title: Evaluation of four molecular methods to detect Leishmania infection in dogs.
abstract: BACKGROUND
  Canine leishmaniasis, a zoonotic disease caused by Leishmania infantum vectored by phlebotomine sand flies, is considered a relevant veterinary and public health problem in various countries, namely in the Mediterranean basin and Brazil, where dogs are considered the main reservoir hosts. Not only diseased dogs but also those subclinically infected play a relevant role in the transmission of L. infantum to vectors; therefore, early diagnosis is essential, under both a clinical and an epidemiological perspective. Molecular tools can be a more accurate and sensitive approach for diagnosis, with a wide range of protocols currently in use. The aim of the present report was to compare four PCR based protocols for the diagnosis of canine Leishmania infection in a cohort of dogs from the Douro region, Portugal.
  
  
  RESULTS
  A total of 229 bone marrow samples were collected from dogs living in the Douro region, an endemic region for leishmaniasis. Four PCR protocols were evaluated for Leishmania DNA detection in canine samples, three single (ITS1-PCR, MC-PCR and Uni21/Lmj4-PCR) and one nested (nested SSU rRNA-PCR). Two of the protocols were based on nuclear targets and the other two on kinetoplastid targets. The higher overall percentage of infected dogs was detected with the nested SSU rRNA-PCR (37.6%), which also was able to detect Leishmania DNA in a higher number of samples from apparently healthy dogs (25.3%). The ITS1-PCR presented the lowest level of Leishmania detection.
  
  
  CONCLUSIONS
  Nested SSU rRNA-PCR is an appropriate method to detect Leishmania infection in dogs. Accurate and early diagnosis in clinically suspect as well as apparently healthy dogs is essential, in order to treat and protect animals and public health and contribute to the control and awareness of the disease.
authors.affiliation: Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Rua da Junqueira 100, 1349-008, Lisbon, Portugal.|Present address: Institut für Zelluläre Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.
authors.forename: Andreia
authors.initials: A
authors.lastname: Albuquerque
authors.affiliation: Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Rua da Junqueira 100, 1349-008, Lisbon, Portugal.|Department of Biomedical Sciences and Medicine, Campus Gambelas, Universidade de Faro, Faro, Portugal.
authors.forename: Lenea
authors.initials: L
authors.lastname: Campino
authors.affiliation: Department of Veterinary Sciences, School of Agrarian and Veterinary Sciences, University of Trás-os-Montes e Alto Douro, UTAD, Quinta de Prados, 5000-801, Vila Real, Portugal. lcardoso@utad.pt.
authors.forename: Luís
authors.identifier: http://orcid.org/0000-0002-6145-7560
authors.initials: L
authors.lastname: Cardoso
authors.affiliation: Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Rua da Junqueira 100, 1349-008, Lisbon, Portugal.
authors.forename: Sofia
authors.initials: S
authors.lastname: Cortes
mesh_terms: D000818:Animals; D058345:Asymptomatic Infections; D001938:Brazil; D004283:Dog Diseases / Q000175:diagnosis* / Q000469:parasitology; D004285:Dogs; D007891:Leishmania / Q000235:genetics / Q000302:isolation & purification; D007896:Leishmaniasis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D011174:Portugal; D015047:Zoonoses
keywords: Canine leishmaniasis; Dogs; Leishmania; Molecular diagnosis; Nested SSU rRNA-PCR; Subclinical infection