In the last decades, wildlife diseases and the health status of animal populations have gained increasing attention from the scientific community as part of a One Health framework. Furthermore, the need for non-invasive sampling methods with a minimal impact on wildlife has become paramount in complying with modern ethical standards and regulations, and to collect high-quality and unbiased data. We analysed the publication trends on non-invasive sampling in wildlife health and disease research and offer a comprehensive review on the different samples that can be collected non-invasively. We retrieved 272 articles spanning from 1998 to 2021, with a rapid increase in number from 2010. Thirty-nine percent of the papers were focussed on diseases, 58% on other health-related topics, and 3% on both. Stress and other physiological parameters were the most addressed research topics, followed by viruses, helminths, and bacterial infections. Terrestrial mammals accounted for 75% of all publications, and faeces were the most widely used sample. Our review of the sampling materials and collection methods highlights that, although the use of some types of samples for specific applications is now consolidated, others are perhaps still underutilised and new technologies may offer future opportunities for an even wider use of non-invasively collected samples.

The Open Field Test (OFT) and Mirror Image Stimulation (MIS) are used to measure behaviours related to an individual’s personality. These tests, carried out in a same novel arena, have been used for different taxa, but only a few papers underline the importance of method validation. Here we investigate how Eurasian red squirrels (Sciurus vulgaris) and Eastern grey squirrels (Sciurus carolinensis) behave during OFT and MIS. Next, we compare the performance between three analytical methods: the Principal Component Analysis (PCA), the Factor Analysis (FA) and an expert-based (EB) method. The EB approach classifies behaviours in groups relating on researchers’ knowledge and returns personality-trait values for each individual facilitating comparisons over studies and/or with new datasets. The comparison between the three methods gave similar results and high repeatabilities in some expert-based personality traits as well as PCA components and FA factors, showing that all three methods were valid to measure activity using OFT (both species) and sociability using MIS (grey squirrel). Repeatabilities of the other traits were less strong. Proportion of time spent in different behaviours did not differ with test duration, since shorter tests yielded valid measures of individual differences in personality. Shorter tests reduce operator time in the field, and are likely to reduce stress and arena-habituation of the animals. Test sequence affected the outcome of OFT: squirrels tested for the first time were more active than squirrels tested a second time. For the two squirrel species investigated, we recommend an OFT of 4 min and a MIS test of three and suggest to test an individual no more than 2 times per season with at least 2 months between repetitions.

Competition between animal species can cause niche partitioning and shape an individual’s phenotype, including its behaviour. However, little is known about effects of interspecific competition on personality, the among-individual variation in behaviour that is consistent across different spatial and temporal contexts. We investigated whether alien grey squirrels (Sciurus carolinensis) influenced the expression of personality traits in native red squirrels (Sciurus vulgaris). In Italy, alien grey squirrels replaced native reds through competition for food resources and space, reducing breeding and recruitment in the native species. We compared personality of red squirrels in red-only (no interspecific competition) and red-grey (with interspecific competition) sites, using arena-tests. The trait activity was measured by Open Field Test while sociability and avoidance were quantified by Mirror Image Stimulation test. Red squirrels co-occurring with the alien species had higher sociability scores and higher between-individual variation in sociability than in red-only sites. Differences in activity and avoidance were not significant. Personality – fitness relationships were not affected by presence or absence of grey squirrels, suggesting that the expression of sociability in red squirrels was not due to short-term selection, but was likely the result of context-related advantages when co-occurring with the competing species.

The EU Regulation no.1143/2014 on invasive species requires the EU Member States to respond promptly and efficiently in eradicating the invasive alien species listed as species of Union concern. The Northern raccoon (Procyon lotor) was introduced in Northern Italy along the river Adda and we carried out its eradication campaign between 2016 and 2019. We used an integrated approach of camera traps and live traps. A standardized approach to the camera trap survey (2904 camera trap-nights) revealed that the detectability of raccoons (0.27 ± 0.07 SD) increased in the proximity of the river and their occupancy (0.15 ± 0.09 SD) was higher in urban areas. These results allowed us to increase the trapping success whilst reducing the effort. During the captures, systematic collection of catch and effort data allowed to estimate population size (65 ± 7.8 raccoons) and thus to have a continuous update on how far the campaign was from the complete eradication of the species, allowing to adjust the trapping effort accordingly. We captured 69 raccoons (7279 cage trap nights) thanks to both systematic trapping and opportunistic activation of traps following stakeholders’ reports. Stakeholders supported the campaign and some of them played an important role in reporting sightings, signs, and allowing access to their private land. Camera trap data also allowed us to make a Rapid Eradication Assessment that estimated an eradication probability of 99% after 6 months without detections. We finally give indications for future control campaigns on the species to increase removal success, reducing the effort.

Eurasian red squirrels (Sciurus vulgaris) in the British Isles are the most recently discovered animal reservoir for the leprosy bacteria Mycobacterium leprae and Mycobacterium lepromatosis. Initial data suggest that prevalence of leprosy infection is variable and often low in different squirrel populations. Nothing is known about the presence of leprosy bacilli in other wild squirrel species despite two others (Siberian chipmunk [Tamias sibiricus], and Thirteen-lined ground squirrel [Ictidomys tridecemlineatus]) having been reported to be susceptible to experimental infection with M. leprae. Rats, a food-source in some countries where human leprosy occurs, have been suggested as potential reservoirs for leprosy bacilli, but no evidence supporting this hypothesis is currently available. We screened 301 squirrel samples covering four species (96 Eurasian red squirrels, 67 Eastern grey squirrels (Sciurus carolinensis), 35 Siberian chipmunks, and 103 Pallas’s squirrels (Callosciurus erythraeus)) from Europe and 72 Mexican white-throated woodrats (Neotoma albigula) for the presence of M. leprae and M. lepromatosis using validated PCR protocols. No DNA from leprosy bacilli was detected in any of the samples tested. Given our sample-size, the pathogen should have been detected if the prevalence and/or bacillary load in the populations investigated were similar to those found for British red squirrels.