Experimental evaluation of pathogenicity and acquired immunity of Eimeria species, E. uekii and E. raichoi, infecting Japanese rock ptarmigans in a subspecies of the birds.

Japanese rock ptarmigans (Lagopus muta japonica) are birds that inhabit only alpine regions of central Honshu Island, Japan, known as the Japanese Alps. The number of these birds has recently declined, and in situ and ex situ national conservation programs for Japanese rock ptarmigans have been initiated. The infections of Eimeria spp. as protozoan parasites of the phylum Apicomplexa, E. uekii and E. raichoi, were frequently reported in the birds. However, the virulence of these Eimeria parasites has not been determined. Here, we analyzed the pathogenicity of these Eimeria parasites using experimental infections of a subspecies model of Japanese rock ptarmigans, Svalbard rock ptarmigans (Lagopus mutus hyperboreus), and evaluated acquired protective immunity against challenge in birds tolerant of low-dose inoculation with Eimeria parasites. Following inoculation with two Eimeria parasites derived from Japanese rock ptarmigans (dose range of 4 × 104 to 4 × 102 for E. uekii and 1.7 × 104 to 4 × 101 for E. raichoi), oocysts were detected at 6-8 days post-inoculation (PI), and the maximum number of oocysts per gram of feces was observed 7-10 days PI and then gradually decreased. The mortality rate and reduction in weight gain of chicks increased following high-dose inoculation of oocysts with abnormal feces (soft and diarrhea). Developmental zoites were detected histopathologically in epithelial tissues and sometimes the lamina propria from the duodenum to the colon. Chicks that survived low-dose inoculation did not show clear clinical symptoms after challenge inoculation. Our results suggest that the pathological characteristics of Eimeria parasites infecting Japanese rock ptarmigans include abnormal feces and reduction in weight gain, resulting in mortality in cases of heavy infection due to high-dose inoculation. These findings provide helpful data for Japanese rock ptarmigan conservation efforts.

Morphological and molecular identification of Eimeria tetartooimia oocysts from a Japanese green pheasant (Galliformes; Phasianidae; Phasianus versicolor) at a zoo in Japan.

We detected Eimeria oocysts from Japanese green pheasants (Phasianus versicolor) at a zoo in Osaka, Japan. The oocyst isolates were subspherical or ovoidal shaped and measured 17.2 (range 14.7-20.0) µm in length and 14.8 (13.3-16.7) µm in width with a length/width (L/W) ratio of 1.2 (1.0-1.4) and each had one polar granule. The oocysts lacked a residuum and micropyle. Sporocysts measured 9.8 (6.7-13.3) µm in length and 5.9 (4.7-7.3) µm in width, with a L/W ratio of 1.2 (1.1-1.4). Compared to previously published values, this strain shows morphological similarities with an isolate of E. teetartooimia from ring-necked pheasants from other countries. Phylogenetic analysis of the 18S rRNA and mitochondrial cytochrome c oxidase subunit I genes places the isolate in a clade related to chicken Eimeria spp., such as E. acervulina or E. brunetti. Although further analysis is needed, this information can be helpful for the diagnosis and determination of virulence of Eimeria spp. in pheasants.

Distribution of Eimeria uekii and Eimeria raichoi in cage protection environments for the conservation of Japanese rock ptarmigans (Lagopus muta japonica) in the Japanese Alps.

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.

Parasitic development in intestines and oocyst shedding patterns for infection by Eimeria uekii and Eimeria raichoi in Japanese rock ptarmigans, Lagopus muta japonica, protected by cages in the Southern Japanese Alps.

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.