We confirm the presence of Echinococcus canadensis genotypes G8 and G10 in gray wolves (Canis lupus) and cervids in Idaho, US. Our results demonstrated that cystic echinococcosis remains a potential public health issue, indicating the need for regular deworming of domestic dogs, who often act as potential bridge hosts.
Foreyt et al. (2009) reported Echinococcus granulosus in gray wolves (Canis lupus) in Montana and Idaho and in various wild ungulates in Idaho. At that time, it was suggested that the E. granulosus variant present was the northern biotype. With advances in molecular genetics, the northern biotype is now recognized as incorporated within the species Echinococcus canadensis, which encompasses two previously identified E. granulosus genotypes: G8 and G10 (Thompson 2017). These genotypes have a Holarctic distribution with sylvatic cycles involving wolves as definitive hosts and moose (Alces alces), elk (Cervus canadensis), and caribou (Rangifer tarandus) as intermediate hosts (Lymbery et al. 2015; Romig et al. 2015; Cucher et al. 2016). Echinococcus canadensis was also identified in domestic dogs (Canis lupus familiaris; Himsworth et al. 2010). Echinococcus canadensis genotypes cause human cystic echinococcosis (CE); however, the disease appears to be less severe than that associated with E. granulosus (G1–3) and Echinococcus intermedius (G6/7; Oksanen and Lavikainen 2015; Cucher et al. 2016).
Because the different species and genotypes of Echinococcus have distinct biological and epidemiologic significance, molecular characterization is critical to understanding transmission patterns, assessment of public health risks, and the design/implementation of control and prevention programs. Given that Foreyt et al. (2009) did not provide the genotypes present, our purpose was to determine which genotypes are present in wildlife in Idaho.
Intestinal tracts from seven wolves, 11 coyotes (Canis latrans), and three red foxes (Vulpes vulpes); lungs from five elk and one mule deer (Odocoileus hemionus); and lungs, spleen, and liver from one mule deer were submitted from hunter-killed animals from central and northern Idaho between 2011 and 2014. Samples were frozen before shipment and stored at −80 C for >2 wk before processing. Once thawed, fecal samples were obtained from the rectum. The tracts were processed for tapeworm recovery, as previously described (Foreyt et al. 2009). The lungs, liver, and spleen were examined grossly. Cysts were excised from the tissues, and contents and sections of cyst walls were frozen at −80 C.
Genomic DNA was isolated from Echinococcus tapeworms, cyst walls/contents, and fecal samples, as previously described (da Silva et al. 1999), except Lysing Matrix A (MP Biomedicals, Santa Ana, California, USA) was substituted for Lysing Matrix F (MP Biomedicals), and the run time was increased to 6.0 M/s for 40 s. We stored DNA at −20 C until it was used in a multiplex PCR for the simultaneous detection of E. granulosus (G1–10), Echinococcus multilocularis, and other tapeworm genera, including Taenia (Trachsel et al. 2007). Because sequencing of amplicons obtained from the multiplex procedure was unsuccessful, a second PCR targeting the NADH1 gene was performed (Obwaller et al. 2004). Amplicons were gel-purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, California, USA), following manufacturer's instructions, and products submitted to Colorado State University's Proteomic and Metabolomic Laboratory (Fort Collins, Colorado, USA) for direct sequencing. Sequences were edited and aligned using Lasergene 10 software (DNASTAR, Madison, Wisconsin, USA) and compared with GenBank database (National Center for Biotechnology Information, Bethesda, Maryland, USA).
All seven wolves were infected with Taenia; however, only four wolves were infected with Echinococcus. Echinococcus canadensis G10 was identified in three wolves, with G8 identified in the fourth. Echinococcus was not found in any of the coyote or fox samples, although three coyotes harbored Taenia sp. Neither E. granulosus (G1–3) nor E. multilocularis were identified in any animal.
Four sets of elk lungs harbored two hydatid cysts each, with the fifth harboring eight cysts. Sequencing results of four cysts from the latter elk showed both G8 and G10 were present (two cysts each). Sequencing results of single cysts from the other four elk showed G10 was present in three, with G8 in the fourth. One set of mule deer lungs had two cysts, both of which were identified as G8. The second mule deer had no cysts in the lungs but did have a single cyst in the spleen, which was identified as G10.
Echinococcus canadensis G8 and G10 was confirmed in wolves and cervids of Idaho. Previously, E. granulosus–infected sheep (Ovis aries) from Idaho were identified in the 1960s (Sawyer et al. 1969). Although that was before molecular genetics, given our current state of knowledge regarding the biology and systematics of the group, it is likely those were E. granulosus G1. There are no reports documenting infected wildlife in Idaho before that of Foreyt et al. (2009). Those authors proposed several potential sources for the current foci, including spillover from domestic sheep; however, any conclusions on the source of the present cycle are speculative.
Echinococcus granulosus (G1) and E. intermedius (G6/7) are the most important causative agents of CE worldwide (Hämäläinen et al. 2015; Cucher et al. 2016). In circumpolar regions, human CE infections identified before genetic typing are thought to be caused by the same genotypes currently identified in the local wildlife (G8/10); however, molecular confirmations of human infections in those regions are rarely performed. Thus far, G10 has been confirmed in four people (southern Mongolia, northwest Russia, northeast China, eastern Finland) and G8 in one person in Alaska (Hämäläinen et al. 2015; Yang et al. 2015).
In the northern latitudes of North America, CE remains a public health concern (Schurer et al. 2013). Because of the severity of human CE caused by E. granulosus (G1), control has focused on the domestic cycle of that species. For E. canadensis, sylvatic (wolf–cervid), semisynanthropic (hunting or sled dogs–cervid), and synanthropic (herding dogs–reindeer) cycles exist with human infections associated primarily with the latter two (Oksanen and Lavikainen 2015). Because dogs are key hosts bridging sylvatic cycles with human exposure, control efforts should target all at-risk animals in endemic regions. Thus, to prevent transmission to dogs, control of their diet is essential (Schurer et al. 2013; Hämäläinen et al. 2015; Oksanen and Lavikainen 2015). However, this is not always possible (e.g., free-roaming dogs). Therefore, deworming at-risk dogs is warranted. The frequency of treatments depends on the parasite species present. If the only concern is E. canadensis, deworming every 6 wk is appropriate. However, if E. multilocularis or other helminths are also present, monthly treatment may be necessary because of the shorter prepatent periods.