Zhang, Chunbin

A novel bacterium that infects laboratory rats was isolated from wild Rattus norvegicus rats in Japan. Transmission electron microscopy of the spleen tissue revealed small cocci surrounded by an inner membrane and a thin, rippled outer membrane in a membrane-bound inclusion within the cytoplasm of endothelial cells. Phylogenetic analysis of the 16S rRNA gene sequence of the bacterium found in R. norvegicus rats and Ixodes ovatus ticks in Japan revealed that the organism represents a novel clade in the family Anaplasmataceae, which includes the Schotti variant found in Ixodes ricinus ticks in the Netherlands and the Ehrlichia-like Rattus strain found in R. norvegicus rats from China. The novel clade was confirmed by phylogenetic analysis of groESL sequences found in R. norvegicus rats and Ixodes ovatus ticks in Japan. No serological cross-reactivity was detected between this bacterium and members of the genera Anaplasma, Ehrlichia or Neorickettsia in the family Anaplasmataceae. It is proposed that this new cluster of bacteria should be designated ‘Candidatus Neoehrlichia mikurensis’.

Summary ‘ Aegyptianella ranarum ’ (order Rickettsiales), an ultrastructurally defined small, Gram‐negative rod, is known to replicate in the red blood cells of frogs. Heretofore, this bacterium has not been characterized genetically. We cloned and sequenced the 16S rRNA (1310 bp) and gyrB (718 bp) genes of ‘ A. ranarum ’ from a Canadian frog blood specimen. In situ hybridization (with an ‘ A. ranarum ’ 16S rRNA gene polymerase chain reaction product as probe) and electron microscopy confirmed that ‘ A. ranarum ’ forms cytoplasmic inclusions in frog erythrocytes. blast comparisons with GenBank 16S rRNA and gyrB sequences showed that both ‘ A. ranarum ’ genes were most similar (91% and 67% identity) to those of Chryseobacterium meningosepticum , a bacterium in the family Flavobacteriaceae . In contrast, ‘ A. ranarum ’ 16S rRNA shared only 61% identity with Aegyptianella pullorum . Phylogenetic analyses of these genes using phylip supported ‘ A. ranarum ’ as a member of Flavobacteriaceae, but suggested that its cladistic sibling may be Bergeyella zoohelcum or Weeksella virosa , rather than C. meningosepticum. We propose to classify ‘ Aegyptianella ranarum ’ as ‘ Candidatus Hemobacterium ranarum’ in the family Flavobacteriaceae . Our results provide a starting point for studies of related intraerythrocytic bacterial infections in frogs.