Prevalence and Risk Factors for Hepatitis E Virus (HEV) in Wild Boar (Sus scrofa) and Red Deer (Cervus elaphus) in Portugal

1. Introduction

The most common cause of acute enterically transmitted hepatitis in developing countries is the hepatitis E virus (HEV) [1]. HEV is a foodborne zoonotic virus with a worldwide infection prevalence of 20 million human cases, causing severe implications for public health and economic progress in developed and developing nations [2,3]. In Europe, more than 20,000 confirmed hepatitis E cases were reported between 2005 and 2015 [4]. Besides humans, HEV infects various mammalian hosts [5]. HEV belongs to the family Hepeviridae, further subdivided into the subfamilies Parahepevirinae and Orthohepevirinae. Members of the Parahepevirinae subfamily infect salmon and trout. In contrast, mammals and birds are infected by members of the Orthohepevirinae subfamily, which is further divided into four genera: Paslahepevirus, Avihepevirus, Rocahepevirus, and Chirohepevirus [6].

HEV is a small virus quasi-enveloped with an icosahedral capsid enclosing the viral genome [7]. The positive-sense, single-stranded RNA comprising the genome is 6.4–7.3 kb long and has four partially overlapping open reading frames [8,9].

HEV is today referred to as Paslahepevirus balayani. Eight genotypes of HEV are known, namely HEV-1 through HEV-8, with genotypes 1 and 2 infecting exclusively humans, genotypes 3, 4, and 7 infecting both humans and animals, and genotypes 5, 6, and 8 infecting only animals [10]. HEV1 and HEV2 are prevalent in developing countries and can cause significant gastrointestinal symptoms/signs in humans and animals. These symptoms/signs are often attributed to consuming contaminated water and food [11,12]. Zoonotic genotypes 3 and 4 are transmitted primarily through consumption of contaminated pork and meat products, or contact with infected animals, particularly pigs [13]. In industrialized countries, HEV3 and HEV4 typically cause sporadic and pauci-symptomatic illnesses. In humans HEV typically manifests with mild clinical characteristics, involving increased liver enzyme levels, jaundice, and non-specific symptoms like loss of appetite, fatigue, and abdominal discomfort. These clinical signs often closely resemble symptoms observed in various other liver disorders, and they usually persist for a duration of 1 to 6 weeks. While most cases resolve on their own, instances of chronic hepatitis E infection have been documented in individuals with weakened immune systems, especially among organ transplant recipients using immunosuppressive drugs [14,15].

After being identified in domestic pigs in the United States in 1997, swine HEV strains have been recognized across the globe in both domestic and wild pig populations, showing considerable variations in their prevalence [16,17].

HEV3 and HEV4 genotypes have been detected in humans, as well as in Sus scrofa domesticus (domestic pigs) and Sus scrofa (wild boars) [12]. The HEV3 and HEV4 genotypes, found in various animal species such as wild ungulates exhibit significant similarities with the genotypes found in humans. These similarities support the zoonotic nature of these two genotypes [17-19]. Genotype 4 is predominantly common in China and is the predominant genotype [20], whereas genotype 3 is common in all other parts of the world and is the most common HEV genotype in Europe [21-24]. On the other hand, HEV genotypes 5 and 6 have only been identified in Japanese wild boar [22], and HEV genotypes 7 and 8 have recently been detected in camels in China and the Middle East [25,26].

The unequivocal link between the consumption of infected wild ungulate meat and human infection has been described [27]. Including, more specifically, the establishment that eating contaminated wild boar meat can result in human infection [28]. Cases of HEV have been directly connected to the consumption of raw deer meat by the presence of identical HEV strains in the consumed deer meat and the respective patients [29]. In immunocompetent human subjects, HEV causes sporadic self-limiting infections presenting a low mortality. Conversely, in immunocompromised patients, HEV can induce several and dangerous clinical signs [30,31]. It may also cause neurological manifestations [32] and is responsible for a self-limiting infection with an overall low mortality. However, mortality can reach higher levels in pregnant women [33]. The main HEV reservoirs are domestic pigs and wild boar [34,35]. However, zoonotic strains have also been identified in deer, rabbits, chickens, mongooses, rats, ferrets, fish and camels. This host range continues to expand over time, showcasing instances of infections crossing between different species [36,37]. The development of HEV antibodies in pigs occurs after the natural decrease in maternal antibody levels, typically around 8–10 weeks of age. Initially, IgM anti-HEV antibodies reach their highest levels alongside the peak of viral shedding through feces, followed by IgG anti-HEV antibodies peaking when the virus is being eliminated from the fecal matter. The liver is the targeted organ, from which HEV spreads to various tissues and organs through hematic diffusion [38].

Although HEV infection has been detected on wild ungulates in many countries around the world [39-43], with only one seroepidemiological study on HEV in wild ungulates in Portugal, and only focusing on wild boar. As such, this study aimed to assess the seroprevalence, and risk factors associated with HEV in wild boar and red deer in Central Portugal.

2. Materials and Methods

From 2016 to 2023, a survey was conducted on serum samples randomly collected from free-ranging wild ungulates hunted and legally killed by hunters in east-central Portugal to investigate the presence of HEV. The first hunted animals were sampled up to a total of 10 per year at each side and this sampling process was repeated in each year.

Sampled municipalities included Alcafozes (n =16), Castelo Branco (n =30), Cegonhas (n = 8), Crato (n = 41), Fratel (n = 35), Granja (n = 10), Idanha-a-Nova (n = 29), Lousa (n = 44), Marvão (n = 31), Mata (n = 40), Monforte (n = 10), Monte Fidalgo (n = 76), Niza (n = 26), Ponte de Sor (n = 25), Portalegre (n = 49), Rosmaninhal (n = 39), Sarnadas do Ródão (n = 40), Tostão (n = 9), Vila Velha de Ródão (n = 64), Vale de Figueira (n = 6), and Vale Pousadas (n = 22).

These regions are home to the largest population of wild ungulates in Portugal. A veterinarian conducted a comprehensive examination of a total of 650 wild ungulates, representing two distinct species: 352 wild boar (S. scrofa) and 298 red deer (Cervus elaphus). Available data on age, sex, body condition, and capture location were utilized to provide insights into the distribution of seropositive individuals.

Blood samples were obtained from the heart or thoracic cavity of the animals during the hunting season. Blood was allowed to clot at environmental temperature and afterwards transported to the laboratory. Blood samples were then centrifuged at 1500×g for 10 min and the separated serum samples kept at −20°C until further testing. Sera were checked for the presence of antibodies to HEV in wild ungulates using an ELISA kit (ID Screen® Hepatitis E Indirect Multi-species ID.vet, Montpellier, France), under the manufacturer’s recommendations and following the guidelines for the interpretation of results. Optical densities (OD) of the tested samples and positive and negative controls were measured by an ELISA plate reader at 450 nm. The OD ratio of the sample and positive control (S/P) was calculated for each sample as follows:

[(OD sample–OD negative) / (OD positive–OD negative)] ×100.

2.1. Statistical analysis

The outcome variable was dichotomized as positive versus not positive to identify any risk factor associated with seropositivity. The Chi-square test was used to assess significant differences among the groups. Multiple logistic regression was used to model the odds ratio (OR) and its 95% confidence interval (CI) of being seropositive related to the variables. Significant potential risk factors at p < 0.05 (two-tailed; alpha = 0.05) were then evaluated using stepwise regression to construct a multiple model (Wald test stepwise p-Wald value to enter p < 0.05). The multiple logistic model was developed using a stepwise approach. Backward elimination followed by a forward selection for each variable at a time was done using a likelihood ratio test at each step with 0.05 (two-tailed; alpha = 0.05) as a significant level for removal or entry. The fit of the models was assessed using the Hosmer and Lemeshow goodness-of-fit test [44]. The model was rerun until all remaining variables presented statistically significant values (p < 0.05). All statistical analyses were performed using SPSS^® 25.0 software for Windows.

3. Results

The studied population of wild ungulates comprised 352 (54.2%) wild boar and 298 (45.8%) red deer. In age groups, adults represented 64.2% (n = 417) and juveniles 35.8% (n = 233). Following the European Regulations (EU Reg.), each animal received ante and post-mortem examinations, respectively performed by hunters (in agreement with the EU Reg. No. 853/2004) and by veterinary sanitary authorities (EU Reg. No. 625/2017).

The global seroprevalence of HEV infection was 5.1% (n = 33; 95% CI: 3.5−7.1%).

Among the positive species, the prevalence in red deer (9.1%; 95% CI: 6.1-12.9%) was higher than in wild boar (1.7%; 95% CI: 0.6−3.7%), with a statistically significant value observed (p < 0.001).

Serologic reactivity data according to species, sex, age, and clinical signs examined are presented in Table 1. The seroprevalence values among males and females were 6.6 % (95% CI: 4.2-9.7%) and 3.3% (95% CI: 1.6-6.0%), respectively (p = 0.054) (Table 1). Regarding age, the lowest value of seroprevalence (1.3%; CI: 0.3−3.7%) was found in juveniles and the highest (7.2%; CI: 4.9−10.1%) in adults. Statistical significant differences were observed between these groups (p < 0.001). Furthermore, there was significant difference in seropositivity results among clinical signs related to the presence (1.7%; CI: 0.2−6.0%) or absence (5.8%; CI: 4.0−8.2%) in the studied species (p = 0.037) (Table 1).

Regarding distribution according to municipalities, anti-HEV antibodies were found in eight of them: three red deer from Castelo Branco (10.0%; 3/30 wild ungulates), four red deer from Crato (9.8%; 4/41 wild ungulates), one red deer from Fratel (2.9%; 1/35 wild ungulates), two wild boar from Lousa (4.5%; 2/44 wild ungulates), four red deer from Marvão (12.9%; 4/31 wild ungulates), two wild boar from Niza (7.7%; 2/26 wild ungulates), four red deer and two wild boar from Rosmaninhal (15.4%; 6/39 wild ungulates), eight red deer from Vila Velha de Ródão (12.5%; 8/64 wild ungulates), and three red deer from Vale Pousadas (13.6%; 3/22 wild ungulates).

Three variables were associated (p < 0.05) with seropositivity in wild ungulates. Seropositivity significantly correlated with the following factors (Table 1): age, species, and animals with clinical signs. These variables were included in the multiple model. A backward stepwise conditional logistic regression was employed using all the above statistically significant variables. Multiple logistic regression analysis of the OR for being seropositive to the potential risk factors listed above is presented in Table 2. At the individual level, the odds of HEV seropositivity were found to be higher for adult animals, OR = 3.66 (95% CI: 1.72−18.11%), and also for in red deer, OR = 4.2 (95% CI: 1.64−10.69%).

4. Discussion

Each year, foodborne pathogens, including the HEV, result in numerous infections across various continents. Ingestion of contaminated food of animal origin exposes the final consumers to the risk of infection. HEV has been identified as an emergent public health risk in several European countries [45].

Annually, HEV leads to a considerable number of infections through its zoonotic transmission. Moreover, it has gained recognition as an occupational disease due to the substantial prevalence of antibodies found in the blood samples of professionals like veterinary workers, slaughterhouse employees, hunters, and similar occupations [12]. Individuals such as veterinary foods inspectors, pig breeders, hunters, pork product sellers, and meat processing workers are frequently exposed to the risk of HEV infection [46]. Even in developed countries where the origins of human hepatitis E cases were previously thought to be rare, detection of HEV in sewage, water sources, coastal, surface waterways, drinking water, and produce raises environmental safety concerns [36,47].

Epidemiological studies utilize molecular biology techniques like real-time PCR assays, along with seroprevalence screening methods involving the detection of immunoglobulin G (IgG) and/or immunoglobulin M (IgM), as the established and highly reliable approaches for diagnosing and assessing the epidemiological situation [48].

This study shows that approximately 5.1% of wild game that are hunted for human consumption have antibodies for hepatitis E virus. As far as we know, the present study is the first serological study conducted on red deer in Portugal, and the largest seroprevalence study on wild boar from Portugal to date.

HEV human infections caused around 21,000 clinical cases in Europe between 2005 and 2015. Most were limited to specific countries (e.g., Germany, France, Italy, and Spain [4]. Serological studies have shown that people are at danger of infection, as reported by Raji et al. [49], who found substantial seroprevalence titres in hunters and in blood donors [31]. Consumption of infected (raw or undercooked) pork liver or wild boar meat products is the mode of HEV infection in humans, and traditional home-cooked foodstuffs, in particular, allow HEV persistence in specific geographical locations [18,31,50,51]. Occupational exposure of workers to swine and sheep represents other risk factors associated with seropositivity [52,53].

In the Portuguese human and animal population, there are reports of evidence of infection detected by molecular and serological methods [54-60]. In Portugal, HEV has previously been detected by molecular methods in wild ungulates. Namely, in two red deer with a prevalence of 2.1% [54], 20 wild boar livers (25.0%), and four wild boar stools in two different studies (10% and 2.8%) [55,56].

In the Iberian Peninsula, serological reports of HEV in wild ungulates have ranged between 8.2−57.4% in wild boar [11,39,61,62,63,64,65] and 4%−13.9% in red deer [62-67]. The seroprevalence in wild ungulates found in the present study is lower than in previous studies from the Iberian Peninsula.

Moreover, the values found in this serological study are lower than those found in other similar European studies. In Italy, a study found seroprevalences between 87.3% and 100% in pigs [68], and similar results have been found in white-tailed deer (Odocoileus virginianus) in Finland (1.4%) [69]. Interestingly, other studies in Italy also detected HEV RNA in 14.3−83.7% of liver samples [11,40,41,42,70,71,72], and in farmed ruminants the majority of HEV genotypes discovered until to date are from the zoonotic HEV3 and HEV4 [73].

Although the seroprevalence shown in this study appears to be low, the etiological agent seems to infect the wild ungulates under evaluation. Seropositive variance between red deer and wild boars indicates the presence of current infections and previous exposures in the examined animals.

For the statistical analysis, only two factors were associated in the final model: age and species. The high seropositivity level reported in adult wild ungulates supports prior research and could be explained by longer-term exposure to the HEV in the environment with similar studies on wild ungulates from other countries of Europe also finding the same pattern [74,75]. Furthermore, this tendency has been observed in humans as well, with anti-HEV seroprevalence increasing with age [76,77] and seropositivity associated with the female gender, probably due to the potential role of sexual hormones peaks which may increase host receptivity to infection [78,79]. Interestingly, in the present study, the test chosen to detect HEV infection proved to be practical and rapid in comparison to the expensive and time-consuming molecular testing.

Domestic pigs play a prominent role as a reservoir for the HEV among wildlife and domestic animal species. HEV surveillance is critical over the world to close the knowledge gap about its transmission and reservoirs, especially given its zoonotic potential [80]. They are closely linked, from an epidemiological standpoint, with other important wild species, such as S. scrofa (wild boars) and wild ruminants, which serve as additional sources for the environmental spread of the virus [18,28,81,82,83]. Results of this study should be interpreted from a One Health perspective. The interaction between different species, particularly wild ungulates, and domestic ruminants, in their shared habitats, can facilitate the transmission of infectious agents such as HEV. Previous studies reveal a similar HEV RNA detection rate in red deer and wild boars inhabiting the same regions, implying that the same HEV-3 variant is frequently transmitted among various ungulate species [64,84] and, ungulate species like deer serve as true host for HEV. However, wild and domestic pigs are still the principal cause of infection for ruminants living in the same regions [84]. Additionally, the proximity of small ruminants and wild animals to humans increases the risk of pathogen transmission to people [85].

However, the results, it is important to interpret these findings cautiously as the present study has some limitations. Specifically, this study involved a cross-sectional design with self-selection animals and the designs constraints prevent the establishment of a definitive cause-and-effect relationship.

The results of this ELISA-based survey indicate that HEV infection is widely distributed among red deer and wild boar from the central region of Portugal. Considering the lack of similar studies in the country, particularly in red deer, our results could contribute to the effective control of HEV in wildlife. These results also confirm that the seroprevalence of HEV infection in red deer and wild boar has been underestimated in Portugal.

The present study aims to contribute to the detection and identification of potential future threats related to HEV infections in wild ungulates. Wildlife monitoring plays a crucial role as it enables the identification of changes in disease occurrence among wild populations [86-88]. Regularly monitoring and studying the health status of wild ungulates can detect any shifts or patterns in transmission dynamics in the wildlife population [88]. This information is essential for understanding the potential risks and impacts associated with HEV infections.

5. Conclusions

In conclusion, the present study emphasizes the importance of a One Health, multidisciplinary approach for assessing wild boar and red deer exposure to HEV in the Centre of Portugal and also for controlling HEV disease. Further research about the role of wildlife in the epidemiology of HEV infection should be conducted. By addressing the concerns associated with wildlife reservoirs and integrating them into disease control, better efforts may be conducted to safeguard animal and human health in the face of HEV.