Detection and quantification of a novel bacterium of the genus Midichloria (family Midichloriaceae, order Rickettsiales) in the hard tick Hyalomma marginatum

INTRODUCTION. Ticks are considered the most important vector of microbial agents maintained in nature by several reservoir hosts like birds, which contribute to the spread of microorganisms across countries and continents (Wallménius et al., 2014, Parasit. Vectors, 7:318). The detection of Midichloria bacteria (family Midichloriaceae) in Hyalomma marginatum ticks collected from migrating birds (Di Lecce et al., 2018, Parasit. Vectors, 11:106) contributes to define the geographic distribution of these bacteria and to increase the knowledge about their potential transmission to vertebrate hosts (as occurs for Midichloria mitochondrii, the best-known representative of the Midichloriaceae family; Serra et al., 2018, J. Wildl. Dis. Mar 16). The aims of this study are: 1) to determine the genetic variability of Midichloria bacteria in H. marginatum ticks collected from trans-Saharan migratory birds arriving in Europe from Africa in spring; 2) to quantify the amount of Midichloria in ticks through a novel quantitative PCR (qPCR) approach. MATERIALS AND METHODS. H. marginatum nymphs were collected from three different migratory bird species (Phoenicurus phoenicurus, Saxicola rubetra and Sylvia communis) on Ventotene Island (Central Italy). DNA was extracted from all nymphs and molecular analyses (qualitative PCR) were performed in order to evaluate the presence of Midichloria and to determine the genetic variability of this bacterium through the amplification of four specific genes. In order to quantify Midichloria in H. marginatum samples, two sets of primers for the amplification of the gyrB (gyrase-B) gene of Midichloria and the cal (calreticulin) gene of H. marginatum were designed and used in a qPCR approach. Results were expressed as gyrB/cal copy numbers ratio. RESULTS AND CONCLUSIONS. The molecular analyses suggest that Midichloria bacteria carried by H. marginatum ticks constitute a previously undescribed Midichloria strain within the Midichloriaceae family. Furthermore, the quantification of Midichloria in H. marginatum nymphs showed a higher bacteria load after the blood meal, similarly to what has been observed for M. mitochondrii in Ixodes ricinus ticks. Future studies should investigate the presence of this Midichloria bacterium in adult H. marginatum ticks, in order to increase the knowledge concerning its role in the biology of this tick species and its transmission mode during the tick bite.