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A colpodean ciliate was found in the faeces of experimental rabbits. It was initially cultivated in medium mixed with 2% (w/v) rabbit faeces. Subsequently, two chemically defined media, designated CA-1 and CA-2, were found to be suitable for axenical cultivation of the ciliate. The maximum abundance of the ciliate isolate in the CA media was 1–2×105 cells/ml. The ciliate isolate was further identified with silver impregnation and molecular analysis. Features of the left oral polykinetid, somatic dikinetids, and sliverline pattern were similar to those of Colpoda aspera as described by Foissner (1993). The 18S small subunit ribosomal RNA gene of the ciliate isolate shared 99% sequence identity with that of C. aspera, with 100% coverage, and formed a sister clade in the phylogenetic tree with the reference C. aspera isolate. In addition, the trophozoite of C. aspera could proliferate over a temperature range from 25–37°C. When resting cysts were cultivated in CA-1 medium at 30–35°C, 98.2% of the trophozoites were detached from the cyst wall after 7h. [ABSTRACT FROM AUTHOR]

Abstract: Genetic manipulation of Apicomplexan parasite Eimeria tenella is only in its earliest stages. In the current study, transfection of E. tenella was conducted by electroporating sporozoites along with linear or circular plasmid DNA, and with or without restriction enzyme. Transfection system containing both linear DNA and restriction enzyme resulted in a transfection efficiency of 2.2×10−3 in vitro, which is 200-fold higher than that using circular plasmid DNA alone. In another transfection strategy, PCR amplicons of expression cassette, instead of whole plasmid DNA, were subjected to transfection, and it was also found successful. These results suggest that linear DNA and restriction enzyme together in the transfection system greatly improve the transfection efficiency of E. tenella. The high transfection efficiency makes possible the establishment of stable transfection in vivo; and the success of PCR-based, restriction enzyme-mediated transfection will further simplify the transfection process for E. tenella and other Apicomplexan parasites. [Copyright &y& Elsevier]

Abstract: Eimeria tenella and Toxoplasma gondii are Apicomplexan protozoa and share many similarities in biology and genomics. While the latter parasites are easily cultured in vitro and genetically manipulated, many Eimeria species are difficult to grow in vitro. We hypothesised that molecular tools for the genetic manipulation of T. gondii could be applied to the study of Eimeria parasites. Here we show that three different promoter sequences originating from E. tenella could function effectively not only in other species of the Eimeria genus (histone H4) but also in T. gondii (histone H4, actin and tubulin). Similarly, promoters of the “housekeeping” gene (tubulin) and differentially regulated gene (surface antigen gene, sag1) of T. gondii were effective in driving the expression of the yellow fluorescent protein (YFP) maker gene in E. tenella. The transfection efficiency with heterologous regulatory sequences was similar to that with homologous promoters; while the promoter strength of heterologous vectors is slightly weaker than the homologous vectors in both E. tenella and T. gondii. The results suggest that 5′ regulatory sequences are functionally conserved not only among the Eimeria species, but also between T. gondii and E. tenella, and that T. gondii could be used as a novel transfection check system for Eimeria-rooted vectors, accelerating the development of reverse genetics in Eimeria spp. [Copyright &y& Elsevier]

Abstract: The obligate intracellular apicomplexan parasite Eimeria tenella, one of seven species of Eimeria that infect chickens, elicits protective cell-mediated immunity against challenge infection. For this reason, recombinant E. tenella parasites could be utilised as an effective vaccine vehicle for expressing foreign antigens and inducing immunity against heterologous intracellular microbes. A stable line of E. tenella expressing foreign genes is a prerequisite, and in this work an in vivo stable transfection system has been developed for this parasite using restriction enzyme-mediated integration (REMI). Two transgenic populations of E. tenella have been obtained that express YFP-YFP constitutively throughout the parasite life cycle. Southern blotting and plasmid rescue analyses show that the introduced exogenous DNA was integrated at random into the parasite genome. Although the life cycle of the transgenic populations was delayed by at least 12h and the output of oocysts was reduced 4-fold relative to the parental BJ strain of E. tenella, the transgenic parasites were sufficiently immunogenic to protect chickens against challenge with either transgenic or parental parasites. These results are encouraging for the development of transgenic E. tenella as a vaccine vector and for more detailed investigation of the biology of the genus Eimeria. [Copyright &y& Elsevier]

Chicken coccidiosis can cause severe enteritis with high mortality, which causes serious economic losses to the global breeding industry each year. The most virulent species is Eimeria tenella ( E. tenella ) , but the infectivity of different E. tenella varies among geographic strains. At present, there are no reports related to the pathogenicity and drug resistance of E. tenella in Yiwu, Zhejiang province, China. A total of 600 fecal samples were collected from 10 farms in Zhejiang province, the overall oocyst prevalence was 54.2% (325/600). The prevalence was significantly higher (P < 0.01) in chickens under 40 d (97.5%) than that in chickens between 60 and 85-days-old (40.5%) and chickens over 90-days-old (24.5%). E. tenella stain was isolated from fecal samples of chickens in Yiwu and the pathogenicity of this isolate was determined, and then we recorded the survival rate, bloody stool score, lesion score, average weight gain. The results showed that all of the chickens infected with 5 × 105 sporulated oocysts of E. tenella died after the seventh day of infection, the bloody stool score and average lesion score of chickens from group 1 (5 × 105), group 2 (5 × 104), group 3 (5 × 103) and group 4 (5 × 102) decreased successively; the average weight gain (g) and relative weight gain (%) increased successively; the weight gain of the low-dose E. tenella infection groups (5 × 103 and 5 × 102) were higher than the other 2 groups (5 × 105 and 5 × 104) (P < 0.05). Finally, The E. tenella isolate was tested for sensitivity to 6 anticoccidial drugs (sulfachloropyrazine sodium, amproline, toltrazuril, clopidol, salinomycin, and nicarbazine) using 4 indexes including anticoccidial index(ACI), percent of optimum anticoccidial activity (POAA), reduction of lesion scores (RLS), and relative oocyst production (ROP). The results showed that this isolate has developed severe resistance to drugs of salinomycin and nicarbazine, moderate resistance to amproline and clopidol, slight resistance to toltrazuril, while the E. tenella isolate performed more sensitive to sulfachloropyrazine sodium. [ABSTRACT FROM AUTHOR]