title: First report of Anaplasma platys infection in red foxes (Vulpes vulpes) and molecular detection of Ehrlichia canis and Leishmania infantum in foxes from Portugal.
abstract: BACKGROUND
  The bacteria Anaplasma platys and Ehrlichia canis and the protozoan Leishmania infantum are vector-borne agents that cause canine vector-borne diseases, some of which are zoonotic. The present survey investigated the prevalence of Anaplasma, Ehrlichia and Leishmania in red foxes (Vulpes vulpes) from Portugal by molecular analysis, in order to evaluate the epidemiological role of these canids as reservoirs of infection.
  
  
  METHODS
  Blood and/or bone marrow samples were collected from 78 red foxes obtained in eight districts of northern, central and southern Portugal. Real-time polymerase chain reactions (PCR) amplified a 123 bp fragment of the 16S rRNA gene of Anaplasma spp. and Ehrlichia spp. and a 265 bp fragment of the L. infantum internal transcribed spacer one (ITS1) region of the rRNA operon evaluated by PCR-high resolution melt analysis (PCR-HRM), with sequencing of the DNA products. A phylogenetic analysis was carried out to compare these to other sequences from Anaplasma spp. and Ehrlichia spp. deposited in GenBank.
  
  
  RESULTS
  A. platys was detected in 10 (14.5%) and E. canis in two (2.9%) out of 69 foxes; and L. infantum was detected in one (1.3%) of the 78 foxes. The prevalence of A. platys was significantly different from the prevalence of E. canis (p=0.016) and from that of L. infantum (p=0.002). No co-infections were found in any one of the 78 foxes. No statistically significant differences were found between the type of sample (blood and bone marrow), geographic regions (north/centre and south), age (<2 years and ≥2 years) and gender for any one of the agents.
  
  
  CONCLUSIONS
  This is the first known report of A. platys in red foxes worldwide, as well as the first molecular evidence of E. canis in foxes from Portugal. The moderate prevalence of A. platys suggests that red foxes may play a role in the epidemiology of infection with this bacterium and serve as a reservoir for domestic dogs.
authors.affiliation: Department of Veterinary Sciences, School of Agrarian and Veterinary Sciences, Universidade de Trás-os-Montes e Alto Douro (UTAD), Vila Real, Portugal. lcardoso@utad.pt.
authors.forename: Luís
authors.initials: L
authors.lastname: Cardoso
authors.affiliation: Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, Rehovot, Israel. matangilaad@gmail.com.
authors.forename: Matan
authors.initials: M
authors.lastname: Gilad
authors.affiliation: Victor Caeiro Laboratory of Parasitology, Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM), University of Évora, Évora, Portugal. heldercortes@gmail.com.
authors.forename: Helder C E
authors.initials: HC
authors.lastname: Cortes
authors.affiliation: Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, Rehovot, Israel. yaarit.biala@mail.huji.ac.il.
authors.forename: Yaarit
authors.initials: Y
authors.lastname: Nachum-Biala
authors.affiliation: Department of Veterinary Sciences, School of Agrarian and Veterinary Sciences, Universidade de Trás-os-Montes e Alto Douro (UTAD), Vila Real, Portugal. aplopes@utad.pt.|Animal and Veterinary Research Centre, UTAD, Vila Real, Portugal. aplopes@utad.pt.
authors.forename: Ana Patrícia
authors.initials: AP
authors.lastname: Lopes
authors.affiliation: Victor Caeiro Laboratory of Parasitology, Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM), University of Évora, Évora, Portugal. mjoaovv@uevora.pt.
authors.forename: Maria João
authors.initials: MJ
authors.lastname: Vila-Viçosa
authors.affiliation: Victor Caeiro Laboratory of Parasitology, Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM), University of Évora, Évora, Portugal. maggiesimo@gmail.com.
authors.forename: Margarida
authors.initials: M
authors.lastname: Simões
authors.affiliation: Department of Veterinary Sciences, School of Agrarian and Veterinary Sciences, Universidade de Trás-os-Montes e Alto Douro (UTAD), Vila Real, Portugal. pavelar@utad.pt.
authors.forename: Paula A
authors.initials: PA
authors.lastname: Rodrigues
authors.affiliation: Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, Rehovot, Israel. gad.baneth@mail.huji.ac.il.
authors.forename: Gad
authors.initials: G
authors.lastname: Baneth
mesh_terms: D000709:Anaplasma / Q000235:genetics / Q000302:isolation & purification*; D000711:Anaplasmataceae Infections / Q000453:epidemiology / Q000469:parasitology / Q000662:veterinary*; D000712:Anaplasmosis / Q000453:epidemiology / Q000469:parasitology; D000818:Animals; D001483:Base Sequence; D016054:DNA, Protozoan / Q000235:genetics; D041061:Ehrlichia canis / Q000235:genetics / Q000302:isolation & purification*; D016873:Ehrlichiosis / Q000453:epidemiology / Q000469:parasitology / Q000662:veterinary; D005589:Foxes / Q000469:parasitology*; D018314:Leishmania infantum / Q000235:genetics / Q000302:isolation & purification*; D007898:Leishmaniasis, Visceral / Q000453:epidemiology / Q000469:parasitology / Q000662:veterinary*; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011174:Portugal / Q000453:epidemiology; D012336:RNA, Ribosomal, 16S / Q000235:genetics
chemical_list: D016054:DNA, Protozoan; D012336:RNA, Ribosomal, 16S