Coccidiosis is an enteric disease caused by protozoan parasites called coccidium. Most coccidian parasite display a high degree of host specificity and can infect a wide variety of animals, including mammals, birds, and reptiles (3). There are many species of coccidian in which Eimeriorina eimeriidae comprises the highest proportion and is typically associated with high host pathogenicity (8). Clinical signs of coccidiosis are mainly diarrhea, weight loss, and inappetence with varying degrees ranging from asymptomatic to sudden death depending on patients or magnitude of infection. Coccidia is mainly infected through feces or ingestion of infected tissues. It is also known to be transmitted mechanically by wild animals (1). Like other infectious diseases, increased contact with wildlife has led to the discovery of new coccidium species and increased interest in the possibility of transmission between wildlife and livestock (2).

Coccidium is mainly diagnosed through fecal examination. Although morphological identification using microscope is a useful method to diagnosis it, it has a low sensitivity. In addition, the results of this method vary depending on the magnitude of infection, skilled manpower, and it is difficult to know cryptic diversity among species. Thus, molecular identification might be needed to estimate the impact coccidium isolated from wildlife on other species such as livestock and human (7). The pathogenicity of coccidian in wild animals is also important wild animals might play an important role as carriers of coccidian. Therefore, it is important to estimate the impact they can have on livestock and human through molecular identification. Research on wildlife is difficult to do for reasons such as sample collection due to wildlife aggression. Thus, research studies on coccidium isolated from wildlife are rarely conducted despite their importance.

This study was conducted to analyze the prevalence and genetic diversity of coccidial pathogens in rescued wild animals with diarrhea.

Sample collection

From January 2020 to December 2021, among rescued wild animals with diarrhea, animals with coccidium observed in fecal examination were included in this study. Those who were diagnosed with coccidian by fecal examination were treated according to usual medical procedures including administration of anticoccidial drug and fluid therapy. Their medical records (species, diagnosis, treatment progress, and prognosis) were collected and analyzed retrospectively. After diagnoses, residual fecal samples were stored in –20°C for molecular analysis.

Nucleic acid extraction

Nucleic acid extraction was conducted using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions after thawing fecal samples. Extracted nucleic acids were stored at –20°C until the next procedure was conducted.

Molecular analysis for coccidiosis

A nested polymerase chain reaction (PCR) was performed for confirmation and phylogenetic analysis of preserved feces of individuals diagnosed with coccidia. PCR targeting partial 18S ribosomal RNA gene sequence was performed using a HotStarTaq Master Mix Kit (Qiagen). Total volume of reagent was 50 µL including 2 µL of nucleic acid from fecal samples. PCR parameters were: denaturation at 95°C for 15 min; 40 cycles of 94°C for 45 s, 60°C for 45 s, and 72°C for 90 s; and a final extended step at 72°C for 7 min before maintaining at 4°C. The first PCR product was used as a template for the second PCR. For nested PCR, EIF1 (5’ - GCT TGT CTC AAA GAT TAA GCC - 3’) and EIR3 (5’ - ATG CAT ACT CAA AAG ATT ACC - 3’) primers were used for the first PCR and EIF3 (5’ - CTA TGG CTA ATA CAT GCG CAA TC - 3’) and EIR3 primers were used for the second PCR. Expected sizes of PCR amplicons were 1399-1407 base pairs (15). If PCR was positive, PCR product sequencing was performed for phylogenetic analysis.

Phylogenetic analysis was conducted for comparing genetic diversity of each species. Sequence alignments were performed using ClustalX version 1.8. Neighbor-joining genetic analysis was performed using MEGA-X version 10.1. Numbers on branches of a phylogenetic tree indicate bootstrap values based on 1,000 replicates. Various Eimeria species were included for phylogenetic analysis (Table 1). Sequence of Toxoplasma gondii (X68523) isolated from a cat (Felis catus, X68523) was used as an outgroup. All sequences detected in this study were deposited in GenBank database (accession numbers: OP824768 to OP824780).

Table 1 . Species information of coccidium such as host and accession number of 18S rRNA gene included for phylogenetic analysis.

Species	Common name (scientific name)	Accession number	Coccidium
Bats	Little brown bat (Myotis lucifugus)	AF324213	Eimeria catronensis
	Western small-footed bat (Myotis ciliolabrum)	AF324215	Eimeria pilarensis
		AF307877	Eimeria rioarribaensis
Primates	Guerea (Colobus guereza)	AF111186	Cyclospora colobi
	Human (Homo sapiens)	AF111183	Cyclospora cayetanensis
Birds	Domestic pigeon (Columba livia domestica)	KT305927	Eimeria sp. RY-2016a
	Red junglefowl (Gallus gallus)	U67116	Eimeria brunetti
		U67118	Eimeria mitis
		U67117	Eimeria maxima
		U76748	Eimeria mivati
		U67115	Eimeria acervulina
		U67119	Eimeria necatrix
		U67121	Eimeria tenella
	Turkey (Meleagris gallopavo)	AF041437	Eimeria meleagrimitis
Rodents	Brown rat (Rattus norvegicus)	AF311643	Eimeria separata
		U40263	Eimeria nieschulzi
	Deer mice (Peromyscu spp)	AF307878	Eimeria arizonensis
	Hispid pocket mouse (Chaetodipus hispidus)	AF339489	Eimeria chaetodipi
	House mouse (Mus musculus)	AF311641	Eimeria papillata
		AF080614	Eimeria falciformis
	Merriam’s kangaroo rat (Dipodomys merriami)	AF324214	Eimeria chobotari
	Northern grasshopper mouse (Onychomys leucogaster)	AF311644	Eimeria sevilletensis
		AF307879	Eimeria onychomysis
	Phillips’s Kangaroo Rat (Dipodomys phillipsii)	AF339490	Eimeria dipodomysis
	Pinyon mouse (Peromyscus truei)	AF339492	Eimeria peromysci
	Silky pocket mouse (Perognathus flavus)	AF311642	Eimeria reedi
	White-footed mouse (Peromyscus leucopus)	AF311640	Eimeria langebarteli
		AF339491	Eimeria leucopi
	White-throated woodrat (Neotoma albigula)	AF307880	Eimeria albigulae
Ruminant	Cattle (Bos taurus)	AF291427	Eimeria alabamensis
		U77084	Eimeria bovis

Fecal examination and molecular diagnosis

From January 2020 to December 2021, 2,276 wild animals were rescued in Jeonbuk province, Korea. Among them, 120 showed diarrhea (Table 2). Of them, 21 showed coccidium through routine fecal examination (Table 3). Regarding fecal examination results, the detection rate of coccodium was 100% in Azure-wingled magpie (1/1), Moorhen (1/1), and Pale thrush (1/1), 59.1% in domestic pigeon (13/22), 33.3% in Eurasian eagle owl (2/6), 10% in Oriental turtle dove (1/10), and 9.1% in raccoon dog (2/22). Since only 13 samples including 8 Domestic pigeon (Columba livia domestica), 1 Oriental turtle dove (Streptopelia orientalis), 1 Eurasian eagle owl (Bubo bubo), 1 Azure-winged magpie (Cyanopica cyanus), 1 Moorhen (Gallinula chloropus), and 1 raccoon dog (Nyctereutes procyonoides) were kept, molecular analysis was performed for them. Comparison of sequences of PCR amplicon and BLAST data showed that sequences of coccidium species in eight domestic pigeons, an Oriental turtle dove, a Eurasian eagle owl, and a Moorhen were similar to those of Eimeria spp. In addition, sequences of coccidium species in Azure-winged magpie were similar to those of Isospora spp. Moreover, sequences of coccidium species in a raccoon dog were most similar to those of Cystoisospora spp. The rescued area and rescue time for the 13 wild animals are shown in Fig. 1.

Table 2 . Information on animals diagnosed with coccidia among wild animals with diarrhea.

Species	Common name (scientific name)	Number of animal	Number of coccidium-positive animal
Mammals	Raccoon dog (Nyctereutes procyonoides)	22	2
	Water deer (Hydropotes inermis)	7	0
	Roe Deer (Capreolus pygargus)	1	0
	Leopard cat (Prionailurus bengalensis)	2	0
	Eurasian Otter (Lutra lutra)	5	0
	Eurasian Badger (Meles meles)	1	0
	Siberian Flying Squirrel (Pteromys volans)	1	0
Birds	Baikal teal (Anas formosa)	1	0
	Black-tailed gull (Larus crassirostris)	1	0
	Magpie (Pica pica)	7	0
	Cinereous vulture (Aegypius monachus)	2	0
	Common buzzard (Buteo buteo)	1	0
	Peregrine Falcon (Falco peregrinus Tunstall)	1	0
	Oriental turtle dove (Streptopelia orientalis)	10	1
	Azure-winged magpie (Cyanopica cyanus)	1	1
	Grey Frog Hawk (Accipiter soloensis)	1	0
	Great crested grebe (Podiceps cristatus)	1	0
	Eurasian hobby (Falco subbuteo)	2	0
	Brown hawk-owl (Ninox scutulata)	3	0
	Moorhen (Gallinula chloropus)	1	1
	Little egret (Egretta garzetta)	2	0
	Eurasian eagle owl (Bubo bubo)	6	2
	Grey heron (Ardea cinerea)	3	0
	Japanese Sparrow Hawk (Accipiter gularis)	1	0
	Intermediate egret (Mesophoyx intermedia)	1	0
	Brown-eared Bulbul (Microscelis amaurotis)	3	0
	Domestic pigeon (Columba livia domestica)	22	13
	Collared scops owl (Otus bakkamoena ussuriensis)	1	0
	Broad-billed roller (Eurystomus orientalis)	1	0
	Cattle egret (Bubulcus ibis)	2	0
	Kestrel (Falco tinnunculus interstinctus)	4	0
	Pale thrush (Turdus pallidus)	1	1
	Spot-billed duck (Anas poecilorhyncha)	1	0
	Pochard (Aythya ferina)	1	0

Table 3 . Rescued year and rescued area of wildlife species (scientific name) with coccidium detected through fecal examination.

Rescued year	No.*	Species	Rescued area
2020	2020-0008	Raccoon dog	Gunsan
	2020-0603	Domestic pigeon	Gunsan
	2020-0701	Domestic pigeon	Jeonju
	2020-0708	Oriental turtle dove	Gunsan
	2020-0735	Azure-winged magpie	Gunsan
	2020-0776	Domestic pigeon	Jeonju
	2020-0856	Raccoon dog	Jinan
2021	2021-0172	Domestic pigeon	Jeonju
	2021-0594	Domestic pigeon	Jeonju
	2021-0782	Domestic pigeon	Jeonju
	2021-0857	Domestic pigeon	Gunsan
	2021-0885	Domestic pigeon	Jeongeup
	2021-0948	Moorhen	Gunsan
	2021-0983	Domestic pigeon	Gunsan
	2021-0986	Domestic pigeon	Jeonju
	2021-0987	Domestic pigeon	Gunsan
	2021-1008	Eurasian eagle owl	Namwon
	2021-1014	Pale thrush	Jeonju
	2021-1027	Eurasian eagle owl	Imsil
	2021-1070	Domestic pigeon	Gunsan
	2021-1085	Domestic pigeon	Jeonju

*Patient identification number..

Figure 1. The rescued area and rescued year of 13 patients were indicated on the map. Yellow line shows rescued year 2020 and red line indicates rescued year 2021. Domestic pigeon was divided into three group (green, red, blue) according to genetic similarity of detected coccidium. Green group, detected in Gunsan, Jeonju and Jeongeup; Blue group, detected in Gunsan and Jeonju; Red group, only detected in Jeonju. Coccidium detected in Oriental turtle dove had high genetic similarities with green group’s rescued in Gunsan.

Phylogenetic analysis

Phylogenetic analysis showed that all coccidia detected in domestic pigeons formed one clade regardless of detection year or region (Fig. 2). The clade was further divided into three. One (2021-0782) of them detected in the city (Jeonju) formed a genetically identical subgroup with coccidium reported in Australia (accession number KT305927). Five coccidian (2020-0603, 2021-0885, 2021-0983, 2021-0986, and 2021-0987) that showed 100% identities to each other regardless of detection year or region formed a subgroup with coccidium (2020-0708) detected in an Oriental turtle dove. In terms of regional distribution, two coccidia that showed 100% sequence identities and formed one subgroup were identified in different years (2020, 2021) and different regions (Gunsan, Jeonju, Korea). Coccidium 2021-0782, which was genetically similar to coccidium reported in Australia (accession number KT305927) the most, was detected in Jeonju, Korea. Interestingly, the coccidium detected in the Eurasian eagle owl (2021-1008) and the Azure-winged magpie (2020-0735) appeared to belong to the same clade as the coccidium reported in Galliformes birds (junglefowl and turkey). The coccidium (2021-1008) detected in the Eurasian eagle owl was included in the same subgroup as seven coccidia (E. brunetti, E. mitis, E. maxima, E. mivati, E. acevulina, E. necatrix, E. tenella) isolated from the junglefowl. Coccidium (2020-0735) detected in Azure-winged magpie was shown to form a subgroup with E. meleagrimitis detected in turkey. The coccidium (2021-0948) detected in Moorhen was confirmed to be genetically distant from others compared in this study. Coccidium detected from raccoon dog was more similar to Toxoplasma gondii than to Eimeria spp.

Figure 2. Phylogenetic analysis of Eimeria spp. identified in wildlife in this study. Sequences obtained from this study are written in terms of patient identification number (rescued year), host species, and gene bank accession number (OP824768 to OP824780) next to the mark (◆). Next to the reference sequences, isolated host species were written. Neighbor-joining genetic analysis was performed using MEGA-X. Bootstrap percentage values are shown on branches.

This study confirmed the incidence and species distribution of coccidia in distressed and diarrheal wildlife especially wild birds. The genetic diversity of coccidia detected was also analyzed. During the survey period, 5.3% (120/2276) of wildlife had diarrhea. However, 17.5% (21/120) them were infected with coccidium, indicating a high prevalence of coccidium. The largest number of coccidium was detected through fecal examination in domestic pigeons (13/22, 59.1%) in this study. Research studies conducted in other countries such as China, India and United States of America have shown that the prevalence rate of coccidium in domestic pigeons varies from at least 10% up to 90% (4,9,10,11). Such a high prevalence rate of coccidium in domestic pigeons might be due to the low pathogenicity of coccidium and life styles of pigeons living in groups (10,11). In this study, four domestic pigeons (2020-0603, 2020-0701, 2020-0708, 2021-0857) showed mild clinical signs with greenish diarrhea. They were well treated. However, four domestic pigeons (2021-0885, 2021-0983, 2021-0986, 2021-0987) were euthanized because it was judged that it was impossible to release them due to irreversible head trauma, bone necrosis, and joint damage not related to enteric disease. Except one patient euthanized on the day of rescue, the rest of the patients showed well-recovered from diarrhea before euthanized. It suggested coccidiosis in domestic pigeons showed mild clinical signs and readily curable.

Unlike domestic pigeon cases, Azure-winged magpie, Moorhen, and Pale thrush died with severe diarrhea with intestinal hemorrhage. Since coccdium was detected through fecal examination and there were no other factors that can cause diarrhea, coccidum is likely to cause severe diarrhea that could lead to death in these species. However, these species showed diarrhea in only one individual, were diagnosed with coccidium (1/1), and there are no other studies involved, so it is not clear that coccidium is highly pathogenic in those species. This needs to be confirmed through follow-up investigations targeting a larger number of individuals.

Coccidium species are generally different depending on the host animal genus (6). However, in the case of domestic pigeons and Oriental turtle dove, it has been reported that they have the same coccidium species even though the genus is different. It is not clear whether it is like a coccidium species that infects domestic pigeon or Oriental turtle dove, or whether either of them accidentally shared another species (5). As with previous reports, our study found similar genotypes of coccidium infection in domestic pigeons (n = 8) and Oriental turtle dove (n = 1). The fact that domestic pigeons and Oriental turtle dove were diagnosed coccidiosis on the day of rescue indicated that there is no possibility of horizontal transmission in the hospital, and high genetic similarity of coccidium detected from domestic pigeons rescued in different regions and different years indicates that these coccidium were dominant coccidium species in the domestic pigeons in Jeonbuk province. In the case of Oriental turtle dove, it was rescued in the city, but considering the characteristics of living in rural area, it cannot be ruled out that the infection occurred by accidentally obtaining the coccidium species of domestic pigeon as the Oriental turtle dove flowed into the city. However, since only one Oriental turtle dove was evaluated in this study, it is necessary to examine and confirm whether the dominant coccidium species infected with Oriental turtle dove are the same as domestic pigeons.

Domestic pigeons are resident birds known to use a small home range (12). Phylogenetic analysis showed that coccidium detected from domestic pigeons shared high genetic similarities to those separated from Austrian (accession number KT305927) species (14). Coccidium detected domestic pigeons rescued in different areas at different time also showed genetic similarities. All of them were diagnosed on the day of rescue. They had different hospitalization periods. These results suggest that domestic pigeon’s home range might be at a level that could cross cites or countries or there might be another vehicle other than host that could transfer coccidium between countries and cities.

Phylogenetic analysis conducted in this study showed that the genetic characteristics of coccdium detected from Eurasian eagle owl was included in same clade such as E. tenella and E. necatrix, which known to have high pathogenicity in poultry (13). Considering food habit of Eurasian eagle owl, its increased contact with farms due to habitat destruction, the rescue area of the Eurasian eagle owl which was a rural area, it might have spread to each other around the farm likely through food hunting. However, the fact that the Eurasian eagle owl had diarrhea caused by coccidium also suggested the possibility that the Eurasian eagle owl shared a coccidium species similar to that of poultry. In this study, only one Eurasian eagle owl did a genetic analysis, it was difficult to determine whether genetic similarity of coccidium detected Eurasian eagle owl and poultry was a coincidence, a possibility of mechanical transmission, or an infection. In order to clarify this, it is necessary to conduct additional studies on larger groups.

This study was supported by the National Institute of Wildlife Disease Control and Prevention through a “Specialized Graduate School Support Project for Wildlife Disease Specialists.”

The authors have no conflicting interests.