Tiawsirisup, Sonthaya

Abstract Background Hemotropic Mycoplasma spp. (hemoplasmas) parasitize erythrocytes and cause hemolytic anemia in several mammalian species, including cats. Mycoplasma haemofelis ( Mhf ), “ Candidatus Mycoplasma haemominutum” ( C Mhm) and “ Candidatus Mycoplasma turicensis” ( C Mt) are the three main feline hemoplasma species. A species closely related to C Mhm 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 C Mhm and at least two groups within C Mt. Notably, we identified C Mhm as well as two putative species closely related to C Mhm from 23S rDNA analysis, including one that has been previously identified. In contrast, the identity of the C Mt-derived 23S rDNA sequence ntST#11 remains unclear due to the lack of C Mt reference sequences, highlighting the need for more comprehensive C Mt data in public databases. Conclusions Our data suggested the presence of two putative species related to C Mhm 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. Graphical abstract

Objectives This study examined feline haemoplasmas ( Mycoplasma haemofelis , ‘ Candidatus Mycoplasma haemominutum’ [ C Mhm] and ‘ Candidatus Mycoplasma turicensis’) infecting Thai domestic cats, using the 16S and 23S rRNA genes as genetic markers. Methods Blood samples from 20 cats were obtained from a diagnostic laboratory and nucleic acids were extracted from each sample using a commercial kit. PCR targeting the 16S rRNA gene was used to screen haemoplasmas in the samples. Positive PCR samples were further sequenced using the 16S and 23S rRNA genes. The sequences from each genetic marker were analysed using Nucleotide BLAST, phylogeny and genetic network analyses. Results Among the 20 samples, five were infected with haemoplasmas. In the 16S rRNA gene sequencing, four sequences were assigned to C Mhm and the remaining sequence was likely to be a closely related species of C Mhm. In the 23S rRNA gene sequencing, four sequences from the same samples used for 16S rRNA gene sequencing were identified as C Mhm and one sequence could be a putative novel haemoplasma species closely related to C Mhm. Conclusions and relevance Only C Mhm and its closely related species were identified in this study. Although C Mhm has been recognised as a low-virulence parasite, cases of severe anaemia in cats infected with C Mhm have been found. Thus, such cases could be confirmed via the analysis of 16S and 23S rRNA genes. Furthermore, molecular detection and genetic analyses of feline haemoplasmas in additional cat blood samples should be conducted using PCR assay and DNA sequencing based on universal primers of 16S rRNA and 23S rRNA genes to enable more specific identification.