cultures, but only few permanently growing isolates Q2 are existing worldwide (Bigalke et al, 1967;Bigalke, 1968;Neuman, 1974;Gö bel et al, 1985;Shkap et al, 1987;Cortes et al, 2006;Ferná ndez-García et al, in press). For isolation purposes, a variety of cell lines have been tested.…”

“…For isolation purposes, a variety of cell lines have been tested. Vero cells are often used for the in vitro isolation and the long-term cultivation of B. besnoiti (Bigalke et al, 1974;Neuman, 1974;Gö bel et al, 1985;Shkap et al, 1987;Cortes et al, 2006). In the case reported here, however, the isolation and long-term cultivation of B. besnoiti from a total number of 13 tissue samples from eight cattle and from two of three peritoneal washings of infected GKO mice failed in Vero cells in all but one case, although numerous viable cystozoites (Table 2) or tachyzoites were visible in many inocula.…”