Echinococcus Granulosus in the Endangered Patagonian Huemul (Hippocamelus bisulcus)

Cystic echinococcosis (CE) is a zoonotic parasitic disease associated with larval stages of Echinococcus granulosus, which negatively affects livestock and public health worldwide (Craig et al. 2007). The parasite is maintained by domestic and wild canids as definitive hosts and by several ungulate species as intermediate hosts in domestic and peridomestic transmission cycles (Cardona and Carmena 2013; Onac et al. 2013; Chaligiannis et al. 2015). Domestic dogs (Canis lupus familiaris) commonly become infected through the consumption of offal with hepatic or pulmonary hydatid cysts of sheep from home slaughtering. Echinococcus granulosus has been classified into three genotypes, with the G1 genotype being the most frequent strain globally, and sheep (Ovis aries) are its primary domestic intermediate host (Craig et al. 2015; Cucher et al. 2016).

Despite successful long-term control programs, CE is endemic and common in several countries of South America, including Chile (Craig et al. 2007; Larrieu and Zanini 2012). Throughout rural Chile, the role of livestock as intermediate hosts and dogs as definitive hosts of the G1 genotype, as well as the occurrence of human hosts incidentally infected through ingesting parasite eggs from dog feces, is widely documented (Acosta-Jamett et al. 2010a, b, 2014; Alvarez Rojas et al. 2017). However, the role of wild herbivores in the wild cycles or the potential transmission of CE from livestock is not known in Chile and the rest of South America.

The Patagonian huemul (Hippocamelus bisulcus), considered one of the most endangered deer species, is endemic to the southern Andes Mountains of Chile and Argentina in a highly fragmented population of fewer than 2,000 individuals (Black-Decima et al. 2016). In addition to hunting pressure, destruction of habitat, and predation by dogs, the transmission of pathogens from domestic ungulates to huemul may constitute another serious conservation threat for this native deer. For instance, livestock diseases, such as bovine viral diarrhea and caseous lymphadenitis, are reported to cause high morbidity and mortality in free-ranging huemul (Corti et al. 2013; Morales et al. 2017). However, further studies are warranted to elucidate the conservation implications posed by CE for the huemul and its epidemiologic connections with other domestic and wild species potentially involved in the wild cycle.

During December 2016, an adult, wild, female huemul, treated for caseous lymphadenitis and later found dead in the Cerro Castillo National Reserve, Aysén region, Chile (46°01′58″S, 71°58′37″W), was necropsied by veterinarians at the National Animal Health Service (Coyhaique, Chile). At necropsy, the animal had multiple abscesses in the pleural and abdominal cavities. A hydatid cyst (8×5 cm) was found in the pulmonary tissue by inspection and palpation. In addition, 10 larvae of Taenia hydatigena were found in the greater omentum in the abdominal cavity. The hydatid cyst was collected and placed in 70% ethanol for further laboratory analyses.

The cyst was examined by microscopy (40×) for the presence of protoscoleces. Total DNA was extracted from the membrane of the pulmonary cyst with a commercial kit (High Pure PCR Template preparation kit, Roche, Germany), which used for amplification of the 1,608 base pairs (bp) of the cytochrome c oxidase subunit 1 (cox1) gene, 471 bp of the nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) gene, and 255 bp of the small subunit ribosomal RNA (ssu-rRNA) gene, with primers cox1-F/cox1-R, JB11/JB12, and Eg1f/Eg1r, respectively (Bowles and McManus 1993; Štefanić et al. 2004; Hüttner et al. 2008). Each of the 25-µL PCR mixtures contained 12.5-µL of double-strength SapphireAmp Fast PCR Master Mix (Takara, Japan), 0.5 mM of each primer, 5–10 µL of DNA template, and ultrapure water. The PCR conditions started with an initial denaturation at 94 C for 5 min, 30 cycles of 94 C for 30 s, 50 C for 30 s, 49 C for 30 s, and 51 C for 30 s for cox1, nad1, and ssu-rRNA, and then, 72 C for 30 s, followed by a final extension at 72 C for 5 min. Each amplicon of the expected size was purified and sequenced in both directions. Sequences were deposited in GenBank (accession nos. MH685111, MH686292, and MH686293). A maximum-likelihood tree was inferred to determine the relationship with Echinococcus species with PhyML software (Guindon et al. 2010) after 1,000 bootstrap replicates. To infer the intraspecific phylogeny, a data set of representative sequences from G1 and G3 genotypes of E. granulosus were used to perform a median joiningnetwork analysis in Popart (Leigh and Bryant 2015) with ɛ=0 and 5,000 iterations.

Microscopically, several protoscoleces were found in the cyst (Fig. 1). The PCR analysis of the DNA samples successfully amplified fragments of the expected size. The maximum-likelihood phylogeny from single and concatenated sequences confirmed that the cysts found in the huemul were E. granulosus (Fig. 1). Further, the median joining-network analysis classified the parasite to the genotype group G1 (Fig. 2). Although the CE found in this huemul was different by at least two mutations from previous isolates of E. granulosus G1 worldwide, the haplotype was closely related to other G1 samples collected from sheep and cattle of Argentina, Chile, Mexico, Brazil, Spain, Italy, and France (Fig. 2).

Herein, we provide evidence of a fertile cyst of a G1 genotype of CE in the endangered Patagonian huemul. In Chilean Patagonia, CE is common among livestock, with prevalences of 19% and 40% in slaughtered sheep and cattle, respectively (Seremi Salud Región de Aysén 2018). Further, the parasite seems to persist locally in peridomestic cycles because fecal samples from 19% of domestic dogs from urban areas in the region were positive by PCR (Seremi Salud Región de Aysén 2018). The Cerro Castillo National Reserve is one of the most important refuges of huemul in the northern Chilean Patagonia, containing close to 40 individuals counted in 2015 and 2016. However, there is a geographic overlap with almost 6,000 sheep and domestic dogs in the border areas of the reserve (Morales et al. 2017), likely increasing the risk of parasite spillover into wild deer. Domestic dogs (i.e., shepherd and stray dogs) and wild foxes (Lycalopex culpaeus), which scavenge sheep carcasses, may act as definitive hosts of E. granulosus G1 genotype (Acosta-Jamett et al. 2010a, 2015), thus disseminating contaminated feces and increasing the risk of infection in huemul.

From a conservation point of view and because this parasite species may produce fertile cysts, low population numbers suggest it is unlikely that huemul could have a major role in the maintenance of the E. granulosus cycle. However, CE infection may cause pathology and morbidity in the huemul, similar to other species of endangered ungulates worldwide (e.g., Boufana et al. 2015). Thus, CE could represent a novel threat of disease transmission from livestock to this native deer through either domestic or wild canids as bridge hosts. Further studies are warranted to characterize the E. granulosus strains in sympatric canids in the region and, thus, enhance knowledge about the potential effects on Huemul populations.

We thank all the rangers from Corporación Nacional Forestal and the laboratory personnel from the Servicio Agrícola y Ganadero who helped during this study. This work was funded by Programa de Protección del Huemul, Región de Aysén. Código BIP: 30136357-0 (SEREMI de Agricultura/SAG/CONAF).