title: In vitro cultivation of Hammondia heydorni: Generation of tachyzoites, stage conversion into bradyzoites, and evaluation of serologic cross-reaction with Neospora caninum.
abstract: Hammondia heydorni was in vitro isolated from oocysts shed by three dogs using a finite cell line from embryonal bovine heart (KH-R). The oocysts were purified and suspended in 2% potassium dichromate or 2% sulphuric acid for sporulation for 2-5 days at room temperature. The parasites were confirmed as H. heydorni by PCR using specific primers (JS4/JS5) and by negative reaction for Neospora caninum employing the primers Np6+/Np21+. H. heydorni sporulated oocysts (1 × 10(6)) from each dog were initially treated with sodium hypochlorite. For excystation of sporozoites, oocysts from one dog were lysed by ultrasound followed by incubation with 0.75% taurocholate. Excystation of sporozoites from the other two dogs was achieved by oocyst fragmentation with glass beads with no further chemical treatment. Tachyzoites were clearly seen in the cultures at three days post inoculation (dpi). Bradyzoite conversion and cyst formation were evaluated at different time points by using a polyclonal rabbit serum against a bradyzoite-specific antigen (anti-BAG1), and a rat monoclonal antibody (mAbCC2) against a cyst wall protein. Bradyzoites were firstly detected at 7 dpi. Between 18 and 21 dpi most of cultured parasites consisted of encysted bradyzoites. The H. heydorni cysts increased in size during cultivation and reached a length of up to 135 μm. The parasite was maintained in the bovine heart cells up to 4.5months. Sera from mice and sheep experimentally infected with H. heydorni oocysts reacted with H. heydorni by IFAT, but did not cross-react with N. caninum antigens using IFAT or immunoblot. These findings suggest that serological cross-reactivity between H. heydorni and N. caninum seems to be of minor importance.
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, Südufer 10, 17493 Greifswald-Insel Riems, Germany; Universidade Federal da Bahia, Escola de Medicina Veterinária e Zootecnia, Departamento de Anatomia, Patologia e Clínicas, Av. Adhemar de Barros, 500, Ondina, 40170-110 Salvador, Bahia, Brazil. Electronic address: pita@ufba.br.
authors.forename: L F P
authors.initials: LF
authors.lastname: Gondim
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Meyer
authors.affiliation: Chemisches und Veterinäruntersuchungsamt Westfalen, Zur Taubeneiche 10-12, 59821 Arnsberg, Germany; Department of Pathology, University of Veterinary Medicine Hannover, Foundation, Bünteweg 17, 30559 Hannover, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Peters
authors.affiliation: Universidade Federal da Bahia, Escola de Medicina Veterinária e Zootecnia, Departamento de Anatomia, Patologia e Clínicas, Av. Adhemar de Barros, 500, Ondina, 40170-110 Salvador, Bahia, Brazil.
authors.forename: M M
authors.initials: MM
authors.lastname: Rezende-Gondim
authors.affiliation: Vet Med Labor GmbH, Division of IDEXX Laboratories, D-71636 Ludwigsburg, Germany.
authors.forename: M G
authors.initials: MG
authors.lastname: Vrhovec
authors.affiliation: Vet Med Labor GmbH, Division of IDEXX Laboratories, D-71636 Ludwigsburg, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Pantchev
authors.affiliation: Institute of Parasitology, Justus Liebig University Giessen, Schubertstrasse 81, 35392 Giessen, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Bauer
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
authors.forename: F J
authors.initials: FJ
authors.lastname: Conraths
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Schares
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology*; D002417:Cattle; D002460:Cell Line; D003048:Coccidiosis / Q000276:immunology / Q000469:parasitology / Q000662:veterinary*; D003429:Cross Reactions; D016054:DNA, Protozoan / Q000235:genetics; D004283:Dog Diseases / Q000276:immunology* / Q000469:parasitology; D004285:Dogs; D005243:Feces / Q000469:parasitology; D051379:Mice; D018688:Neospora / Q000276:immunology*; D033661:Oocysts; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011817:Rabbits; D051381:Rats; D021745:Sarcocystidae / Q000235:genetics / Q000254:growth & development / Q000276:immunology* / Q000302:isolation & purification; D012756:Sheep
chemical_list: D000913:Antibodies, Protozoan; D016054:DNA, Protozoan
keywords: Cell culture; Cyst; Hammondia; Isolation; Neospora