Exploring the epidemiological role of the Eurasian lynx (Lynx lynx) in the life cycle of Toxoplasma gondii Author Scherrer, Patrick Institute for Fish and Wildlife Health, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty University of Bern, L ¨ anggassstrasse 122, 3012, Bern, & Institute of Parasitology, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty University of Bern, L ¨ anggassstrasse 122, 3012, Bern, Switzerland Author Ryser-Degiorgis, Marie-Pierre Institute for Fish and Wildlife Health, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty University of Bern, L ¨ anggassstrasse 122, 3012, Bern, Author Marti, Iris A. Institute for Fish and Wildlife Health, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty University of Bern, L ¨ anggassstrasse 122, 3012, Bern, Author St Author Borel, ephanie Institute for Fish and Wildlife Health, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty University of Bern, L ¨ anggassstrasse 122, 3012, Bern, Author Frey, Caroline F. Institute of Parasitology, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty University of Bern, L ¨ anggassstrasse 122, 3012, Bern, Switzerland Author Mueller, Norbert Institute of Parasitology, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty University of Bern, L ¨ anggassstrasse 122, 3012, Bern, Switzerland Author Ruetten, Maja PathoVet AG, Buckstrasse 2, 8317, Tagelswangen, Switzerland Author Basso, Walter Institute of Parasitology, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty University of Bern, L ¨ anggassstrasse 122, 3012, Bern, Switzerland text International Journal for Parasitology: Parasites and Wildlife 2023 2023-08-31 21 1 10 http://dx.doi.org/10.1016/j.ijppaw.2023.03.005 journal article 10.1016/j.ijppaw.2023.03.005 2213-2244 PMC10074407 37032843 10668262 4.4. Occurrence of T. gondii tissue stages Tissue stages were rarely found in this study. Tissue cyst abundance in felids is indeed known to be low. At the FIWI , lynx organs are systematically screened by histology for general health evaluation ( Ryser-Degiorgis et al., 2021 ) but tissue cysts of any kind were rarely observed ( FIWI , unpublished data). Re-evaluation of selected cases in this study revealed a higher detection of protozoan tissue stages than initially recorded in routine diagnostics. Tissue cysts are small in early stages of infection and therefore easily missed. While mature T. gondii and Sarcocystis tissue cysts and Hepatozoon schizonts are easily identifiable in histological sections, early stages of these parasites might be occasionally misidentified, which makes diagnosis solely based on morphological criteria challenging. PCR techniques represent an adequate aid for parasite identification in histological sections. However, it has a low sensitivity, as parasite stages may be not present in all histological sections. Moreover, formalin fixation may cause DNA 7 damage and a molecular diagnosis might be not possible if tissue samples were fixed for long periods. Immunohistochemical techniques also aid in the morphological diagnosis of protozoan tissue stages. In this study, T. gondii was specifically detected by IHC staining in one animal. Sarcocystis tissue cysts (morphologically identified in H&E stained sections) did not stain by IHC for T. gondii ; therefore, cross-reactions with early stages of Sarcocystis were unlikely. In two further IHC-negative cases with protozoan parasite stages that were not clearly identifiable in H&E, subsequent PCR and sequencing enabled their attribution to H. silvestris . This Hepatozoon species was already identified as cause of fatal myocarditis in a domestic cat in Switzerland before ( Kegler et al., 2018 ). Hepatozoon is frequently found in European wild cats (Hodˇzic´ et al., 2017), which share lynx habitat in Switzerland . Hepatozoon spp. were also reported in free-ranging Iberian lynx, bobcats and ocelots ( Felis pardalis ) ( Mercer et al., 1988 ; Metzger et al., 2008 ; Allen et al., 2011 ). In this study, observed tissue cysts/parasite stages were not associated with inflammatory or necrotic processes. This is in accordance with felids not being highly susceptible for clinical toxoplasmosis. Only a few cases of toxoplasmosis with fatal outcome are reported in wild felids, all in captive individuals, including a newborn bobcat that died one week after birth ( Dubey et al., 1987 ) and juvenile Pallas’ cats ( Otocolobus manul ) ( Riemann et al., 1974 ; Kenny et al., 2002 ; Basso et al., 2005 ).