Molecular confirmation of Fasciola hepatica infection in capybaras (Hydrochoerus hydrochaeris) from the state of Espírito Santo, Brazil Confirmação molecular de infecção por Fasciola hepatica em capivaras (Hydrochoerus hydrochaeris) do Estado do Espírito Santo, Brasil

Braz J Vet Parasitol 2021; 30(2): e000221 | https://doi.org/10.1590/S1984-29612021027 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Molecular confirmation of Fasciola hepatica infection in capybaras (Hydrochoerus hydrochaeris) from the state of Espírito Santo, Brazil

The common liver fluke, Fasciola hepatica, is a digenean trematode of worldwide occurrence (Howell & Williams, 2020). Domestic livestock, in special cattle and small ruminants, are among the most affected hosts, and infection cause a significant economic impact due to subclinical impacts, acute or chronic clinical disease, mortality, and liver condemnation at slaughter house (Howell & Williams, 2020). Non-ruminant domestic livestock (e.g., pigs, camelids), wildlife, and human may also serve as definitive hosts for F. hepatica (Arias et al., 2013). Human fascioliasis is a zoonotic disease endemic to various parts of Latin America and other continents, and it is considered a neglected tropical disease by the World Health Organization (Mas-Coma et al., 2018).
Various wildlife species may serve as reservoir for F. hepatica in endemic areas of South America. In Brazil, capybaras (Hydrochoerus hydrochaeris Linnaeus, 1766) have been proven susceptible to infection by F. hepatica, and may play an important epidemiological role for this parasite (Santarém et al., 2006;El-Kouba et al., 2008;Bellato et al., 2009;Dracz et al., 2016). Reports of natural infection by F. hepatica in capybaras are common in the southern states of Brazil, where fascioliasis is endemic (El-Kouba et al., 2008;Bellato et al., 2009). However, there have been recent reports of F. hepatica infecting capybaras in states of southeastern Brazil, including cases of mortality (Dracz et al., 2016;Labruna et al., 2018).
Capybaras are semi-aquatic, herbivores rodents and are the world's largest rodent species. These rodents have a widespread distribution across the Neotropical region, comprising most South American countries (Reid, 2016). Across Brazil, capybaras are commonly found sharing pasture areas with domestic ruminants, and therefore prone to share potential pathogens (Labruna et al., 2018). With the expansion of urban areas, interactions between human populations and domestic and wild animals are increasing, which provides favorable situations for dissemination of diseases and pathogens between hosts and environments (El-Kouba et al., 2008).
Considering the susceptibility of capybaras to this fluke, the objective of the present study was to assess the presence of F. hepatica in capybaras at conservation units located (CUs) in the State of Espírito Santo, southeastern Brazil, by means of classical and molecular techniques. During the period from April to July 2015, fecal samples morphologically consistent with that of capybaras were collected from the environment and were kept refrigerated. Subsequently, samples were taken for processing to the Parasitology Laboratory of the Veterinary Hospital, Federal University of the State of Espírito Santo.
The fecal samples were processed for detection of F. hepatica eggs using a simple sedimentation technique validated by Martins et al. (2008). The eggs were collected and observed under a compound microscope (Olympus CX41RF) at 100x magnification. Photomicrographs were captured and eggs were measured using the Dino-Eye ® system and the Dino Capture ® image analysis software.
Descriptive data analysis was performed, with estimation of infection frequency, considering the total number of processed samples. Mapping of location of each CU, was performed using the Quantum GIS software 2.0.
Genomic DNA was isolated from eggs isolated via fecal sedimentation and fixed in molecular-grade 96-99% ethanol, using the DNeasy Blood & Tissue kit (QIAGEN, Hilden, Germany), following the manufacturer's instructions. The internal transcribed spacers, including the 5.8S ribosomal unit (ITS1-5.8S-ITS2), were amplified using the forward primer BD1 (5-GTC GTA ACA ACG TTT CCG TA-3) and the reverse primer BD2 [5-TAT GCT TAA (G/A) TT CAG CGG GT-3], as previously used by Luton et al. (1992). The PCR reaction components (in a total volume of 25 μL) consisted of 2.5 μL of 10× PCR buffer minus Mg, 1.5 μL of MgCl 2 (50 mM), 2 μL of each dNTP (2.5 mM), 1.25 μL of each oligonucleotide primer (10 mM), 0.2 μL of Platinum Taq DNA polymerase (5 U/μL) (Invitrogen, Carlsbad, California), 13.8 μL of H 2 O, and 2.5 μL of genomic DNA. The PCR thermocycling protocol consisted of 94 °C for 3 min followed by 30 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min, and then a final elongation step at 72 °C for 10 min. Among the localities with positive samples (Figure 1, shown in red), three were in municipalities in the southern region of the state (Alegre and Castelo) and three were from municipalities in the metropolitan region (Guarapari and Cariacica). No eggs were observed in feces obtained from CUs in the central region (Sooretama) or north region of the state (Conceição da Barra) (shown in green). No fecal samples were obtained (shown in yellow) from CUs in Domingos Martins and Santa Teresa (central region) and Dores do Rio Preto (southern region).
To our knowledge, this is the first report of F. hepatica infection in capybaras in the endemic region of Espírito Santo, southeastern Brazil. Only a handful of reports are known from southern states, and other states in southeastern Brazil. In a study conducted by El-Kouba et al. (2008), in three public parks in the municipality of The 28S region of the rDNA was amplified using the primers digl2 (5-AAG CAT ATC ACT AAG CGG-3) and L0 [5-GCT ATC CTG AG (A/G) GAA ACT TCG-3] (Tkach et al., 2000). PCR reactions were conducted as described above and PCR cycling parameters for amplification were performed as described by Tkach et al. (2000).
The PCR products were purified through an enzymatic treatment with ExoProStar TM (GE Healthcare) and sent for sequencing at ACTGene (Ludwig Biotec, Rio Grande do Sul, Brazil), using the same PCR primers. Subsequently, the sequences were analyzed in MEGA X (Kumar et al., 2018) and compared with other sequences available from GenBank via BLAST.
Among the 11 CUs visited, sampling for capybara feces was found in eight of them. A total of 62 samples were collected and the presence of large, golden-brown operculated eggs, characteristic of F. hepatica were found in 37.1% (23/62) of the samples. The mean length and width of F. hepatica eggs were 118.49 μm and 64.65 μm, respectively. Positive samples were present in six CUs (75%) with frequency ranging from 28.57 to 85.71% (Table 1). Curitiba, state of Paraná, F. hepatica eggs were found with a prevalence of 54.5% (18/33) in one of the parks, slightly higher than the overall prevalence found in our study. Bellato et al. (2009) conducted a study in the municipality of Timbó, state of Santa Catarina, Brazil, and found the prevalence of F. hepatica infection to be 18.12% for cattle and 8.96% for capybaras; whereas in Dracz et al. (2016), F. hepatica eggs were retrieved in only one out of 11 fecal samples of capybaras from the state of Minas Gerais. El-Kouba et al. (2008) and Dracz et al. (2016) reported mean dimensions for F. hepatica eggs retrieved from capybara feces of 138.56 µm in length and 72.96 µm in width, and 142.74µm and 75.74µm, respectively. However, according to Valero et al. (2001), the morphometry of F. hepatica eggs is highly variable among different host species and geographic locations.
Studies in the state of have indicated that F. hepatica cause significant economic impact to cattle industry. A mean prevalence of 22.91% is reported based on inspection of bovine livers at slaughterhouses in the southern region of the state (Bernardo et al., 2011). A more recent study, revealed an increased frequency of F. hepatica in cattle , and infection has been reported in sheep, goat and water buffalo from the same region (Carneiro et al., 2013). Moreover, the presence of the snail intermediate host, Pseudossuccinea columella, has been confirmed in Espírito Santo, enabling the F. hepatica life cycle to be completed autochthonously in this endemic area. (Medeiros et al., 2014).
The occurrence of F. hepatica in capybaras from CUs in the metropolitan region corroborates the findings of Freitas et al. (2014), who observed presence of this fluke in livers at a slaughterhouse between 2009 and 2011. Furthermore, these authors proposed a bioclimatic zoning scheme for occurrences of F. hepatica in the state of Espírito Santo, considering factors such as altitude, air temperature, rainfall and topography, and their respective risks for potential occurrence of F. hepatica. They found that 52.22% of the entire territory of the state is located in areas that are favorable for occurrence of F. hepatica, including the metropolitan region of the State, an other region assessed in our study .
Capybaras are herbivores with strong association with aquatic habitats (Reid, 2016), which possibly increase the likelihood of infection by ingesting metacercariae of F. hepatica, and also of water contamination, since defecation may occur next to or inside water bodies. Capybaras and cattle are frequently found grazing in the same areas, which may suggest that F. hepatica can be disseminated from cattle to wildlife and vice versa, (Arias et al., 2013). In addition, capybaras are highly adaptable to many habitats, and this facilitates their establishment in urban areas subject to anthropic activity (Reid, 2016). This proximity with human populations may provide favorable conditions for the dissemination of F. hepatica (El-Kouba et al., 2008). Altogether, these factors highlight the capybara's potential as wild reservoirs of F. hepatica throughout their vast geographic range, which encompasses most countries in continental South America (Reid, 2016).
Zoonotic fascioliasis occurs when humans ingest metacercariae of F. hepatica along contaminated leafy greens, in special watercress. In Brazil, human fascioliasis is not a reportable disease, and therefore its prevalence is likely underestimated (Pritsch et al., 2019). While it remains unclear whether capybaras could play an important role to human infection, avoiding contamination of water sources used for irrigation with feces of susceptible hosts and fencing off areas of cultures that require an aquatic environment remain as relevant control measures. This is the first report of capybaras naturally infected by F. hepatica in the state of Espírito Santo, southeastern Brazil. Our findings emphasize the importance of epidemiological studies in this region, with the objective of estimating and locating areas where F. hepatica is present in the state. Such studies may contribute towards adoption of more comprehensive control measures, covering not only domestic ruminants but also wildlife. Susceptible wildlife reservoirs may play a significant epidemiological role for this emerging, neglected zoonosis.