Histopathological findings of Fasciola hepatica infection in non ‐ native European hare ( Lepus europaeus ) in Southern Chile Achados histopatológicos da infecção por Fasciola hepatica em lebre europeia não-nativa ( Lepus europaeus ) no Sul do Chile

Fasciola hepatica is a parasite introduced in America from Europe, being considered of concern for public health and animal production, especially in countries with endemic areas as occur in Chile. In this country, the research has been focused mainly in domestic ruminants, with native and introduced wildlife being largely ignored. Thus, the aim of this study was to report the infection by Fasciola hepatica in an introduced mammal, the European hare, which is distributed along a vast area of the country. One in 42 hares hunted showed histological lesions compatible with F. hepatica, and harbored a parasitic load of 6 liver flukes. This is the first report of this lagomorph acting as host of this helminth in the country and the third in South America. Yet, we found no previous reports regard the pathology of this fluke in the species. Thus, we emphasize the importance of more studies regarding introduced mammals which could be acting as reservoirs for this important fluke.

Fasciola hepatica Linnaeus, 1758 (Platyhelminthes: Fasciolidae) is responsible of fascioliasis, an important parasitic disease that causes important economic losses by organ condemnation, decreases in milk and meat production and mortality of livestock, moreover is a public health concern disease.The indirect lifecycle involves lymnaeid snails as intermediate hosts, releasing cercariae which encyst as metacercariae, the infective stage, in vegetation.The latter is ingested by the definitive hosts; e.g.cattle, sheep, camelids, pigs, even humans.The fluke reaches the liver, where it matures and release eggs through bile, being eliminated to the environment through the feces.This fluke was originated in the Eurasian near east region, reaching an almost worldwide distribution, being present in several countries from Africa, Americas, Asia, Europe and Oceania (MAS-COMA et al., 2007, 2009;VALERO et al., 2012).In Chile, domestic mammals and wild mammals such as the European rabbit (Oryctolagus cuniculus), Huemul (Hippocamelus bisulcus) and Southern pudu (Pudu puda) have been reported as F. hepatica hosts (ALCAÍNO et al., 1988;APT et al., 1993;POVILITIS, 1998;ALCAÍNO & GORMAN, 1999;FUGASSA, 2015).Furthermore, the World Health Organization (WHO) consider Chile as an endemic country for human fascioliasis (WHO, 2018).
The European hare Lepus europaeus Pallas, 1778 (Mammalia: Lagomorpha) is a non-native mammal introduced from Europe during the XIX century for commercial activities, currently present in some countries of South America, e.g.Argentina, Brazil, Chile and Uruguay.In Chile, the species has a wide distribution with a well stablished population from Copiapó to Magallanes, thus being considered a pest (JAKSIC et al., 2002).Regard local parasitological surveys, there is a unique study attempted by González-Acuña et al. (2005) with no positive results regard the liver fluke.
A total of 42 European hares were hunted between June 2015 and June 2016 in an area of pasture used for grazing livestock in Futrono commune, Los Ríos region, Chile (40°08'00" S; 72°24'00" O).The location presents a rainy temperate climate with Mediterranean influence, mountain weather and icy climate by the effect of height due to being Cordillera Pre-Andean zone.The rate of bovine liver seized by F. hepatica in slaughtering industries in the region is approximately 30% (SALAS, 2003).The hare hunters were authorized by the governmental service SAG (Servicio Agrícola y Ganadero), according to the national hunting law (SAG, 2015).Necropsy, liver dissection and histopathological analysis to determine mature and immature forms of F. hepatica and lesions compatible were performed at Unidad de Anatomía Patológica Veterinaria, Universidad Austral de Chile.After being fixed in 4% tamponed formaldehyde, sections of liver were processed according to routine histology techniques and then stained with hematoxylin-eosin (HE).The isolated parasites were dehydrated by a series of alcohol concentrations, 10% to absolute alcohol, then stained with Semichon's acetocarmine stain, cleared in methyl salicylate and finally mounted on slides with Canada balsam.The parasites were photographed and measured with the software ScopeImage v9.0 associated to a light microscope SudeLab.All measurements were expressed in mm and then compared with Valero et al. (2001Valero et al. ( , 2005Valero et al. ( , 2012)), Jones (2005), Periago et al. (2006Periago et al. ( , 2008)), Mezo et al. (2013) and Cuervo et al. (2015).The parameters of prevalence (P) and infection intensity (I) were calculated and interpreted according to Bush et al. (1997).All parasites were deposited in the collection of Unidad de Parasitología Veterinaria, Universidad Austral de Chile (1185-1188Parasitol.UACh).
Only one in 42 hares was infected with helminths and evidenced hepatic lesions compatible with the liver fluke.In the macroscopic inspection of the carcass of the infected hare, peritonitis with a marked hepatomegaly was evident along with notorious dilatation of bile ducts and output of liver flukes.The hepatic flukes were identified as F. hepatica (P= 2.4%; I= 6) (Figure 1a-e).All isolated specimens were gravid flukes with morphological and morphometric characters coincident with previous authors (Table 1).The histopathological findings corresponded to hyperplasia and hypertrophy of bile ducts, portal fibrosis and sections of adult flukes F. hepatica-like and trematode-like eggs.In some areas, the wall of biliary ducts was infiltrated with inflammatory cells together with multiple fluke-like eggs, and a fibroblastic response suggesting chronicity of lesions (Figure 1f-h).No migration tracks, granulomatous response nor immature flukes were detected at histopathology.
The present finding suggests European hare as an additional definitive host for F. hepatica in Chile, considering previous local reports from European rabbit (COURTIN et al., 1975(COURTIN et al., , 1979;;ALCAÍNO et al., 1988;APT et al., 1993;ALCAÍNO & GORMAN, 1999).Furthermore, there are only two previous records from South America, both surveys restricted to Argentina (KLEIMAN et al., 2004;CUERVO et al., 2015).Therefore, to our knowledge, the present finding would be the first for Chile and the third in South America.
The lesions reported in the present study are characteristic for the biliary or chronic infection period with inflammation and hyperplasia   Invasive hare: new host for Fasciola hepatica, pathology of biliary ducts walls.Lesions were mostly around portal spaces with characteristic dilatation of bile ducts which were hyperplastic and filled with cellular debris, eggs and flukes as was reported by Urquhart (1956), Santarém et al. (2006), Mendes et al. (2012), Ashrafi et al. (2014) and Labruna et al. (2018).Alcaíno et al. (1988) suggested that mild parasitic load, i.e. 4-10 flukes, could be equally pathogenic to the lagomorph host, agreeing with the parasitic load found in this study.On the other hand, the peritonitis observed in the hare affected could be associated to track-like lesions associated to young flukes or to migration of adult parasites to peritoneal cavity and other organs in pursuit for feeding (MENDES et al., 2012;LABRUNA et al., 2018).
Regarding morphometric data, the values are similar in comparison to those from hares reported by Cuervo et al. (2015).Unfortunately, descriptions of flukes from wild mammals are focused mainly in length and width (COURTIN et al., 1979;MÉNARD et al., 2001;SANTARÉM et al., 2006;GAYO et al., 2011), probably because its typical shape which is used as the most important diagnosis criteria.In spite of the above, there is evidence of environmental and host influence over the morphology of flukes and its eggs (VALERO et al., 2001(VALERO et al., , 2012;;PERIAGO et al., 2006;MAS-COMA et al., 2007), thus is important to record these data to a better understanding of the parasite development.In the present study, it was notorious the small size of flukes in comparison to those from domestic animals, which could be consequence of the microhabitat given by biliary ducts (VALERO et al., 2001(VALERO et al., , 2012;;PERIAGO et al., 2008;CUERVO et al., 2015).Eggs count was not performed in the present study, however, the presence of ovigerous specimens and biliary ducts with eggs at histopathology suggest that hare is a potential definitive host (CUERVO et al., 2015).
The relevance of this finding lies in the potential of this mammal acting as reservoir of fascioliasis both to human, domestic and wild animals just like has been proposed by other authors (e.g., OLSEN, 1948;BAILENGER et al., 1965;RONDELAUD et al., 2001;KLEIMAN et al., 2004;WALKER et al., 2011;CUERVO et al., 2015).Furthermore, according to Muñoz-Pedreros and Yañez (2009) this lagomorph has a wide distribution in the country, consequently it could favor the spread of this parasite into a wide geographic area considering a daily release between 410 and 1772 eggs of F. hepatica per hare (CUERVO et al., 2015).However, it is necessary to evaluate the hatchability of released eggs, capacity of miracidiae to infect local lymnaeid snails and developing of intramolluscan stages to finally form the metacercariae.Then, experimental infections in other mammal hosts different from hares would be necessary to assess its infectivity (MÉNARD et al., 2001;KLEIMAN et al., 2004).
The present survey together with previous reports from European rabbits, highlight the role of both lagomorphs as wild reservoirs for animal and human fascioliasis in the country (COURTIN et al., 1975;ALCAÍNO et al., 1988).On the other hand, there is a need of parasitological surveys from protected native ruminants such as Taruca (Hippocamelus antisensis), Huemul (H.bisulcus) and Pudu (P.puda), and threatened native marsupials and rodents (see JAKSIC et al., 2002;MUÑOZ-PEDREROS & YAÑEZ, 2009) which could act as definitive hosts as well (MÉNARD et al., 2000(MÉNARD et al., , 2001;;SANTARÉM et al., 2006;GAYO et al., 2011;MENDES et al., 2012;FUGASSA, 2015;LABRUNA et al., 2018).This information, combined with histopathological analysis when possible, would allow to assess the potential threat of this fluke to the health of these native mammals.Also is important to consider the potential role as reservoirs of feral introduced mammals such boars and deers, which also acts as hosts for this parasite (SHIMALOV & SHIMALOV, 2000;MEZO et al., 2013).Finally, this finding constitute a precedent for future research concerning the epidemiology and control programs, considering Chile as an endemic country for this important fluke (WHO, 2018) which has implications for human, livestock and wildlife health.