Introduction

Small mammals are recognized hosts of bacteria of the Anaplasmataceae family and of apicomplexans with importance in veterinary and public health (Goodman et al. 2005; Perles et al. 2019). Due to their ubiquity, high population densities, and implications in food webs, small mammals play an important role in the dissemination of pathogens (Han et al. 2015). However, knowledge in relation to whether these vertebrates naturally carry pathogenic tick-borne bacteria or protozoans in South America is still emerging and requires further study.

The Anaplasmataceae family of bacteria includes intracellular obligate gram-negative alphaproteobacteria that infect ticks, and some strains cause disease in humans (Rar and Golovljova 2011). While pathogenic species of Anaplasma, Ehrlichia, and “Candidatus Neoehrlichia” are known to infect wild rodents in the Northern Hemisphere (Rar and Golovljova 2011), significantly less is known regarding vertebrate hosts of these bacteria in Southern latitudes of the globe. In particular, research on tick-borne bacterial pathogens is still incipient in Chile.

The Apicomplexa are obligate parasites with more than 6,000 described species, some of which may cause severe disease to their hosts and provoke significant economic losses worldwide (Votýpka et al. 2017). Decades ago, the genus Babesia attracted the attention of scientists because of a clinical disease that affected humans and domestic animals (Homer et al. 2000; Schreeg et al. 2016). In this context, it is currently known that species in the Babesia microti complex are zoonotic agents in Asia, Europe, and North America, with rodents carrying pathogenic strains in some cases (Goethert 2021). Although B. microti has never been detected in wild animals in South America, a B. microti-like agent was identified using molecular methods in humans from Bolivia (Gabrielli et al. 2016), suggesting that these parasites may represent neglected pathogens in the region.

Animals can also develop illness when infected with apicomplexan parasites such as species in the genus Hepatozoon (Merino et al. 2009; de Sousa et al. 2017; Perles et al. 2019). Hepatozoon spp. are intraerythrocytic and intraleukocytic parasites with a heteroxenous life cycle that involves vertebrates and invertebrates as intermediate and definitive hosts, respectively (Smith 1996). In South America, studies based on the detection of the Hepatozoon 18S rRNA structural locus (18S hereafter) suggested that in nature, these parasites use rodents and other small mammals as intermediate and paratenic hosts (Smith 1996; Merino et al. 2009; Wolf et al. 2016; Muñoz-Leal et al. 2019; Perles et al. 2019; Alabí et al. 2021). Hepatozoon spp. are transmitted to vertebrates through the ingestion of infected ectoparasites (Smith 1996). Interestingly, ticks of the genus Ixodes associated with rodents have be suggested as potential vectors in Chile (Muñoz-Leal et al. 2019).

Chilean small mammals are represented by 72 species: 67 belong to the order Rodentia and five to Marsupialia (D’elía et al. 2020; Mejías et al. 2021). Although the role of Chilean rodents and marsupials as reservoirs of pathogenic microorganisms is still obscure, DNA of Anaplasmataceae, Bartonella, Borrelia, Hepatozoon, and Mycoplasma has been detected previously (Merino et al. 2009; Müller et al. 2018, 2020; Muñoz-Leal et al. 2019; Thomas et al. 2020; Alabí et al. 2020, 2021). Therefore, it seems likely that widening the repertoire of species submitted to molecular analyses searching for those agents would contribute to the understanding of eco-epidemiological cycles. To address this objective, we targeted DNA of bacterial and apicomplexan agents in a range of small mammals captured in northern Chile.

Material and methods

Study area and collection of samples

Collections were performed during July of 2018 (Austral winter) in seven localities of northern Chile roughly between latitudes 19 and 31°S as follows: Parinacota and Socoroma (Arica and Parinacota Region), Enquelga, Chusmiza, and Pampa del Tamarugal National Reserve (PTNR) (Tarapacá Region), Llanos de Challe National Park (LCNP), and Bosque Fray Jorge National Park (BFJNP) (Atacama Region and Coquimbo regions, respectively) (Fig. 1). This area of northern Chile is characterized by constant dryness along the year, with small peaks of humidity in summer (Luebert and Pliscoff 2017), and includes the core of the Atacama Desert, the driest ecosystem on earth (Clarke 2006).

Fig. 1
figure 1

Map of Northern Chile with the localities where collections were performed, highlighted with black and white symbols. Administrative regions: I, Tarapacá; III, Atacama; IV, Coquimbo; XV, Arica and Parinacota. Abbreviations: NP, National Park; NR, National Reserve

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To capture rodents and marsupials, we set an average of 113 Sherman-like traps baited with oats along transects that remained active for two nights per locality (100 h in total), totalizing 1139 traps/night. Animals were manipulated as recommended by Sikes (2016). Briefly, we used a 300 g scale (Pesola) to weight the animals, and an intraperitoneal injection with a ketamine (60 mg/kg)-xylazine (3 mg/kg) solution to restrain them (Carpenter and Marion 2018). Approximately 20 μL of blood was obtained by nicking the caudal vein. Samples were preserved in sterile tubes with 96% ethanol (Sigma-Aldrich). Animals were identified morphologically in situ using a taxonomic guide (Patton et al. 2015), and released at their capture sites after recovering from anaesthesia.

DNA extraction and gene amplification

Total genomic DNA was extracted from blood samples employing the DNeasy Blood & Tissue Kit (QIAGEN, Germany), and eluted in 40 μL of Buffer AE (10 mM Tris–Cl; 0.5 mM EDTA, pH 9.0). Successful DNA extractions were checked through a conventional PCR targeting the mammalian gapdh (glyceraldehyde-3-phosphate dehydrogenase) gene (Birkenheuer et al. 2003). Positive samples were subsequently screened for Anaplasmataceae (16S rRNA), Piroplasmida spp. (18S and COI), and Hepatozoon spp. (18S) DNA using conventional PCR protocols. A touchdown PCR to amplify a fragment of the mammalian cytb gene was used to genetically identify positive animals (Leite and Patton 2002). Primers and PCR thermal conditions used in this study are provided in Table 1.

Table 1 Primers and thermal conditions for PCR were used for the detection and genetic characterization of Anaplasmataceae, Piroplasmida, Hepatozoon, and mammals
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Each PCR reaction was performed by adding 2 μL genomic DNA into a mix of 2.5 μL DreamTaq Buffer, 0.5 μL dNTPs (0.2 mM), 0.3 μL DreamTaq Polymerase, 1 μL of each primer (10 pmol), and 17.7 μL of ultra-pure water. Amplicons were submitted to electrophoresis into 2% agarose gels, stained with SYBR Safe (Life Technologies/Thermo Fisher Scientific, Carlsbad, CA), and visualized by UV transillumination. Products of the expected size were purified and sequenced in both directions at the AUSTRAL-omics facility, in Universidad Austral de Chile (Valdivia, Chile). Positive controls included DNA of Ehrlichia canis, Hepatozoon canis, and Babesia canis previously obtained from infected dog blood.

Phylogenetic analyses

Sequences were quality-checked and edited with Geneious Prime® version (v) 2021.2.2 (www.geneious.com). Pairwise comparisons were performed with BLASTn (https://blast.ncbi.nlm.nih.gov), and similar sequences were downloaded from GenBank (https://www.ncbi.nlm.nih.gov) to construct alignments with MAFFT (Katoh and Standley 2013).

Phylogenies were built using Bayesian Inference (BI) and Maximum Likelihood (ML) methods with MrBayes 3.2.2. (Ronquist et al. 2012) and IQ-TREE v 1.6.12 (Nguyen et al. 2015), respectively. BI best evolutionary models were selected using the MrBayes command “lset nst = mixed rates = gamma” (Huelsenbeck 2004; Ronquist et al. 2012). Two independent tests of 107 generations and four MCMC chains were run, with sampling of trees every 1,000 generations removing the first 25% as burn-in. Tracer v 1.7.1 was used to confirm the effective sample size values (ESS) and the correlation of Markov chains (Rambaut et al. 2018). Bayesian posterior probabilities (BPP) with values ≥ 0.70 were considered to represent strong statistical support (Huelsenbeck and Rannala 2004). ModelFinder was used to select best nucleotide substitution model for ML analyses (Kalyaanamoorthy et al. 2017). We used rapid hill-climbing and stochastic disturbance methods to evaluate the robustness of the inferred tree with 1,000 ultrafast bootstrapping pseudo-replicates. Ultrafast bootstrap values ≥ 70%, between 70 and 94%, and > 95% were interpreted as low, medium, and strong statistical support values respectively (Minh et al. 2013).

Results

A total of 102 small mammals belonging to 10 species and three families were captured (Table 2). The production of amplicons of the expected size for gapdh corroborated successful DNA extractions in all samples. While PCR screenings for Anaplasmataceae yielded negative results, 15/102 (14.7%), samples were positive for apicomplexan DNA (Table 2). Sequences of mammalian cytb were obtained for 13/15 positive animals, confirming the identities of five species of rodents and one marsupial (Table S1). GenBank accession numbers for nucleotide sequences generated in this study are available in Table S2.

Table 2 Sampled small mammal species, localities of collection, and PCR assays. The GenBank accession numbers are indicated for the positive samples. Every sequence was obtained from a different specimen. Abbreviations: BFJNP, Bosque Fray Jorge National Park; LCNP, Llanos de Challe National Park; PTNR, Pampa del Tamarugal National Reserve
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Babesia

Two genotypes of Babesia 18S were obtained (Babesia sp. LC87, and Babesia sp. LC77, hereafter). The Babesia sp. LC87 genotype (537 bp) was obtained from two specimens of Phyllotis darwini (Rodentia: Cricetidae) and two Abrothrix jelskii (Rodentia: Cricetidae) in LCNP and Parinacota, respectively. BLASTn comparisons for this genotype yielded an identity of 94.8% (439/463 bp, 83% query cover, 7 gaps, 0 E value) with Theileria sp. ex Damaliscus lunatus clone TS23-6 (HQ179765), amplified from the common tsessebe (Damaliscus lunatus, Artiodactyla: Bovidae) in South Africa (Brothers et al. 2011). The Babesia sp. LC77 genotype (546 bp) was retrieved from a single specimen of P. darwini captured at LCNP. BALSTn comparisons for Babesia sp. LC77 revealed an identity of 98.6% (489/496 bp, 90% query cover, 0 gaps, 0 E value) with B. microti isolate Kv21 (MG062780), detected in Ixodes persulcatus from Russia (Livanova et al. 2018).

ML and BI phylogenetic reconstructions for 18S showed 11 discrete phylogenetic clades (Fig. 2). With strong support for Babesia sp. LC87 clustering into a monophyletic clade with a Babesia sp. detected in Thrichomys pachyurus (Rodentia: Echymidae) and a Babesia sp. detected in Phyllostomus discolor (Chiroptera: Phyllostomidae) from Brazil (clade XI, Fig. 2). In contrast, the Babesia sp. LC77 genotype is grouped within the B. microti complex, branching basally to several strains of this group detected in the Northern Hemisphere (clade I, Fig. 2).

Fig. 2
figure 2

Bayesian and Maximum Likelihood phylogenies for a subset of Piroplasmida spp. inferred using an alignment (1,721 bp) of the gene encoding 18S rDNA. Calculated substitution models for BI and ML were M125, M191, M134, M200, M136, M40, and M189, and TIM3 + F + R6, respectively. Best models were chosen using the Bayesian Information Criterion (BIC) (Schwarz 1978). Values of Bayesian Posterior probability/ML Bootstrap are indicated above or below main branches. The position of Babesia spp. characterized in the present study is highlighted in bold. Roman numbers indicate phylogenetic lineages of the Piroplasmida order according to Jalovecka et al. (2019) and Ikeda et al. (2021); I: B. microti group, II: Monotremata group, III: Western group, IV: Marsupialia group, V: Percei group, VI: Rhinocerotidae group, VII: Cytauxzoon, VII: Equus group, IX: Theileria sensu stricto, X: Babesia sensu stricto, XI: Babesia spp. detected in South American mammals

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Amplicons of COI target (922 bp) were obtained only in the sample from which Babesia sp. LC77 was retrieved, showed an identity of 86.7% (794/916 bp, 98% query cover, 6 gaps, 0 E value) with B. microti strain RI (LN871600) isolated from humans (Cornillot et al. 2012). In accordance with this genetic similarity, ML and BI phylogenies for COI confirmed that Babesia sp. LC77 belongs to the B. microti group (clade I; Fig. 3).

Fig. 3
figure 3

Bayesian Inference and Maximum Likelihood phylogenies for a subset of Piroplasmida spp. inferred using an alignment (1,401 bp) of the gene encoding cytochrome c oxidase I (COI). Calculate substitution models for BI and ML were M136, M40, M125, M191, and M198 (part1), M93, M184, and M155 (part2), M201, M162, M200; M189, M203, M134, M138, and M198 (part3), and TN + F + I + G4 (part1), TVM + F + R3 (part2), GTR + F + I + G4 (part3), respectively. Best models were chosen using the Bayesian Information Criterion (BIC) (Schwarz 1978). Values of Bayesian Posterior probability/ML Bootstrap are indicated above each branch. The position of Babesia spp. characterized in the present study is highlighted in bold

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Hepatozoon

Four genotypes of Hepatozoon 18S were identified. Three identical sequences were recovered from three different specimens of P. darwini captured in BFJNP. A consensus of 544 bp designated as Hepatozoon sp. BFJ69 represents those sequences. Hepatozoon sp. BFJ69 was identical to Hepatozoon sp. isolate HepIxo-281 (MH174345), detected in ticks of the Ixodes sigelos group collected at the same locality (Muñoz-Leal et al. 2019). A second genotype of 544 bp (Hepatozoon sp. LC82 hereafter) was obtained from two specimens of P. darwini collected in BFJNP and LCNP. BLASTn comparisons of this second haplotype yielded 99.8% of identity with Hepatozoon sp. isolate HepIxo-284 (MH174344), obtained from the same Ixodes ticks at BFJNP (Muñoz-Leal et al. 2019).

Two additional Hepatozoon genotypes of 566 and 577 bp were recovered from Abrothrix olivacea (Rodentia: Cricetidae) collected in BFJNP (Hepatozoon sp. BFJ7 hereafter) and Oligoryzomys longicaudatus (Rodentia: Cricetidae) from Socoroma (Hepatozoon sp. Soc48 hereafter), respectively. Hepatozoon sp. BFJ7 showed an identity of 99.8% (565/566 bp, 100% query cover, 0 gaps, 0 E value) with Hepatozoon sp. AS7 (FJ719819) detected in Abrothrix sanborni (Rodentia: Cricetidae) from Chiloé Island, in southern Chile (Merino et al. 2009). On the other hand, Hepatozoon sp. Soc48 showed an identity of 98.6% (569/577 bp, 100% query cover, 1 gap, 0 E value) with Hepatozoon sp. HepIxo-281 (MH174345) recovered from ticks of the I. sigelos group in BFJNP (Muñoz-Leal et al. 2019).

Phylogenetic analyses performed for Hepatozoon 18S yielded a logic topology, separating the genus into two large clades: one composed by Hepatozoon spp. detected in amphibians, small mammals, reptiles, and ticks, and a second one conformed by species detected in canids. The genotypes characterized herein formed four independent lineages within a large clade composed by Hepatozoon spp. detected in South American rodents and their ticks (Fig. 4).

Fig. 4
figure 4

Bayesian and Maximum Likelihood phylogenies for a subset of Hepatozoon spp. inferred using an alignment (1,662 bp) of the gene encoding 18S rRNA gene. Calculate substitution models for BI and ML were M85, M15, M177, M147, M134, and M179, and HKY + F + G4, respectively. Best models were chosen using the Bayesian Information Criterion (BIC) (Schwarz 1978). Values of Bayesian Posterior probability/ML Bootstrap are indicated above, below, or arrowing major branches. The position of Hepatozoon spp. characterized in the present study is highlighted in bold

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Sarcocystidae

Interestingly, the primers targeting DNA of Piroplasmida order amplified a 577-bp fragment of 18S that upon BLASTn analyses did not match any piroplasmids species. The amplicons were produced form two specimens of Thylamys elegans (Didelphimorphia: Didelphidae) and a single specimen of Thylamys pallidior (Didelphimorphia: Didelphidae), captured at BFJNP and Socoroma, respectively. Sequences obtained from the two species of Thylamys were identical and showed 100% sequence similarity with a undetermined apicomplexan denominated as Sarcocystidae sp. TE1 (577/577, 100% query cover, 0 gaps, 0 E value, EU443095), detected in the blood of T. elegans captured in Chile (Merino et al. 2010). Considering the high similarity with the sequence reported by Merino et al. (2010), and that the identity of these apicomplexans remains unsolved, we opted to designate it as Sarcocystidae sp. BFJ35 until additional genes can be analysed.

From a phylogenetic point of view, Sarcocystidae sp. BFJ35 formed a monophyletic clade with sequences of sarcocystids detected in the Australian marsupials Petaurus australis (Diprotodontia: Petauridae) and Acrobates pygmaeus (Diprotodontia: Acrabatidae) (Fig. 5) (Zhu et al. 2009; Holz et al. 2020). Collectively, the undetermined apicomplexan parasites detected in Acrobates, Petaurus, and Thylamys form an independent lineage within the Sarcocystidae family.

Fig. 5
figure 5

Bayesian and Maximum Likelihood phylogenies for a subset of apicomplexan protozoans. inferred using an alignment (1,992 bp) of the gene encoding 18S rRNA. Calculate substitution models for BI and ML were M40, M134, M136, M162, M138, and M125, and TIM2 + F + R3, respectively. Best models were chosen using the Bayesian Information Criterion (BIC) (Schwarz 1978). Values of Bayesian Posterior probability/ML Bootstrap are indicated above, below, or arrowing major branches. The position of Sarcocystidae sp. BFJ35 characterized in the present study is highlighted in bold

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Discussion

To elucidate the role of wild mammals in the tick-host-microorganism reservoir system is important for recognizing areas where outbreaks of tick-borne pathogens might occur in nature (Mills 1998). Although previous research in Chile looking for tick-borne Anaplasmataceae identified “Candidatus Neoehrlichia chilensis” in Abrothrix sp., Mus musculus (Rodentia: Murinae), and Ixodes ticks collected upon P. darwini and Octodon degus (Rodentia: Octodontidae) (Müller et al. 2018; Muñoz-Leal et al. 2019), in our study, none of the tested animals was positive. Importantly, we used blood samples to perform the screenings whereas previous studies extracted DNA from the spleen to detect these agents (Müller et al. 2018), likely increasing the sensitivity of the assays. While our samples were negative for Anaplasmataceae, we detected novel genotypes of Babesia and Hepatozoon in rodents and expanded the distributional and host range of an apicomplexan of the Sarcocystidae family that infects marsupials.

Babesia

Babesia species are transmitted by ticks and merge their life cycles with wild mammals that maintain the infection in nature (Karshima et al. 2021). The B. microti group is of medical importance in the Northern Hemisphere, and five clades currently define its diversity (Goethert and Telford 2003). Human pathogenic strains belong to B. microti sensu stricto (Goethert and Telford 2003), and are represented mostly by Eurasian and North American isolates (Goethert 2021). Remarkably, a Babesia sp. 99% identical with B. microti sensu stricto detected in blood from inhabitants of the Bolivian Chaco (Gabrielli et al. 2016) represents the sole genetic evidence of this blood parasite in South America. Despite being a human pathogen, the identity of the vector or vertebrate reservoir of this agent is yet to be elucidated. Herein, we characterized amplicons derived from the 18S and COI of a Babesia sp. (Babesia sp. LC77) belonging to the B. microti group in Phyllotis rodents from Chile, suggesting that these mammals could act as a reservoir. Our results, combined to the record of Gabrielli et al. (2016) from Bolivia, indicate that the B. microti group is likely underrepresented in South America, and that neglected human-pathogenic strains circulate in the region.

The role of rodents as hosts for Babesia spp. has been barely assessed in South American ecosystems. However, Babesia spp. have been detected in T. pachyurus (Wolf et al. 2016), Thrichomys fosteri (de Sousa et al. 2018), Rattus norvegicus, and Oligoryzomys nigripes from Brazil (Gazeta et al. 2004). Herein, we characterized an additional genotype, Babesia sp. LC87, detected in P. darwini and A. jeslkii, therefore adding novel hosts for these tick-borne apicomplexans in the continent. Interestingly, Babesia sp. LC87 clusters phylogenetically within a recently described array of babesias associated with bats and rodents from Brazil (Wolf et al. 2016; Ikeda et al. 2021) (Fig. 2). It is important to note that we are aware that our sequences are relatively short, and that analyses employing longer sequences of 18S might yield a different tree topology. However, the phylogenies derived from previous studies do mirror our analyses (de Sousa et al. 2018; Ikeda et al. 2021), and support the occurrence of a South American lineage of Babesia spp. (Fig. 2). Although the vectors of these agents in the region remain unidentified, it is well known that Ixodes ticks transmit Babesia in the Northern Hemisphere (Karshima et al. 2021). In Chile, the Ixodes sigelos group of ticks are common parasites of Abrothrix and Phyllotis rodents (Muñoz-Leal et al. 2019; Landaeta-Aqueveque et al. 2021), so attempts to understand the ecoepidemiology of Babesia sp. LC77 and LC87 should consider those ticks as potential vectors.

Hepatozoon

Ten years ago, Merino et al. (2009) reported an Hepatozoon sp. in A. olivacea and A. sanborni at Chiloé island. Recently, Ixodes ticks collected upon Abrothrix longipilis (Rodentia; Cricetidae) and P. darwini were positive to this apicomplexan as well (Muñoz-Leal et al. 2019). Herein, we report P. darwini as new host for Hepatozoon in Chile, therefore expanding the distribution of agents of this genus within the country. Collectively, these results point that rodent of genera Abrothrix, Oligoryzomys, and Phyllotis are common intermediate hosts for Hepatozoon along Chilean ecosystems. Importantly, the four genotypes of Hepatozoon characterized in this study were retrieved from cricetid rodents, and clustered into a large monophyletic group including sequences retrieved from other rodents of this family and their ticks (Fig. 4a). This fact supports the hypothesis that cricetid rodents and their ectoparasites could maintain enzootic cycles of a natural group of Hepatozoon spp. in the region (Muñoz-Leal et al. 2019). Although the detection of Hepatozoon in ticks feeding on small mammals does not demonstrate any transmission capacity (Giannelli et al. 2013), these ectoparasites should not be ruled out as potential vectors (Muñoz-Leal et al. 2019).

Recently, Alabí et al. (2021) reported DNA of Hepatozoon in synanthropic rodents (M. musculus and R. rattus) and O. longicaudatus from Valdivia, in southern Chile. Given that the 18S sequences generated by Alabí et al. (2021) corresponded to a different region of the locus, we were unable to include them in our phylogenies. However, according to our phylogenetic inferences (Fig. 4), and those of Alabí et al. (2021), discrete lineages of Hepatozoon would be associated with specific genera of native rodents, likely denoting events of coevolution (Poulin and Keeney 2008). A similar pattern of association between Hepatozoon spp. and their rodent hosts was also reported by Merino et al. (2009), yet this trend seems to be an exception, since Hepatozoon species are considered by some authors to be rather generalist parasites with low specificity for their vertebrate hosts (Smith 1996; Telford et al. 2001). The fact that in Chile, Hepatozoon species appear to be associated with specific genera of rodents is intriguing and suggests that the evolutionary history of these mammals may be shaping the diversity of the parasites (Hoberg and Brooks 2010). To test this hypothesis, future research should aim to retrieve complete Hepatozoon 18S sequences together with data for fast-evolving loci such as COI, and target previously unstudied rodent species.

Sarcocystidae

The detection of apicomplexans in mouse opossums of genus Thylamys is an incidental finding that reflects the low specificity exhibited by the primers used in this study to amplify 18S of piroplasmid species. Moreover, our findings are not a novelty in Chile. In fact, Merino et al. (2010) reported an undescribed apicomplexan species related to the Sarcocystidae family in T. elegans. Although the number of Thylamys analysed in our study was low (n = 3), it is noteworthy that all the specimens were positive. In this context, the previous detection of a Sarcocystidae sp. in Chilean opossums also showed a high prevalence of infection (Merino et al. 2010). In contrast, all rodents (n = 99) sampled in our study were negative to this apicomplexan, indicating a degree of specificity for Thylamys opossums. Indeed, specific associations between Sarcocystidae parasites and their hosts have been reported to occur in other ecosystems (Šlapeta et al. 2003).

The relatedness of Sarcocystidae sp. BFJ35 genotype detected in Chilean opossums with apicomplexans from Australian marsupials is particularly interesting (Fig. 5), and suggests that a common ancestor that infected marsupials diverged with them 45–50 million years ago during the Eocene era, after the split of Antarctica and Australia (Nilsson et al. 2004; Merino et al. 2010). It is pertinent to note that there is evidence that this novel apicomplexan taxon can disseminate and cause severe liver, spleen, and lung infection, and as such, it would represent a threat to marsupial populations with conservation issues (Holz et al. 2020). In the meantime, the Sarcocystidae sp. associated with Australian and Chilean marsupials remains as an understudied taxon that clearly deserves more attention (Duszynski 2016).

Parasites of the Sarcocystidae family have heteroxenous life cycles involving carnivores as definitive hosts, and other vertebrates such as marsupials, as intermediate hosts (Votýpka et al. 2017). Oocysts of these parasites are released into the environment through faeces and reach novel hosts after ingestion of contaminated food or water (Votýpka et al. 2017; Holz et al. 2020). While the ecology of Sarcocistidae sp. BFJ35 is still unclear, the study of owls and foxes that usually prey on Thylamys spp. (Jaksic Andrade 1993; Carevic et al. 2013; Valladares Faúndez et al. 2018) should shed light on the definitive hosts of this novel apicomplexan species in Chile.