Hidden hemoplasma species within the "Candidatus Mycoplasma haemominutum" lineage in Thai cats revealed by analyses of two independent genetic markers.

BACKGROUND: Hemotropic Mycoplasma spp. (hemoplasmas) parasitize erythrocytes and cause hemolytic anemia in several mammalian species, including cats. Mycoplasma haemofelis (Mhf), "Candidatus Mycoplasma haemominutum" (CMhm) and "Candidatus Mycoplasma turicensis" (CMt) are the three main feline hemoplasma species. A species closely related to CMhm was recently proposed as a putative novel species based on the 23S ribosomal RNA (rRNA) gene. METHODS: In this study, 16S and 23S rRNA genes were used to investigate hemoplasma diversity in cats. Blood samples from 388 cats were obtained and screened for hemoplasma infection based on a PCR assay targeting the 16S rRNA gene. Positive samples were sequenced based on the 16S and 23S rRNA genes. All obtained sequences were analyzed by the nucleotide Basic Local Alignment Search Tool (BLASTn), the DnaSP6 computer program, phylogenetic construction, genetic network and pairwise identity matrix. RESULTS: The 388 blood samples collected from the cats were screened for hemoplasma infection. The tests showed that 68 cats (17.5%, 95% confidence interval [CI] 13.9-21.7%) were positive for hemoplasmas. Of these 68 positive samples, 49 were successfully sequenced for both the 16S and 23S rRNA genes and the sequences subsequently assigned to 11 nucleotide sequence types (ntSTs). The 16S rDNA analysis revealed one Mhf group, at least three groups within CMhm and at least two groups within CMt. Notably, we identified CMhm as well as two putative species closely related to CMhm from 23S rDNA analysis, including one that has been previously identified. In contrast, the identity of the CMt-derived 23S rDNA sequence ntST#11 remains unclear due to the lack of CMt reference sequences, highlighting the need for more comprehensive CMt data in public databases. CONCLUSIONS: Our data suggested the presence of two putative species related to CMhm identified in domestic cats in Thailand. Integrating analyses of independent genetic markers, such as 16S and 23S rRNA genes, would enhance hemoplasma species identification and novel species discovery.