Cytauxzoon europaeus infections in domestic cats in Switzerland and in European wildcats in France: a tale that started more than two decades ago.

BACKGROUND: Cytauxzoon spp. infection is believed to be a newly emerging tick-borne disease in felids in Europe, with three species of the haemoparasite having recently been differentiated in wild felids. In Switzerland, rare infections have been documented in domestic cats in the west and northwest of the country, the first of which was in 2014. The aims of the present study were: (i) to characterize a Cytauxzoon spp. hotspot in domestic cats in central Switzerland; (ii) to elucidate the geographic distribution of Cytauxzoon spp. in domestic cats in Switzerland; (iii) to assess suspected high-risk populations, such as stray and anaemic cats; and (iv) to investigate the newly emerging nature of the infection. Cytauxzoon spp. were further differentiated using mitochondrial gene sequencing. METHODS: The overall study included samples from 13 cats from two households in central Switzerland (study A), 881 cats from all regions of Switzerland (study B), 91 stray cats from a hotspot region in the northwest of Switzerland and 501 anaemic cats from across Switzerland (study C), and 65 Swiss domestic cats sampled in 2003 and 34 European wildcats from eastern France sampled in the period 1995-1996 (study D). The samples were analysed for Cytauxzoon spp. using real-time TaqMan quantitative PCR, and positive samples were subjected to 18S rRNA, cytochrome b (CytB) and cytochrome c oxidase subunit I (COI) gene sequencing. RESULTS: In study A, six of 13 cats from two neighbouring households in central Switzerland tested postive for Cytauxzoon spp.; two of the six infected cats died from bacterial infections. In studies B and C, only one of the 881 cats (0.1%; 95% confidence interval [CI]: 0-0.3%) in the countrywide survey and one of the 501 anaemic cats (0.2%; 95% CI: 0-0.6%) tested postive for Cytauxzoon spp. while eight of the 91 stray cats in the northwest of Switzerland tested positive (8.8%; 95% CI: 3.0-14.6%). In study D, Cytauxzoon spp. was detected in one of the 65 domestic cat samples from 2003 (1.5%; 95% CI: 0-4.5%) and in ten of the 34 European wildcat samples from 1995 to 1996 (29%; 95% CI: 14.2-44.7%). The isolates showed ≥ 98.6% sequence identities among the 18S rRNA, CytB and COI genes, respectively, and fell in the subclade Cytauxzoon europaeus based on CytB and COI gene phylogenetic analyses. CONCLUSIONS: The study challenges the newly emerging nature of Cytauxzoon spp. in central Europe and confirms that isolates from domestic cats in Switzerland and European wild felids belong to the same species.

De novo genome sequencing and comparative stage-specific transcriptomic analysis of Dirofilaria repens.

The zoonotic mosquito-borne filarial nematode Dirofilaria repens causes subcutaneous and ocular infections in dogs, cats and humans. From infected vertebrate hosts, microfilariae are taken up by mosquitoes and develop into infective L3. These are transmitted to new vertebrate hosts and develop over two further moults to adult worms. The aims of the project were (i) the de novo sequencing and annotation of the D. repens genome and (ii) comparative transcriptomic analyses of the two developmental stages, mf and L3. Genomic DNA was obtained from adult male D. repens. RNA was extracted from mf from naturally infected dogs and from L3 produced in Aedes aegypti mosquitoes fed on blood spiked with mf. The 99.59 MB genome was approximately 17% larger than that of the related species Dirofilaria immitis (dog heartworm) and contained 8.9% fewer predicted genes (10,357). Approximately 1.8% of identified proteins (206/11,262) could not be mapped to D. immitis. Out of these, six (2.9%) presented an ortholog in all other considered filarial nematodes (e.g. Loa loa) and Caenorhabditis elegans. A significantly higher number of D. repens proteins, compared with D. immitis, mapped to the filarial nematode L. loa, reflecting the similarity in biology of D. repens and L. loa. A total of 876 genes were differentially expressed, of which 591 could be annotated in UniProtKB/Swiss-Prot. In particular, 155 genes with a UniProtKB/Swiss-Prot annotation to C. elegans and filarial nematodes were upregulated in the L3 and 57 in the mf stage, respectively. Fifteen Gene Ontology Biological Processes were significantly enriched for the L3 group and 12 for the mf. To our knowledge these data provide the first insight into the differential gene expression profiles of this filarial nematode and can serve future investigations of metabolic processes and stage-specific diagnostics.