Drivers of infection with Toxoplasma gondii genotype type II in Eurasian red squirrels (Sciurus vulgaris)

Abstract

Background: In September 2014, there was sudden upsurge in the number of Eurasian red squirrels (Sciurus vulgaris) found dead in the Netherlands. High infection levels with the parasite Toxoplasma gondii were demonstrated, but it was unclear what had caused this increase in cases of fatal toxoplasmosis. In the present study, we aimed to gain more knowledge on the pathology and prevalence of T. gondii infections in Eurasian red squirrels in the Netherlands, on the T. gondii genotypes present, and on the determinants of the spatiotemporal variability in these T. gondii infections. The presence of the closely related parasite Hammondia hammondi was also determined.

Methods: Eurasian red squirrels that were found dead in the wild or that had died in wildlife rescue centres in the Netherlands over a period of seven years (2014-2020) were examined. Quantitative real-time polymerase chain reaction was conducted to analyse tissue samples for the presence of T. gondii and H. hammondi DNA. Toxoplasma gondii-positive samples were subjected to microsatellite typing and cluster analysis. A mixed logistic regression was used to identify climatic and other environmental predictors of T. gondii infection in the squirrels.

Results: A total of 178 squirrels were examined (49/178 T. gondii positive, 5/178 H. hammondi positive). Inflammation of multiple organs was the cause of death in 29 squirrels, of which 24 were also T. gondii polymerase chain reaction positive. Toxoplasma gondii infection was positively associated with pneumonia and hepatitis. Microsatellite typing revealed only T. gondii type II alleles. Toxoplasma gondii infection rates showed a positive correlation with the number of days of heavy rainfall in the previous 12 months. Conversely, they showed a negative association with the number of hot days within the 2-week period preceding the sampling date, as well as with the percentage of deciduous forest cover at the sampling site.

Conclusions: Toxoplasma gondii infection in the squirrels appeared to pose a significant risk of acute mortality. The T. gondii genotype detected in this study is commonly found across Europe. The reasons for the unusually high infection rates and severe symptoms of these squirrels from the Netherlands remain unclear. The prevalence of T. gondii in the deceased squirrels was linked to specific environmental factors. However, whether the increase in the number of dead squirrels indicated a higher environmental contamination with T. gondii oocysts has yet to be established.

Keywords: Microsatellite typing; Monitoring; Oocyst; Parasite; Population structure; Sentinel; Squirrel; Toxoplasmosis; Zoonoses.

Fig. 1

Geographical distribution of the squirrels that were quantitative real-time polymerase chain reaction (qPCR) positive or negative for Toxoplasma gondii (A) or Hammondia hammondi (B)

Fig. 2

Cause of death determined from pathological examination of quantitative real-time polymerase chain reaction (qPCR) Toxoplasma gondii-positive squirrels (A) and real-time qPCR T. gondii-negative squirrels (B). Connections between dots indicate co-occurring conditions

Fig. 3

Quantification cycle (Cq) values of Toxoplasma gondii by quantitative real-time polymerase chain reaction (qPCR) for cause of death (COD) [comparison of Trauma and other (Remaining) CODs; these included inflammation of multiple organs, pneumonia, and undetermined causes] for individual organs [lung (n = 35) (A); liver (n = 35) (B); heart (n = 35) (C)]

Fig. 4

A–E Results of affinity propagation clustering (APC) using microsatellite (MS) genotyping data (n = 39), based on Bruvo's distance. A Variation in the number of parasite clusters indicated by APC. B Results of the principal coordinate analysis (colours correspond to the two clusters identified by APC). C Minimum spanning network of haplotypes (colour denotes parasite clusters according to APC; circle size corresponds to the total number of individuals with the same MS type; branch thickness is proportional to inferred genetic distance between haplotypes). D Geographic distribution of Toxoplasma gondii-positive squirrels (n = 39) harbouring the T. gondii MS types (n = 32; colours correspond to those of the clusters in B). E Representatives of the parasite clusters over time. Representatives of each of the clusters were observed for all of the periods, except for 2018–2019, for which there were only four typed samples