Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Mon, 03 Dec 2018 09:31:52 Proposal for ‘Candidatus Mycoplasma haemomuris subsp. musculi’ in mice, and ‘Candidatus Mycoplasma haemomuris subsp. ratti’ in rats Ryo ˆ Harasawa, 1 Hiromi Fujita, 2 Teruki Kadosaka, 3 Shuji Ando 4 and Yasuko Rikihisa 5 Correspondence Ryo ˆ Harasawa harasawa-tky@umin.ac.jp 1 The Iwate Research Center for Wildlife Diseases, Morioka 020-0816, Japan 2 Mahara Institute of Medical Acarology, Anan 779-1510, Japan 3 Department of Microbiology and Immunology, Aichi Medical University, Nagakute 480-1195, Japan 4 Department of Virology 1, National Institute of Infectious Diseases, Tokyo 162-8640, Japan 5 Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, USA Mycoplasma haemomuris is causative of infectious anaemia or splenomegaly in rodents. We examined the nucleotide sequences of the non-ribosomal genes, rnpB and dnaK, in strains of the species M. haemomuris detected in small field mice and black rats. rnpB nucleotide sequences in strains of the species M. haemomuris isolated from small field mice and black rats had only 89 % sequence similarity, suggesting their separation into two distinct subgroups. dnaK had a nucleotide sequence similarity of 84 % between the subgroups. These results support the classification of M. haemomuris into two genetically distinct subgroups. Here we propose the establishment of these subgroups as ‘Candidatus Mycoplasma haemomuris subsp. musculi’, detected in small field mice (Apodemus argenteus), and ‘Candidatus Mycoplasma haemomuris subsp. ratti’, detected in black rats (Rattus rattus). The group of bacterial species know as haemotropic myco- plasma or haemoplasma are pathogens that have been recognized as agents of infectious anaemia in various mammalian species (Messick, 2004). This group includes not only species formerly classified as Haemobartonella and Eperythrozoon, but also newly identified haemotropic species of the genus Mycoplasma. Haemoplasma strains have been detected in rodents, including mice, rats and hamsters, and have been classified primarily based on nucleotide sequences of the 16S rRNA or RNase P RNA (rnpB) genes (Peters et al., 2008; Tasker et al., 2003). The two haemotropic species of the genus Mycoplasma currently recognized in rodents, Mycoplasma haemomuris and Mycoplasma coccoides, were transferred from Haemobartonella muris (formerly Bartonella muris) and Eperythrozoon coccoides, respectively, based on their 16S rRNA gene sequences (Neimark et al., 2001, 2002, 2005; Rikihisa et al., 1997). Recent studies have suggested that M. haemomuris consists of two subgroups based on differences in the 16S–23S rRNA intergenic spacer (ITS) sequence (Sashida et al., 2013). In the present study, we further investigated nucleotide sequences of the non- ribosomal genes rnpB and dnaK to evaluate these two subgroups among strains of the species M. haemomuris. Anti-coagulated blood or spleen homogenates were obtained from small field mice (Apodemus argenteus) and black rats (Rattus rattus) previously diagnosed with M. haemomuris infection, based on a 99 % nucleotide sequence similarity of the 16S rRNA gene (Sashida et al., 2013). Details regarding the source of the samples examined in this study are provided in Table 1. Total DNA was extracted from 200 ml of the spleen or blood cells of these mice and rats using the QIAamp DNA Blood Mini kit (Qiagen) according to the manufacturer’s instructions. Eight DNA samples extracted from small field mice and black rats were subjected to PCR to amplify the rnpB and dnaK. PCR was carried out with 50 ml reaction mixtures, each containing 2 ml of DNA solution, 24 ml of EmeraldAmp PCR Master Mix and water to a final volume of 50 ml. Primer sequences for PCR and sequencing were designed as described in previous reports (Hicks et al., 2014; Steer et al., 2011) and as shown in Table S1 (available in the online Supplementary Material). The rnpB was amplified using The GenBank/EMBL/DDBJ accession numbers for rnpB and dnaK, appearing for the first time in this study, are AB973078 to AB973092. Two figures and one table are available with the online Supplementary Material. International Journal of Systematic and Evolutionary Microbiology (2015), 65, 734–737 DOI 10.1099/ijs.0.069856-0 734 069856 G 2015 IUMS Printed in Great Britain