Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures.
Neospora caninum, like Toxoplasma gondii, undergoes stage conversion in chronically infected animals, and forms tissue cysts which contain the slowly proliferating bradyzoite stage. These tissue cysts are delineated by a cyst wall, protect the parasite from physiological and immunological reactions on part of the host, and bradyzoites remain viable within an infected host for many years. However, unlike T. gondii, N. caninum bradyzoites have been difficult to obtain using in vitro culture techniques, and current protocols, based on those developed for T. gondii, have been shown to be not very efficient in promoting tachyzoite-to-bradyzoite stage conversion. We report here an alternative in vitro culture method to obtain stage conversion of N. caninum from the proliferative to the cystic stage by using the Nc-Liverpool isolate, murine epidermal keratinocytes as host cells, and continuous treatment of infected cultures with 70 microM sodium nitroprusside for up to 8 days. This treatment significantly reduced parasite proliferation as assessed by Neospora-specific quantitative real-time PCR. The expression of bradyzoite markers was analysed by immunofluorescence following 4 and 8 days of in vitro culture using antibodies directed against bradyzoite antigen 1, the mAbCC2, and the lectin Dolichos biflorus agglutinin. Expression of the tachyzoite-specific immunodominant antigen NcSAG1 and the tachyzoite antigen NcMIC1 was also assessed. Transmission electron microscopy revealed that the majority of parasitophorous vacuoles were in the process of forming a distinct cyst wall through accumulation of granular material at the periphery of the vacuole, and parasites exhibited the typical features of bradyzoites. These findings demonstrate the usefulness of this culture technique as a promising way to study tachyzoite-to-bradyzoite stage conversion in N. caninum in vitro.
Institute of Parasitology, Faculties of Veterinary Medicine and Medicine, University of Berne, Länggass-Strasse 122, CH-3012 Berne, Switzerland. vonlaufen@ipa.unibe.ch
Nathalie
N
Vonlaufen
Norbert
N
Müller
Nadine
N
Keller
Arunasalam
A
Naguleswaran
Wolfgang
W
Bohne
Milton M
MM
McAllister
Camilla
C
Björkman
Eliane
E
Müller
Reto
R
Caldelari
Andrew
A
Hemphill
D000818:Animals; D000953:Antigens, Protozoan / Q000032:analysis; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003560:Cysts / Q000737:chemistry / Q000648:ultrastructure; D000078404:Epidermal Cells; D004817:Epidermis / Q000033:anatomy & histology; D006790:Host-Parasite Interactions; D006801:Humans; D015603:Keratinocytes / Q000469:parasitology*; D008018:Life Cycle Stages; D051379:Mice; D018688:Neospora / Q000254:growth & development* / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology*; D009599:Nitroprusside / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014709:Vero Cells
D000953:Antigens, Protozoan; D009599:Nitroprusside; D009569:Nitric Oxide