Ultrastructure and phylogenetic analysis of ‘Candidatus Neoehrlichia mikurensis' in the family Anaplasmataceae, isolated from wild rats and found in Ixodes ovatus ticks

Abstract

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'.The family Anaplasmataceae currently includes five genera: Ehrlichia, Anaplasma, Neorickettsia, Aegyptianella and Wolbachia (Dumler et al., 2001;Rikihisa et al., 2003). These are all obligately intracellular bacteria that are capable of infecting invertebrates and/or vertebrates. Species of the genera Ehrlichia and Anaplasma include the emerging tick-borne human pathogens, such as Ehrlichia chaffeensis and Anaplasma phagocytophilum. As it is difficult to isolate and culture this group of bacteria, the ultrastructure and sequences of conserved genes, such as 16S rRNA, groEL and gltA (citrate synthase gene), are primarily used to identify and classify this group of bacteria. The present study describes a novel bacterium in the family Anaplasmataceae that was isolated from wild Rattus norvegicus rats by using laboratory rats. The bacteria were found in R. norvegicus rats and Ixodes ovatus ticks in Japan and represent a novel genetic cluster, based on phylogenetic analysis of 16S rRNA gene sequences that included the bacteria found in R. norvegicus rats in China (Pan et al., 2003) and in Ixodes ricinus ticks in the Netherlands (Schouls et al., 1999). Of note, infection of I. ricinus ticks with similar bacteria in Baltic regions of Russia was reported by Alekseev et al. (2001). A similar 16S

Phylogenetic characterisation of two novel Anaplasmataceae from Australian Ixodes holocyclus ticks: ‘Candidatus Neoehrlichia australis' and ‘Candidatus Neoehrlichia arcana'

“…'Candidatus Neoehrlichia' is a sister genus to Anaplasma and Ehrlichia, and currently contains two species, 'Candidatus N. mikurensis' and 'Candidatus N. lotoris' (Kawahara et al, 2004;Yabsley et al, 2008b). As members of these genera are notoriously difficult to culture, these novel species were detected and differentiated based on the sequences of the conserved 16S rRNA (16S), citrate synthase (gltA) and groESL genes (Kawahara et al, 2004;Yabsley et al, 2008b).…”

“…The Anaplasmataceae currently consists of five established genera; Aegyptianella, Anaplasma, Ehrlichia, Neorickettsia and Wolbachia (Dumler et al, 2001), as well as two recently described genera with Candidatus status; 'Candidatus Xenohaliotis' (Friedman et al, 2000), and 'Candidatus Neoehrlichia' (Kawahara et al, 2004;Yabsley et al, 2008b). All species within the genera Anaplasma, Ehrlichia and 'Candidatus Neoehrlichia' are tick-borne mammalian blood parasites, and include numerous animal pathogens and several emerging human pathogens, such as Anaplasma phagocytophilum, Ehrlichia chaffeensis, and 'Candidatus Neoehrlichia mikurensis' (Dumler & Bakken, 1995;Silaghi et al, 2016).…”

Molecular Identification of Ehrlichia Species and ‘Candidatus Neoehrlichia mikurensis’ from Ticks and Wild Rodents in Shizuoka and Nagano Prefectures, Japan

“…Eleven (Fuji6/9/15/16/18-1/18-2/25, Izu8/9/13, and Nagano6) out of 17 'Candidatus N. mikurensis' sequences from rodents were found to be identical to that of the IS58 variant (Table 3), which was previously identified from I. ovatus ticks in Hokkaido (9), by the BLAST search and they were designated as the 'Candidatus N. mikurensis' FIN686 sequence. The remaining 6 sequences (Nagano21/22/28/30/32/33) were similar to that of 'Candidatus N. mikurensis' Schotti variant (similarity of 99.6%), which was identified from I. ricinus ticks in the Netherlands and Russia (1,19), and they were designated as the 'Candidatus N. mikurensis' 48 H. NAITOU ET AL Table 3.…”

“…Most recently, 'Candidatus Neoehrlichia mikurensis' has been proposed as the novel genus and species (9), and this genus is located at an intermediate position between the genera Ehrlichia and Anaplasma on phylogenetic classification. Previously, this species was found only from Rattus norvegicus (wild rat) on Mikura Island (TK4456 R variant) and from I. ovatus ticks in Hokkaido (IS58 variant) (9), but not on the main island of Japan (Honshu).…”

“…Only one species 'Candidatus Neoehrlichia mikurensis' belongs to this genus and it consists of three genetic variants; TK4456 R (or Rattus) from wild rats, Rattus norvegicus, in Mikura Island, Japan (or in China) (9,17); IS58 from I. ovatus ticks in Hokkaido, Japan (9); and Schotti from I. ricinus in the Netherlands and Russia (1,19).…”