Changes of cecal microflora in chickens following Eimeria tenella challenge and regulating effect of coated sodium butyrate.

Eimeria tenella, one of the most important parasitic protozoa in the genus Eimeria, is responsible for chicken caecal coccidiosis resulting in huge economic losses to poultry industry. The present study investigated the changes in caecal microflora of E. tenella-infected chickens and the regulating effect of coated sodium butyrate, a potential alternative to antibiotics. Using high-throughput sequencing of 16S rRNA V3-V4 region of bacteria we found significant changes in caecal microflora of E. tenella-infected chickens indicated by an increase of Firmicutes (mainly Ruminococcaceae, Lachnospiraceae and vadin BB60) and Proteobacteria (mainly Enterobacteriaceae) and a decrease of Bacteroidetes (predominantly Bacteroidaceae). Inclusion of coated sodium butyrate in the diet of chickens per se had no significant effect on caecal microflora of normal healthy chickens but significantly prevented the increase in Firmicute abundance and decrease of Bacteroidetes abundance in E. tenella-infected birds. No significant changes to caecal microflora were observed at the phylum level between control and E. tenella-infected birds given coated sodium butyrate. In conclusion, our results show that coated sodium butyrate can balance the disorders of cecal microflora caused by E. tenella; thus, it can be a useful supplement for the control of avian coccidiosis.

Molecular characterization and analysis of a novel calcium-dependent protein kinase from Eimeria tenella.

The calcium-dependent protein kinases (CDPKs) are unique enzymes found only in plants, green algae, ciliates and apicomplexan parasites. In this study, a novel CDPK gene of Eimeria tenella, designed EtCDPK3, was cloned using rapid amplification of cDNA ends (RACE) based on the expressed sequence tag (EST). The entire cDNA of EtCDPK3 contained 1637 nucleotides encoding 433 amino acids and the deduced EtCDPK3 protein had canonical characteristic domains identified in other CDPKs, including a well-conserved amino-terminal kinase domain and a carboxy-terminal calmodulin-like structure with 4 EF-hand motifs for calcium binding. The expression profiles of the EtCDPK3 gene in different development stages were investigated by real-time quantitative PCR. Messenger RNA levels from the EtCDPK3 gene were higher in sporozoites than in other stages (unsporulated oocysts, sporulated oocysts and merozoites). Western blot analysis showed that rabbit antiserum against recombinant EtCDPK3 could recognize a native 49 kDa protein band of parasite. Indirect immunofluorescent antibody labelling revealed dispersed localization of EtCDPK3 during the first schizogony and intense specific staining. EtCDPK3 was located at the apical end of the sporozoites after early infection of DF-1 cells and the protein was highly expressed. Inhibition of EtCDPK3 function using specific antibodies reduced the ability of E. tenella to invade host cells. These results suggested that EtCDPK3 may be involved in invasion and survival of the parasite intracellular stages of E. tenella. Because this kinase family is absent from hosts, it represents a valid target that could be exploited for chemotherapy against Eimeria spp.

Identification of differentially expressed genes in early stages of Eimeria tenella by suppression subtractive hybridization and cDNA microarray.

Avian coccidiosis, a major parasitic disease of poultry, is caused by Eimeria spp. infection. It inflicts severe economic losses on the poultry industry worldwide. To further understand the molecular basis of sporulation and invasion of Eimeria spp., suppression subtractive hybridization and microarray approaches were combined to identify novel and important genes involved in the development and invasion of the early stages of Eimeria tenella . Three subtractive cDNA libraries were constructed for 3 stages of E. tenella including unsporulated oocysts, sporulated oocysts, and sporozoites. A subset of clones was selected from the 3 subtractive libraries to construct cDNA microarrays. Microarray analysis was used to assay expression changes of these clones. A total of 657 valid expressed sequence tags (ESTs) was obtained, including 119 unique sequences, 31 from sporulated oocysts and 88 from sporozoites. Homology searches of the public sequence databases showed that, among the 119 ESTs, 32 genes encoded proteins homologous with previously reported proteins including microneme proteins and surface antigen proteins of E. tenella , small heat shock proteins, rhoptry proteins of Toxoplasma gondii , and calcium-dependent protein kinase of Plasmodium spp. Thus, the remaining 87 ESTs have not previously been reported. Further characterization of these differentially expressed genes will be useful in understanding those responsible for sporulation, invasion, growth, and development of E. tenella .

Eimeria tenella: genetic recombination of markers for precocious development and arprinocid resistance.

A mating was made between two populations of Eimeria tenella possessing the complementary traits of normal development (virulence) + resistance to the anticoccidial drug arprinocid or precocious development (attenuation) + drug-sensitivity. A small number of "recombinant" oocysts was recovered. The inheritance of markers from both parents into 22 cloned lines derived from the recombinant oocysts was confirmed by analyses of restriction fragment length polymorphisms of four repetitive DNA sequences.