Eimeria spp . ( Apicomplexa : Eimeriidae ) of marsupials ( Mammalia : Didelphimorphia ) in southern Bahia , Brazil Eimeria spp . ( Apicomplexa : Eimeriidae ) de marsupiais ( Mammalia : Didelphimorphia ) na região sul da Bahia , Brasil

The occurrence of Eimeria Schneider, 1875 in mammals of the order Didelphimorphia indicates the infection-predisposition of these animals, which in turn is mainly determined for their eating habits. The objective of this work was to evaluate the parasitism of Eimeria spp. in marsupials of the Atlantic Forest of the southern region of Bahia. Fecal samples were collected from marsupials captured in the regions of Ilhéus, Una, Belmonte and Mascote, with traps of the Sherman model (23 × 8 × 9 cm), Tomahawk (50 × 17 × 17 cm) and pitfall and analyzed by Sheather’s modified centrifugal-flotation method. Oocysts were identified by microscopical evaluation of their morphology and morphometry. Didelphis aurita Wied-Neuwied, 1826, Gracilinanus agilis Burmeister, 1854, Monodelphis americana Müller, 1776, Marmosa demerarae O. Thomas, 1905 and Marmosa murina Linnaeus, 1758 were parasitized by Eimeria philanderi Lainson & Shaw, 1989 and Eimeria gambai Carini, 1938. Mixed parasitism for these two coccidia was observed in two of the 56 marsupials sampled. In conclusion, this work registers new hosts for E. philanderi and E. gambai, as well as the state of Bahia as a new distribution site for these coccidia.


Introduction
Eimeria Schneider, 1875 is an intracellular protozoan that infects a wide range of vertebrate hosts.Transmission occurs by ingestion of sporulated oocysts, by direct fecal/oral contact, or by ingestion of contaminated food or water (URQUHART et al., 1987;ZANETTE et al., 2008).Infection by this protozoan can be asymptomatic or cause mild to moderate disease in domestic and wild animals (ZANETTE et al., 2008).
The samples were processed and examined by the modified flotation technique with 1,43 (g/L) density sucrose solution (500 g sucrose, 350 ml water) by centrifugation (5 min at 2000 rpm) described by Sheather (1923) and modified by Duszynski & Wilber (1997).All samples were observed under an Olympus BX51 microscope on the 20× objective and confirmed with a 40× objective.Positive samples were kept at 20-24ºC, allowing aeration for seven days to ensure complete oocyst sporulation.Sporulated oocysts were observed, photomicrographed and measured using a 100× objective microscope and immersion oil, coupled to a digital camera connected to a computer using the Image -Pro Express 6.0 program.For specific identification, morphological and morphometric guidelines described by Duszynski & Wilber (1997) and Berto et al. (2014) were used.
Eimeria philanderi (Figure 2A, B) was identified from D. aurita, M. americana, M. demerarae and M. murina, at Una, Belmonte and Mascote in Southern Bahia, Brazil (Figure 1).Their oocysts sporulated in 7-8 days and were observed as sub-spherical, with a double-layered wall (the inner one being dark and occupying about 1/4 of the wall, and the outer one brown, with striations and projections occupying around 3/4 of the wall).Micropyle and oocyst residue were absent, but a polar granule was present.The sporocysts were ovoid, with prominent Stieda bodies, although the sub-Stieda was barely discernible.Parastieda bodies were absent.Sporocyst residue was present in the form of a few dispersed granules.The accompanying sporozoites had two refractile bodies.The morphometries of E. philanderi isolated from each host species are compared in Table 2.
Eimeria gambai (Figure 2C, D) was identified from G. agilis, M. demerarae and M. murina, at Una, Belmonte and Mascote in Southern Bahia, Brazil (Figure 1).Their oocysts sporulated in 7-8 days and were observed as ellipsoids, with a double-layered Table 1.Distribution of Eimeria species related to marsupials and their respective collection points as indicated in Figure 1, in southern Bahia, Brazil.wall (the inner one being dark and occupying about 1/3 of the wall and the outer one brown, with striations and projectoins occupying about 2/3 of the wall).Micropyle, polar granule and oocyst residue were absent.The sporocysts were ovoid, with discrete Stieda bodies and sub-Stieda.Parastieda bodies were absent.Sporocyst residue was present in the form of a few dispersed granules.The accompanying sporozoites had two refractile bodies.The morphometries of E. gambai isolated from each host species are compared in Table 2.

Discussion
To the best of our knowledge, the marsupials captured in this study have not been reported previously as hosts for E. philanderi and E. gambai.This represents the first novel finding of the current report.The oocysts of both species had morphological and morphometric characteristics that were very similar to the original descriptions, and to subsequent follow-up studies (CARINI, 1938;LAINSON & SHAW, 1989;TEIXEIRA et al., 2007).
Eimeriaphilanderi was identified in four species of marsupials in the present study.Their oocysts had morphometries (Table 2) similar to those originally described by Lainson & Shaw (1989).The morphology also corroborates with the studies of Lainson &Shaw (1989) andHeckscher et al. (1999), although in the present study, the presence of sub-Stieda and some differences in the intensity of roughness, striations, and wall projections were observed.
Eimeria gambai had only previously been isolated from Didelphis aurita (CARINI, 1938;TEIXEIRA et al., 2007).Our current study now extends the host range to G. agilis, M. demerarae and M. murina.The E. gambai oocysts had morphometry (Table 2) similar to that originally described by Carini (1938) and later by Teixeira et al. (2007).The presence of sub-Stieda in the sporocyst, the absence of polar granules, and differences in the intensity of roughness in the wall were observed in oocysts of this study.However, these differences were insufficient to warrant the announcement of a new species.
The delimitation of coccidian species has been discussed since the beginning of parasitology.Traditionally, the description of the new species follow the guidelines of Duszynski & Wilber (1997), who consider that the oocysts should be compared with coccidian species that are feature-similar and belong to the same host family.
Currently with the advent of genetic sequencing techniques, the 18S and 28S rRNA genes and the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene have been recognized as useful loci to complementation of species characterization and in phylogenetic studies (YANG et al., 2015;SILVA-CARVALHO et al., 2018).However, this molecular tool has still limited ability to delimit species because there is no consensus on which genes are primordial and which percentage of genotypic difference separates the species ( SILVA et al., 2016).Anyway, none of these Eimeria spp.from marsupials have ever been sequenced in these loci.
In this sense, we endorse Kunz (2002), who states that the criterion for identification of new species can not be just on the basis of a certain number of base exchanges within DNA sequence; therefore, we intend in this work to identify the coccidian species evaluating all the morphological, biological and ecological (host specificity) aspects.
It is also worth mentioning that this paper shows the inconclusive and imprecise taxonomy of marsupial coccidia, since our study prioritized the identification of the species primarily described (CARINI, 1938;LAINSON & SHAW, 1989).This was to the detriment of more recently described species, which were not clearly differentiated from the predecessors (HECKSCHER et al., 1999;TEIXEIRA et al., 2007).In this sense, we emphasize the difficulty in delimiting Eimeria spp.from marsupials and correctly assigning them to new or already described species.Such difficulty is associated to the paucity of studies concerning Eimeria taxonomy, especially regarding some hosts, such as marsupials.

Conclusion
To the best of our knowledge, this is the first identification of Eimeria infection in marsupials captured in the State of Bahia.Although E. philanderi and E. gambai have already been described in marsupials, there are no records in the scientific literature reporting these two species parasitizing the marsupials D. aurita, G. agilis, M. americana, M. demerarae, and M. murina.Therefore, this paper has identified new hosts for E. philanderi and E. gambai, and indicates that the state of Bahia is a new distribution site for these coccidia.

Table 2 .
Morphometry of oocysts and sporocysts of Eimeria spp.identification of fecal samples of marsupials, from southern Bahia, Brazil.