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Department of Clinical Veterinary Science, University of Bristol, Langford, Bristol BS40 5DU,1 Cytopath, Ledbury HR8 2YD, United Kingdom,6 Department of Clinical Science, Koret School of Veterinary Medicine, Hebrew University of Israel, Rehovot 76100, Israel,2 Bryanston Veterinary Hospital, Bryanston 2021, South Africa,3 Faculty of Veterinary Science, The University of Sydney, Sydney, New South Wales 2003, Australia,4 Clinique Vétérinaire, 30250 Sommieres, France5
Received 3 March 2003/ Returned for modification 23 March 2003/ Accepted 16 May 2003
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Blood samples were obtained from cats and dogs in the United Kingdom, Israel, South Africa, Australia, France, and Germany that were believed to be hemoplasma infected. Following DNA extraction (DNeasy Tissue Kit; Qiagen, Crawley, United Kingdom), amplification of nearly complete 16S rRNA gene sequences was carried out using universal 16S rRNA gene primers (10, 22). Successfully amplified products of the appropriate size (approximately 1,500 bp) were purified (QIAquick gel extraction kit; Qiagen) and then cloned by using a TOPO pCR 2.1 kit (Invitrogen, Groningen, The Netherlands). Plasmid DNA was purified (Qiaprep Plasmid Spin Miniprep Kit; Qiagen) and submitted for fluorescent dideoxynucleotide sequencing (University of Dundee Sequencing Service, Dundee, Scotland). Sequences of the RNA subunit of the RNase P gene were generated using two primer pairs (RNasePFor1 [5'-CTGCGATGGTCGTAATGTTG-3'] plus RNasePRev1 [5'-GAGGAGTTTACCGCGTTTCA-3'] and RNasePFor2 [5'-TATTTAAAGTAGAGGAAAGTC-3'] plus RNasePRev1). Following PCR amplification, PCR products (approximately 160 to 210 bp) were purified as described above and submitted to the same sequencing facility. The sequence data derived from all PCR products amplified from each hemoplasma isolate were aligned (AssemblyLIGN software; Oxford Molecular, Oxford, United Kingdom) and combined to generate a final sequence. Sequences were aligned using CLUSTAL X (version 1.8 EMBL) (20), and further manual adjustment was performed if visual observation showed regions of misalignment. Phylogenetic trees were constructed with the PHYLIP package (3), using the neighbor-joining program (18), from a distance matrix corrected for nucleotide substitutions by the Kimura two-parameter model (9), and parsimony analysis by the method of Fitch (4) was used to count the number of base changes required on a given tree. The data set was resampled 100 times to generate bootstrap percentage values.
In total, 12 nearly complete 16S rRNA gene sequences were generated for different feline and canine hemoplasma isolates. These sequences showed 97 to 99% identity to United States "Candidatus M. haemominutum," M. haemofelis, or M. haemocanis sequences. Phylogeny studies using both distance and discrete methods yielded similar results (data not shown), with no obvious geographical or host specificity grouping of isolates evident (Fig. 1). Partial sequences of the RNA subunit of the RNase P gene were generated for six hemoplasma isolates. Phylogenetic analyses of the RNase P gene sequences available for representatives of the class Mollicutes and those sequenced in this study gave similar results with both distance (Fig. 2) and discrete (data not shown) methods in that all hemoplasmas fell within one clade, with the closest relatives being species in the Mycoplasma pneumoniae group. No obvious geographical grouping of isolates was observed, and although host specificity grouping was seen when the distance method was employed, this was not as conclusive with the discrete method.
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Analysis of 16S rRNA gene phylogeny of hemoplasmas from different countries revealed grouping of these organisms into one of two distinct clades, one clade comprising the "Candidatus M. haemominutum" isolates and related species and the other consisting of M. haemocanis and M. haemofelis isolates along with M. haemomuris, as reported previously with sequences derived in the United States (8, 11, 12). Although M. haemocanis and M. haemofelis isolates grouped together consistently, no reliable division of M. haemocanis and M. haemofelis was seen using either distance or discrete phylogenetic methods. This posed the question of whether the M. haemocanis and M. haemofelis isolates truly represent different species. Host specificity of canine and feline hemoplasmas had been suggested in an early study in which attempts to transmit feline hemoplasma infection to dogs failed (5). Although 16S rRNA sequences have proved to be a very effective tool in determining the phylogeny and taxonomy of the mollicutes, additional phylogenetic markers would be helpful to support the conclusions based on 16S rRNA gene data (15). It was, therefore, decided to determine the sequences of non-16S rRNA genes for further evaluation of the relationship between M. haemocanis and M. haemofelis as well as other hemoplasmas.
In this study, partial sequences of the RNA subunit of the RNase P gene from a number of hemoplasma isolates were determined. The RNase P gene sequences available for mycoplasmas were limited in number, but preliminary phylogenetic analysis of those available did not generate the distinct five groupings of mycoplasmas described in studies of 16S rRNA gene phylogeny (23). Reports of studies of phylogeny of the genus Mycoplasma based on RNase P sequences have not been published to date, and further analysis of a larger number of Mycoplasma species would be helpful in defining these relationships. Based on 16S rRNA phylogeny studies, the hemoplasmas are most closely related to the M. pneumoniae group (14); this is supported by the findings of our studies using RNase P sequence data. Distance method phylogeny analysis of the derived RNase P sequences showed division of the M. haemofelis and M. haemocanis isolates into two distinct clusters, accompanied by high bootstrap percentages. However, the discrete method yielded no such division, although the bootstrap percentages associated with this method were lower. It appears that the RNase P gene may more reliably differentiate between M. haemofelis and M. haemocanis than 16S rRNA gene sequencing and supports the concept that these sequences represent distinct species infecting cats and dogs, respectively. A previous study, which looked at sequence identity only, also suggested that RNase P gene data might be more reliable for distinguishing M. haemofelis and M. haemocanis (1). Future research should focus on determining the RNase P gene sequences of other hemoplasma isolates for use in phylogenetic analyses.
There is no specific definition of exactly what constitutes a species (17). A polyphasic approach to taxonomy and species description, using phenotypic, genotypic, and phylogenetic information to describe an organism and deduce its status, has been suggested (21). The inability to culture hemoplasmas means that phenotypic characteristics cannot be described adequately to differentiate among species, resulting in a reliance on molecular methods (6, 11-13). DNA-DNA hybridization has been used to distinguish species (isolates from a single species having at least 70% DNA relatedness) (7, 17) and would be a useful technique to apply to hemoplasmas, although culturing of organisms is usually required to generate the large quantities of DNA needed for such studies. Phylogenetic analysis provides valuable information on the taxonomy of unculturable organisms such as the hemoplasmas. The studies reported here provide additional information on the relationship of the hemoplasmas and support the existence of distinct hemoplasma species in dogs and cats. Additionally, these studies report the existence in Germany, the United Kingdom, France, Australia, South Africa, and Israel of canine and feline hemotropic pathogens with 16S rRNA gene sequences nearly identical to those described in the United States. These organisms should be considered possible etiological agents in cases of anemia in recognized host species.
Nucleotide sequence accession numbers. The GenBank accession numbers of the nucleotide sequences derived in this study are
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We kindly thank E. Thein for providing hemoplasma positive blood samples from German dogs. We thank A. Radford for help with the phylogenetic analysis.
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