title: Cultivation and molecular identification of Ehrlichia canis and Ehrlichia chaffeensis from a naturally co-infected dog in Venezuela.
abstract: BACKGROUND
  Diagnosis of canine ehrlichiosis in Venezuela is normally performed by examination of buffy coat smears (BCS). Characteristic inclusion bodies are frequently observed in leukocytes and platelets from dogs with clinical signs of the disease.
  
  
  OBJECTIVE
  The purpose of this study was to investigate the co-infection of a dog with Ehrlichia canis and E hrlichia chaffeensis using microbiological and molecular techniques.
  
  
  METHODS
  Primary cultures of monocytes from a dog showing signs of ehrlichiosis were performed. Ehrlichial inclusions in blood cells were demonstrated by BCS and in cultured cell smears with direct immunofluorescence and Dip Quick staining. Nested PCR analysis was performed with DNA from blood samples and cultures, using primers specific for E. canis and E. chaffeensis. The amplified DNA fragments were sequenced to confirm the specificity of the amplifications.
  
  
  RESULTS
  The BCS of the naturally infected dog contained intracellular morulae. Ehrlichial inclusions were observed 9 days after inoculation of the primary cultures. After 3 passages with monocytes from a healthy dog, 65% of infected cells, and cells with >60 morulae were observed. A healthy female German Shepherd dog, seronegative for E. canis and E. chaffeensis antigens and without contact to ticks, was inoculated with an infected culture. The animal developed signs of canine monocytic ehrlichiosis and became seropositive. Nested PCR results and sequencing of amplified DNA fragments demonstrated the simultaneous presence of E. canis and E. chaffeensis in both dogs.
  
  
  CONCLUSIONS
  This is the first report of E. chaffeensis in dogs in South America. This organism was previously identified in dogs by PCR only in the United States.
authors.affiliation: Biomedical Research Institute (BIOMED-UC), Univresity of Carabobo campus Aragua, Maracay, Venezuela.
authors.forename: Clara N
authors.initials: CN
authors.lastname: Gutiérrez
authors.forename: María
authors.initials: M
authors.lastname: Martínez
authors.forename: Erlinda
authors.initials: E
authors.lastname: Sánchez
authors.forename: Marisol
authors.initials: M
authors.lastname: De Vera
authors.forename: Marìa
authors.initials: M
authors.lastname: Rojas
authors.forename: Joahnny
authors.initials: J
authors.lastname: Ruiz
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Triana-Alonso