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1 Department of Medicine and Epidemiology, University of California, Davis, CA 95616, USA
2 Veterinary Medical Teaching Hospital, University of California, Davis, CA 95616, USA
Correspondence
Jane E. Sykes
jesykes{at}ucdavis.edu
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and rnpB gene sequences of ‘Candidatus Mycoplasma haematoparvum’ are AY383241 and AY380803, respectively.
A figure showing a larger phylogenetic tree of 16S rRNA gene sequences, using Asteroleplasma anaerobium as the outgroup and including non-haemotropic mollicutes, is available as supplementary material in IJSEM Online.
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; Neimark et al., 2001, 2002); these are haemoplasmas of cats, pigs, cattle, alpacas and opossums, respectively. In contrast, haemoplasmas belonging to the haemofelis subcluster include Mycoplasma haemofelis, Mycoplasma haemomuris and Mycoplasma haemocanis, which infect cats, mice and dogs, respectively.
Until recently, the only canine haemoplasma identified worldwide was M. haemocanis (previously Haemobartonella canis), which was first reported in 1928 (Kikuth, 1928). This is a relatively large haemoplasma (0·3–2·0 µm in diameter), which characteristically forms long chains across the erythrocyte surface (Messick et al., 2002; Venable & Ewing, 1968). Chronic, subclinical infection may develop in immunocompetent dogs, which might then develop signs of haemolytic anaemia following splenectomy. Signs have occasionally been reported in dogs that are immunocompromised for other reasons, and, rarely, disease has been reported in immunocompetent dogs (Austerman, 1979; McNaught et al., 1935; North, 1978).
We have recently identified a novel haemotropic mycoplasma in a splenectomized dog with haemic neoplasia (Sykes et al., 2004). Of the canine and feline haemoplasmas, this organism most closely resembled ‘Candidatus M. haemominutum’ in that it was uniformly small in size (0·3 µm in diameter) and chains of organisms were not seen. Splenectomy was performed, together with multiple blood-product transfusions, just prior to diagnosis of acute lymphocytic leukaemia. Chemotherapy with prednisone and chlorambucil was instituted; 2 weeks after chemotherapy was instituted, numerous organisms were seen on the surfaces of approximately 70 % of the erythrocytes. Reticulocytosis was identified 1 week later, and a few haemoplasma-like organisms were detected. Treatment with doxycycline was initiated; 11 days later, no organisms were visible on blood smears. The aim of this work was to characterize this novel haemoplasma in more detail by sequencing the partial 16S rRNA gene and partial rnpB (RNase P) gene, which have been used to examine the relationship between other haemoplasmas (Birkenheuer et al., 2002; Messick et al., 2002; Neimark et al., 2001; Tasker et al., 2003). On the basis of these results, we propose a candidate species name for this organism, ‘Candidatus Mycoplasma haematoparvum’.
DNA was extracted from 200 µl anticoagulated whole blood in EDTA collected when organisms were first visualized (3 weeks after splenectomy) using the QIAamp DNA blood mini kit (Qiagen) according to the manufacturer's instructions. DNA was eluted with 200 µl elution buffer. Previously extracted DNA from cat blood containing M. haemofelis and cat blood containing ‘Candidatus M. haemominutum’ was used as a positive control. DNA extracted from healthy dog blood was used as a negative control. A 1457 bp fragment of the 16S rRNA gene was amplified using primers 8F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') (Pitulle et al., 1999). These primers amplify almost the complete length of the 16S rRNA gene. Each 50 µl reaction volume contained 1x reaction buffer, 1·25 U Taq DNA polymerase (Brinkmann Instruments), 1·5 mM MgCl2, 0·4 µM each primer, 200 µM each dNTP and 10 µl DNA template. Cycling conditions were as follows: 95 °C for 10 min, followed by 35 cycles of amplification (1 min at 95 °C, 1 min at 48 °C and 2 min at 72 °C), and a final extension of 5 min at 72 °C in a Dyad thermocycler (MJ Research).
A 188 bp fragment of the rnpB gene was also amplified using primers 80F1 [5'-GAGGAAAGTCCRYGCTWGCAC-3' (R=A or G, Y=C or T, W=A or T)] and 290R1 (5'-TCCCYTACCRAAATTTRGGTTTCT-3') (Birkenheuer et al., 2002). Each 50 µl reaction volume contained 1x reaction buffer, 1·25 U Taq DNA polymerase, 1·5 mM MgCl2, 0·4 µM each primer, 200 µM each dNTP and 5 µl DNA template. Cycling conditions were as follows: 95 °C for 5 min, followed by 45 cycles of amplification (1 min at 95 °C, 1 min at 45 °C and 1 min at 72 °C), and a final extension of 5 min at 72 °C.
PCR amplicons were cloned into the pGEM-T Easy vector system (Promega) and transformed into Escherichia coli, according to the manufacturer's instructions. Plasmid DNA was isolated using standard methods. DNA sequencing was performed by using the dideoxynucleotide chain-termination method with an ABI 377 automated sequencer (Perkin-Elmer–Applied Biosystems), using M13 primers. Internal sequencing primers 5'-GCTGCGGTAAAACATAGGTC-3' and 5'-CCTGTCATTCTGATAGCCTCC-3' were used to obtain the partial sequence of the large 16S rRNA gene fragment.
DNA sequence editing, analysis and construction of phylogenetic trees were performed with MacVector 7 and AssemblyLIGN (Accelrys) software. Phylogenetic trees were created from the distance matrices by using the neighbour-joining method. The dataset was resampled 1000 times and bootstrap percentage values are given at the nodes of the phylogenetic tree shown in Fig. 1. A figure showing a larger tree, using Asteroleplasma anaerobium as the outgroup and including other mollicutes, is available as supplementary material in IJSEM Online.
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). Comparison of the nucleotide sequence of the rnpB gene fragment with those of ‘Candidatus M. haemominutum’ (GenBank accession no. AY150990), M. haemofelis (AF407210) and three M. haemocanis isolates (AF407209, AF407211 and AF407213) revealed nucleotide sequence identities of 75, 65 and 65 %, respectively.
On the basis of the results of 16S rRNA gene sequence analysis, organisms belonging to the genera Haemobartonella and Eperythrozoon have now been reclassified as haemotropic Mycoplasma species (‘haemoplasmas’) (Neimark et al., 2001, 2002). These organisms are most closely related to the fastidious, glucose-fermenting mycoplasmas Mycoplasma cavipharyngis and Mycoplasma fastidiosum (Johansson et al., 1999). Until recently, only one canine haemoplasma had been described, and was recently renamed M. haemocanis (Messick et al., 2002). Acute and severe anaemia has been described in infected dogs, especially those that are immunocompromised or splenectomized; chronic, subclinical infection is typical in immunocompetent dogs. In this report, we have characterized an additional canine haemoplasma. The infected dog was both splenectomized and immunocompromised due to concurrent lymphoid neoplasia and immunosuppressive drug therapy, and haemolysis was suggested by the appearance of marked reticulocytosis following the appearance of organisms on blood smears (Sykes et al., 2004).
Analysis of the 16S rRNA gene sequence of this novel haemoplasma revealed that it was most closely related to ‘Candidatus M. haemominutum’, a small, low-virulence feline haemoplasma (Foley & Pedersen, 2001). This organism can induce anaemia in cats that are concurrently infected with feline leukaemia virus, and there is some evidence that it may enhance the oncogenicity of this virus (George et al., 2002). Whether this novel canine haemoplasma is capable of inducing disease in immunocompetent dogs, and the extent to which it may cause disease in immunosuppressed dogs, await further study. Preliminary results of experimental infection of cats suggest that this organism may be capable of infecting cats (J. E. Sykes and others, unpublished).
The 16S rRNA gene of M. haemofelis is >99 % similar to that of M. haemocanis (previously H. canis), an organism that causes anaemia in splenectomized dogs. In fact, using this method of classification, some M. haemofelis isolates are more similar to M. haemocanis than they are to other M. haemofelis isolates, so it has been suggested that M. haemofelis and M. haemocanis may, in reality, be the same organism infecting different species of animals (Birkenheuer et al., 2002; Brinson & Messick, 2001). However, cats inoculated with blood from a dog infected with M. haemocanis have remained asymptomatic, but blood from these cats, when inoculated into dogs, reproduced the disease (Lumb, 2001). Blood from M. haemofelis-infected cats, when inoculated into splenectomized and non-splenectomized dogs, did not result in anaemia or cytologically detectable parasitaemia, and inoculation of susceptible cats with this blood also did not result in anaemia or parasitaemia (Lumb, 2001; Splitter et al., 1956). Because of these results, and morphological differences between these organisms, discrimination of these organisms on the basis of their rnpB gene sequences has been suggested (Birkenheuer et al., 2002; Tasker et al., 2003). Using this method, sequence identity between M. haemofelis and M. haemocanis is lower (94·3–95·5 %) than that noted within strains of M. haemofelis (99·4 %) and M. haemocanis (97·7–100 %). The rnpB gene sequence identity between the novel haemoplasma and ‘Candidatus M. haemominutum’, M. haemocanis and M. haemofelis was considerably lower (75, 65 and 65 %, respectively).
According to the guidelines for the naming of uncultivated prokaryotes (Murray & Stackebrandt, 1995), we propose a Candidatus designation for this newly recognized canine haemoplasma, and recommend that it be named ‘Candidatus Mycoplasma haematoparvum’.
Description of ‘Candidatus Mycoplasma haematoparvum’
‘Candidatus Mycoplasma haematoparvum’ [hae.ma.to.par'vum. Gr. neut. n. haema -atos blood; L. neut. adj. parvum small; N.L. neut. adj. haematoparvum small (mycoplasma) from blood] [(Mollicutes) NC; NA; O, wall-less; NAS (GenBank accession no. AY383241)].
In Wright–Giemsa-stained peripheral blood smears, cells appear as small (approx. 0·3 µm) coccus-shaped organisms present singly or in doublets and attached to erythrocytes. The light-microscopic appearance resembles that of ‘Candidatus M. haemominutum’ (Foley & Pedersen, 2001). Although further study is required, the organism appears to induce a parasitaemia and haemolysis in dogs; immunocompromise and/or splenectomy may be necessary for disease to occur. On the basis of sequence analysis of the 16S rRNA gene, ‘Candidatus M. haematoparvum’ is most closely related to ‘Candidatus M. haemominutum’, a low-virulence haemoplasma of cats.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Birkenheuer, A. J., Breitschwerdt, E. B., Alleman, A. R. & Pitulle, C. (2002). Differentiation of Haemobartonella canis and Mycoplasma haemofelis on the basis of comparative analysis of gene sequences. Am J Vet Res 63, 1385–1388.[CrossRef][Medline]
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Johansson, K.-E., Tully, J. G., Bolske, G. & Pettersson, B. (1999). Mycoplasma cavipharyngis and Mycoplasma fastidiosum, the closest relatives to Eperythrozoon spp. and Haemobartonella spp. FEMS Microbiol Lett 174, 321–326.[CrossRef][Medline]
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