Molecular identification of two Eimeria species, E. uekii and E. raichoi as type B, in wild Japanese rock ptarmigans, Lagopus muta japonica.

Thus far, two types of Eimeria parasites (E. uekii and type B) have been morphologically identified in wild Japanese rock ptarmigans, Lagopus muta japonica. Although high prevalences were reported for these parasites, genetic analyses have not been conducted. We first clarified the phylogenetic positions of two eimerian isolates using genetic analyses of 18S rRNA and mitochondrial cytochrome c oxidase subunit I gene regions. Consequently, of 61 samples examined, 21 and 11 samples were positive for E. uekii and type B, respectively. Additionally, the infection rate increased in the summer. Molecular analyses revealed both Eimeria isolates formed their own clusters; E. uekii was included in clades of chicken Eimeria and type B was include in clades of turkey Eimeria. Based on our findings in this study and previous data, we herein propose type B as E. raichoi. These genetic data will be helpful to conduct detailed classification and understand the impact of these parasites for conservation of endangered Japanese rock ptarmigans.

Introduction

Species in the genus Eimeria are protozoan parasites belonging to the phylum Apicomplexa. More than 4000 species of the parasites have been described, and they infect a wide range of vertebrate and invertebrate hosts (Levine, 1982), and are considered to be highly host-specific. Infection by eimerian parasites causes coccidiosis, which is mainly characterized as watery or bloody diarrhea, weight loss, and increased mortality rates of infected hosts (Fitzgerald, 1980; McDonald and Shirley, 2009). Although Eimeria spp. are the most important and prevalent parasites due to their economic impact, especially in livestock, e.g., chicken and cattle, most Eimeria spp. causing disease in wild animals remain largely unknown and poorly characterized.

The study of Eimeria spp. from the rock ptarmigan (Lagopus muta) (Montin, 1781), in the order Galliformes, is important because of specificity of the host. The rock ptarmigan is a cold-adapted bird that inhabits alpine areas of the Northern Hemisphere. To date, seven Eimeria spp. have been identified in the rock ptarmigan: E. lagopodi from Switzerland (Galli-Valerio, 1929), E. brinkmanni and E. fanthami from Canada (Levine, 1953), E. uekii and type B from Japan (Kamimura and Kodama, 1981; Ishihara et al., 2006), and E. muta and E. rjupa from Iceland (Skirnisson and Th Thorarinsdottir, 2007). Descriptions of these species were primarily based on morphological (sporulated oocysts) and biological characteristics. However, these parameters can be insufficient for reliable differentiation among species owing to overlapping morphometric and biological features (Long et al., 1977). Therefore, molecular analyses are necessary to classify and precisely identify the species and to assess phylogenetical developments of the parasites as well as the hosts.

In terms of taxonomy, rock ptarmigans are currently divided into 23–30 subspecies, e.g., L. m. evermanni, L. m. kurilensis, and L. m. pleskei (Johnsgard, 1983; del Hoyo et al., 1994; Holder et al., 2000). The Japanese rock ptarmigan (L. m. japonica) inhabits only the timberline regions of the Japanese alpine zone at approximately 3000 m above sea level. This subspecies is endemic to Japan and is thought to be endangered due to a decline in the overall population (estimated population: ≤2000 individuals). The Japanese rock ptarmigan was designated as a special natural monument of Japan in 1955 and is listed as vulnerable in the Japanese Red Data Book (Murata et al., 2007; Wildlife Division of the Ministry of the Environment, 2017). Recently, Eimeria infections were reported to be associated with a decrease in overall body condition and an increase in mortality in rock ptarmigans (L. m. islandorum) in Iceland (Stenkewitz et al., 2016). Although two types of Eimeria parasites, namely E. uekii and type B, highly infect the Japanese rock ptarmigan (Kamimura and Kodama, 1981; Ishihara et al., 2006; Matsubayashi et al., 2018b), molecular analyses of these parasites have not been conducted. In the present study, we genetically examined E. uekii and type B to determine their precise phylogenic positions and classifications.

Materials and methods

Examined areas and birds

In the present study, we collected fecal samples at Hida Mountains of the Northern Japanese Alps and Akaishi Mountains of the Southern Japanese Alps from May 2016 to August 2017; the areas extend over Toyama, Gifu, Nagano, and Niigata prefectures and Nagano, Yamanashi, and Shizuoka prefectures, respectively. We collected a total of 61 fresh Japanese rock ptarmigan fecal samples, including 6 chick samples as described in Table 1. Samples were collected from 3 sites: Mt. Tateyama (36°35′N, 137°36′E), Norikuradake (36°6′N, 137°33′E), and Kitadake (35°40′N, 138°14′E) (Fig. 1). In Kitadake, a temporary cage protection procedure was conducted for three families for 3 weeks starting at the end of June 2016. Three families (hen and her chicks) were individually housed during nighttime to avoid eventual predation by predators and sometimes during daytime when severe weather (strong wind and rain) occurred. Otherwise, the families were allowed to graze the natural food outside of the cage. In cages, food such as Vaccinium ovalifolium, Oxytropis japonica var. japonica, Polygonum viviparum, and Stellaria nipponica was collected from surrounding areas to offer to the birds. Previously collected fruits of Vaccinium vitis-idaea and commercially available worms (Tenebrio spp.) were also supplied as supplemental food. Fecal samples were placed in a cooler box, transported to our laboratory, and stored at 4 °C until analyses.

Table 1

Summary of examined Japanese ptarmigans and infection of Eimeria spp.

Sampling date		No. of positive samples/examined samples	Eimeria spp.		Average OPG (No. of samples)a3	Location
			E. uekii	type B
2016	May	0/9	–	–
	June	6/10	6	2	1133.6 (1)	Tateyama
	July	2/6a1	2	–	240.0 (2)	Norikuradake
	August	4/5	3	4	2883.1 (3)	Kitadake
	November	0/5	–	–		Tateyama
2017	May	3/15	3	1		Tateyama and Norikuradake
	June	1/5	1	–		Tateyama
	August	6/6a2	6	4	7244.7 (6)	Kitadake
Total		22/61 (36.1%)	21	11

Three chicks were included: two chicks in a1 (positive for E. uekii) and three chicks in a2 (positive for E. uekii and type B).

3, OPG was calculated only in birds that shed countable numbers of oocysts in feces.

Fig. 1

Location of three sampled areas in Japan, Mt. Tateyama (36°35′N, 137°36′E), Norikuradake (36°6′N, 137°33′E), and Kitadake (35°40′N, 138°14′E) (triangle boxes 1–3).

Fecal examinations

The parasites were examined by sucrose centrifugal flotation methods (Uga et al., 2000). The number of oocysts per gram (OPG) was determined by counting after purification of the parasites. Several Eimeria-positive samples were incubated in a 2.5% potassium dichromate (K2Cr2O7) solution at 25 °C to allow the oocysts to sporulate. Sporulated oocysts were observed under a differential interference contrast microscope under oil immersion at 1000 × magnification. Internal structures of 50 oocysts were then analyzed.

Experimental infections

Purified sporulated oocysts from sample No. 60 were used for experimental infection. Four conventional Japanese quails (Coturnix japonica) (Delight Base, Aichi, Japan) were housed in wire-floored cages in coccidia-free rooms with free access to feed and water, which contained no anticoccidial drugs or antibiotics. Then, chicks (34-days-old) were orally inoculated with 2 × 104 of E. uekii and 0.5 × 104 of type B oocysts in 500 μl. Their feces were collected from 3 days post-inoculation (dpi) to 20 dpi and examined by sugar floatation methods.

Molecular identification

For genetic analysis of one Eimeria species, one oocyst of E. uekii or type B was isolated using a disposable glass capillary micropipette as previously described (Matsubayashi et al., 2005) and transferred to 8 μl of phosphate-buffered saline (pH 7.4) in a PCR tube. Isolated oocysts were treated by five freeze-thaw cycles, heated at 99 °C for 8–10 min, and then used as DNA template.

Molecular identification of Eimeria spp. was performed by PCR using the primer pair 1FE-4RB (approximately 560 bp), which targets the 18S ribosomal RNA (rRNA) gene (Jinneman et al., 1999; Matsubayashi et al., 2005). Additionally, nested PCRs the targeting mitochondrial cytochrome c oxidase subunit I (COI) gene (approximately 780 bp) (Ogedengbe et al., 2011; Yang et al., 2013) and 28S rRNA gene (approximately 1420 bp) (Schrenzel et al., 2005; Yang et al., 2014) were conducted. PCR products were sequenced, and phylogenetic trees were constructed as described previously (Matsubayashi et al., 2018a).

Histopathological examinations

We obtained five chicks (Nos. a–e), which were protected in the cage at Kitadake and unintentionally dead in 2016. They were preserved at −20 °C before necropsy; the heart, liver, kidneys, stomach, duodenum, jejunum, ileum, cecum, and colon were collected, fixed in 10% neutral-buffered formalin, and processed for routine histological examinations. Tissue sections were stained with hematoxylin and eosin (HE) and Periodic acid-Schiff. Stained histological sections were examined under a light microscope (400 × ). Histological scores were determined based on the number of parasites (+, a few parasites in some fields; ++, 1–5 parasites per field; +++, >5 parasites per field).

Results

We detected oocysts of Eimeria spp. in 22 (36.1%) of 61 examined samples at three areas of the Japanese Alps (Table 1) and two types of the oocysts were morphologically found (Fig. 2) (Table 2). No other parasites were found. E. uekii-type oocysts were ellipsoidal in shape, had no oocyst residuum, and contained 1–2 ovoid polar granules. The micropyle was indistinct or absent. Sporulated oocysts (n = 50; length × width) measured 23.7 ± 2.0 μm (22.0–27.1 μm) × 15.3 ± 1.2 μm (13.3–18.1 μm) and had a shape index (L/W) of 1.56 ± 0.18 (1.26–1.89). Sporocysts (n = 50) measured 11.7 ± 1.3 μm (9.1–14.3 μm) × 6.9 ± 0.7 μm (5.7–8.1 μm) and had a shape index (L/W) of 1.71 ± 0.22 (1.29–2.25). Stieda body and sporocyst residuum were present and observed in sporozoites.

Fig. 2

Eimeria oocysts detected in the feces of Japanese rock ptarmigans. (A) E. uekii and (B) type B. Scale bars indicate 10 μm.

Table 2

Comparative morphologies of Eimeria spp. from wild Japanese rock ptarmigans and rock ptarmigans from other countries.

Hosts (countries)	Oocysts					Sporocysts					ST	Reference
	length × width (range)	L/W	Mi	OR	No. of PG(range)	length × width (range)	L/W	SB	RB	SR
L. m. japonica (Japan)
This study(E. uekii)	23.7 (20.0–27.1)×15.3 (13.3–18.1)	1.56 (1.26–1.89)	±	–	1–2	11.7 (9.1–14.3)×6.7 (5.7–8.1)	1.71 (1.29–2.25)	+	ND	ND	24–48 h
E. uekii	24 (19–29) × 16 (13–21)	1.5 (1.2–1.7)	+	–	1–2 (2 × 1)	12 (11–15) × 6 (5–7)	ND	+	2.2–3	+	24 h	Kamimura and Kodama, 1981
E. uekii	23.7 (20.2–28.8) × 15.6 (13.7–18.8)	1.5 (1.2–1.7)	±	–	(1.5–3.1)	12.4 (9.7–14.4) × 6.7 (5.8–7.8)	1.8 (1.4–2.3)	+	1.3–4	+	<24 h	Ishihara et al., 2006
This study (type B)	20.6 (18.6–21.8) × 17.5 (14.6–19.1)	1.18 (1.08–1.26)	–	–	1	11.8 (10.0–13.6) × 7.1 (5.9–8.2)	1.65 (1.38–2.00)	+	ND	ND	24 h
Type B of Eimeria	21.3 (13.6–25.9) × 19.1 (13.1–24.5)	1.1 (1.0–1.4)	–	–	(1.5–2.8)	12.1 (9.6–13.7) × 7.4 (5.8–9.2)	1.6 (1.2–2.0)	+	1.4–4	+	<48 h	Ishihara et al., 2006
L. m. islandorum (Iceland)
E. muta	24.9 (19.5–30) × 16.6 (14.5–19)	1.5 (1.2–1.8)	＋	–	1–3	14.3 (12–16.5) × 6.3 (5.5–7)	2.3 (1.9–2.7)	＋	3.9–2.9	+	ND	Skirnisson.and Th Thorarinsdottir, 2007
E. rjupa	24.7 (20–28) × 22.2 (18–24.5)	1.1 (1.0–1.3)	–	–	1–2	14.4 (12–15.5) × 8.0 (6.5–9)	1.8 (1.6–2.2)	+	3.9–2.9	+	ND	Skirnisson.and Th Thorarinsdottir, 2007
L. m. rupestris (Canadian arctic)
E. brinkmanni	28.6 (26.0–29.7) × 18.8 (18.0–19.6)	1.52 (1.4–1.6)	–	–	1–2	13 × 7	ND	+	ND	–	ND	Levine, 1953
E. fanthami	28.3 (27.0–29.2) × 18.8 (18.0–20.1)	1.52 (1.4–1.6)	–	–	1–3	ND	ND	+	ND	–	ND	Levine, 1953
L. m. helveticus (Switzerland)
E. lagopodi	24 × 15	ND	＋	ND	ND	12	ND	ND	ND		ND	Galli-Valerio, 1929

Mi, micropyle; OR, oocyst residuum; PG, polar granules; SB, stieda body; RB, refractile body; SR, sporocyst residuum; ST, sporulation time. Sizes are in μm.

Type B-like oocysts were subspherical, had no oocyst residuum or micropyle, and contained one ovoid polar granule. Sporulated oocysts (n = 50; length × width) measured 20.6 ± 0.9 μm (18.6–21.8 μm) × 17.5 ± 0.7 μm (14.6–19.1 μm) and had a shape index (L/W) of 1.18 ± 0.05 (1.08–1.26). Sporocysts (n = 50) measured 11.8 ± 0.7 μm (10.0–13.6 μm) × 7.1 ± 0.5 μm (5.9–8.2 μm) and had a shape index (L/W) of 1.65 ± 0.12 (1.38–2.00). Stieda body and sporocyst residuum were present. After incubation at 25 °C, E. uekii and type B were sporulated for 24 h and 24–48 h, respectively. After comparisons with previous studies (Kamimura and Kodama, 1981; Ishihara et al., 2006), we identified the oocysts isolated in this study as E. uekii and type B oocysts. Regarding experimental infections using quails, we could not confirm any clinical symptoms or detect any oocysts of Eimeria spp. during 20 dpi.

Specific fragments of the 18S rRNA gene using DNA prepared from one oocyst were successfully amplified by PCR analyses with primer pairs 1FE-4RB. Partial sequences of the products for E. uekii (Accession Nos. LC380046- LC380054) and type B (Accession Nos. LC380055- LC380068) were determined, and we constructed a phylogenetic tree of the gene (Fig. 3). E. uekii and type B isolates formed a sister group to chicken Eimeria spp. and turkey Eimeria spp., respectively, and separated from other Eimeria spp. from mammals such as rodents and cattle. Although the 28S rRNA gene region was not amplified, COI gene sequences from some isolates (Accession Nos. LC380069- LC380076) could be analyzed (Fig. 4). Phylogenetic analyses of COI gene showed that both Eimeria isolates formed their own clusters; E. uekii was included in chicken Eimeria spp. and type B was included in turkey Eimeria spp. Based on these findings and those from previous studies (Ishihara et al., 2006; Matsubayashi et al., 2018b), we propose type B of Eimeria from Japanese rock ptarmigans as E. raichoi, as described below.

Fig. 3

Phylogram of E. uekii, type B, other Eimeria spp., and related parasites (Cyclospora spp.) inferred by the neighbor-joining method using partial 18S rRNA gene sequences. Accession numbers and derived hosts are shown in parentheses. Scale bar represents substitutions per nucleotide, and bootstrap values are indicated (>1000). Cystoisospora spp. are used as an outgroup taxon.

Fig. 4

Phylogram of E. uekii, type B, and other related Eimeria spp. inferred by the neighbor-joining method using partial mitochondrial cytochrome c oxidase subunit I gene sequences. Accession numbers and derived hosts are shown in parentheses. Scale bar represents substitutions per nucleotide, and bootstrap values are indicated (>1000). Toxoplasma gondii is used as an outgroup taxon.

Descriptions

E. raichoi n. sp. (Fig. 5)

Fig. 5

Composite line drawing of oocyst of Eimeria raichoi n. sp (previously referred as type B). Scale bars indicate 10 μm.

Host: Japanese rock ptarmigans (Lagopus muta japonica).

Locality: Hida Mountains of the Northern Japanese Alps and Akaishi Mountains of the Southern Japanese Alps, Japan.

Prevalence: Seasonally variable.

Other hosts: Unknown.

Prepatent period: Unknown.

Patent period: Unknown.

Site of infection: Unknown.

Sporulation time: 24–48 h.

Note: Representative purified oocysts from infected birds have been deposited as syntype in the Meguro Parasitological Museum (4-1-1 Shimomeguro, Meguro, Tokyo 153–006, Japan; accession numbers MPM Col. 21449-a).

In histopathological examinations, all organs were severely degraded after death. However, we could observe some parasites in the small and large intestines of two chicks (Nos. a and b), namely, the duodenum (+and +), jejunum (+++ and +), ileum (+++ and +++), and cecum (+++ and +++), respectively. There were no parasites in the stomach and colon. The parasites were mainly gamete, zygote, or oocysts and a few schizonts (Fig. 6) and were estimated as E. uekii and type B based on the morphologies of ellipsoidal and subspherical shapes, respectively. However, we could not determine the species and the infected location, such as epithelium cells or lamina propria, or the cause of death.

Fig. 6

Histopathologic sections of ceca which were obtained from dead chicks infected with Eimeria spp. Arrowhead indicates macrogametocytes with a prominent wall-forming body (A), and arrows indicate zygotes or early oocysts, which are surrounded by an oocyst wall (A and B). Pathological lesions could not be observed because of severe degradation after death. Scale bars indicate 20 μm.

Discussion

Thus far, two types of Eimeria have been reported in wild Japanese rock ptarmigans. Their prevalence increases up until August (summer) and then decreases as the temperature decreases until November (winter) (Ishihara et al., 2006; Matsubayashi et al., 2018b). In the present study, infection rates were in accordance with previous findings although examined numbers were small (Table 2). Morphologies of the two type oocysts in our study were almost identical to those of E. uekii and type B as described previously (Kamimura and Kodama, 1981; Ishihara et al., 2006; Matsubayashi et al., 2018b). Although we could determine the phylogenetic positions of these isolates and type B was characterized as E. raichoi, sporulated oocysts of E. uekii and type B were similar to those of E. muta and E. rjupa from Iceland. Japanese rock ptarmigans are not migratory birds and inhabit only the timberline regions of the Japanese alpine zone at approximately 3000 m above sea level. Thus, genetical analyses of Eimeria spp. from other rock ptarmigans are needed to clarify the classification and assess the evolution of Eimeria spp. according to adapted progression of rock ptarmigans.

The pathogenicity of Eimeria spp. infecting Japanese rock ptarmigans remains unknown. The developmental stages of parasites including gametes were detected in the intestines although detailed histopathological findings could not be determined. Therefore, there is a possibility that these parasites can affect the health status of rock ptarmigans. Previously, it has been reported that E. uekii may cause lesions in the host (Kamimura and Kodama, 1981). Furthermore, this species appeared to be phylogenetically closely related to chicken Eimeria spp., which contain highly virulent or chronically pathogenic species. Thus, further study is necessary to clarify the pathogenicity and assess these parasites as the key factor for conservation of Japanese rock ptarmigans as endangered species.

Compliance with ethical standards

Ethics statement

All experiments in the wild were carried out without using live animals. Thus, ethical approval of animal experimentation was not necessary. All examinations in the field study were permitted by the Ministry of the Environment Government of Japan. Fecal collection was performed in a non-invasive manner. No animals were scarificed for the purpose of the field study. In the experimental infection study, the animals were treated in accordance with the protocols approved by the Animal Care and Use Committee in accordance with the Animal Experimentation Guidelines of Osaka City University (approval No. 15003). Human participants were not involved in this study.

Conflicts of interest

The authors declare that they have no conflict of interest.