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Japanese rock ptarmigans, Lagopus muta japonica, are classified as an endangered species in Japan and are found only in the Japanese Alps. The number of birds has decreased in the last half century and cage protection projects have been undertaken as in situ conservation strategies (one of the projects for the recovery plan of Japanese rock ptarmigan) in the mountains. During the period with cage protections, some chicks died and two Eimeria spp., E. uekii and E. raichoi, were identified in the chicks. Here, we examined the soil within the cages and in the surrounding environment to assess potential sources of infection between July to August 2020. We found high numbers of oocysts in the cages, especially at the back sides where the ptarmigan family frequently congregated, but soils in other areas outside the cages were less contaminated or not contaminated at all. The time required for more than 50% of the oocysts to sporulate at 15, 20 and 25 °C for E. uekii was 20, 11, and 5 h, respectively, and 72, 48 and 18 h, respectively, for E. raichoi. Our results cast some doubt that coprophagia by chicks is the source of infection because chicks consumed fresh cecal feces (approximately within 1 h) as far as we know, and instead, the protected chicks might be directly or indirectly infected by oocysts in soils or the environment., Graphical abstract Image 1, Highlights • Cage protection is effective for protecting chicks of Japanese rock ptarmigans. • Soils at the back sides in the cages were highly contaminated with Eimeria spp. • E. uekii can rapidly be sporulated at 15 °C in timber regions. • Protected chicks might be infected by oocysts in soils or the environment.

The population of Japanese rock ptarmigan (Lagopus muta japonica), an endangered species with a habitat above the timberline of the southern Japanese Alps, has declined. As one of the recent conservation strategies for this species, cage protection for broods (hens and chicks) has been introduced in their habitats. Two species of Eimeria have frequently been detected in these birds, but little is known about the parasitic circulation in the region, including among birds and in the environment. Here, we conducted histopathology examinations of dead chicks collected under cage protection in 2018, and examined the feces of the hens and chicks of three broods and environmental soils for parasites in 2019 in order to assess the potential sources of infection and pathogenicity. Developmental zoites were found in the epithelial mucosa and/or the submucosa from the duodenum to the colon of all dead chicks. Fecal examination revealed oocysts of E. uekii and/or E. raichoi in all hens and chicks. Oocysts of Eimeria spp. per gram of feces in chicks increased within 2 weeks after hatching and then gradually deceased. Following infection of the chicks, oocysts could accumulate within the cage areas, and oocyst density exceeded more than 1000 oocysts per gram of cage soils. Based on having sporulated morphologies, oocysts could be infective and therefore, be direct or indirect potential sources of infection. However, based on our findings that not all chicks were clinically affected by the infections, other factors such as microbial flora in the chicks established by coprophagy or from the habitat environment, including climate, might be associated with the pathogenicity of Eimeria spp., although further studies are needed to assess these correlations., Graphical abstract Image 1, Highlights • Cage protection is effective on conservation of Japanese rock ptarmigans. • Hens caring chicks were highly infected with Eimeria spp. and shedded the oocysts. • Potential sources of the infection could be contaminated soils but not coprophagy behaviors of chicks. • Pathogenicity of Eimeria might be also associated with factors like establishment of gut microbiota or habitat conditions.

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., Graphical abstract Image 1, Highlights • Molecular analyses revealed Eimeria isolates from Japanese rock ptarmigans formed own clades. • The species was phylogenetically closely related to virulent chicken Eimeria spp. • Based on morphological data and sequences, Eimeria type B was proposed as a new species E. raichoi. • These parasites could be the key factor for conservation of the endangered birds.