title: The first isolation and molecular characterization of Toxoplasma gondii from horses in Serbia.
abstract: BACKGROUND
  Consumption of undercooked or insufficiently cured meat is a major risk factor for human infection with Toxoplasma gondii. Although horsemeat is typically consumed rare or undercooked, information on the risk of T. gondii from infected horse meat to humans is scarce. Here, we present the results of a study to determine the presence of T. gondii infection in slaughter horses in Serbia, and to attempt to isolate viable parasites.
  
  
  METHODS
  The study included horses from all regions of Serbia slaughtered at two abattoirs between June 2013 and June 2015. Blood sera were tested for the presence of specific IgG T. gondii antibodies by the modified agglutination test (MAT), and samples of trypsin-digested heart tissue were bioassayed in mice. Cyst-positive mouse brain homogenates were subjected to DNA extraction and T. gondii strains were genotyped using 15 microsatellite markers (MS).
  
  
  RESULTS
  A total of 105 slaughter horses were sampled. At the 1:6 cut-off 48.6% of the examined horses were seropositive, with the highest titre being 1:400. Viable parasites were isolated from two grade type mares; both parasite isolates (RS-Eq39 and RS-Eq40) were T. gondii type III, and both displayed an increased lethality for mice with successive passages. These are the first cases of isolation of T. gondii from horses in Serbia. When compared with a worldwide collection of 61 type III and type III-like strains, isolate RS-Eq39 showed a combination of MS lengths similar to a strain isolated from a duck in Iran, and isolate RS-Eq40 was identical in all markers to three strains isolated from a goat from Gabon, a sheep from France and a pig from Portugal. Interestingly, the source horses were one seronegative and one weakly seropositive.
  
  
  CONCLUSIONS
  The isolation of viable T. gondii parasites from slaughter horses points to horsemeat as a potential source of human infection, but the fact that viable parasites were isolated from horses with only a serological trace of T. gondii infection presents further evidence that serology may not be adequate to assess the risk of toxoplasmosis from horsemeat consumption. Presence of T. gondii type III in Serbia sheds more light into the potential origin of this archetypal lineage in Europe.
authors.affiliation: National Reference Laboratory for Toxoplasmosis, Centre of Excellence for Food- and Vector-Borne Zoonoses, Institute for Medical Research, University of Belgrade, Dr Subotića 4, 11129, Belgrade, Serbia.
authors.forename: Ivana
authors.initials: I
authors.lastname: Klun
authors.affiliation: National Reference Laboratory for Toxoplasmosis, Centre of Excellence for Food- and Vector-Borne Zoonoses, Institute for Medical Research, University of Belgrade, Dr Subotića 4, 11129, Belgrade, Serbia.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Uzelac
authors.affiliation: Centre National de Référence de la Toxoplasmose, Laboratoire de Parasitologie-Mycologie, CHU Maison Blanche, EA 3800 SFR CAP-SANTE, UFR Médecine Université de Reims Champagne-Ardenne, 45 rue Cognacq-Jay, 51092, Reims, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Villena
authors.affiliation: INSERM, UMR_S 1094, Neuroépidémiologie Tropicale, Université de Limoges, 2 rue du Docteur Marcland, 87025, Limoges, France.|Toxoplasma Biological Resource Center (BRC), Centre Hospitalier-Universitaire Dupuytren, 87042, Limoges, France.
authors.forename: Aurélien
authors.initials: A
authors.lastname: Mercier
authors.affiliation: National Reference Laboratory for Toxoplasmosis, Centre of Excellence for Food- and Vector-Borne Zoonoses, Institute for Medical Research, University of Belgrade, Dr Subotića 4, 11129, Belgrade, Serbia.
authors.forename: Branko
authors.initials: B
authors.lastname: Bobić
authors.affiliation: National Reference Laboratory for Toxoplasmosis, Centre of Excellence for Food- and Vector-Borne Zoonoses, Institute for Medical Research, University of Belgrade, Dr Subotića 4, 11129, Belgrade, Serbia.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Nikolić
authors.affiliation: National Reference Laboratory for Toxoplasmosis, Centre of Excellence for Food- and Vector-Borne Zoonoses, Institute for Medical Research, University of Belgrade, Dr Subotića 4, 11129, Belgrade, Serbia.
authors.forename: Irena
authors.initials: I
authors.lastname: Rajnpreht
authors.affiliation: National Institute for Public Health and the Environment (RIVM), Antonie van Leeuwenhoeklaan 9, 3520BA, Bilthoven, Netherlands.
authors.forename: Marieke
authors.initials: M
authors.lastname: Opsteegh
authors.affiliation: Centre National de Référence de la Toxoplasmose, Laboratoire de Parasitologie-Mycologie, CHU Maison Blanche, EA 3800 SFR CAP-SANTE, UFR Médecine Université de Reims Champagne-Ardenne, 45 rue Cognacq-Jay, 51092, Reims, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Aubert
authors.affiliation: Ecole Nationale Vétérinaire d'Alfort, UMR BIPAR, ANSES, INRA, Université Paris-Est, Laboratoire de santé animale de Maisons-Alfort, Maisons-Alfort, France.
authors.forename: Radu
authors.initials: R
authors.lastname: Blaga
authors.affiliation: National Institute for Public Health and the Environment (RIVM), Antonie van Leeuwenhoeklaan 9, 3520BA, Bilthoven, Netherlands.
authors.forename: Joke
authors.initials: J
authors.lastname: van der Giessen
authors.affiliation: National Reference Laboratory for Toxoplasmosis, Centre of Excellence for Food- and Vector-Borne Zoonoses, Institute for Medical Research, University of Belgrade, Dr Subotića 4, 11129, Belgrade, Serbia. olgicadj@imi.bg.ac.rs.
authors.forename: Olgica
authors.initials: O
authors.lastname: Djurković-Djaković
mesh_terms: D000003:Abattoirs; D000372:Agglutination Tests; D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D001681:Biological Assay; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D005838:Genotype; D006734:Horse Diseases / Q000175:diagnosis* / Q000469:parasitology*; D006736:Horses / Q000469:parasitology*; D007074:Immunoglobulin G / Q000097:blood; D051379:Mice; D018895:Microsatellite Repeats; D055771:Serbia; D014122:Toxoplasma / Q000302:isolation & purification*; D014124:Toxoplasmosis, Animal / Q000175:diagnosis* / Q000469:parasitology
chemical_list: D000913:Antibodies, Protozoan; D016054:DNA, Protozoan; D007074:Immunoglobulin G
keywords: Genotyping; Horse; Isolation; Microsatellite markers; Prevalence; Serbia; Strain; Toxoplasma gondii; Type III