Helminth fauna in roe deer (Capreolus capreolus Linnaeus, 1758) in the province of Grosseto (central Italy)

The roe deer (Capreolus capreolus L., 1758) is the most abundant and widespread species of deer in Italy and one of the most important species from a hunting perspective. After a historical population contraction over the last few decades, which affected Italy and most of Europe and led the species to near extinction, the roe deer is now widespread throughout the continent's forests (Lovari et al., 2016). In Italy, it is found in two main sub-areas: the Alps, and Apennine chain with small, isolated populations in the south of the peninsula (Carnevali et al., 2009). The causes of its recovery in Italy are thought to be the reduction in agricultural practices in hilly and mountainous areas, and the progressive increase in wooded and forest areas, as well as regulated hunting management (Perco, 2011).

Several studies have shown how the roe deer is host to numerous parasites, some of which are important not only in terms of its own health but also for domestic ruminants with which it is increasingly coming into contact (Vetyska, 1980; Ladini, 1989; Aguirre et al., 1999; Rehbein et al., 2000; Diaz et al., 2010; Pato et al., 2013; Beaumelle et al., 2022).

Helminth infections have been documented in several studies in Europe, but data on the taxonomic composition of helminths in C. capreolus in Italy are out of date, not homogeneous, and not continuous (Genchi et al., 1987; Poglayen et al., 1990; Rossi et al., 1997; Zaffaroni et al., 2000).

The most relevant investigations on the helminth fauna of roe deer in Italy have mostly been carried out in the Alpine region. Twenty-two species of gastrointestinal helminths have been found, of which the genus Spiculopteragia, Ostertagia, and Nematodirus were the most abundant (Rossi et al., 1997). Sixteen species were detected in the abomasa of C. capreolus, many of which are shared with other wild ruminants (Zaffaroni et al., 2000). An older investigation on gastrointestinal nematodes in Trento, showed a high prevalence of Ostertagia leptospicularis and Spiculopteragia spiculoptera, Haemonchus contortus, and the species Nematodirus filicollis and Nematodirus rupicaprae, the latter as a consequence of the promiscuity of receptive hosts (wild and domestic ruminants) in their habitat (Genchi et al., 1987).

Although Tuscany hosts one of the most abundant Italian roe deer populations, epidemiological investigations carried out to date are scarce. A survey on the helminth fauna of 60 roe deer (Capreolus capreolus) was conducted in the Apennines in the province of Florence (Italy). Two helminth species were found in the lungs, and 16 in the digestive system (Poglayen et al., 1996). Pacini et al., 2020 found samples positive for gastrointestinal strongyle eggs, Eimeria spp. Oocysts, and numerous nematode larvae of the species Varestrongylus capreoli.

The aim of the current study is to provide first insights into the helminth fauna of roe deer in the province of Grosseto (the southernmost part of Tuscany, Italy), an area that has never been investigated before, and to analyse the data collected in order to test whether sex, age, or territory differences lead to statistically significant differences in these infections.

This survey was carried out in several municipalities in the province of Grosseto (Tuscany, Italy), an area of 4503km² which are primarily agro-silvopastoral.

During the 2018 – 2019 / 2019 – 2020 hunting seasons, the gastrointestinal and bronchopulmonary organs of 54 roe deer were collected and analysed at the Department of Veterinary Sciences of the University of Pisa, Italy. From the two hunting seasons, 31 adults (23 females, 8 males) and 23 juveniles (11 females, 12 males) were sampled. Samples were also collected in two different climatic areas of Grosseto: a coastal area characterized by an annual mean temperature of 15 – 16°C and annual mean precipitation of 600 – 700 mm; and a hilly-mountain area with an annual mean temperature of 12 – 14°C and annual mean precipitation of 800 – 1000mm (Sforzi & Ragni, 1997).

The samples were not always received complete with all the organs, and thus the total number of samples examined varied according to the type of analysis performed. In terms of the gastrointestinal tract, the abomasum, small intestine, and large intestine were analysed by the sedimentation and counting technique (SCT) (Eckert et al., 2003). The liver and cardiorespiratory system tract, which includes the trachea and the lungs, were visually inspected under a stereomicroscope. The lungs were dissected and washed with tap water in a conical beaker to collect the parasites. Each nematode was clarified with lactophenol, and morphologically identified.

Coprological analyses were conducted on faeces extracted directly from the rectal ampulla of the sample using the Mini-Flotac technique (Cringoli et al., 2017) with a solution of ZnCl₂ (p.s. 1.200).

For the adult parasites, the following epidemiological indices were calculated (Margolis et al., 1982; Bush et al., 1997): prevalence, mean intensity, intensity, and range.

Data were analysed using OpenOffice Calc 4.1.6 and R software version 3.1.2. (R Development Core Team, 2014).

We use the term prevalence (P) to indicate the ratio between the number of hosts infected and the sample size, mean intensity (MI) to indicate the ratio between the total number of parasites and the number of hosts infected.

The aim of the statistical analysis was to compare the prevalence and mean intensity between animals of different ages (young vs adults), sex (males vs females), and habitats (coast vs hilly-mountain). To better highlight any differences and understand whether these may be due to the single parasite taxon or are linked only to the difference between the three parameters mentioned above, it would be useful to extend the statistical comparisons to each parasite taxon. However, the high quantity of different parasite taxons infesting the animals analyzed, combined with a sample of about 50 units, would make it statistically irrelevant to test each taxon.

Before carrying out the tests, we thus put the individual parasites into groups linked by some affinities and tested the age, sex, habitat for each family. Any differences found in the single-family tests could therefore highlight a greater vulnerability of the animals to that particular family. On the other hand, any differences in the global tests (which include all parasites taxon at once) may highlight a particular vulnerability of animals of a certain age, sex, or habitat to infestations in general.

Based on the data collected, several tests (Pearson chi-squared and Mann-Whitney U test) were performed to check for differences between male and female specimens and between young and adult animals. Two-tailed Pearson's chi-square test was used for the prevalence, as all the hypotheses required were met. A median comparison test was used on mean intensity (namely, the Mann-Whitney, two-tailed U test: H0 = the distribution in the population is the same vs H1 = the distributions are different) since the distribution of values was far from normal, the sample sizes were relatively small (from 9 to 30 sample units) and no specific hypothesis was made regarding the distribution of the mean intensity values.

The different areas (coast or hilly-mountain) were also evaluated to investigate any statistical differences (H0 = no difference vs H1 = difference).

Since the number of non-parasitized animals was too low, a global (i.e. on the whole sample) check for prevalence on the entire sample would be statistically irrelevant. Only global tests on mean intensity values were thus run. According to our targets, the parasites found in the sample were then divided according to the families they belong to: Chabertiidae (Popova, 1952) (Oesophagostomum venulosum and Chabertia ovina), Trichostrongylidae (Leiper, 1912) (Trichostrongylus colubriformis, Trichostrongylus spp., and Ostertagia ostertagi), and Trichuridae (Ransom, 1911) (Trichuris spp.). A test for prevalence and mean intensity for each family was then run.

Since the number of total tests across all families was 18, Bonferroni's correction was applied to reduce the risk of false positives at the cost of increasing the chance of false negatives (reducing the tests’ power).

All animal procedures used in this study agreed with the ethical and animal welfare concerns of the Committee on the Ethics of Animal Experiments of Minimally Invasive Surgery Centre and fully complied with recommendations outlined by Italian law. Animals were hunted following regional hunting laws (Regolamento di attuazione della legge regionale 12 gennaio 1994 n 3 DPGR 48/R/2017).

In terms of gastrointestinal helminths in the small intestine, the nematode Trichostrongylus colubriformis (Trichostrongylidae) in our study (41.2 %) was found with a higher prevalence than in other studies carried out in Italy (Poglayen et al. 1990, 1996; Zaffaroni et al., 2000; Rossi et al., 1997) and in Europe (Table 2).

It is not easy to understand the reasons for the high prevalence found in our study compared to other surveys in Italy and Europe. One hypothesis is that our study area is a favorable environment for the spread of this parasitosis in roe deer and more generally for the genus Trichostrongylus spp. in other animal species. In Lepus europeus for example, in the same area as this survey, the species Trichostrongylus retortaeformis showed a higher prevalence than other parasites (Sergi et al., 2017 and Macchioni et al., 2022). A prevalence of 7.8 % (4/51) of the cestode Moniezia expansa was found in the present study which is, to the best of our knowledge, the first report of the species in roe deer in Italy, while it is common in domestic ruminants. A similar prevalence was found in roe deer in Poland (Tomczuk et al., 2017), while higher prevalences have been reported in Romania (Hora et al., 2017) and Switzerland (Andrews et al., 1974). Lower prevalences, however, have been found in Sweden (Aguirre et al., 1999), and Ukraine (Kuzmina et al., 2010) (Table 2).

To the best of our knowledge, the prevalence of the genus Trichuris spp. (53.8 %), reported in our study is the highest among previous surveys carried out to date in Italy on roe deer (Table 2). Oeosphagostomum venulosum was the second most frequent parasite found in the large intestine (50 %). In previous studies, carried out in Italy on roe deer, lower prevalences have been found (Poglayen et al., 1990; Poglayen et al., 1996). In Europe, on the other hand, equal prevalences have been found in Germany (Rehbein et al., 2000), Spain (Pato et al., 2013), Turkey (Umur et al., 2011), and slightly lower, again in Turkey (Bolukbas et al., 2012). Lower prevalences have been found in the Czech Republic (Vetyska, 1980), Austria (Schwarz et al., 2011), and Poland (Tomczuk et al., 2017; Balicka-Ramisz et al., 2003)

Chabertia ovina showed the lowest prevalence in the large intestine 26.9 % (41.2 %) in the present study. The only Italian report on roe deer showed a higher prevalence of 71 % (Poglayen et al. 1990), whereas, in Europe, the prevalence varies according to the region considered (Table 2).

In the present study, the only parasite found in the abomasum was Ostertagia ostertagi, which has already been reported in Italy (Poglayen et al., 1990; Poglayen et al., 1996; Rossi et al., 1997; Zaffaroni et al., 2000) and also throughout Europe (Andrews et al., 1974; Vetyska, 1980; Borgsteede et al., 1990; Drozdz et al., 1992; Korsholm et al., 1992; Aguirre et al., 1999; Rehbein et al., 2000; Kusak et al., 2012).

The liver examination was conducted on 33 samples. Nematodes belonging to Setaria tundra species (Onchocercidae) were found only on the surface of two livers. The first report in Italy for Setaria tundra was in the province of Turin (Piedmont region, Favia et al., 2003) in the abdominal cavity of four roe deer. More recently Setaria tundra was reported in the province of Gorizia in an analysis conducted on roe deer with a prevalence of 47.6 % (Beraldo et al., 2016), and its presence was associated with severe forms of peritonitis in the animals examined. Currently, Setaria tundra is widespread in 15 European countries, with Finland being the northernmost limit and Spain the westernmost and furthest from the site of the first report in Russia in 1928 (Oloś et al., 2021). In Europe, Setaria tundra has recently been reported in roe deer in Croatia (Čurlík et al., 2019) and Spain (Angelone-Alaasad et al., 2016).

Of the 34 bronchopulmonary samples analysed, only the lung of an adult female was positive for the presence of Dictyocaulus spp. In Italy, reports regarding lung helminths in roe deer are scarce. A low prevalence of 6.6 %, but still higher than in our study, was found in the province of Florence by Poglayen et al. (1996).

As far as Europe is concerned, a prevalence not higher than 30 % has been reported for the genus Dictyocaulus (Vazquez et al., 2009; Morrondo et al., 2017) (Table 2).

Some studies have highlighted differences in prevalence and intensities of gastrointestinal helminths between adult roe deer and fawns (Borgsteede et al., 1990; Segonds-Pichon et al., 2000), while another study also reported no significant differences in both parameters (Pato et al., 2013).

The susceptibility to parasites may be due to the influence of morphological, hormonal, and behavioral factors that characterize both sexes in certain periods of the year (Zuk & Mckean, 1996), which thus makes it difficult to highlight significant differences between the sexes. The differences between hunting policies, which can differ a lot from country to country, brings to different sampling types and makes even more difficult to compare different surveys. In our study, only the global test on mean intensity highlighted a statistically significant difference and only between male and female roe deer, while no statistically significant difference between mean intensities in young and adult animals, or in animals living in coastal or mountain areas (p-value > 0.05) were found. It is likely that the variations in temperature and rainfall in the two areas were not relevant to the parasite's spread and development. Pato et al., (2013) found statistically significant differences in different climatic areas in Spain only for the genera Trichuris, Oesophagostomum, and Spiculopteragia, which were more prevalent in the coastal area.

Our survey involved an exploratory study of the parasites in roe deer in the Grosseto area, for which there are no data in the literature. The results of the present study indicate that roe deer harbor several species of parasites.

In our study the parasite intensity found was relatively low. However, it should be highlighted that in the case of massive infection, these parasites play an important role in the health of roe deer and domestic ruminants. In fact, our results indicate that the roe deer hosts several species of parasites, which are also common in other cervids and domestic ruminants. It is thus crucial to address the problem within the One Health approach to prevent harm to farms and wild animals.