Eimeria stiedai is an apicomplexan protozoan parasite that invades the liver and bile duct epithelial cells in rabbits and causes severe hepatic coccidiosis, resulting in significant economic losses in the domestic rabbit industry. Hepatic coccidiosis lacks the typical clinical symptoms and there is a lack of effective premortem tools to timely diagnose this disease. Therefore, in the present study we cloned and expressed the two microneme proteins i.e., microneme protein 1 (EsMIC1) and microneme protein 3 (EsMIC3) from E. stiedai and used them as recombinant antigens to develop a serodiagnostic method for an effective diagnosis of hepatic coccidiosis. The cDNAs encoding EsMIC1 and EsMIC3 were cloned and the mRNA expression levels of these two genes at different developmental stages of E. stiedai were determined by quantitative real-time PCR analysis (qRT-PCR). The immunoreactivity of recombinant EsMIC1 (rEsMIC1) and EsMIC3 (rEsMIC3) proteins were detected by Western blotting, and indirect enzyme-linked immunosorbent assays (ELISAs) based on these two recombinant antigens were established to evaluate their serodiagnostic potential. Our results showed that the proteins encoded by the ORFs of EsMIC1 (711 bp) and EsMIC3 (891 bp) were approximately 25.89 and 32.39 kDa in predicted molecular weight, respectively. Both EsMIC1 and EsMIC3 showed the highest mRNA expression levels in the merozoites stage of E. stiedai. Western blotting analysis revealed that both recombinant proteins were recognized by E. stiedai positive sera, and the indirect ELISAs using rEsMIC1 and rEsMIC3 were developed based on their good immunoreactivity, with 100% (48/48) sensitivity and 97.9% (47/48) specificity for rEsMIC1 with 100% (48/48) sensitivity and 100% (48/48) specificity for rEsMIC3, respectively. Moreover, rEsMIC1- and rEsMIC3-based indirect ELISA were able to detect corresponding antibodies in sera at days 6, 8, and 10 post E. stiedai infection, with the highest positive diagnostic rate (62.5% (30/48) for rEsMIC1 and 66.7% (32/48) for rEsMIC3) observed at day 10 post infection. Therefore, both EsMIC1 and EsMIC3 can be used as potential serodiagnostic candidate antigens for hepatic coccidiosis caused by E. stiedai.

BACKGROUND: All dogs imported into Iceland must undergo mandatory quarantine in a special station before introduction into the country. A faecal sample is collected from the first stool passed by the dog in this station and subsequently examined for the presence of intestinal parasite stages.
METHODS: In May 2019 unsporulated oocysts were detected in faeces from a 7-year-old household dog that had been imported from Sweden. Most of the oocysts studied strongly resembled those of Eimeria canis Wenyon, 1923. As this species is not valid, the purpose of the present article was to identify the correct species and examine their possible origin. Studies confirmed the presence of two distinct unsporulated oocyst morphotypes in the faeces; measurements and photomicrographs confirmed their identification as Eimeria magna Pérard, 1925 and Eimeria stiedai (Lindemann, 1865) Kisskalt and Hartmann, 1907, both common parasites of European rabbits, Oryctolagus cuniculus (L., 1758). When the owner of the dog was questioned about the food administrated to the dog prior to its import to Iceland, it turned out that it had exclusively been fed dry dog food pellets. However, the owner also reported that on the morning prior to transportation to Iceland, the dog was allowed to move freely in a grassland area where rabbits are common and heaps of their faeces are present. Furthermore, the owner confirmed that the dog consumed rabbit faeces that morning.
CONCLUSIONS: It is believed that this coprophagic behaviour can explain the detection of rabbit eimerids in the dog\'s faeces, and that such behaviour must be taken into consideration by veterinarians and other diagnostic personnel when they detect atypical cysts or eggs during coprological examinations.

To study the pathogenicity of Eimeria stiedai, sporulated oocysts were given orally to coccidian-free two-month-old New Zealand rabbits(1000±20g). After 30days, blood samples from the rabbit hearts were collected for routine blood tests, liver functions and four characteristics of blood coagulation. Additionally, specimens of the liver, bile duct and duodenum were collected to observe the changes in pathology and ultrastructure. E. stiedai severely restricted the growth and development of rabbits. Blood tests showed that glutamine transferase (GGT) and serum cholinesterase (ChE) were significantly different from the non-infected controls. Other extremely significant differences were observed in the biochemical indices of routine blood tests, liver function and four blood coagulation characteristics, indicating that the liver functions were significantly affected. Staining showed that, compared with the negative control group, the liver, bile duct and duodenum contained significant numbers of lesions, and organs and cell structures suffered severe damage in ultrastructure, which greatly affecting bodily functions. E. stiedai-infected rabbits model was successfully established, which might provide a theoretical basis for research on the pathogenesis of rabbit coccidia, and the diagnosis and prevention of coccidiosis in rabbits.