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

Abstract

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.

Preliminary evaluation of the protective effects of recombinant AMA1 and IMP1 against Eimeria stiedae infection in rabbits

“…The rabbits were housed in pairs in flame-sterilized steel cages, with a plastic partition placed at the bottom to prevent contact with feces. The rabbits were raised based on the method described by Wei’s research [ 22 ]. Anticoccidial drugs were discontinued 1 week before the challenge infection was performed, and pathogenic examination was performed every other day to ensure that no coccidia oocysts were detected.…”

Evaluation of the immune protective effects of rEmMIC2 and rEmMIC3 from Eimeria magna in rabbits

“…Forty-eight coccidia-free New Zealand White rabbits were born and bred in this laboratory. Rabbits were weaned after 18 days and then fed infant formula milk powder and high-temperature formula pellets (made in our laboratory), during which time drinking water was boiled and fed (Shi et al 2016 ; Wei et al 2020 ). Existing feeds were replaced with anticoccidial drug-free feed 5 days before the coccidia challenge.…”

Eimeria proteins: order amidst disorder

“…After secretion, MICs persistently appear on parasite membrane and host cell surfaces [ 85 , 86 ] to enable Eimeria sporozoites to bind a diverse range of host cell glycan epitopes [ 87 ]. Functionally, MICs are critical for cellular processes including gliding motility, active cell invasion, migration [ 88 ] and parasite adhesion [ 89 ] (Table 1 ). Specifically, E. tenella MIC2 plays a crucial role in host cell identification and binding [ 60 ] just as MIC8 is a key protein in E. tenella metabolic processes [ 71 ].…”

“…Meanwhile, obvious challenges with mass spectrometry include decoy search strategy [ 146 ], correct peptide identification [ 91 , 112 ] and intractable genome annotations [ 144 ]. Expression of protein can be hindered in situ by lack of correlation between transcription and translation [ 39 , 89 , 113 ]. As well, RNA degradation can cause transcriptional suppression [ 147 ] of protein mRNAs [ 148 ] and hence obstruct translational events [ 149 ].…”