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The red fox acts as reservoir for several helminthic infections which are of interest for both public and animal health. Huge efforts have been made for the assessment of the sensitivity of coprological tests for the detection of Echinococcus multilocularis, while less attention has been paid to other helminthic species. This study aimed at assessing the performance of two copromicroscopic techniques in the detection and prevalence estimation of gastrointestinal helminths in the red fox. Helminths were isolated from the small intestines of 150 red foxes from Bolzano province, Italy, with a scraping, filtration and counting technique (SFCT) and morphologically identified. Rectal contents were collected and submitted to simple flotation (FT) and, only for Taenids, a method based on the concentration of eggs and identification with multiplex PCR (CMPCR). Using SFCT as a reference standard, we estimated the sensitivity of the copromicroscopic tests. Three species of nematodes (namely, Toxocara canis, Uncinaria stenocephala and Pterygodermatites sp.) and five species of cestodes (E. multilocularis, Taenia crassiceps, T. polycantha, Hydatigera taeniaeformis, Mesocestoides sp.) were identified with SFCT, whereas eggs referable to the same taxa were detected with fecal diagnostics, except for Pterygodermatites sp. and Mesocestoides sp. The sensitivity of FT was low for all taxa, ranging from 9.8 to 36.3%, with lower values for Taeniidae. CMPCR was confirmed to perform better for the detection of Taeniidae eggs (23.5%) and the multiplex PCR on retrieved eggs was effective in the identification of the species. A meta-analysis of literature also suggested that our results are consistent with existing data, indicating that copromicroscopy tends to underestimate the prevalence of helminthic infections. The extent of such underestimation varies with taxon, being higher at high prevalence levels, in particular for cestodes. Irregular dynamics of egg shedding, and routine deep freezing of red fox feces may explain the frequency of false negatives with copromicroscopy. Low sensitivity of copromicroscopic tests should be accounted for when estimating prevalence and when defining the correct sample size for the detection of the parasites.

Surveillance of Echinococcus multilocularis at the edge of its range is hindered by fragmented distributional patterns and low prevalence in definitive hosts. Thus, tests with adequate levels of sensitivity are especially important for discriminating between infected and non-infected areas. In this study we reassessed the prevalence of E. multilocularis at the southern border of its distribution in Province of Bolzano (Alto Adige, northeastern Alps, Italy), to improve surveillance in wildlife and provide more accurate estimates of exposure risk. We compared the diagnostic test currently implemented for surveillance based on coproscopy and multiplex PCR (CMPCR) to a real-time quantitative PCR (qPCR) in 235 fox faeces collected in 2019 and 2020. The performances of the two tests were estimated using a scraping technique (SFCT) applied to the small intestines of a subsample (n = 123) of the same foxes as the reference standard. True prevalence was calculated and the sample size required by each faecal test for the detection of the parasite was then estimated. True prevalence of E. multilocularis in foxes (14.3%) was markedly higher than reported in the last decade, which was never more than 5% from 2012 to 2018 in the same area. In addition, qPCR showed a much higher sensitivity (83%) compared to CMPCR (21%) and agreement with the reference standard was far higher for qPCR (0.816) than CMPCR (0.298) meaning that for the latter protocol, a smaller sample size would be required to detect the disease. Alto Adige should be considered a highly endemic area. Routine surveillance on definitive hosts at the edges of the E. multilocularis distribution should be applied to smaller geographic areas, and rapid, sensitive diagnostic tools using directly host faeces, such as qPCR, should be adopted.

The assessment of red fox population density is considered relevant to the surveillance of zoonotic agents vectored by this species. However, density is difficult to estimate reliably, since the ecological plasticity and elusive behavior of this carnivore hinder classic methods of inference. In this study, red fox population density was estimated using a non-invasive molecular spatial capture-recapture (SCR) approach in two study areas: one in a known hotspot of the zoonotic cestode Echinococcus multilocularis, and another naïve to the parasite. Parasitological investigations on collected samples confirmed the presence of the parasite exclusively in the former area; the SCR results indicated a higher fox population density in the control area than in the hotspot, suggesting either that the relationship between fox density and parasite prevalence is not linear and/or the existence of other latent factors supporting the parasitic cycle in the known focus. In addition, fox spotlight count data for the two study areas were used to estimate the index of kilometric abundance (IKA). Although this method is cheaper and less time-consuming than SCR, IKA values were the highest in the areas with the lower molecular SCR density estimates, confirming that IKA should be regarded as a relative index only.