Nucleofection and In Vivo Propagation of Chicken &lt;em&gt;Eimeria&lt;/em&gt; Parasites

Duan, Chunhui; Tang, Xinming; Hu, Dandan; Zhang, Sixin; Liu, Jie; Bi, Feifei; Hao, Zhenkai; Suo, Jingxia; Yu, Ying; Wang, Min; Sun, Pei; Li, Du; Suo, Xun; Liu, Xianyong

doi:10.3791/60552

Cited by 8 publications

(5 citation statements)

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“…Sporozoites were purified through a diethylaminoethyl-52 cellulose column after releasing from sporulated oocysts ( 36 ). The two new plasmids, linearized with restriction enzyme SnaB I (New England BioLabs, Inc.), were transfected into 1 × 10 7 EacWT sporozoites by REMI as previously described ( 37 ).…”

Section: Methodsmentioning

confidence: 99%

Microneme-located VP2 in Eimeria acervulina elicits effective protective immunity against infectious bursal disease virus

Yu,

Tang,

Duan

et al. 2024

Infect Immun

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Using transgenic Eimeria spp. to deliver exogenous antigens is a viable option for developing multivalent live vaccines. Previous research revealed that the location of antigen expression in recombinant Eimeria dictates the magnitude and type of immune responses. In this study, we constructed genetically modified Eimeria acervulina that expressed VP2 protein, a protective antigen from infectious bursal disease virus (IBDV), on the surface or in the microneme of sporozoites. After vaccination, VP2-specific antibody was readily detected in specific pathogen-free chickens receiving transgenic E. acervulina parasites expressing VP2 in microneme, but animals vaccinated with which expressing VP2 on surface failed to produce detectable antibody after two times immunizations. Moreover, the bursal lesion of microneme-located VP2 transgenic E. acervulina immunized chickens was less severe compared with un-immunized animals after IBDV challenge infection. Therefore, genetically modified E. acervulina that express IBDV-derived VP2 in micronemes are effective in inducing specific antibody responses against VP2, while parasites that have VP2 expression on cell surface are not suitable. Thus, the use of Eimeria parasites as vaccine vectors needs to consider the proper targeting of exogenous immunogens. Our results have implications for the design of other vector vaccines.

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Section: Methodsmentioning

confidence: 99%

Microneme-located VP2 in Eimeria acervulina elicits effective protective immunity against infectious bursal disease virus

Yu,

Tang,

Duan

et al. 2024

Infect Immun

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“…The resistant strains were generated under halofuginone (30 mg/kg) through an experimental evolution strategy and verified through a drug resistance test ( Sun et al, 2023 ). The procedures for collection, sporulation and purification of the parasite were carried out as the previous report ( Duan et al, 2020 ). The cervical dislocation was performed for chickens necessary for sacrifice, which aims to lose consciousness of chickens rapidly.…”

Section: Methodsmentioning

confidence: 99%

Transcriptome profile of halofuginone resistant and sensitive strains of Eimeria tenella

et al. 2023

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The antiparasitic drug halofuginone is important for controlling apicomplexan parasites. However, the occurrence of halofuginone resistance is a major obstacle for it to the treatment of apicomplexan parasites. Current studies have identified the molecular marker and drug resistance mechanisms of halofuginone in Plasmodium falciparum. In this study, we tried to use transcriptomic data to explore resistance mechanisms of halofuginone in apicomplexan parasites of the genus Eimeria (Apicomplexa: Eimeriidae). After halofuginone treatment of E. tenella parasites, transcriptome analysis was performed using samples derived from both resistant and sensitive strains. In the sensitive group, DEGs associated with enzymes were significantly downregulated, whereas the DNA damaging process was upregulated after halofuginone treatment, revealing the mechanism of halofuginone-induced parasite death. In addition, 1,325 differentially expressed genes (DEGs) were detected between halofuginone resistant and sensitive strains, and the DEGs related to translation were significantly downregulated after halofuginone induction. Overall, our results provide a gene expression profile for further studies on the mechanism of halofuginone resistance in E. tenella.

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“…A third E. tenella strain, the SC strain, is a field-isolated strain isolated from a broiler farm in Sichuan province, China with a history of prolonged decoquinate use. The protocols for oocyst collection, sporulation and purification were as previously described [ 20 ]. Euthanasia was performed to chickens using cervical dislocation to ensure a rapid and painless loss of consciousness.…”

Section: Methodsmentioning

confidence: 99%

Distinct non-synonymous mutations in cytochrome b highly correlate with decoquinate resistance in apicomplexan parasite Eimeria tenella

Hao,

Chen,

Sun

et al. 2023

Parasites Vectors

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Background Protozoan parasites of the genus Eimeria are the causative agents of chicken coccidiosis. Parasite resistance to most anticoccidial drugs is one of the major challenges to controlling this disease. There is an urgent need for a molecular marker to monitor the emergence of resistance against anticoccidial drugs, such as decoquinate. Methods We developed decoquinate-resistant strains by successively exposing the Houghton (H) and Xinjiang (XJ) strains of E. tenella to incremental concentrations of this drug in chickens. Additionally, we isolated a decoquinate-resistant strain from the field. The resistance of these three strains was tested using the criteria of weight gain, relative oocyst production and reduction of lesion scores. Whole-genome sequencing was used to identify the non-synonymous mutations in coding genes that were highly associated with the decoquinate-resistant phenotype in the two laboratory-induced strains. Subsequently, we scrutinized the missense mutation in a field-resistant strain for verification. We also employed the AlphaFold and PyMOL systems to model the alterations in the binding affinity of the mutants toward the drug molecule. Results We obtained two decoquinate-resistant (DecR) strains, DecR_H and XJ, originating from the original H and XJ strains, respectively, as well as a decoquinate-resistant E. tenella strain from the field (DecR_SC). These three strains displayed resistance to 120 mg/kg decoquinate administered through feed. Through whole-genome sequencing analysis, we identified the cytochrome b gene (cyt b; ETH2_MIT00100) as the sole mutated gene shared between the DecR_H and XJ strains and also detected this gene in the DecR_SC strain. Distinct non-synonymous mutations, namely Gln131Lys in DecR_H, Phe263Leu in DecR_XJ, and Phe283Leu in DecR_SC were observed in the three resistant strains. Notably, these mutations were located in the extracellular segments of cyt b, in close proximity to the ubiquinol oxidation site Qo. Drug molecular docking studies revealed that cyt b harboring these mutants exhibited varying degrees of reduced binding ability to decoquinate. Conclusions Our findings emphasize the critical role of cyt b mutations in the development of decoquinate resistance in E. tenella. The strong correlation observed between cyt b mutant alleles and resistance indicates their potential as valuable molecular markers for the rapid detection of decoquinate resistance. Graphical abstract

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Nucleofection and In Vivo Propagation of Chicken <em>Eimeria</em> Parasites

Cited by 8 publications

References 0 publications

Microneme-located VP2 in Eimeria acervulina elicits effective protective immunity against infectious bursal disease virus

Microneme-located VP2 in Eimeria acervulina elicits effective protective immunity against infectious bursal disease virus

Transcriptome profile of halofuginone resistant and sensitive strains of Eimeria tenella

Distinct non-synonymous mutations in cytochrome b highly correlate with decoquinate resistance in apicomplexan parasite Eimeria tenella

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