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Journal of Clinical Microbiology, November 2004, p. 5397-5399, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.5397-5399.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Demonstration of Two Distinct Hemotropic Mycoplasmas in French Dogs

Martin J. Kenny,1 Susan E. Shaw,1 Fréderic Beugnet,2 and Séverine Tasker1*

Department of Clinical Veterinary Science, University of Bristol, Langford, United Kingdom,1 Merial Animal Health Ltd., Lyon, France2

Received 9 February 2004/ Returned for modification 4 April 2004/ Accepted 29 June 2004


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ABSTRACT
 
In North America it has been shown that distinct hemotropic mycoplasmas exist in dogs. Blood samples from 460 French dogs were analyzed by PCR to evaluate hemoplasma infection status. Seventy-one dogs (15.4%) were positive; of these, 44 (9.6%) were infected with an organism closely related to "Candidatus Mycoplasma haemoparvum" only, 15 (3.3%) were infected with Mycoplasma haemocanis only, and 12 dogs (2.6%) were dually infected with both organisms.


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TEXT
 
Epierythrocytic bacteria associated with anemia have been intermittently reported in dogs, with a widespread geographical distribution, over the past 60 years (1-3, 5, 12, 15). In these reports, it is suggested that dogs are latently infected until other factors, such as splenectomy or immunosuppression, trigger overt disease. Immune-mediated hemolytic anemia can result from infection (1, 4). Canine epierythrocytic bacteria were considered to be closely related to the feline pathogen Haemobartonella felis, and accordingly were named Haemobartonella canis. In the absence of a culture system for members of the genus Haemobartonella, these organisms were originally classified as rickettsiae on the basis of their morphological characteristics and response to antibiotic treatment (23). However, recently 16S rRNA gene-based molecular studies have shown that the genus Haemobartonella is, in fact, more closely related to the pneumoniae group of mycoplasmas, leading to the reclassification of these organisms within the genus Mycoplasma (13). In line with this, H. canis has been renamed Mycoplasma haemocanis (11) and H. felis is now named Mycoplasma haemofelis (13, 14). These molecular studies have also documented the existence of an additional, distinct feline epierythrocytic organism designated "Candidatus Mycoplasma haemominutum" (8). These hemotropic mycoplasmal species have collectively been given the trivial name "hemoplasmas" (13).

M. haemocanis isolates have been reported in both North American and European (German) dogs (11, 20). One recent report (17) and two submitted DNA sequences (GenBank accession numbers AY383241 and AY297712) from North America suggest that heterogeneity is present among canine hemoplasma isolates based on the results of 16S rRNA gene sequencing. These sources describe the existence of novel canine hemoplasma sequences which are distinct from M. haemocanis and show greater sequence identity to the feline species "Candidatus Mycoplasma haemominutum." In GenBank these canine sequences have been named "Candidatus Mycoplasma haemoparvum" (AY383241) and "Candidatus Mycoplasma haemominutum" (AY297712). Phylogenetic studies and further descriptions of these isolates have not yet been published.

The greater sensitivity, specificity, and robustness of PCR-based assays for detecting hemoplasmas, in comparison with microscopy (21), have allowed us to assess, for the first time, the carriage and heterogeneity of hemoplasmas in a population of French dogs.

Anticoagulated blood samples were collected from 460 dogs in the south of France. The blood samples were taken for clinical diagnostic purposes, and the remainder was used for this study. Data on age and sex were obtained for all animals, but clinical information was incomplete. Blood was stored at –20°C until analysis. DNA was extracted (QIAamp DNA Blood Mini kit; QIAGEN, West Sussex, United Kingdom) from 200 µl of blood and was eluted in 100 µl of elution buffer before storage at –80°C until use.

Initially, the DNA from 90 animals was analyzed using the conventional generic hemoplasma 16S rRNA PCR reported by Criado-Fornelio et al. (6). Sequencing of the resulting 595- or 618-bp amplicons from six positive samples (performed by GRI-Genomics, Essex, United Kingdom) showed that these sequences had 100% identity with the primers and Taqman probes used in a real-time PCR assay recently reported to detect, and distinguish between, both feline hemoplasma species (19). Subsequently, this real-time assay was used to analyze all 460 samples. The identity of the isolates detected by the real-time PCR assay was confirmed by subjecting more than half of the positive DNA samples (n = 40) to the conventional generic hemoplasma 16S rRNA PCR and DNA sequencing the resultant 595- or 618-bp amplicons. Sequences were decoded using the BioEdit suite of programs (www.mbio.ncsu.edu/BioEdit/bioedit.html), and their identity was evaluated by checking against existing sequences using BLAST (http://www.ncbi.nlm.nih.gov/BLAST). Sequencing of the 16S rRNA gene fragments showed a clear division of sequences into two groups, one with 100% identity to M. haemocanis and one with greater identity to "Candidatus Mycoplasma haemoparvum."

In order to derive maximal phylogenetic information from the "Candidatus Mycoplasma haemoparvum" subset of sequences, we attempted to clone the complete 16S rRNA gene from representative samples. However, we failed to obtain full-length hemoplasma amplicons using several universal eubacterial 16S rRNA primers (10); amplicons from contaminating bacteria were cloned or no PCR products were obtained. Finally we made the reactions more specific to derive a complete 16S rRNA gene sequence by cloning two overlapping fragments of the 16S rRNA gene generated by combining universal 16S rRNA gene primers (10) and the generic 16S rRNA hemoplasma primers (6). The derived consensus complete 16S rDNA sequence of the "Candidatus Mycoplasma haemoparvum" isolate was deposited in GenBank. This sequence was subjected to phylogenetic analysis with related canine and feline hemoplasma sequences derived from NCBI BLAST searches. Mycoplasma pneumoniae was used as an outgroup. Sequences were aligned using CLUSTALX (version 1.81; EMBL) with further adjustment made manually by eye as necessary (22). Phylogeny was performed using the algorithms implemented in the PAUP v. 4 program (PAUP Phylogenetic Analysis, version 4; Sinauer Associates, Sunderland, Mass.) using the neighbor-joining method. The data set was resampled 100 times to generate bootstrap percentage values.

The relationship between infection status and age or sex was determined using the Student's t test and chi-square tests (SPSS v. 10.1.0; SPSS, Woking, Surrey, United Kingdom), respectively; significance was taken as a P of <0.05.

A total of 71 of the 460 dogs (15.4%) were positive for hemoplasma infection using the real-time PCR assay. A positive result was generated with the M. haemofelis Taqman probe alone in 15 of the 460 samples (3.3%), while 44 of the 460 samples (9.6%) were positive with the "Candidatus Mycoplasma haemominutum" Taqman probe only. Twelve dogs (2.6%) were dually positive with both probes. Confirmatory DNA sequence analysis and NCBI BLAST searches of over half of the positive real-time PCR samples, derived following amplification with the generic 16S rRNA hemoplasma primers, showed that those positive with the M. haemofelis Taqman probe showed 100% identity with a German M. haemocanis isolate, while those positive with the "Candidatus Mycoplasma haemominutum" Taqman probe showed near-identity to the recently submitted (but unpublished) DNA sequence of "Candidatus Mycoplasma haemoparvum." No significant association was found between infection status and either age or sex. Unfortunately, the clinical data from the sampled animals were incomplete so any disease association with infection cannot be investigated.

Phylogenetic analysis (Fig. 1) showed the newly derived French "Candidatus Mycoplasma haemoparvum" sequence from the present study to consistently group with a "Candidatus Mycoplasma haemoparvum" canine isolate from North America in GenBank (AY383241) which has yet to be fully described. Together these isolates consistently grouped within a clade containing feline "Candidatus Mycoplasma haemominutum" isolates and a "Candidatus Mycoplasma haemominutum" isolate from a dog (AY297712). Further studies will be required, including phylogeny with non-16S rRNA gene sequences, to more fully describe the relationship between the canine and feline "Candidatus Mycoplasma haemominutum" and "Candidatus Mycoplasma haemoparvum" isolates.



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  		FIG. 1. Phylogeny of hemoplasma 16S rRNA gene sequences and related species. The phylogenetic tree was constructed using the neighbor-joining program from a distance matrix corrected for nucleotide substitutions by the Kimura two-parameter model. M. pneumoniae was included as an outgroup. Evolutionary distances are to the scale shown. Bootstrap percentage values are given to the left of the tree nodes. GenBank accession numbers are shown. *, indicates the isolate for which 16S rDNA sequence was derived in the present study.

These results report, for the first time, the prevalence of canine hemoplasma species in a sample of French dogs and represent the first survey of its type in Europe using real-time PCR.

Additionally, this study represents the first report of the presence of a European canine hemoplasma isolate distinct from M. haemocanis. This isolate showed greater identity with "Candidatus Mycoplasma haemoparvum" and is present in twice as many animals as the recognized M. haemocanis organism. The prevalences of the canine hemoplasmas seen in the present study are similar to those previously reported for feline hemoplasmas in cats (9, 18) in that "Candidatus Mycoplasma haemominutum"-infected cats are found most commonly while M. haemofelis-infected and dual-positive cats are less common. It will be interesting to compare the infection rates of the different canine hemoplasmas in different climatic zones as the tick Rhipicephalus sanguineus, which has a limited geographical distribution, has been shown to transmit H. canis (16). Further work is needed to compare the relative pathogenicities of the two species, since in cats it is apparent that M. haemofelis is significantly more pathogenic than "Candidatus Mycoplasma haemominutum" (7).

Nucleotide sequence accession number. The GenBank accession number of the nucleotide sequence derived in this study is AY532390.


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ACKNOWLEDGMENTS
 
This work was funded by the Department of Clinical Veterinary Science, University of Bristol. Séverine Tasker held a position funded by Waltham/Royal Canin.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Clinical Veterinary Science, University of Bristol, Langford House, Langford BS40 5DU, United Kingdom. Phone: 44 117 928 9558. Fax: 44 117 928 9559. E-mail: s.tasker{at}bristol.ac.uk. Back


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Journal of Clinical Microbiology, November 2004, p. 5397-5399, Vol. 42, No. 11
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.11.5397-5399.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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