Toxoplasma gondii, (Nicolle & Manceaux, 1908)

Scherrer, Patrick, Ryser-Degiorgis, Marie-Pierre, Marti, Iris A., St, Borel, ephanie, Frey, Caroline F., Mueller, Norbert, Ruetten, Maja & Basso, Walter, 2023, Exploring the epidemiological role of the Eurasian lynx (Lynx lynx) in the life cycle of Toxoplasma gondii, International Journal for Parasitology: Parasites and Wildlife 21, pp. 1-10 : 8

4.5. Genotyping T. gondii in lynx

Recent studies, especially in Brazil, have shown that there is greater genetic variability in T. gondii than initially believed ( Vitaliano et al., 2014; Witter et al., 2020). An overview of the known allele-type combinations is provided by the ToxoDB database (https://toxodb.org/ toxo/app, ToxoDB, accessed 09.08.2022).

This is the first T. gondii genotyping study in wild carnivores from Switzerland. A complete multilocus genotyping could only be achieved for four animals, though T. gondii was identified in more individuals. The main difficulty was to obtain samples with sufficient DNA amount. The well-known and widespread genotype #3 (type II variant; Shwab et al., 2014) was detected in three of the animals. Toxo DB #3 has previously been isolated from European wildlife several times. This genotype was detected in arctic foxes from Norway, wildcats and Eurasian beavers ( Castor fiber ) from Germany and even dolphins from the Mediterranean Sea ( Prestrud et al., 2008; Herrmann et al., 2013; Fern´andez-Escobar et al., 2022b). Also in Switzerland, genotype ToxoDB #3 was already detected in domestic cats and voles ( Berger-Schoch et al., 2011; Spycher et al., 2011; Pardo Gil et al., 2023). Genotype II is typically considered intermediate to non-virulent in mice, causing mostly subclinical infections ( Sibley and Boothroyd, 1992; Wendte et al., 2011). Nevertheless, genotype II was associated with fatal infections in 32 animals from altogether five different species ( Jokelainen, 2012), including a fatal case in a domestic cat from Switzerland ( Spycher et al., 2011). Genotype III is strongly represented and largely considered of low virulence ( Sibley and Boothroyd, 1992).

Furthermore, a new, unknown genotype was found in skeletal muscle of a juvenile lynx. In the newly discovered allele pattern, six of the 10 tested markers corresponded to type III alleles, two to type II, one to type I (Apico), as well as one marker ( SAG1 ) where a type II or III allele was possible. The obtained sequences for all markers were of a very good quality and did not show the presence of double peaks, which could suggest a coinfection with two T. gondii genotypes in the same analysed tissue sample. Therefore, this genotype could represent a II x III recombinant strain, as it was observed in several studies in Europe (Fern´andez-Escobar et al., 2022b).

It is known that RFLP is a good tool for tracing ancestry from historical archetypes, as certain sequences are required for enzyme digestion. However, SNPs can only be detected with complete sequencing of the marker sequence. Strictly speaking, occurring SNPs would lead to a new classification or to description of more non-clonal genotypes ( Wendte et al., 2011). In this study, due to sequencing and in silico digestion, complete marker sequences were available for analysis and were compared to reference sequences of archetypal T. gondii types I ( GT1 ; ToxoDB genotype #10), II ( ME49 ; ToxoDB genotype #1) and III ( VEG; ToxoDB genotype #2). The observed SNP in the SAG3 sequence of a lynx (ID: W21_0845; OQ230332) has also been detected in sheep in Spain (Fern´andez-Escobar et al., 2020). There , it was also noticed that this mutation leads to a change in the codon at this position. Whether this results in a change in pathogenicity has not been investigated in detail, but abortions with this genotype and mutation have been observed in the same sheep. Whether the occurrence of this SNP might be frequent throughout Europe is currently unknown because most of the studies performed traditional RFLP analysis without sequencing of the obtained amplicons. There were no deviations from the original types I, II and III in the rest of the complete sequences.

An important question is the relationship between Toxoplasma detection (whether in faeces or tissue) and the animal clinical status. In the case of the young lynx with the novel genotype, T. gondii cysts were also detected in the heart but they were not associated with inflammatory reaction. This animal was an orphan in a wildlife rescue centre that died of a severe purulent peritonitis with concomitant Yersinia pseudotuberculosis infection ( Morend et al., 2022). This infection may have happened either via preying on rodents or eating contaminated food. It is unclear whether Toxoplasma found its way into the organism via the same route. In this case, the infection was an incidental finding. Pathological changes associated with Toxoplasma cysts or DNA were not found in the other animals in this study either. These findings suggest that despite the presence of the parasite, clinical and pathological changes would be exceptional. Congenital toxoplasmosis can occur ( Dubey et al., 1987) and therefore neonates would be most likely to develop toxoplasmosis but there were no neonatal animals in this study. Indeed, such young animals are hardly ever found under free-ranging conditions.

In conclusion, this study showed that the Eurasian lynx can act as an intermediate and final host of T. gondii by demonstrating oocyst shedding and tissue cyst occurrence. Genotyping revealed the presence of both a locally common T. gondii lineage and a previously undescribed genotype. Investigation of lynx prey in the same study area including genotyping would increase the understanding of T. gondii epidemiology in Swiss wildlife.

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Supplementary data associated with this article.