Download Candidatus Mycoplasma haemomuris subsp. ratti

International Journal of Systematic and Evolutionary Microbiology (2015), 65, 734–737
DOI 10.1099/ijs.0.069856-0
Proposal for ‘Candidatus Mycoplasma haemomuris
subsp. musculi’ in mice, and ‘Candidatus
Mycoplasma haemomuris subsp. ratti’ in rats
Ryô Harasawa,1 Hiromi Fujita,2 Teruki Kadosaka,3 Shuji Ando4
and Yasuko Rikihisa5
Correspondence
1
Ryô Harasawa
2
[email protected]
The Iwate Research Center for Wildlife Diseases, Morioka 020-0816, Japan
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 mycoplasma 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)
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.
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sequence (Sashida et al., 2013). In the present study, we
further investigated nucleotide sequences of the nonribosomal 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
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Mycoplasma haemomuris
Table 1. Details of the sources of the samples examined in this study
All samples were collected from animals killed under anaesthesia.
Sample designation
Shizuoka
S151-2
S152-2-4
S152-5-7
S159-11-13
S154
Ikemajima5-1
Ikemajima14-1
Host
Place animal was trapped
Year of sampling
Sample conditions
Small field mouse
Small filed mouse
Small field mouse
Small field mouse
Small filed mouse
Black rat
Black rat
Black rat
Shizuoka
Fukushima
Fukushima
Fukushima
Aomori
Fukushima
Okinawa
Okinawa
1985
1985
1986
1986
1988
1987
2010
2010
Spleen homogenate
Erythrocyte suspension
Spleen homogenate
Spleen homogenate
Spleen homogenate
Spleen homogenate
Whole blood
Whole blood
primers RNP-F and RNP-R, and the dnaK was amplified
using primers DNK-F1 and DNK-R1. After initial denaturation at 94 uC for 5 min, the reaction cycle was carried
out 30 times, with each cycle consisting of denaturation at
94 uC for 30 s, annealing at 55 uC for 60 s and extension at
72 uC for 60 s in a thermal cycler. The PCR products were
fractionated on horizontal, submerged 1.0 % SeaKem ME
agarose gels (FMC Bioproducts) in TAE buffer (40 mM Tris,
pH 8.0, 5 mM sodium acetate and 1 mM disodium ethylenediaminetetracetate) at 100 V for 30 min. After electrophoresis, the gels were stained in GelRed solution (Biotium)
for 15 min and visualized under a UV transilluminator.
DNA in a clearly visible band was extracted using (model
C-61 Cultra-Violet Products, San Gabriel, CA) a NucleoSpin
Extract II kit (Macherey-Nagel) and was subjected to direct
sequencing in both directions using the same primers used
for PCR, in a 3500 Genetic Analyzer (Applied Biosystems).
The nucleotide sequences of rnpB from the eight strains of M.
haemomuris along with the 16 established species of the
genus Mycoplasma were aligned using CLUSTAL W (Thompson
et al., 1994). A phylogenetic tree based on nucleotide
sequences of rnpB was generated using the neighbour-joining
method (Saitou & Nei, 1987) from a distance matrix
corrected for nucleotide substitutions according to the
Kimura two-parameter model (Kimura, 1980). The eight
strains of M. haemomuris were divided into two subgroups
with a high bootstrap value; subgroup A consisted of S151-2,
S152-2-4, S152-5-7, S159-11-13 and Shizuoka strains detected
in small field mice, and subgroup B consisted of S154,
Ikemajima5-1 and Ikemajima14-1 strains detected in black
rats (Fig. 1). In addition, we examined the secondary
structure predicted for the P12 portion of RNase P RNA,
which is a product of rnpB and a ribozyme responsible for
processing the 59 end of tRNA molecules. This could provide
a key piece of information with which to distinguish
between closely related species of the genus Mycoplasma ,
such as M. haemocanis and M. haemofelis (Sasaoka et al.,
2011), which have 99 % similarity in their 16S rRNA
sequences. The secondary structures of the P12 helix were
predicted according to the algorithm of Zuker & Stiegler
(1981). The GAAA tetra-nucleotide at the top of the P12
helix on the RNase P RNA of M. haemomuris was conserved in both subgroups, though there was a palindromic
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nucleotide substitution on the stem region due to a
transversion between adenine and uracil (Fig. S1).
Amino acid sequences predicted from the dnaK of the eight
strains from the two subgroups of M. haemomuris were
also aligned using CLUSTAL W (Thompson et al., 1994).
Although the dnaK nucleotide sequences showed 84 %
similarity between the subgroups (data not shown), the
amino acid sequences showed 96 % similarity (Fig. S2).
Thus, dnaK analysis, including both nucleotide and amino
acid sequences, supported separation of the two subgroups
of M. haemomuris.
The strains of the species M. haemomuris examined in the
present study have previously been reported to be divisible
into two subgroups based on their ITS sequences (Sashida
et al. 2013). Our study further confirmed the existence of
two distinct subgroups among the strains of the species M.
haemomuris that may not be attributable to the differences
in the geographical locations of strain collection, because
subgroup A included strains from Aomori, Fukushima and
Shizuoka Prefectures, and subgroup B included strains
from Fukushima and Okinawa Prefectures. This variation,
thus, most likely depends on species differences between
the natural hosts of these strains of the species M. haemomuris.
Specifically, strains of subgroup A were detected in small
field mice, while strains of subgroup B were detected in black
rats. Our analyses confirmed the existence of two genetically
distinct subgroups among strains of the species M.
haemomuris.
M. haemomuris was first observed in the blood of rats. At
that time, it was named Bartonella muris ratti (Mayer, 1921),
and was confirmed to be the causative agent of infectious
anaemia in splenectomized albino rats (Ford & Eliot, 1928).
Subsequently, another type of Bartonella, called at that time
Bartonella muris musculi, was found in the blood of albino
mice (Schilling, 1929). It was discovered later that the
previous scientific name Bartonella muris (currently M.
haemomuris) was more accurately separated into Bartonella
muris subsp. ratti in rats and Bartonella muris subsp. musculi
in mice (Noguchi, 1928; Regendanz & Kikuth, 1928), despite
the possibility of cross-transmission between these rodent
species by experimental infection. This raises the question of
whether M. haemomuris Shizuoka, a strain isolated from a
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R. Harasawa and others
Ca. M. haemominutum (EU078614)
753
M. suis (EU078611)
545
Ca. M. haematoparvum (EU078616)
406
Ca. M. haemolamae (EU078613)
505
M. wenyonii (EU078610)
899
994
Ca. M. haemocervae Sika152 (AB561882)
M. ovis (EU078612)
Ca. M. kahaneii (EU078615)
875
Ca. M. aoti Aotus (HM123756)
M. haemofelis (EU078617)
999
M. fermentans
PG18 (U41756)
1000
M. haemocanis (EU078618)
872
Ca. M. turicensis UK (EF212002)
M. coccoides (EU078619)
779
Ca. M. haemohominis Plymouth (GU562825)
861
875
0.1
S159-11-13
Subgroup A 1000
M. haemomuris Shizuoka (GU562824)
665
S151-2, S152-2-4, S152-5-7
999
Ikemajima5-1
Subgroup B
1000
S154
994
Ikemajima14-1
Fig. 1. A neighbour-joining phylogenetic tree based on a nucleotide sequence comparison of rnpB among haemotropic species
of the genus Mycoplasma including eight strains of the species M. haemomuris. Nucleotide sequences obtained from the DNA
databases are shown with accession numbers in parentheses. A strain name was not available for the accession numbers
EU078610 to EU078619. Mycoplasma fermentans PG18 (U41756) was included as an out-group. Numbers at the relevant
branch points refer to values of bootstrap probability of 1000 replications. Bar, the estimated evolutionary distance (0.1 nt
substitutions per site), computed in CLUSTAL W (Thompson et al., 1994) using the neighbour-joining method (Saitou & Nei,
1987).
small field mouse, would formerly have been classified as B.
muris subsp. musculi. This allows us to propose that the
species M. haemomuris be separated into ‘Candidatus Mycoplasma haemomuris musculi’, detected in small field mice,
and ‘Candidatus Mycoplasma haemomuris ratti’, detected in
black rats, based on the accumulated findings.
Description of ‘Candidatus Mycoplasma
haemomuris subsp. musculi’ (basonym
Mycoplasma haemomuris (Mayer 1921) Neimark
et al. 2002)
‘Candidatus Mycoplasma haemomuris subsp. musculi’
(mus’cu.li. L. masc. gen. n. musculi of the mouse).
In Giemsa-stained peripheral blood smears, tiny round
bodies appear as deep purple projections from the erythrocyte surface. Susceptible to chlortetracycline and oxytetracycline. Most infections are latent, but may become
apparent following splenectomy. Causes infectious anaemia
or splenomegaly in mice. Nucleotide sequence of the 16S
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rRNA gene are deposited in DNA databases under
accession numbers U82963 and AB918692, and PCR
primers to amplify the 16S rRNA gene are described by
Rikihisa et al. (1997).
Description of ‘Candidatus Mycoplasma
haemomuris subsp. ratti’ (basonym Mycoplasma
haemomuris (Mayer 1921) Neimark et al. 2002)
‘Candidatus Mycoplasma haemomuris subsp. ratti’ (rat’ti.
L. gen. n. ratti of the rat).
Tiny coccoid bodies sometimes in chains are found on the
erythrocytes in Giemsa-stained peripheral blood smears.
Susceptible to chlortetracycline and oxytetracycline. Latent
infection is common among rats including laboratory rats.
Causes infectious anaemia or splenomegaly in rats. The rat
louse (Polypax spinulosa) is a putative vector. Nucleotide
sequences of the 16S rRNA gene, ITS and partial 23S rRNA
gene are deposited in DNA databases under accession
numbers AB758434, AB758435 and AB758439, and PCR
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Mycoplasma haemomuris
primers to amplify the sequence are described by Sashida
et al. (2013).
Hemoplasmas and other Mycoplasma species. J Clin Microbiol 46,
1873–1877.
Regendanz, P. & Kikuth, W. (1928). Sur la Bartonella muris ratti. CR
Soc Biol (Paris) 98, 1578–1579.
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