Expression Analysis and Serodiagnostic Potential of Microneme Proteins 1 and 3 in Eimeria stiedai

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.

A Comparative Investigation of Methods for the Discovery of Helminth Eggs and Coccidia Oocysts in Soil Samples

A comparative investigation of two methods of soil study was performed - a modified by us and a classic flotation method, using standard media and soil. Different flotation solutions and cultures of eggs of Trichuris vulpis, Toxocara canis and Taenia pisisformis and oocysts of Eimeria stiedai were used. A method for use in diagnostic practice is proposed.

Body Weight, Oocyte Elimination and Blood Profile of Rabbit After Challenge Test Using Eimeria stiedai

The objective of the research was to investigate body weight, oocyte elimination and blood profile of rabbits infected with various doses of Eimeria stiedai isolates. The observed rabbits’ blood profile included erythrocyte, hemoglobin, hematocrit, leucocyte, thrombocyte, total protein plasma (TPP) and fibrinogen. Twenty-five male New Zealand White rabbits aged 3 months and weighed approximately 2 kg were provided with pellet and boiled drinking water and Eimeria stiedai isolates. The experiment used Completely Randomized Design to analyze 5 treatments with five replicates. The examined variables included D0: Infection 0 (control of infection without challenge test), D1: Infection 101 with challenge test 103, D2: infection 102 with challenge test 103, D3: infection 103 with challenge test 103, D4: infection 0 with challenge test 103 (control of infection). Data were subject to analysis of variance followed by Honestly Significant Difference Test (HSD). Analysis of Variance result showed that there was no significant difference on body weight, oocyte elimination and blood profile including erythrocyte, hemoglobin, hematocrit, leucocyte, thrombocyte, and fibrinogen. However, total protein plasma (TTP) was significantly different at 5% HSD. It can be concluded that challenge test with Eimeria stiedai has not been used as an alternative in increasing rabbits’ body immune against coccidiosis infection.

Observations on oocyst development of Eimeria stiedai in rabbits

The formation of the oocyst wall was examined in Eimeria stiedai in the bile duct epithelium of the rabbit and was found to follow the general eimerian pattern. However from the beginning of the formation of the outer layer of the oocyst wall the parasite was surrounded by a rarely reported veil membrane. Cell damage of the bile ducts at the gamogony stage of parasite development is depicted.

Systemic inflammatory response indicators in rabbits (Oryctolagus cuniculus) experimentally infected with sporulated oocysts of Eimeria stiedai (Apicomplexa: Eimeriidae)

Hemograms and acute-phase proteins in adult male New Zealand White rabbits that had been experimentally infected orally with sporulated oocysts of Eimeria stiedai were evaluated over a 28-day period. Fifty animals were used, divided into two groups: group A infected with 1 × 10(4) sporulated oocysts of E. stiedai and group B inoculated with distilled water. On the seventh day after infection, the infected animals presented anemia and leukocytosis with neutrophilia and monocytosis. Protein fractionation by means of electrophoresis identified 19 acute-phase proteins with molecular weights ranging from 24 to 238 kD. Ceruloplasmin, transferrin and haptoglobin showed high levels on the seventh day after infection, with gradual increases in their concentrations until the end of the experimental period. Thus, from the data of the present study, E. stiedai is considered to be a pyogenic etiological agent for which the infection level can be monitored through the leukocyte count and serum concentrations of ceruloplasmin, transferrin and haptoglobin, and these can be recommended as complementary tests.