INTRODUCTION

Wildlife disease surveillance is crucial to identify changes in wildlife disease occurrence and epidemiology, and it is an essential part of the One-Health approach (Yon et al. 2019). Surveillance is required to identify new and reemerging pathogens to recognize possible changes in disease occurrence in host and vectors species, adopt appropriate measures to protect domestic animals and human health, and safeguard the wildlife ecosystem. A wildlife surveillance program targeting several pathogens has been running in the Lombardy Region of northern Italy for several years. The populations of wild boar (Sus scrofa), European brown hare (Lepus europaeus), fallow deer (Dama dama), red deer (Cervus elaphus), and roe deer (Capreolus capreolus) are sampled for the presence of antibodies against Brucella spp. Brucellosis is a worldwide zoonosis caused by a gram-negative facultative intracellular bacterium belonging to the genus Brucella. To date, 12 Brucella species have been identified from a wide spectrum of hosts (Khurana et al. 2021). Some species infect terrestrial mammals: Brucella abortus, Brucella melitensis, Brucella suis, Brucella ovis, Brucella canis, Brucella neotomae, and Brucella microti. Brucella ceti and Brucella pinnipedialis affect marine mammals. Novel species named Brucella papionis, Brucella vulpis, and Brucella inopinata were respectively isolated from baboons (Papio spp.), red foxes (Vulpes vulpes), and human breast implant infection, although the natural reservoir of these species remains uncertain (Scholz et al. 2010, 2016). Brucellosis causes significant economic losses in animal production, due to diminished milk yield, abortion, infertility, and other reproductive disorders. Considering its impact on human and animal health, brucellosis is a notifiable disease in many countries, including Italy (Khurana et al. 2021). Within the European Union (EU), livestock brucellosis caused by B. abortus, B. melitensis, and B. suis has been eradicated in many European Member States. Croatia and Spain are close to achieving eradication; conversely, Greece, Italy, and Portugal, although with declining incidence rates, still report the infection in their livestock populations. In the Lombardy, as well as in some other Italian regions, bovine, ovine, and caprine brucellosis have been eradicated for several years (European Food Safety Authority and European Centre for Disease Prevention 2021).

In addition, brucellosis represents a human health problem because Brucella spp. can infect humans as an incidental host. Generally, Brucella is transmitted to humans through the consumption of contaminated animal products, especially unpasteurized milk and cheeses, or through direct contact with infected tissues or secretions (Moreno 2014). Certain occupations, such as slaughterhouse workers, meat-packing employees, veterinarians, hunters, and laboratory workers are characterized by a higher risk of brucellosis (Pereira et al. 2020). The most pathogenic and invasive species for humans is B. melitensis, followed by B. abortus, B. suis, and B. canis (Khurana et al. 2021). Human brucellosis cases are rarely reported in Europe (Bagheri Nejad et al. 2020).

Despite their respective host preferences, various Brucella species have been reported in several wild animal species. In Europe, B. suis biovar 2 has been reported in wild boars and hares; B. melitensis and B. abortus (rarely reported in wildlife) have been reported in Alpine ibex (Capra ibex), chamois (Rupicapra sp.), Spanish ibex (Capra pyrenaica), and red deer (European Food Safety Authority and European Centre for Disease Prevention 2021). Native wild ruminants are mostly considered as dead-end hosts rather than as true reservoirs for Brucella spp. (Ferroglio et al. 1998; Muñoz et al. 2010) and may serve as an epidemiological sentinel for the presence of B. melitensis in domestic livestock (Godfroid et al. 2013).

The presence of B. suis has been reported in wild boar and hares in some EU states for decades, and these two species have been identified as reservoir of B. suis in Europe (European Food Safety Authority 2009). Data on B. suis infection in European brown hares are limited and reported seroprevalences range from 0 to 17% in different parts of Europe (Winkelmayer et al. 2005; Tsokana et al. 2020). Seroprevalence of Brucella spp. infection in wild boars in Europe spans from 0 to 60% (Cvetnic et al. 2004; Wu et al. 2011; Grégoire et al. 2012; Hälli et al. 2012; Risco et al. 2014). There are no available official data suggesting that B. suis is currently present in any of the indoor commercial pig holdings in the EU (European Food Safety Authority 2009). Nevertheless, the prevalence observed in wild boar suggests that this species might act as a potential source of transmission of B. suis biovar 2 to domestic pigs in outdoor farming systems or backyard herds. Brucella suis biovar 2 has been isolated from a semi–free-range pig farm in Italy (Barlozzari et al. 2015).

The aim of our study was, through the analysis of the data collected during the wildlife surveillance program, to describe Brucella exposure and diffusion in the sampled wildlife, information useful for risk analysts to carry out a rapid qualitative-quantitative risk assessment, and to estimate the risk of livestock and human exposure to Brucella spp., to inform consistent and effective control measures against brucellosis in a One Health perspective.

MATERIALS AND METHODS

Data analysis

Considering the nonprobabilistic nature of sampling for data generation, which exposes it to an unpredictable selection bias in magnitude and direction, only the overall proportion of positive samples and the proportions in the different factors of interest were calculated and are expressed as percentages. A map (Fig. 1) was drawn using leaflet (Cheng et al. 2023) and sf (Pebesma 2018) packages in R (R Core Team 2023).

Figure1

Map showing the spatial distribution of backyard swine farms and Brucella seropositivity percentages in wild boar (Sus scrofa) in Cremona and Brescia provinces, Lombardy Region, northern Italy, 2017–20. The inset shows the position of the study region within Italy.

RESULTS

During 2017–20, 8,129 serum samples were tested for the presence of B. abortus, B. melitensis, and B. suis antibodies. Wild boar and hare were species most commonly represented in the samples, with 6,440 and 1,502 tested animals, respectively. The geographical distribution of the samples of these two species is summarized in Table 1. Except for wild boar, all the tested wild ungulates were seronegative (data not shown). We found three seropositive European brown hares (0.2%). In the wild boar, 2,304 animals were male (1,098 adults; 585 yearlings; 470 juveniles; 151 age unknown) and 2,541 were female (1,130 adults; 740 yearlings; 515 juveniles; 156 age unknown); sex information was not available for 1,595 carcasses (42 adults; 25 yearlings; 25 juveniles; 1,503 age unknown). The overall percentage positivity was 5.9%, ranging from a low of 0% to a maximum of 13.9% (Table 2). The number of tested and positive samples for each class is shown in Table 2. Regarding the sex classes, the positivity observed was 171/2,304 males, 119/2,541 females, and 88/ 1595 animals for which sex was unknown. By age classes, positivity was observed in 125/ 2,270 adults, 90/1,350 yearlings, 48/1,010 juveniles, and 115/1,810 animals with undefined age. The differences observed in the different sex and age groups were considered not to be epidemiologically relevant.

Table 1

Sample size by province of wild boar (Sus scrofa) and European brown hare (Lepus europaeus) populations in the Lombardy Region of northern Italy, 2017–20, tested for the presence of antibodies against Brucella spp.

Table 2

Number of tested (tot) and Brucella spp.–seropositive (pos) wild boars (Sus scrofa) and respective percentage of seropositive samples in Lombardy Region, northern Italy, 2017–20, separated by age and sex.

Results on Brucella serological tests of wild boars are shown by area in Table 3. Higher percentages of positivity were observed in samples of the southern provinces of Lombardy Region (Cremona, Pavia, Lodi) that are located near the course of the Po River. Conversely, wild boars of the pre-Alpine area of Brescia and Como provinces showed lower percentages of positive results. The spatial distribution of the backyard pig farms in the province of Brescia and Cremona and the Brucella serological results observed in wild boar in the municipalities of these two provinces are both depicted in Figure 1. Backyard pig farming is rare in the municipalities of Cremona where a high percentage of positive samples was recorded. Conversely, in the northern area of the province of Brescia, backyard farms are common in municipalities where low seropositivity was detected in wild boar.

Table 3

Number of the tested and positive wild boars (Sus scrofa) and respective percentage of Brucellaseropositive samples by province within the Lombardy Region, northern Italy, 2017–20.

Of 95 animals tested for Brucella spp. by culture of submandibular lymph nodes, 18 resulted positive for Brucella spp. (18.9%). All the isolates were confirmed as B. suis by qPCR; two were analyzed by PCR-RFLP and characterized as B. suis biovar 2 by the National Reference Centre for Brucellosis.

No lesions related to brucellosis were identified in any spleens examined. Conversely, of 92 wild boars' reproductive organs, 3 showed macroscopic pathological findings. Specifically, severe unilateral chronic orchitis was observed in two male yearlings (1–2 yr old) and miliary metritis, characterized by the presence of 2–3-mm nodules seeded on the uterine mucosa, was found in one female of the same age class (Fig. 2). Microbiological culture of the organs with pathological lesions and of the submandibular lymph nodes from the same individuals were negative for Brucella spp., whereas qPCR from testes and uterus with lesions was positive for B. suis. For a summary of the pathological, microbiological, and PCR findings, see Table 4.

Figure2

Severe unilateral chronic orchitis (A) and miliary metritis with 2–3-mm nodules (indicated by arrows) seeded on the uterine mucosa (B) caused by Brucella suis in 1–2-yr-old wild boars (Sus scrofa) from the Lombardy Region, northern Italy.

Table 4

Pathological, microbiological, and PCR findings in organs from wild boar (Sus scrofa) collected in the Cremona Province (Lombardy Region) of northern Italy in the 2018 hunter season.

DISCUSSION

We used serological data collected during the wildlife monitoring plan of the Lombardy Region, Italy, to describe the exposure of the wildlife species to Brucella spp. Given the nonprobabilistic nature of sampling characterizing this retrospective study, our results are biased and therefore should be evaluated with caution when interpreted as seroprevalence estimation.

Serology is an effective and inexpensive tool to carry out surveillance in wildlife and especially wild ungulates. Some wild ungulate species, such as wild boar, roe deer, and red deer, have the potential to colonize new territories of southern Europe (Vingada et al. 2010; Linnell et al. 2020) and thus may potentially carry zoonoses into new areas. All the wild ungulates tested in the current study, with the exception of wild boar, were seronegative for Brucella spp. Low Brucella seroprevalence in wild ungulates (0.4%), with the exception of wild boar, has also been reported in Spain (Muñoz et al. 2010).

The hare population included in the Lombardy Region wildlife surveillance program showed 0.2% seropositive samples. Similarly, low values were reported in other investigations: 3.54% in Austria with considerable regional variations (Winkelmayer et al. 2005); 0% in the Czech Republic in the period 1986–91 (Hubálek et al. 1993) and in 2003 (Winkelmayer et al. 2005); 0% in a German Region (Schleswig-Holstein) during 1998–2000 (Frölich et al. 2003); and 1.6% in the Czech Republic during 2004–06 (Treml et al. 2007).

Notwithstanding their low seroprevalence, hares are known to be a reservoir of B. suis (Godfroid 2018). In Italy in 1995, B. suis biovar 2 was detected in a male hare imported from Hungary (Quaranta et al. 1995). Over the time, several studies have proposed that B. suis was introduced in Italy through the importation of hares, for repopulating hunting areas, from Eastern Europe, where the infection was endemic in wild species (Ebani et al. 2003; Gennero et al. 2004; Bergagna et al. 2009; De Massis et al. 2012).

The higher detection rate of anti-Brucella immunoglobulins in wild boar (5.9%) than in other wildlife species is not surprising considering that this species is a well-known animal B. suis reservoir (Godfroid et al. 2013). The similar Brucella serological results observed in male and female wild boar are in accordance with those of previous studies (Montagnaro et al. 2010; Pilo et al. 2015). In contrast with previously reported data (Risco et al. 2014), we found that percentages of seropositive samples did not increase with the age of the animals; this difference may be attributed to the low number of positives samples for each age class. Notably, when considering data disaggregated by area, the southern provinces of Lombardy Region (Cremona, Pavia, Lodi), along the Po River, presented the highest values. This might be explained by considering the land morphology of this territory, characterized by a dense network of natural watercourses and canals bordered by a riparian forest. These natural areas are surrounded by rich and specialized crops, mainly intended for production of cereals and fodder for livestock. The availability of shelter, the abundance of fresh water and food, make this relatively small area particularly attractive for wild boar. Family groups are closely linked to these resource-rich environments, favoring potential infectious contacts between individuals, which may explain the higher exposure observed compared to the other investigated area.

The seropositive results observed in wild boar in our study are lower than those reported in other European countries such as Switzerland (35.8%; Wu et al. 2011) and various regions of Spain (25–46%; Muñoz et al. 2010). Conversely, our data are mostly in agreement with previous Italian records: antibodies against Brucella were found in wild boar in a range from 5.74% to 19.76% in different regions of Italy (Bergagna et al. 2009; Montagnaro et al. 2010; Pilo et al. 2015; Cilia et al. 2021; Fabbri et al. 2022; Jamil et al. 2022). However, the comparison of values obtained in different studies and areas should be interpreted with caution due to the different sampling strategies and characteristics of the diagnostic tests used.

As a result of microbiological analyses from wild boars of Cremona Province, B. suis was isolated from 18.9% of submandibular lymph nodes. Other authors have previously reported similar prevalence: 17.7% in the Iberian Peninsula (from cranial and iliac lymph nodes, spleen, and sexual organs; Muñoz et al. 2010) and 17% in Croatia (from uteri; Cvetnic et al. 2004), 10.8% in the Regional Park of Piedmont, northwest Italy (from uteri, spleen, and testicles; Bergagna et al. 2009). Isolation of B. suis biovar 2 from lymph nodes of a female wild boar has been reported also in the Abruzzo Region of central Italy (De Massis et al. 2012).

Our bacteriology data confirm the specificity of serological results and therefore the circulation of the infection in this species. Moreover, the isolation of B. suis from submandibular lymph nodes suggests the importance of the oral transmission route in the pathogenesis of B. suis infection, for example, through the ingestion of heavily contaminated aborted fetuses, fetal membranes, or contaminated foodstuffs (European Food Safety Authority 2009), especially in animals that have not reached sexual maturity and for which the venereal transmission route is unlikely (Elmonir et al. 2022). The presence of B. suis in reproductive organs confirms the possible contribution of the venereal transmission. The negative microbiological result yielded from the three reproductive organs with macroscopic lesions and positive PCR might indicate chronic infection associated with low number of bacteria in the analyzed tissues.

The characterization of B. suis as biovar 2 agrees with the situation recently depicted in Italy. Analysis of Brucella field strains submitted for typing to the Italian National Reference Laboratory for Brucellosis in 2007–15 revealed that B. suis biovar 2 was the main strain isolated from wild boars (De Massis et al. 2019). Likewise, in Europe B. suis biovar 2 has previously been identified as the main etiological agent of brucellosis in wild boar (Jamil et al. 2022).

Although the serological and bacteriologic findings suggest a potential risk of transmission of B. suis to domestic pigs, to date no spillover from wild boar to domestic pigs has been reported. A significant number of large commercial pig farms are present in the Cremona Province; however, the consistent application of biosecurity measures reduces the risk of introduction of infectious agents in this kind of farms. Noncommercial farms, also known as backyard farms, are generally recognized as being at greater risk of contact between pig and wild boar population due to lower biosecurity levels and because backyard farming often provides outdoor access to domestic animals. Previous studies on B. suis have identified population density and spatial overlapping of wild boar and domestic pig farms, together with fence characteristics, as risk factors for disease transmission (Wu et al. 2011; Risco et al. 2014).

In this light, the low density of backyard farms (Fig. 1, blue dots) in Cremona province, suggests that spillover from wild board to domestic pigs is unlikely to occur, even with the high percentages of Brucella seropositive results in wild boar, but attention should still be paid to identifying possible spillover to livestock.

Higher risk for human Brucella exposure is connected to hunting activities of wild animals in infected areas (Kmetiuk et al. 2021). Despite being considered low pathogenic for humans, human infections with B. suis are increasingly reported (Centers for Disease Control and Prevention 2009; Carrington et al. 2012; Franco-Paredes et al. 2017; Mailles et al. 2017; Gowe et al. 2022). Being involved in wild boar hunting–related activities such as field dressing or preparation of raw meat have been shown as risk factors for this infection. The consequences of B. suis infection cannot be underestimated, particularly considering the rising average age of the Italian hunters' population (Istituto Nazionale di Statistica 2003). Hunters also need to be aware that hunting dogs may become infected by B. suis while playing with the animal carcass or eating raw meat (Centers for Disease Control and Prevention 2017). Training on safe handling of carcasses, coordinated surveillance, and research activities may help to control zoonotic foci and minimize public health risks (Martin et al. 2011).

Despite the inevitable limitations inherent in the studies of wildlife surveillance, nonrandom samples as used in this study nevertheless may be useful for monitoring the spread of brucellosis in wildlife (Boyd et al. 2023). Thus, this descriptive epidemiological analysis provides useful data on reservoir species of Brucella for developing risk assessments and adopting preventive measures to limit transmission of the disease to domestic animals and humans.