Co-occurrence of Francisella and spotted fever group Rickettsia in avian-associated Hyalomma rufipes

Introduction: The migratory behaviour of wild birds aids in the geographical spread of ticks and their microorganisms. Ticks are known to harbor both pathogenic and symbiotic bacteria - such as species of the genera Francisella, Rickettsia,and Midichloria - and multiple bacterial species may occur within them. Francisella occurs in different tick taxa andconsists of closely related pathogenic and non-pathogenic species. Spotted fever group Rickettsia are transmitted to humans by different tick genera and are emerging human pathogens in Europe. The aims of this study were to investigate dispersal of Francisella as well as co-occurrence of Francisella and spotted fever group Rickettsia in ticks infesting northward migrating birds in the African-Western Palaearctic region. Materials and methods: Birds were trapped using mist nets at bird observatories in Spain, Italy, Greece, and Israel during their spring migration of 2014 and 2015. Ticks were screened for the genus Francisella, the species Francisella tularensis, and spotted fever group Rickettsia by microfluidic qPCR. Ticks with putative positive results for F. tularensiswere subjected to confirmation analyses, metagenomics analysis, enrichment, and whole genome sequencing. Results: There was a high prevalence of Francisella species (76.7%) and co-occurrence of Francisella species and spotted fever group Rickettsia (50.6%) in the tick species Hyalomma rufipes. Two H. rufipes yielded putative positive test results for the human pathogen F. tularensis during initial screening. Metagenomics analysis revealed presence of Francisella sp., Rickettsia sp., and Midichloria sp. DNA in the two H. rufipes ticks. The levels of Rickettsia and Midichloria DNA were relatively high while the level of Francisella DNA was low and required enrichment for the construction of metagenome-assembled genomes. Phylogenetic inference and calculations of the average nucleotide identity (ANI) indicated that: i) the Francisella genomes belonged to the Francisella-like endosymbiont (FLE) group in Clade 1 of Francisella and had highest sequence identity to an FLE found in Ornithodoros moubata (ANI: 96.7/97.0%), ii) the Rickettsia genomes had highest resemblance to Rickettsia aeschlimannii (ANI: 98.8 - 99.9%), and iii) the Midichloria genomes resembled Midichloria mitochondrii (ANI: 91.5 - 92.3%). Conclusions: The results of this study suggest ticks containing Francisella species, FLEs, and spotted fever groupRickettsia are dispersed by n