One Health 17 (2023) 100619 Contents lists available at ScienceDirect One Health journal homepage: www.elsevier.com/locate/onehlt Epidemiological survey and risk factors associated with Paslahepevirus balayani in equines in Europe Javier Caballero-Gómez a, b, c, Antonio Rivero-Juárez a, c, *, David Cano-Terriza b, c, Tomás Fajardo a, b, c, Francesco Buono d, Eduard Jose-Cunilleras e, Jesús García f, Eduardo Alguacil g, Antonio Rivero a, c, Ignacio García-Bocanegra b, c a Grupo de Virología Clínica y Zoonosis, Unidad de Enfermedades Infecciosas, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Hospital Universitario Reina Sofía, Universidad de Córdoba, Córdoba, Spain b Departamento de Sanidad Animal, Grupo de Investigación GISAZ, UIC Zoonosis y Enfermedades Emergentes ENZOEM, Universidad de Córdoba, Córdoba, Spain c CIBERINFEC, ISCIII – CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, 28029 Madrid, Spain d Department of Veterinary Medicine and Animal Productions, Università degli Studi di Napoli Federico II, Naples, Italy e Servei de Medicina Interna Equina, Departament de Medicina Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Barcelona, Spain f Fethard Equine Hospital, Tipperary, Ireland g Uplands Way Vets, Diss, UK A R T I C L E I N F O A B S T R A C T Keywords: Paslahepevirus balayani (HEV) is an important emerging zoonotic virus in Europe. Although domestic pigs and Hepatitis E wild boar are the main reservoirs of this pathogen, susceptibility to this virus has been confirmed in a growing Emerging number of animal species, including equines. However, their role in the epidemiology of this virus remains Risk factors poorly understood. Our aim was to assess HEV circulation and identify potential risk factors associated with Horse Donkey exposure in equid species in different European countries. A total of 596 equines, including 496 horses, 63 Mule donkeys and 37 mules/hinnies bred in four European countries (Spain, Italy, United Kingdom and Ireland) were sampled. Thirty-three animals (5.5%; 95%CI: 3.7–7.4) had anti-HEV antibodies. Seropositivity was found in 4.6% of horses, 11.1% of donkeys and 8.1% of mules/hinnies tested. By country, 6.3%, 5.4%, 5.0% and 4.0% of the equines sampled in Spain, Italy, United Kingdom and Ireland, respectively, were seropositive, respectively. Statistical analysis showed that “species” and “drinking water from ponds and streams” were potential risk factors associated with HEV seropositivity in equines in Europe. HEV RNA was not detected in any (0.0%; 95%CI: 0.0–1.8) of the 202 equines tested. Our results provide evidence of a low, spatially homogeneous and widespread viral circulation that is not equal across species in equid populations in the European countries analyzed and indicate that these species appear to play a limited role in the epidemiology of this virus. Further studies are required to elucidate the differences in seroprevalence between donkeys, mules/hinnies and horses and to determine the risk of zoonotic transmission of this pathogen from equid species. 1. Introduction Europe. In Europe, the number of autochthonous cases of this genotype has increased considerably in recent years [1–3], with most infections Hepatitis E virus, formally known as Paslahepevirus balayani (HEV; being acquired through consumption of animal products or the contact family Hepeviridae), is the main cause of acute viral hepatitis in humans with infected animals [2,4]. Domestic pigs (Sus scrofa domesticus) and worldwide. Eight different genotypes (HEV-1 to HEV-8) have been wild boar (Sus scrofa) are considered the main reservoirs of HEV, but confirmed to date, of which HEV-1 to HEV-4 are the most important viral circulation has been confirmed in a growing number of mammal from the point of view of public health. HEV-1 and HEV-2 affect only species over the last two decades [5]. humans, mainly in developing countries, whereas HEV-3 and HEV-4 are Throughout history, equines have been closely associated with zoonotic and are frequently reported in high-income regions, including humans not only as a common source of food, but also as working * Corresponding author at: Grupo de Virología Clínica y Zoonosis, Unidad de Enfermedades Infecciosas, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Hospital Universitario Reina Sofía, Universidad de Córdoba, 14004 Córdoba, Spain. E-mail address: arjvet@gmail.com (A. Rivero-Juárez). https://doi.org/10.1016/j.onehlt.2023.100619 Received 16 June 2023; Received in revised form 21 August 2023; Accepted 21 August 2023 Available online 22 August 2023 2352-7714/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). J. Caballero-Gómez et al. One Health 17 (2023) 100619 animals used for transport, agriculture and also for recreation. Previous premixed solution containing Taq DNA polymerase, dNTPs and reaction studies have shown that these species, which are found worldwide, may buffer (Promega, Madison, WI, USA), respectively. The nested RT-PCRs be potential sources of zoonotic pathogens, including HEV [6–8]. amplicons were examined on 1.5% agarose gel stained with RedSafe™ However, even though the European Food Safety Authority has high­ Nucleic Acid Staining solution (iNtRON Biotechnology, Seongnam, lighted the need to develop surveillance programs for HEV in equines Korea). [9], there is very little information on the role of these species in the worldwide epidemiology of this pathogen worldwide. In this context, 3. Statistical analysis the aims of the present study were to assess HEV circulation and to identify potential risk factors associated with viral exposure in equids in The prevalence of antibodies and the prevalence of viremic animals different European countries. to HEV were estimated from the ratio of ELISA- or RT-PCR- positive animals, respectively, to the total number of analyzed animals, with a 2. Material and methods 95%CI. The continuous variable “intra-farm equid count” was catego­ rized using the 33rd and 66th percentiles as cut-off points. Associations A cross-sectional study was conducted in different European coun­ between seroprevalence to HEV and explanatory variables were tries between 2013 and 2021. Blood samples from 496 horses (Equus analyzed using the Pearson’s chi-square or Fisher’s exact tests. Variables ferus caballus) were collected in four countries of Europe: Spain (n = 200; with p < 0.10 in bivariate analysis were included for further analysis. including 100 from Andalusia (southern Spain) and 100 from Catalonia Collinearity between pairs of variables was tested by Spearman’s Rho. (northeastern Spain)), Italy (n = 100), United Kingdom (n = 100) and Finally, a multivariable analysis was performed using a generalized Ireland (n = 96). The total sample size was calculated as 498 horses, estimating equations (GEE) model. The number of seropositive animals assuming a prevalence of 50% with a 97.5% confidence level (97.5%CI) was assumed to follow a binomial distribution and “municipalities” was and a desired precision of ±5% [10]. Whenever possible, at least 99 defined as the subject variable. Variables with p values <0.05 in the GEE horses from each country were sampled to detect exposure with a 95% model were considered statistically significant. Statistical analyses were probability and a minimum within-country seroprevalence of 3%. In performed using SPSS 25.0 software (Statistical Package for Social Sci­ addition, samples from donkeys (Equus africanus asinus) (n = 63: 34 from ences, Inc., Chicago, IL, USA). Spain, 27 from Italy, 2 from Ireland) and two equid hybrids, mules/ hinnies (E. africanus × ferus) (n = 37: 34 from Spain, 2 from Italy, 1 from 4. Results and discussion Ireland) were also gathered during the study period using convenience sampling. Since the first description in animals in 1997 [16], the host range of Blood samples were collected by puncture of the jugular vein and HEV has expanded considerably [17]. Since then, the susceptibility of sera were obtained by centrifugation at 400g for 10 min and kept frozen equines to HEV has been confirmed [7] and contact with horses has been at − 20 ◦ C until laboratory analysis. Individual animal and herd epide­ suggested as a risk factor for human HEV infection [18]. Nevertheless, miological information was collected for each animal whenever possible only a small number of studies have assessed exposure to this zoonotic (Table 2). The presence of anti-HEV antibodies was assessed using the pathogen in equines worldwide [19]. To the best of our knowledge, this commercial double-antigen multispecies sandwich HEV ELISA 4.0 (MP is the first large-scale study to assess HEV exposure in domestic equines Diagnostics, Illkirch, France), following the manufacturer’s instructions. in Europe. A total of 33 (5.5%; 95%CI: 3.7–7.4) of the 596 equids This assay uses the highly conserved recombinant ET2.1 protein of the analyzed showed antibodies against HEV. By species, the frequencies of HEV capsid to coat the plates [11], and detects total antibodies (IgG, IgM seropositivity in horses, donkeys and mules/hinnies were 4.6%, 11.1% and IgA) to the virus in serum from a wide range of animals, including and 8.1%, respectively (Table 2), confirming that these three equine perissodactyls [12]. species are naturally exposed to this zoonotic virus in Europe. The In parallel, a subset of approximately one third of the sampled ani­ seroprevalence detected in horses (4.6%) is lower than that previously mals (165 horses, 24 donkeys and 13 mules/hinnies) was randomly found in this equine species in other parts of the world, including selected for the molecular analyses, including six seropositive and 196 serosurveys carried out in China (16.3%; 8/49) [20], Korea (12.4%; 35/ seronegative samples. Viral nucleic acids were from 400 μl of pooled 283) [21] and Egypt (13.0%; 26/200) [22]. In contrast, the seroposi­ serum (100 μl per animal) using the QIAmp MinElute Virus Spin kit tivity observed in donkeys (11.1%) was similar to that detected in the (QIAGEN, Hilden, Germany). The presence of HEV RNA was determined only large study conducted in this species to date (Rui et al., 2019), using three different RT-PCR assays in parallel (Table 1). A real-time RT- whose authors found that 49 (12.2%) of the 401 donkeys sampled in PCR that is able to detect all HEV genotypes was performed, using 10 μl China had anti-HEV antibodies. Consistent with this, one of the three of RNA template and the One Step PrimeScript III RT-PCR Kit (Takara donkeys tested from a zoo in Germany was exposed to HEV [23]. Besides Bio, Shiga, Japan) [13]. Two nested broad-spectrum RT-PCRs for the horses and donkeys, exposure to this virus has been shown in other detection of hepevirus were also performed [14,15]. For these two as­ equine species, such as Przewalski’s horse (Equus caballus przewalski) says, the first and second PCR rounds were performed using the One Step and the Somali wild ass (Equus africanus somaliensis) [12,23]. The PrimeScript III RT-PCR kit (Takara Bio, Shiga, Japan), and the 2× presence of HEV antibodies in mules in our study is consistent with the Table 1 Molecular assays and list of primers and probe used in the present study. PCR Oligonucleotide Sequence (5′-3′) Reference Real-time RT-PCR FWD-Pangenotypic RGTRGTTTCTGGGGTGAC [13] RVS-Pangenotypic AKGGRTTGGTTGGRTGA Probe-Pangenotypic TGAYTCYCARCCCTTCGC-TAMRA Broad spectrum nested RT-PCR HEV-cs TCGCGCATCACMTTYTTCCARAA [14] HEV-cas GCCATGTTCCAGACDGTRTTCCA HEV-csn TGTGCTCTGTTTGGCCCNTGGTTYC†G HEV-casn CCAGGCTCACCRGARTGYTTCTTCCA Broad spectrum heminested RT-PCR HEV-F4228 ACYTTYTGTGCYYTITTTGGTCCITGGTT [15] HEV-R4598 GCCATGTTCCAGAYGGTGTTCCA HEV-R4565 CCGGGTTCRCCIGAGTGTTTCTTCCA 2 J. Caballero-Gómez et al. One Health 17 (2023) 100619 * Table 2 Missing values omitted. Distribution of Paslahepevirus balayani seroprevalence in equid species by cate­ gory and results of bivariate analysis. previous detection of HEV RNA in this hybrid species in Spain [7] and Variable Categories No. positives/ Seroprevalence P confirms susceptibility to this virus. Overall* (%) Seropositive equids were detected in all four countries analyzed, Donkey 7/63 11.1 with seroprevalences ranging from 4.0% (4/99) in Ireland to 6.3% (17/ Species Horse 23/496 4.6 0.083 268) in Spain (Table 2 and Fig. 1), and in 27 (15.4%) of the 175 mu­ Mule/hinny 3/37 8.1 Geriatric 7/134 5.2 nicipalities sampled. Seropositivity was also found in five of the seven Age Adult 13/252 5.2 0.663 years sampled. These results suggest widespread and homogeneous Foal 7/92 7.6 circulation of HEV in equids in Europe during the last decade. Notably, Female 19/327 5.8 Sex 0.463 antibodies against HEV were detected in a yearling foal sampled in Male 14/265 5.3 Pure 19/312 6.1 Ireland in 2019, which suggests circulation of this virus during that year. Breed 0.352 However, since the ELISA can detect IgG, IgM and IgA and the exact age Crossed 8/168 4.8 Sport 8/163 4.9 of this animal was unknown, the presence of maternal antibodies cannot Milk be ruled out. 1/23 4.3 production The GEE model showed that “species” was a potential risk factor Activity Work 3/43 7.0 0.730 Breeder 9/120 7.5 associated with HEV seropositivity (Table 3). The risk of exposure to Family HEV was 2.1 times higher in donkeys than in horses (p < 0.001), which 7/174 4.0 leisure could be associated with a higher genetic susceptibility to HEV infection Ireland 4/99 4.0 in donkeys or unequal exposure of the two species to the virus in Spain, Italy 7/129 5.4 Country Spain 17/268 6.3 0.846 where the highest seropositivity was found in donkeys. The high envi­ United ronmental stability of the virus [24] and the frequent handling of don­ 5/100 5.0 Kingdom keys in extensive systems in this region may explain the higher risk of Pure 19/312 6.1 contact with this virus. In any case, further studies including a larger Breed 0.352 Crossed 8/168 4.8 number of donkeys from the different countries analyzed are needed to 2013 5/74 6.8 2015 0/9 0.0 confirm this hypothesis. 2016 0/14 0.0 Significantly higher seroprevalence was found in equids that used Sampling year 2017 2/66 3.0 0.819 ponds and streams (13.3%) for drinking compared to those that did not 2018 2/27 7.4 have access to these water sources (4.9%) (p < 0.001) (Table 3). HEV has 2019 17/270 6.3 2021 6/121 5.0 been detected in a wide variety of water sources [25]. Although HEV-1 <18 8/158 5.1 and HEV-2 are responsible for waterborne outbreaks in humans in Intra-farm equid count 18–100 8/159 5.0 0.861 developing countries, contaminated water has also been suggested as a >100 8/152 5.3 source of infections by zoonotic HEV genotypes, not only in humans High 5/88 5.7 [26,27] but also in other mammal species, such as domestic pigs. Density of ticks Low 2/15 13.3 0.243 Absence 9/226 4.0 Walachowski et al. [28] observed a significantly higher prevalence Outdoor 10/188 5.3 among swine that drank water from springs or shallow wells than in Shelter (autumn- Indoor 11/240 4.6 0.754 those whose water sources were tap water or deep wells. Similarly, Holt winter) Indoor/ et al. [29] found that pigs on farms where manure ended up in water 3/41 7.3 Outdoor Outdoor 17/292 5.8 sources were more likely to be exposed to HEV. Our results support the Shelter (spring- Indoor 4/131 3.1 hypothesis that untreated or undertreated water may be a source of HEV 0.442 winter) Indoor/ 3/46 6.5 infection in mammals, but further studies are warranted to assess the Outdoor role of environmental waters in the eco-epidemiology of HEV [30]. Yes 15/258 5.8 Presence of dogs 0.409 Apart from domestic pigs, and to a lesser extent non-human primates No 6/130 4.6 Yes 13/232 5.6 (NHPs), the course of HEV infection in other animal species has scarcely Presence of cats 0.470 No 10/159 6.3 been studied. Experimental studies in these species have shown limited Presence of Yes 4/44 9.0 HEV viraemia lasting between one and four weeks after infection domestic 0.241 [31,32]. In our study, none (0.0%; 95% CI: 0.0–1.8) of the 202 serum No 8/161 5.0 ruminants Yes 6/93 6.5 samples tested were positive for HEV RNA, which suggests absence of Presence of poultry 0.434 active circulation of HEV during the study period. However, given that No 12/226 5.3 Contact with horses Yes 4/84 4.8 the ELISA detects total antibodies and the presence of IgM in seroposi­ 0.561 outside the farm No 14/267 5.2 tive animals cannot be discarded, a recent exposure cannot be ruled out. Contact with Yes 14/242 5.8 0.519 Absence of circulation or a low prevalence of active HEV infection has wildlife No 10/224 4.5 Yes 12/250 4.8 also been reported in horses from Korea (0%; 0/397), Spain (0.4%; 3/ Tap water to drink 0.340 No 16/266 6.0 692), China (2.0%; 1/49) and Egypt (4.0%; 4/100) [7,20–22], in don­ Well water to drink Yes 7/132 5.3 0.572 keys from Spain (1.2%; 1/86) and China (4.2%; 17/401) [7,33], and in No 21/384 5.5 mules from Spain (3.6%; 3/83) [7]. Nevertheless, the high homology Water from ponds Yes 4/30 13.3 and streams to 0.071 between HEV strains detected in humans and equines in previous studies No 24/486 4.9 suggests a potential risk of zoonotic transmission to humans drink Cleaning of Yes 20/419 4.8 [7,12,20,33]. 0.246 facilities No 4/50 8.0 In conclusion, the seropositivity found in the present study provides Disinfection of Yes 9/124 7.3 0.154 evidence of natural exposure to HEV, but low, spatially homogeneous facilities No 15/344 4.4 Deworming Yes 23/449 5.1 and widespread viral circulation that is not equal across species in 0.520 program No 2/51 3.9 different equid populations in Europe. Our results suggest that con­ Rodent control Yes 13/259 5.0 0.509 sumption of contaminated water could be a potential source of HEV in No 11/204 5.4 mammals. Serological and molecular results indicate that equines 3 J. Caballero-Gómez et al. One Health 17 (2023) 100619 Fig. 1. Seropositivity of Paslahepevirus balayani by equid species in different European countries. 0017/0012) integrated into the Plan Nacional de I + D + I and co- Table 3 financed by the ISCIII-Subdirección General de Evaluación and the Results of the generalized estimating equation analysis of potential risk factors Fondo Europeo de Desarrollo Regional (FEDER) and the Fundación para associated with HEV exposure in equid species in different European countries. la Investigación en Salud (FIS) del Instituto Carlos III (Research Project Variable Categories β P OR (95% grant numbers: PI21/00793 and PI22/01098). Antonio Rivero-Juarez is CI) the recipient of a Miguel Servet Research Contract by the Spanish Species Mules/ 1.046 0.057 2.9 Ministry of Science, Innovation and Universities (CP18/00111). Javier hinnies (0.9–8.4) Caballero Gómez is supported by the CIBER -Consorcio Centro de Donkeys 0.746 <0.001 2.1 (1.7–2.6) Investigación Biomédica en Red-(CB21/13/00083), Instituto de Salud Horses a a a Carlos III, Ministerio de Ciencia e Innovación and Unión Europea- Water from ponds and streams Yes 1.073 <0.001 2.9 NextGenerationEU. The funders did not play any role in the design, to drink (2.0–4.3) conclusions or interpretation of the study. a a a No a Reference category. Ethical statement appear to play a limited role in the epidemiology of this virus. Further Horse samples were obtained within the official Epidemiological studies are required to determine differences in HEV exposure among Surveillance System Programs and from specimens subjected to medical donkeys, mules and horses and to determine the risk of zoonotic trans­ check-ups or surgical interventions during the study period. Animal mission of this pathogen from equid species. handling and sampling were performed by qualified and trained veter­ inarians following European (Directive 86/609/CEE). Therefore, no Funding statement ethical approval was necessary. This research was supported by CIBER Consorcio Centro de Inves­ Declaration of Competing Interest tigación Biomédica en Red (CB 2021), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión None of the authors of this study has a financial or personal rela­ Europea–NextGenerationEU, by Secretaría General de Investigación, tionship with other people or organizations that could inappropriately Desarrollo e Innovación en Salud (PI-0287-2019) for grants for the influence or bias the content of the paper. financing of Investigación, Desarrollo e Innovación Biomédica y en Ciencias de la Salud en Andalucía, the Ministerio de Sanidad (RD12/ 4 J. Caballero-Gómez et al. One Health 17 (2023) 100619 Data availability [15] J.F. Drexler, A. 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