title: Theileria annae (syn. Babesia microti-like) infection in dogs in NW Spain detected using direct and indirect diagnostic techniques: clinical report of 75 cases.
abstract: BACKGROUND
  In north-western Spain, piroplamosis caused by Theileria annae is now recognized as a serious problem because veterinarians, despite being aware of the clinical signs of piroplasmosis, lack the necessary information on its epidemiology or specific diagnostic tools for its management. This, along with the fact that T. annae infection is also refractory to current piroplamosis treatments, prompted this study designed to assess the clinical presentation and diagnosis of this largely unknown parasitic disease in dogs.
  
  
  METHODS
  One hundred and twenty dogs in NW Spain suspected clinically of having piroplasmosis were examined and piroplasm species detected by light microscopy (LM) observation of Giemsa-stained blood smears, immunofluorescent antibody test (IFAT), and PCR plus sequencing.
  
  
  RESULTS
  Seventy five of the sick dogs were confirmed to be infected with T. annae by PCR (designated "true infection cases"). Intraerythrocytic ring-shaped bodies morphologically compatible with small piroplasms were observed by LM in 59 (57 true infections) of the 120 blood samples. Anti-Babesia antibodies were detected by IFAT in 59 of the 120 sera (55 of which were "true infections"). Using PCR as the reference method, moderate agreement was observed between positive LM vs PCR and IFAT vs PCR results (kappa values: 0.6680 and 0.6017, respectively). Microscopy examination and IFAT were moderately sensitive in detecting the pathogen (76% and 73.3%, respectively). In the 75 cases of "true infection", the most common clinical signs observed were pale mucous membranes, anorexia and apathy. Blood cell counts consistently revealed severe regenerative anaemia and thrombocytopenia in dogs with piroplasmosis due to T. annae. Young dogs (≤3 year) (p = 0.0001) were more susceptible to the disease.
  
  
  CONCLUSIONS
  Microscopy showed moderate diagnostic sensitivity for acute T. annae infection while IFAT-determined antibody titres were low (1/64 to 1/128). The infecting species should be therefore confirmed by molecular tests. Our results suggest that the disease affects dogs in regions of Spain bordering the endemic Galicia area where this piroplasm has not been previously reported (Asturias, northern Spain). Further epidemiological surveys based on serological and molecular methods are required to establish the current geographical range of T. annae infection.
authors.affiliation: Department of Animal Health, Veterinary Faculty, Universidad Complutense de Madrid, Madrid, Spain. gmiro@ucm.es.
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Miró
authors.affiliation: Department of Animal Health, Veterinary Faculty, Universidad Complutense de Madrid, Madrid, Spain. rocichec@ucm.es.
authors.forename: Rocío
authors.initials: R
authors.lastname: Checa
authors.affiliation: Vector- and Water-Borne Pathogen Research Group, School of Veterinary & Life Sciences, Murdoch University, Murdoch, WA, Australia. a.paparini@murdoch.edu.au.
authors.forename: Andrea
authors.initials: A
authors.lastname: Paparini
authors.affiliation: Department of Animal Health, Veterinary Faculty, Universidad Complutense de Madrid, Madrid, Spain. theileria.annae@gmail.com.
authors.forename: Nieves
authors.initials: N
authors.lastname: Ortega
authors.affiliation: Xarope Veterinary Centre, Laracha, Coruña, Spain. jluisgfraga@yahoo.es.
authors.forename: José Luís
authors.initials: JL
authors.lastname: González-Fraga
authors.affiliation: Vector- and Water-Borne Pathogen Research Group, School of Veterinary & Life Sciences, Murdoch University, Murdoch, WA, Australia. A.Gofton@murdoch.edu.au.
authors.forename: Alex
authors.initials: A
authors.lastname: Gofton
authors.affiliation: Gran Vía Veterinary Centre, Carballo, Coruña, Spain. adribarhus@gmail.com.
authors.forename: Adrián
authors.initials: A
authors.lastname: Bartolomé
authors.affiliation: Department of Animal Health, Veterinary Faculty, Universidad Complutense de Madrid, Madrid, Spain. amontoya@ucm.es.
authors.forename: Ana
authors.initials: A
authors.lastname: Montoya
authors.affiliation: Department of Animal Health, Veterinary Faculty, Universidad Complutense de Madrid, Madrid, Spain. r.galvez@ucm.es.
authors.forename: Rosa
authors.initials: R
authors.lastname: Gálvez
authors.affiliation: Hospital Veterinario Nacho Menes, Gijón, Asturias, Spain. pedropablovet@yahoo.es.
authors.forename: Pedro Pablo
authors.initials: PP
authors.lastname: Mayo
authors.affiliation: Vector- and Water-Borne Pathogen Research Group, School of Veterinary & Life Sciences, Murdoch University, Murdoch, WA, Australia. P.Irwin@murdoch.edu.au.
authors.forename: Peter
authors.initials: P
authors.lastname: Irwin
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D001769:Blood / Q000469:parasitology; D004283:Dog Diseases / Q000175:diagnosis* / Q000469:parasitology / Q000473:pathology*; D004285:Dogs; D005455:Fluorescent Antibody Technique; D008853:Microscopy; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013030:Spain; D016794:Theileria / Q000166:cytology / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D013801:Theileriasis / Q000175:diagnosis* / Q000469:parasitology / Q000473:pathology*
chemical_list: D000913:Antibodies, Protozoan