A new species of Eimeria Schneider, 1875 from the Serra dos Órgãos National Park, Rio de Janeiro, Brazil, with notes on its endogenous development in the montane grass mouse, Akodon montensis Thomas, 1913 (Rodentia: Sigmodontinae)

A total of 53 specimens of the montane grass mouse, Akodon montensis Thomas, 1913 were collected in the Serra dos Órgãos National Park (SONP) in November 2014 and July 2015. The fecal material was analyzed, and a prevalence of 7.5% was recorded for a new coccidian species of the genus Eimeria Schneider, 1875, with part of its endogenous development recorded in the small intestine. The oocysts of a new coccidian species of genus Eimeria are ellipsoidal to subspherical. The wall is bi-layered, c. 1.5 μm (1.3–1.6 μm) thick, outer layer rough. Oocyst (n = 126) mean length is 25.3 μm (21.0–28.0 μm), with a width of 20.2 μm (17.0–22.0 μm) and mean length/width (L:W) ratio of 1.3 (1.2–1.4). Polar granule is present, with the oocyst residuum as a large spherical to subspherical globule. Sporocyst shape (n = 126) is ellipsoidal, with a mean length of 11.8 μm (9.3–14.4 μm), width of 7.9 μm (6.7–9.3 μm), and mean L:W ratio of 1.5 (1.4–1.7). Sporocysts with nipple-like Stieda body and sub-Stieda body are absent. A sporocyst residuum formed by several globules, usually along the sporocyst wall. This is the first record of Eimeria in the montane grass mouse from Brazil.

The rodent family Cricetidae has 130 genera encompassing 681 species, 55 of which are endemic in Brazil. The cricetid genus Akodon Meyen, 1833 has 44 species distributed throughout South America (Musser and Carlenton 2005), with 10 species being found in Brazil, on the east coast between Paraíba and Rio Grande do Sul, and inland in highland areas of Minas Gerais, and in the southwest of the state of Mato Grosso do Sul (Bonvicino et al. 2008). The ecology and taxonomy of some common Akodon species are well known. They are found in a range of different environments, both pristine and impacted, in addition to their epidemiological importance, as reservoirs of zoonoses (Oliveira et al. 2012;Müller et al. 2013;Teixeira et al. 2014 Thomas, 1913(Paglia et al. 2012. These rodents are terrestrial insectivore-omnivores (Graipel et al. 2003). The present study describes a new species of Eimeria, with notes on its endogenous development in the montane grass mouse, Akodon montensis.

Study area
The study area encompasses a continuous tract of wellpreserved Brazilian Atlantic Forest, isolated from human settlements or urban zones. The rodents were captured in the Serra dos Órgãos National Park (SONP), in the municipality of Petrópolis, Rio de Janeiro State, Brazil (22°27′ 49″ S, 43°0 5′ 14.09″ W). This park is located near the center of the Serra do Mar Ecological Corridor (Aguiar et al. 2005) and is one of the principal remnants of Atlantic Forest in the state of Rio de Janeiro.

Capture of the rodents
Rodents were captured in November 2014 and July 2015 along ten transects in each expedition. The specimens were captured in Tomahawk ® (16 × 5 × 5 in.) and Sherman ® traps set on the ground, a total of 90 live traps set per day. Additionally, 20 pitfall traps, made of 100-l buckets, were buried in the ground, along four transects, a capture effort of 80 pitfalls traps set per day. Each trapping session lasted for ten consecutive days. All animals were captured alive and euthanized a posteriori for sample collection. Voucher specimens were deposited in the scientific collection of the National Museum (Museu Nacional) at the Universidade

Collection and morphological analyses of oocysts
Samples were collected during necropsy, directly from the large intestine. The fecal material was placed in a 15-ml conical centrifuge tube containing a 2.5% (w/v) solution of K 2 Cr 2 O 7 and maintained at room temperature. On five consecutive days, the tube was opened and the solution was stirred vigorously to oxygenate it and promote the sporulation of any oocyst present in the faces. The samples were processed in a saturated sugar solution by the centrifugal flotation method (Sheather's method), placed on slides examined under a microscope at a magnification of × 400 (Duszynski and Wilber 1997). Morphological observations and measurements were obtained using a Carl Zeiss Axio Scope.A1 binocular microscope with an apochromatic oil immersion objective lens and AxioVision imaging system. The oocysts were examined with an Imager.A2 light microscope Zeiss equipped with Nomarski interference contrast microscopy × 100 objective lenses, and the images captured with an AxioCam MRc. All measurements are given as the mean value in micrometers, followed by the range of values in parentheses.

Histopathological analysis
To determine the site of infection, the small intestines of specimens positive for oocysts in their feces were segmented and fixed overnight in Carson Millonig formalin. The tissue samples were subsequently dehydrated in a progressive series of ethyl alcohol concentrations, diaphonized using xylol, and Zeiss (Humason 1979). The developmental stages were photographed and measured, and then analyzed qualitatively and described based on the scheme of Kheysin (2013).

Results
Four (7.5%) of the 53 A. montensis specimens analyzed were infected by an undescribed form of Eimeria. The histological analyses revealed the endogenous stages of the parasite in the small intestine of the mice, including the presence of macrogametocytes, microgametocytes, zygotes, and oocysts. Eimeria akodonensis n. sp. Type host: Akodon montensis Thomas, 1913 (Rodentia: Sigmodontinae), Symbiotype host (Frey et al. 1992), skin and skeleton, deposited in the National Museum of Rio de Janeiro (adult males,MNRJ nos. 83768,83774,83776,83777).
Type-material: The oocysts were preserved in 70% ethanol, based on Duszynski and Gardner (1991). The samples were deposited in the Parasite Collection of the Department of Animal Parasitology (http://r1.ufrrj.br/lcc) at the Federal Rural University of Rio de Janeiro, in Seropédica, Rio de  (1.2-1.4). Polar granule is present, with oocyst residuum as a large spherical to subspherical globule.
Endogenous stages Description (Fig. 5-12) The histological analysis revealed the endogenous development of the parasite in the jejunum portion of the small intestine. The infected cells of the lamina propria contained only parasites in the gametogenic phase, with the immature microgametocytes being enveloped by the parasitophorous vacuole, and having a rounded shape, approximately 10.9 μm (5.4-16.8) in length and 11.8 μm (5.5-17.3) in width (Fig. 5). Free immature micro-gametocytes and microgametes were also observed (Figs. 6-7), as were macrogametes at different stages of development, including the formation of the oocyst wall from the granules, approximately 20.9 μm (15.9-25.7) in length and 16.2 μm (12.6-19.9) in width, subspherical to ellipsoidal in shape . Mature and immature oocysts were also observed .

Discussion
In Brazil, the coccidian diversity found in wild rodents is still poorly known, since only three species were reported infecting rodents of the subfamily Sigmodontinae (Rodentia: Cricetidae): Eimeria oryzomysi from specimens of Oryzomyini rodents (Carini 1937), Eimeria zygodontomyis in Necromys lasiurus (Lainson and Shaw 1990), and Besnoitia akodoni in Akodon montensis (Dubey et al. 2003). In Venezuela, Eimeria akodoni was described in the grass mouse Necromys urichi J. A. Allen and Chapman, 1897 (Arcay-de-Peraza 1981), while Eimeria ojastii Table 1 Comparative morphology of Eimeria akodonensis n. sp. and other Eimeria species recorded from rodents from Brazil and Venezuela. The metrical data are given in micrometers as the mean followed by the range in parentheses. When no data were provided, or the data are inadequate for comparisons, the variable is shown as NR = Not Reported  -de-Peraza, 1964). The morphology and morphometry of Eimeria akodonensis n. sp. were compared with data for coccidia of the family Eimeriidae described from other cricetid hosts in Brazil and Venezuela (Table 1). In the case of the study of Arcay-de-Peraza (1964), some data were missing, although they were not crucial to the comparison with Eimeria akodonensis n. sp.. Eimeria oryzomysi (Carini 1937) has the same shape as Eimeria akodonensis n. sp., but is slightly smaller, on average (22-25 μm × 17-19 μm vs. 21-28 μm × 17-22 μm), and does not have a polar granule, its wall is smooth and not very thick, and the residua of the sporocysts are made up of fine granules. Eimeria ojastii (Arcay-de-Peraza, 1964) is considerably smaller (17.4 × 13.2 vs. 25.3 × 20.2), ellipsoidal in shape, and its sporocyst does not have a Stieda body; no data were presented on the presence or absence of a polar granule. Eimeria zygodontomyis (Lainson and Shaw 1990) is also much smaller (16.5 μm × 12 μm vs. 25.3 μm × 20.2 μm), significantly different in shape, and have a fine, single-layered wall (0.6 vs. 1.5 μm) in comparison with the rough double wall of Eimeria akodonensis n. sp.
Morphologically, the most similar of the four species to Eimeria akodonensis n. sp. is E. akodoni, which was described by Arcay- de-Peraza (1981) parasitizing N. urichi. The two species are similar in size (27 μm × 18 μm vs. 25.3 μm × 20.2 μm) and both have a polar granule, although the sporocysts are slightly different in their morphology and size (14 μm × 7 μm vs. 11.8 μm × 7.9 μm). Arcay-de-Peraza (1981) describes the oocysts of E. akodoni as fusiform, and when they were observed in immersion, their thick wall was apparent along their whole length except for the extremities, where they become thinner, taking on the appearance of Bfalse opercules,^in the words of the author. This characteristic is not observed in Eimeria akodonensis n. sp., and the oocysts are not fusiform in shape. Arcay-de-Peraza describes the presence of three walls in E. akodoni and the absence of residuum in the oocyst, whereas in Eimeria akodonensis n. sp., the oocyst has a double wall, with a large residuum. Despite these broad morphological similarities, the endogenous development of Eimeria akodonensis n. sp. occurs in different portions of the intestine in comparison with E. akodoni. Whereas Eimeria akodonensis n. sp. develops in the small intestine, Arcay- de-Peraza (1981) found E. akodoni in the large intestine. According to Arcay-de-Peraza (1981), merogony and gametogony of E. akodoni occurred in epithelial cells of the large intestine. In the case of E. akodonensis n. sp., gametogony was only recorded in the jejunum portion of the small intestine. The microgametocytes of E. akodonensis n. sp. were smaller (10.9 × 11.8 μm) than E. akodoni (30.6 × 20.4 μm). In addition, macrogametocytes of E. akodonensis n. sp. were sub-spherical, 20.9 × 16.2 μm (L/W ratio: 1.2 μm), and those of E. akodoni were ellipsoidal, 19.7 × 11.7 μm (L/W ratio: 1.6 μm). The effects of infection by Eimeria akodonensis n. sp. on the intestinal tissue of Akodon montensis varied considerably, from a total absence to a light and diffuse infiltration distributed in the intestinal mucosa composed of neutrophils and mononuclear cells. However, this infiltration was not found in association with the parasites themselves, but rather in the epithelium of the intestinal villi. Overall, the set of morphological and physiological characteristics found in Eimeria akodonensis n. sp. supports conclusively its description as a new species.