AUTHOR=Grillini Marika , Beraldo Paola , Frangipane di Regalbono Antonio , Dotto Giorgia , Tessarin Cinzia , Franzo Giovanni , Marchiori Erica , Modrý David , Simonato Giulia 

TITLE=Molecular survey of Cytauxzoon spp. and Hepatozoon spp. in felids using a novel real-time PCR approach

JOURNAL=Frontiers in Veterinary Science

VOLUME=10

YEAR=2023

URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2023.1113681

DOI=10.3389/fvets.2023.1113681

ISSN=2297-1769

ABSTRACT=<p>Tick-transmitted apicomplexans of the genera <italic>Cytauxzoon</italic> and <italic>Hepatozoon</italic> affect a wide range of felids worldwide, but little is known about them. Recently, several studies addressed the species circulating in Europe, their distribution, and their hosts. Molecular assays are the method of choice for their detection. Unfortunately, conventional PCRs already described are time- and cost-consuming and specific for either <italic>Hepatozoon</italic> or <italic>Cytauxzoon</italic> detection. This study was developed to evaluate (i) the occurrence of <italic>Cytauxzoon</italic> and <italic>Hepatozoon</italic> in felids using a fast and cost-saving real-time PCR capable of detecting both protozoa simultaneously, (ii) the distribution of <italic>Cytauxzoon</italic> and <italic>Hepatozoon</italic> species in north-eastern Italy, and (iii) the involvement of other susceptible felid hosts in the same area. An SYBR<sup>®</sup> Green-based real-time PCR with primers targeting the 18S-rRNA was validated and applied to 237 felid samples, i.e., whole blood from 206 domestic cats and 12 captive exotic felids, and tissues from 19 wildcats. Positive results were obtained by melting temperature curve analysis due to the specific melting peak (i.e., 81°C <italic>Cytauxzoon</italic> spp.; 78–78.5°C <italic>Hepatozoon</italic> spp.). Positive samples were subjected to conventional PCR, followed by sequencing for species identification. Phylogenetic analyses were performed to assess relatedness among European isolates. Data on domestic cats (age class, sex, origin, management, and lifestyle) were recorded, and statistical analyses were performed to identify potential risk factors. A total of 31 (15%) domestic cats were positive for <italic>Hepatozoon</italic> spp. (i.e., 12 for <italic>H. felis,</italic> 19 for <italic>H. silvestris</italic>), while six (2.9%) for <italic>C. europaeus</italic>. The prevalence of <italic>Hepatozoon felis</italic> was significantly (<italic>p</italic> &lt; 0.05) higher in domestic cats, while <italic>H. silvestris</italic> was higher in strays and animals from the Eastern region (i.e., Friuli-Venezia Giulia). <italic>Cytauxzoon europaeus</italic> was detected only in stray cats from Friuli-Venezia Giulia (province of Trieste). Among captive felids, one tiger was infected with <italic>H. felis</italic> and another with <italic>H. silvestris</italic>; eight out of 19 (42%) wildcats were positive for <italic>Hepatozoon</italic> spp. (i.e., six with <italic>H. felis</italic>, two with <italic>H. silvestris</italic>) and four out of 19 (21%) for <italic>Cytauxzoon europaeus</italic>. Outdoor lifestyle and origin (i.e., Friuli-Venezia Giulia region) were the most relevant risk factors for <italic>H. silvestris</italic> and <italic>C. europeus</italic> infections. Conversely, <italic>H. felis</italic> was most frequently isolated from domestic cats, suggesting different modes of transmission.</p>