Three new species of Eimeria (Apicomplexa: Eimeriidae) from the Amami rabbit, Pentalagus furnessi (Mammalia: Leporidae).

The Amami rabbit, Pentalagus furnessi (Mammalia: Lagomorpha: Leporidae), is a relict and endangered species endemic to the Amami-Oshima and Tokunoshima Islands, located in southwestern Japan. Here, we described three new species of Eimeria (Apicomplexa: Eimeriidae) parasites detected from fecal samples of wild Amami rabbits. Eimeria furnessi n. sp., recorded in 21 (58.3%) samples, has ellipsoidal oocysts with two walls and micropyle, 26.0 × 16.6 μm, and elongate-ovoidal sporocysts, 13.1 × 6.3 μm, with Stieda body. Eimeria hilleri n. sp., recorded in 9 (25.0%) samples, has ellipsoidal oocysts with two walls and micropyle, 34.7 × 21.4 μm, and elongate-fusiform to elongate-ovoidal sporocysts, 15.7 × 8.3 μm, with Stieda and substieda bodies. Eimeria sagentae n. sp., recorded in 13 (36.1%) samples, has ellipsoidal oocysts with two walls and micropyle, 20.9 × 14.5 μm, and elongate-ovoidal sporocysts, 10.4 × 5.0 μm, with Stieda body. The three new species can be distinguished by the size and color of their oocysts. Further studies related to the pathogenicity of these parasites can improve the breeding and propagation procedures of the Amami rabbit.

Introduction

Pentalagus is a lagomorph genus that is comprised of a single species the Amami rabbit, Pentalagus furnessi, and no extinct species are known. It is endemic on the Amami-Oshima and Tokunoshima Islands in the Ryukyu Archipelago, a string of islands in southwestern Japan located between Kyushu and Taiwan (Sugimura et al., 2000). The Amami rabbit primarily inhabits the forest on both islands; however, after deforestation in the 1970s and 1980s, it appeared in both cut-over areas and forest edges (Sugimura et al., 2003; Yamada and Cervantes, 2005). Currently, it is categorized as an endangered species by the International Union for Conservation of Nature Red List of Threatened Species because of its rapid population decline due to diminishing habitats and predation by feral and stray cats and dogs (Yamada, 2015).

Eimeria (Apicomplexa: Eimeriidae) are obligate, typically host-specific, intracellular parasite. More than 50 Eimeria species have been described in the family Leporidae, and each is host specific, at least to a particular genus (Duszynski and Couch, 2013). Kamiya et al. (1987) reported several Eimeria spp. from P. furnessi individuals from the Amami-Oshima Island; however, did not identify to species or provide detailed morphologic descriptions of the oocysts they recovered. In 2020, we identified by histopathological examination a coccidian infection in a wild Amami rabbit that had died following a Staphylococcus aureus infection. This finding led us to collect fecal samples and intestinal contents from wild Amami rabbits to determine species of coccidian present and their prevalence in the wild rabbit population.

Materials and methods

Ethics statement

The Amami rabbits that inhabit the Amami-Oshima and Tokunoshima Islands are designated as a special national treasure and are protected by the Law for the Protection of Cultural Properties. Thus, the sheltering, treatment, and necropsy of rabbit was conducted by veterinarians under the permission of the Ministry of the Environment, Japan. Additionally, fecal samples of Amami rabbits were collected in the field. The sampling in the regulated area (Yamakubiri Forest Road) of the Tokunoshima Island was conducted under the research permission of the Tokunoshima Town office. No animals were harmed during sampling.

Histopathological analysis

In August 2020, one debilitated Amami rabbit was taken into the Animal clinic at the Amami-Oshima Island. Staphylococcus aureus was isolated from an abscess on its body; however, the rabbit died after unsuccessful treatment. A necropsy was performed at the clinic, and tissue samples of the liver, spleen, lungs, kidneys, adrenal grands, heart, and digestive tracts were fixed in 10% neutral buffered formalin and processed into paraffin blocks for routine histopathologic procession at the Nippon Veterinary and Life Science University. Paraffin-embedded tissues were sectioned at 5 μm and stained with hematoxylin and eosin (HE) for microscopic examination with BX53 microscope (Olympus, Japan). Photomicrographs were captured using a DP74 (Olympus, Japan) or NanoZoomer-SQ Digital slide scanner (Hamamatsu Photonics, Japan).

Fecal samples of Amami rabbits

From August 2020 to November 2020, 30 fecal samples of Amami rabbits were collected from the Kinsakubal Forest (28°20′ N, 129°26′ E) (n = 20) and Yamakubiri Forest Road (27°52′ N, 128°56′ E) (n = 10) on Amami-Oshima and Tokunoshima Islands, respectively (Fig. 1). Fecal mass with 10–20 wet fecal pellets was considered as feces discharged by one individual. The sampling localities and dates are listed in Table 1. Additionally, intestinal contents were collected from six Amami rabbits that died at two animal clinics from December 2020 to May 2021 on Amami-Oshima Island.

Fig. 1

Map showing the location of the Amami-Oshima and Tokunoshima Islands. Circles indicate the sites where fecal samples of Amami rabbits were collected in the field.

Table 1

Prevalence of microscopic detection of Eimeria species in fecal samples of the Amami rabbit.

Localities	Origin	Date of sampling	No. of samples examined	No. of oocyst-positive samples
				Total	Type A	Type B	Type C	Mixed
Amami-Oshima Is.	Field	Nov. 2020	5	3	3	2	2	4
	Field	Jan. 2021	15	12	10	4	5	10
	Intestinal contents	Dec. 2020 to May 2021	6	3	2	0	2	2
Tokunoshima Is.	Field	Oct. 2020	10	8	6	3	4	7
		Total (%)	36	26 (72.2%)	21 (58.3%)	9 (25.0%)	13 (36.1%)	23 (63.9%)

Fecal samples and intestinal contents were placed in separate vials, transported at room temperature for 3–5 days, and examined by direct smear and sucrose flotation procedures. The presence of oocysts was examined under a BX41 microscope (Olympus) with 100 × and 400 × magnification. Oocyst-positive specimens were placed in separate vials with a 2.5% (w/v) potassium dichromate (K2Cr2O7) solution. Oocyst sporulation was induced by incubating at 23–26 °C, and the specimens were stored at 4 °C.

Morphological examination

Oocysts in K2Cr2O7 solution were observed under a BX41 microscope (Olympus) with differential interference contrast. Photomicrographs were captured using a DP27 Photomicroscope (Olympus). Sporulated oocysts (n = 30) were measured with ImageJ software ver. 1.53 (Schneider et al., 2012) using pictures taken under an oil immersion objective (1000 × magnification). The measurements were expressed in micrometers (μm) with the ranges followed by the means in parentheses. The oocysts and sporocysts were described following the standard guidelines (Duszynski and Wilber, 1997; Wilber et al., 1998; Berto et al., 2014) including: oocyst length (L) and width (W), their ranges and ratios (L/W), micropyle (M), nucleus (N), oocyst residuum (OR), polar granule (PG), sporocyst (SP), Stieda body (SB), substieda body (SSB), parastieda body (PSB), sporocyst residuum (SR), refractile body (RB), and sporozoite (SZ).

Results

General findings

The histopathological examination of the deceased Amami rabbit revealed coccidial infections in the jejunum and ileum (Fig. 2). In the jejunum, few immature gamonts were found in the epithelial cells. In the ileum, numerous gametocytes, unsporulated oocysts, and few schizont-like structures were observed mixed with debris of the epithelial cells in the lumen. These parasite stages could not be traced to specific tissue sites due to postmortem changes. Nematode infections of Lagostrongylus and Obeliscoides, common nematode parasites of Amami rabbits (Fukumoto, 1986, Yamada et al., 2012), were observed in the small intestine and stomach, respectively. In addition, fungal infections were found in the esophagus, lungs, and liver.

Fig. 2

Endogenous stages of coccidian parasites found in the section of the jejunum and ileum of the Amami rabbit. HE. Scale bar = 20 μm and applies to all parts. (a) Schizont-like structures (arrows) in detached epithelial cells. (b) Immature microgamont (arrow) with basophilic small nuclei. (c) Immature macrogamont (arrow) with eosinophilic bodies. (d) Microgamont (arrow) with peripherally located basophilic nuclei. (e) Macrogamont (arrow) with peripheral arrangement of eosinophilic wall-forming bodies. (f) Unsporulated oocyst (arrow) with a central nucleus and oocyst wall. High-resolution version of these slides for use with the Virtual Microscope are available as eSlide: VM06653.

Nomarski interference contrast photographs of oocysts of Eimeria furnessi from the Amami rabbit stored in a potassium dichromate solution. M: micropyle; N: nucleus; OWo: outer layer of the wall; OWi: inner layer of the wall; RB: refractile body; SB: Stieda body; SO: sporont; SR: sporocyst residuum. Scale bars (all in the same scale) = 10 μm.

Nomarski interference contrast photographs of oocysts of Eimeria hilleri from the Amami rabbit stored in a potassium dichromate solution. M: micropyle; N: nucleus; OWo: outer layer of the wall; OWi: inner layer of the wall; RB: refractile body; SB: Stieda body; SR: sporocyst residuum; SSB: substieda body. Scale bars (all in the same scale) = 10 μm.

Nomarski interference contrast photographs of oocysts of Eimeria sagentae from the Amami rabbit stored in a potassium dichromate solution. M: micropyle; N: nucleus; OWo: outer layer of the wall; OWi: inner layer of the wall; RB: refractile body; SB: Stieda body; SO: sporont; SR: sporocyst residuum. Scale bars (all in the same scale) = 10 μm.

Line drawings of the Eimeria species detected in the Amami rabbit. (a)Eimeria furnessi. (b)Eimeria hilleri. (c)Eimeria sagentae. Scale bar = 10 μ

Microscopic examination of the intestinal contents and fecal samples revealed that 72.2% (26/36, 95% CI 55.9–84.3%) of the samples were positive for Eimeria oocysts (Table 1). The oocysts could be classified into three types (Types A for medium, B for large, and C for small) based on their morphological characteristics, and a mixed infection was found in 63.9% (23/36) of the samples. These three types represented three new species, as described in the following sections.

(Fig. 3, Fig. 6a)

Description

Oocyst shape: ellipsoidal; number of walls: 2; wall thickness: 1.1 (0.9–15); wall characteristics: outer is rough, yellowish, finely granular, irregular thickness, and detached readily and/or can be removed, inner is smooth and transparent; L × W: 26.0 × 16.6 (22.9–30.3 × 14.9–18.5); L/W ratio: 1.6 (1.3–1.7); M: present; M characteristics: present in the outer layer of the wall, 4.0 (3.2–5.7) wide; OR and PG: both absent.

Sporocyst shape: elongate-ovoidal; L × W: 13.1 × 6.3 (12.0–13.7 × 5.1–7.4); L/W ratio: 2.1 (1.8–2.4); SB: present; SB characteristics: knob-like; SSB and PSB: both absent; SR: present; SR characteristics: a compact mass of large granules or a few scattered granules between SZ; SZ: elongate-ovoidal and lies head-to-tail with one large RB at the wider end.

Taxonomic summary

Type host: Mammalia (Lagomorpha: Leporidae), P. furnessi (Stone, 1900), Amami rabbit.

Other hosts: None to date.

Type locality: Amami-Oshima and Tokunoshima Islands, Kagoshima, Japan.

Site of infection: Unknown. Oocysts were recovered from feces and intestinal contents.

Sporulation: Exogenous. The exact hours were unknown, but oocysts became fully sporulated within 5 days at 23–26 °C.

Prevalence: 58.3% (21/36, 95% CI 42.2–72.9%) of the type host.

Prepatent and patent periods: Unknown.

Materials deposited: Photosyntype (Bandoni and Duszynski, 1988) of sporulated oocysts was deposited in the Meguro Parasitological Museum, Meguro, Tokyo, Japan, under MPM Coll. No. 21857.

ZooBank LSID: The ZooBank LSID is 6E429959-1DB0-41ED-AD8B-703423E0A3FE.

Etymology: Specific epithet “furnessi” is adopted from the name of Dr. William Henry Furness III (1866–1920), one of the discoverers of the Amami rabbit; it is given as a noun in the genitive case, in accordance to the Article 31.1. of the International Code of Zoological Nomenclature (ICZN) (International Commission on Zoological Nomenclature, 1999).

Remarks: Sporulated oocysts of E. furnessi resemble, in shape, size, and the presence of two walls and micropyle, those of Eimeria matsubayashii [24.8 × 18.3 (22–30 × 14–22)], recorded from Oryctolagus cuniculus (Tsunoda, 1952), and those of Eimeria neoirresidua [25.7 × 17.9 (19–31 x 15–20)] (Duszynski and Marquardt, 1969) and Eimeria poudrei [26.0 × 18.1 (20–31 × 15–21)] (Duszynski and Marquardt, 1969), detected from Sylvilagus audubonii. Sporulated oocysts of E. furnessi differed from these three species by its rough outer layer. Furthermore, E. matsubayashii oocysts differed from those of E. furnessi by the presence of OR and by having smaller sporocysts (7 × 6). Oocysts of E. neoirresidua and E. poudrei differed from those of E. furnessi by having larger sporocysts (14.5 × 6.4 for E. neoirresidua and 14.4 × 6.4 for E. poudrei).

(Fig. 4, Fig. 6b)

Oocyst shape: ellipsoidal; number of walls: 2; wall thickness: 2.0 (1.5–2.5); wall characteristics: outer is rough, yellowish-brown, and easily detached, inner is smooth and light yellow; L × W: 34.7 × 21.4 (30.6–41.2 × 15.2–24.3); L/W ratio: 1.6 (1.5–2.0); M: present; M characteristics: in the outer layer of the wall, 3.9 (3.3–5.1) wide; OR and PG: both absent.

Sporocyst shape: elongate-fusiform to elongate-ovoidal; L x W: 15.7 × 8.3 (14.5–17.3); L/W ratio: 1.9 (1.7–2.1); SB: present; SB characteristics: nipple-like; SSB: present; SSB characteristics: narrow and rounded; PSB: absent; SR: present; SR characteristics: a compact mass of large granules or a few scattered granules between SZ; SZ: elongated with one end wider than the other, lying length-wide head-to-tail, and with a clear RB posterior to N.

Type host: Mammalia (Lagomorpha: Leporidae), P. furnessi (Stone, 1900), Amami rabbit.

Other hosts: None to date.

Type locality: Amami-Oshima and Tokunoshima Islands, Kagoshima, Japan.

Site of infection: Unknown. Samples were obtained from feces.

Sporulation: Exogenous. The exact hours were unknown, but oocysts became fully sporulated within 5 days at 23–26 °C.

Prevalence: 25.0% (9/36, 95% CI 13.6–41.3%) of the type host.

Prepatent and patent periods: Unknown.

Materials deposited: Photosyntype of sporulated oocysts was deposited in the Meguro Parasitological Museum, Meguro, Tokyo, Japan, under MPM Coll. No. 21858.

ZooBank LSID: The ZooBank LSID is 9038A261-915C-4D4C-BF9F-7160D8A6FE40.

Etymology: Specific epithet “hilleri” is adopted from the name of Dr. Hiram Milliken Hiller (1867–1921), one of the discoverers of the Amami rabbit; it is given as a noun in the genitive case, in accordance to the Article 31.1. of the ICZN.

Remarks: Sporulated oocysts of E. hilleri resemble, in shape, the size, and the presence of two walls and micropyle, of Eimeria gobiensis [38.6 × 24.2 (27–49 × 19–32)] (Gardner et al., 2009), Eimeria pierrecouderti [35 × 24 (33–37 × 23–26] (Aoutil et al., 2005), Eimeria vejdovskyi [32.9 × 19.2 (30–37 × 18–21)] (Pakandl, 1988), and Eimeria stiedai [37 × 20 (31–42 × 17–25)] (Duszynski and Couch, 2013) detected from lagomorphs outside the Pentalagus genus. Oocysts of the first three species have a distinct OR, which those of E. hilleri lack, whereas E. stiedai oocysts have a few small OR, thin outer layer, and its sporocysts [18 × 10 (17–18 × 8–10)] (Duszynski and Couch, 2013) are larger than those of E. hilleri.

(Fig. 5, Fig. 6c)

Oocyst shape: ellipsoidal; number of walls: 2; wall thickness: 1.1 (0.6–1.5); wall characteristics: outer is rough, light yellowish, finely granular, and easily detached, inner is smooth and transparent; L × W: 20.9 × 14.5 (18.8–23.0 × 12.3–16.9); L/W ratio: 1.4 (1.3–1.6); M: present; M characteristics: in the outer layer of the wall, 3.9 (3.2–5.6) wide; OR and PG: both absent.

Sporocyst shape: elongate-ovoidal; L x W: 10.4 × 5.0 (8.7–11.7 × 4.3–5.9); L/W ratio: 2.1 (1.7–2.4); SB: present; SB characteristics: nipple-like; SSB and PSB: both absent; SR: present; SR characteristics: a compact mass of large granules or a few scattered granules between SZ; SZ: elongated with one end wider than the other, lying length-wide head-to-tail, and with a clear RB posterior to N.

Type host: Mammalia (Lagomorpha: Leporidae), P. furnessi (Stone, 1900), Amami rabbit.

Other hosts: None to date.

Type locality: Amami-Oshima and Tokunoshima Islands, Kagoshima, Japan.

Site of infection: Unknown. Samples were obtained from feces and intestinal contents.

Sporulation: Exogenous. The exact hours were unknown, but oocysts became fully sporulated within 5 days at 23–26 °C.

Prevalence: 36.1% (13/36, 95% CI 22.4–52.5%) of the type host.

Prepatent and patent periods: Unknown.

Materials deposited: Photosyntype of sporulated oocysts was deposited in the Meguro Parasitological Museum, Meguro, Tokyo, Japan, under MPM Coll. No. 21859.

ZooBank LSID: The ZooBank LSID is 82404C1B-EC2A-4C53-90BD-876D1548687C.

Etymology: Specific epithet “sagentae” is adopted from the name of Mr. Sagenta Nagoya (1820–1881), the author of “Nanto-Zatsuwa,” an ethnography published in 1855 that describes the nature and culture of the Amami-Oshima Island; it is given as a noun in the genitive case, in accordance to Article 31.1. of the ICZN.

Remarks: Sporulated oocysts of E. sagentae resemble, in shape, the size, presence of two walls, and the lack of OR, of Eimeria audubonii [21.2 × 17.1 (15–25 × 13–20)], detected from Sylvilagus audubonii (Duszynski and Marquardt, 1969), and of Eimeria rowani [22.1 × 15.8 (18–31 × 12–18)], detected from Lepus americanus (Samoil and Samuel, 1977). However, E. rowani oocysts differ from those of E. sagentae by the absence of M.

Discussion

Coccidian parasites of the genus Eimeria commonly infect species of the family Leporidae. This family includes approximately 11 extant genera (Brachylagus, Bunolagus, Caprolagus, Lepus, Nesolagus, Oryctolagus, Pentalagus, Poelagus, Pronolagus, Romerolagus, and Sylvilagus) with over 60 distinct species; 7 of them (Brachylagus, Bunolagus, Caprolagus, Oryctolagus, Pentalagus, Poelagus, and Romerolagus) are monotypic (Hoffmann and Smith, 2005). Rabbit eimerians have homoxenous life cycles and to date, 57 valid Eimeria species are included in the family Leporidae (Pellérdy, 1974; Pakandl, 2009; Duszynski and Couch, 2013; Cui et al., 2017). Most Eimeria species (78.9%; 45/57) are mesostenoxenous (multiple host, but restricted to one host genus) or oioxenous (only one host species), with 29 species recorded from Lepus, 9 species from Sylvilagus, 6 species from Oryctolagus, and one species form Brachylagus. The remaining 12 species (E. exigua, E. irresidua, E. leporis, E. magna, E. matsubayashii, E. media, E. minima, E. perforans, E. pierrecouderti, E. piriformis, E. stiedai, and E. sylvilagi) are euryxenous and were recorded from or have established cross-transmission in Lepus, Sylvilagus, and/or Oryctolagus (Duszynski and Couch, 2013).

Eimeria infections were first reported in Amami rabbits more than 30 years (Kamiya et al., 1987). This is the first report identifying and describing Eimeria species present in this host. The three new Eimeria species described in this study can be distinguished by the size of their oocysts and sporocyst, and color: E. furnessi is medium and yellow; E. hilleri is large and yellowish-brown, and E. sagentae is small and light yellow. Note that the outer layers with these oocysts are easily fragile and become colorless or light yellow, making them difficult to distinguish. Although E. furnessi and E. hilleri have similar sizes after removing the outer layer, they can be distinguished by the size of their mature sporocysts.

The genus Pentalagus diverged from the genus Pliopentalagus, which once inhabited Eurasia (Tomida and Otsuka, 1993). Pliopentalagus became extinct from the continent, whereas Pentalagus survived in the Ryukyu Islands; therefore, P. furnessi is a relict species that is often called a living fossil. The three eimerian species described in this study were commonly found in the Amami-Oshima and Tokunoshima Islands. The rabbits do not migrate between the two islands, suggesting that the eimerian species were already endemic parasites of the Amami rabbit before the islands were split. Molecular and cross-transmission studies will help to determine their phylogenetic position within the genus Eimeria and their relationship with other species parasitizing rabbits that do not belong the Pentalagus.

The pathogenicity of the three new species to Amami rabbits is unknown. Several species, including E. intestinalis, E. flavescens, E. piriformis, E. media, E. magna, and E. stiedai, are mildly to highly pathogenic for the domestic rabbit, O. cuniculus (Pakandl, 2009; Pakandl and Liu, 2020). Since coccidiosis is not commonly observed in the numerous wild Amami rabbits sheltered and treated at clinics in the Amami-Oshima Island (unpublished), these coccidian parasites may not be highly pathogenic to wild individuals. Understanding the pathogenesis may improve the breeding and artificial propagation of endangered animals, as Eimeria species can be horizontally transmitted via oocysts, which can be problematic in closed environments. The Eimeria species described in this study appeared to be parasites of the intestinal tract; however, further studies are needed to determine their life cycle, pathogenicity and treatment options.

Declaration of competing interest

No conflict of interest declared by any author.