Molecular diagnosis of granulocytic anaplasmosis and infectious cyclic thrombocytopenia by PCR-RFLP

Anaplasma phagocytophilum (A. phagocytophilum, formerly Ehrlichia phagocytophila) is a tick-borne pathogen responsible for tick-borne fever in ruminants, equine granulocytic ehrlichiosis (EGE) in horses, canine granulocytic ehrlichiosis (CGE) in dogs, and for human granulocytic ehrlichiosis (HGE). Human cases have been registered in many countries with a broad range of symptoms and pathogenicity. This article focused on Sardinia as the prevalence in humans was almost seven times higher than in the rest of Italy. To evaluate the risk, blood samples were collected from dogs and horses on the island. Genomic DNA was extracted from the buffy coat and amplified by heminested polymerase chain reaction (PCR) using the groEL gene primers. The first PCR reaction amplified a 624-bp fragment for both A. phagocytophilum and A. platys while the second PCR reaction amplified 573-bp and 515-bp fragments for the above two pathogens, respectively. Six A. phagocytophilum samples were PCR positive (3 dogs and 3 horses) while another dog was A. platys PCR positive. A phylogenetic analysis was conducted with A. phagocytophilum sequences in GenBank from the United States, Slovenia, Switzerland, Germany, UK, Austria, and Czech Republic. Surprisingly, the related phylogenetic tree showed that the Sardinian isolates were closer to the American isolates, which were showing highest mortality rates than from the other two European lineages.