Parasites of the genus Entamoeba comprise unicellular anaerobic organism that infect humans (Shirley et al., 2018; Cui et al., 2019), domestic animals (Noble and Noble, 1952; Stensvold et al., 2010; Cui et al., 2019; Ai et al., 2021) and wild animals (Stensvold et al., 2010; Shilton et al., 2018; Cui et al., 2019). Entamoeba parasites develop through a faecal-oral life cycle, and infections with pathogenic species can cause intestinal disease and damage the liver and brain (Ngui et al., 2012). The initial classification of Entamoeba species was established based on the type of host in which the parasites were identified (Noble and Noble, 1952) and on parasite morphological features (Stensvold et al., 2011), such as cyst size and the number of nuclei. Using this approach, Entamoeba species have been classified into four distinct groups, including E. gingivalis-like group (species without cysts), E. bovis-like group (uni-nucleated cysts), E. histolytica-like group (quadri-nucleated cysts), and E. coli-like group (octo-nucleated cysts) (Clark et al., 2006; Stensvold et al., 2011). In recent years, the analysis of Entamoeba 18S ribosomal RNA (18S rRNA) sequences has significantly expanded the repertoire of genetically distinct Entamoeba organisms (Clark et al., 2006; Stensvold et al., 2011; Jacob et al., 2016). Although morphology-based analysis will be required to consolidate such findings, they provide unique insights into variation within species, evolutionary relationships, and host specificity (Clark et al., 2006; Stensvold et al., 2011; Jacob et al., 2016). Moreover, analysis of Entamoeba DNA sequences is an essential tool in endemic countries where microscopy does not allow for the distinction of pathogenic and non-pathogenic Entamoeba species (Nath et al., 2015). The genetic diversity of morphologically identical parasites, and the host promiscuity of Entamoeba organisms, highlights an ongoing need for further characterisation of genetic variants and host range, particularly in pathogenic species and emerging zoonotic species infections.

In animals, ruminants such as cattle and sheep appear to be common hosts of the uni-nucleated cyst Entamoeba species (Noble and Noble, 1952; Clark et al., 2006; Stensvold et al., 2010). Nevertheless, cyst morphology varies greatly within and between uni-nucleated cyst-producing species isolated from different ruminant hosts (Stensvold et al., 2010). The term “ribosomal lineage” (RL) was introduced to name newly discovered Entamoeba 18S rRNA sequences with greater than 5% divergence from known species. These RLs represent organisms not yet described morphologically and not referrable to described species (Jacob et al., 2016). The analysis of Entamoeba 18S rRNA sequences detected in farmed and wild ruminants over the last decade revealed the presence of E. bovis and eight RLs. Of these, four RLs are closely related to E. bovis (Entamoeba RL 1-3 and 8) (Jacob et al., 2016). Besides being detected in cattle (Bos taurus) (Stensvold et al., 2010; Jacob et al., 2016; Nolan et al., 2017; Matsubayashi et al., 2018; Ai et al., 2021) and sheep (Ovis aries) (Stensvold et al., 2010; Jacob et al., 2016; Ai et al., 2021), E. bovis and Entamoeba RLs have also been detected in goats (Capra hircus) (Nolan et al., 2017; Al-Habsi et al., 2017; Ai et al., 2021), horses (Equus ferus) (Ai et al., 2021), camels (Camelus ferus) (Ai et al., 2021), and cervids. Among the studies conducted on cervids, white-tailed deer (Odocoileus virginianus) from the USA (Kingston and Stabler, 1978), fallow deer (Dama dama) from Mauritius (Jacob et al., 2016), and reindeer (Rengifer tarandus) from Iceland (Stensvold et al., 2010) tested positive for E. bovis, while Entamoeba RL 1 was detected in roe deer (Capreolus capreolus) from Sweden (Stensvold et al., 2010). Information about the pathogenicity of E. bovis and Entamoeba RLs in ruminants remains limited; however, their detection in cattle in the absence of clinical symptoms such as diarrhoea, suggests low pathogenicity (Matsubayashi et al., 2018; Ai et al., 2021). E. bovis have a broad ruminant host range and can be transmitted by faecal excretion of cysts followed by oral ingestion of contaminated food or water (Noble and Noble, 1952; Clark et al., 2006; Stensvold et al., 2010). Based on the oral-faecal life cycle of Entamoeba parasites, the transmission of E. bovis between different host taxa that share common land is highly likely.

To date, Entamoeba parasites have only been identified twice in Australian wild animals. E. ranarum was detected and characterised in wild cane toads (Rhinella marina) (Shilton et al., 2018), and E. bovis was detected in feral goats in Western Australia rangeland with a 6.4% prevalence (Al-Habsi et al., 2017). The prevalence and distribution of Entamoeba species in Australian farmed and wild ruminants, such as wild deer, remains yet to be investigated. Wild deer and livestock commonly share grazing areas in agricultural landscapes and are equally susceptible to a wide range of pathogens of agricultural importance (Cripps et al., 2019). Wild deer represent a significant source of pathogen transmission; thus, we hypothesised wild deer to be involved in the transmission of Entamoeba parasites to livestock and vice versa. In recent years, our team has investigated the role of wild deer as carriers of livestock pathogens in Australia (Huaman et al., 2020; Huaman et al., 2021; Huaman et al., 2021a; Huaman et al., 2021b), and here, we report the first detection of Entamoeba parasites in wild deer sampled in Australia. Further, we assess the prevalence, distribution, and characterisation of Entamoeba species and RLs as well as the potential of cross-species transmission.

In recent decades, Australian wild deer populations have significantly increased in abundance and distribution, leading to regular close interactions between deer and livestock, which increases the risk of pathogen transmission (Davis et al., 2016; Cripps et al., 2019). However, little is known about the epidemiology of pathogens that Australian deer may transmit to livestock, other domestic animals, or wildlife. The present study complements our initial work on investigating pathogens in wild deer across multiple geographic locations in Australia (Huaman et al., 2020; Huaman et al., 2021; Huaman et al., 2021a; Huaman et al., 2021b). Here we report the identification of Entamoeba sequences in wild deer and cattle faecal samples collected in south-eastern Australia, with subsequent phylogenetic analysis to evaluate the cross-species transmission of Entamoeba parasites. This baseline information is of value for monitoring the status of parasitic infections in Australian deer and evaluating the risk of disease transmission between wild deer and livestock. Additionally, the data provided by this study increases our knowledge of the host range and distribution of Entamoeba, a group of parasites prevalent in ruminant livestock. Finally, this study represents the first molecular screening and characterisation of Entamoeba in Australian wild deer.

The predominant Entamoeba species identified in the wild deer and cattle samples collected and analysed in this study was Entamoeba bovis, a species recognised to infect ruminants, including livestock animals (Stensvold et al., 2010; Matsubayashi et al., 2018; Ai et al., 2021). The prevalence of Entamoeba infections previously reported in cattle are relatively low [2.5% in Costa Rica (Jimenez et al., 2007), 4.8% in Korea (Ismail et al., 2010)] when detected by microscopic analysis; while higher prevalences have been reported following PCR analysis [72% in Japan (Matsubayashi et al., 2018), 80% in Uganda (Nolan et al., 2017), 100% in China (Ai et al., 2021)]. Similar to these last reports, the present study detected a prevalence of 100% for E. bovis in cattle samples (n=23) using a PCR-based analysis.

E. bovis has also been previously detected in wild cervids, including in wild goats from Western Australia with a prevalence of 6.4% (Al-Habsi et al., 2017). This relatively low E. bovis prevalence contrasts with the much higher prevalence of 74.6% (53/71) reported here in fallow deer and sambar deer from eastern Australia. This difference could be attributed to methodology, as E. bovis was identified in wild goats from Western Australia (Nolan et al., 2017) by microscopy analysis. Indeed, microscopy and morphology-based detection methods are likely to underestimate parasite prevalence, as discussed above for the case of cattle samples, and are less sensitive methods when compared to the molecular detection tools employed in the present study. Although microscopy detection methods might underestimate the number of E. bovis infections, it is not excluded that lower parasite prevalence can exist, for example, due to climatic reasons. The sampling areas of this study are located in south-eastern Australia, where a Mediterranean-like climate prevails with significantly humid winters, which can facilitate the maintenance of parasites in the environment (Shirley et al., 2018). In contrast, the sampling area of Al-Habsi et al. (2017) was the semiarid rangeland area in Western Australia.

Wild deer and cattle-derived Entamoeba 18S rRNA sequences cluster within the E. bovis clade, although different species-specific clades are formed (Figure 3). Pairwise analysis revealed differences within sequences of wild deer origin, indicating high parasite diversity within this host. Polymorphic markers such as serine-rich protein genes and tRNA-linked short tandem repeats (STRs) have been used for the genotyping and correlation with the geographical distribution of other Entamoeba species such as E. histolytica, E. dispar, and E. nuttalli (Tawari et al., 2007; Weedall and Hall, 2011; Feng et al., 2014). However, this approach is yet to be used to identify E. bovis. We employed a methodology previously used in E. histolytica (Ali et al., 2005); however, the sequencing of ten samples (4 wild deer and 6 cattle samples) using primers S^TGA-D5 and S^TGA-D3 did not generate good quality DNA sequences, even when STR amplicons were cloned prior to sequencing. The primers used here were originally designed to amplify E. hystolytica t-RNA STRs (Ali et al., 2005). Therefore, the presence of polymorphisms in the t-RNA gene of E. bovis and/or the sensitivity of the primers could account for the low quality of the sequences generated.

In the absence of STR data and to determine whether a potential E. bovis cross-species transmission was possible, the time of the most common ancestor between E. bovis sequences of wild deer and cattle origin was estimated (Figure 3). The most common ancestor of E. bovis identified in wild deer and cattle hosts was estimated to have existed well before 200 years ago (before cattle and deer were introduced in Australia). Both ML and Bayesian phylogenetic analyses grouped the sequences according to their host species with moderate sequence divergence. Therefore, taken together, these results provide no evidence of E. bovis transmission between wild deer and cattle in Australia. This finding was somewhat unexpected, but it is possible that since wild deer populations have only recently increased in density, they did not play an important role in the transmission of these parasites thus far. However, this may change in the future due to deer density expansion, increasing contact rates (direct or indirect) and transmission events with livestock species.

Phylogenetic analysis of all Entamoeba sequences of deer origin identified that two sequences (VIC89 and NSW304) cluster with two distinct Entamoeba RL. The term “ribosomal lineage” (RL) was proposed to name Entamoeba strains with greater than 5% sequence divergence from known species (Jacob et al., 2016). Sequence VIC89 detected in a sambar deer from Victoria clustered with high genetic similarity with an Entamoeba RL1 sequence from a roe deer from Sweden (FN666253). This RL was also detected in one gazelle and one bighorn sheep in the USA; however, their sequences are not available for comparison (Jacob et al., 2016). Sequence NSW304, detected in a fallow deer from NSW, clustered with an Entamoeba RL8 sequence (KR025406). This ribosomal lineage has been previously identified in a variety of hosts, including cow (Jacob et al., 2016), camel (Ai et al., 2021) and goat (Ai et al., 2021). The sequence NSW304 presents high homology with an RL8 sequence of a cow (99.9%; KR025406) and camel (99.9%; MN749974) origin and lower homology with a sequence of goat origin (95.1%; MN749989). Here we present the first identification of Entamoeba RL1 and Entamoeba RL8 sequences in wild deer. These results broaden the host range of both RLs.

Interestingly, Entamoeba sequences (NSW332, VIC93 and VIC91) detected in three wild deer animals did not cluster with any 18S rRNA reference sequence. One sambar deer-sourced sequence (VIC91) was genetically distinct from other Entamoeba species found in ruminants, suggesting a possible novel Entamoeba species. Sequences NSW332 and VIC93 of fallow deer origin displayed 100% identity with each other and clustered as a sister clade with the VIC89/Entamoeba RL1 clade. Jacob et al. (2016) proposed the classification for RL sequences based on two criteria: i) sequences with ≥ 80% coverage of the 18S rRNA region, and ii) ≥ 5% difference with previously known sequences. Our phylogenetic analysis has identified NSW332 and VIC93 as a putative novel RL; however, the sequences generated here are shorter than 80% (48%) of the full Entamoeba 18S rRNA gene. Therefore, future studies to determine the complete 18S rRNA gene sequence of NSW332 and VIC93 are needed to confirm this finding.

In conclusion, here we present evidence of three Entamoeba RLs in Australian ruminants: E. bovis in wild deer and cattle, Entamoeba RL1 in wild sambar deer, and Entamoeba RL8 in wild fallow deer. Our study represents the first identification of Entamoeba parasites in Australian deer, expanding the host range of Entamoeba parasites. Further, we present evidence of a potential novel Entamoeba species (VIC91) of wild deer origin, closely related to Entamoeba RL1. We detected a high prevalence of E. bovis (100%) in cattle in the absence of clinical signs, which aligns with the low pathogenicity of E. bovis and its alleged commensal relationship with its cattle host (Ai et al., 2021). Finally, our study suggests a lack of current E. bovis transmission between wild deer and cattle in Australia. However, considering the ongoing expansion of wild deer populations in Australia, both in size and distribution, this scenario is likely to change in the future.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

The work presented in this manuscript required no specific ethical approval. Deer culling was carried out as management operations and independently from this research. Similarly, cattle samples were collected as part of clinical investigations independently from this research. In both cases, we accessed the samples opportunistically.

Conceptualisation, CP and TC. Methodology, JH, CP, and TC. Formal analysis, LK-E and JH. Investigation, JH, CP, and TC. Funding: CP, KH, and TC. Resources, CP, MD and TC. Data curation, JH. Writing—original draft preparation, JH and TC. Writing—review and editing, CP, MD, KH and TC. All authors have read and agreed to the published version of the manuscript.

This study was funded by the Centre for Invasive Species Solutions (Grant PO1-L-002).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.