Single oocyst infection: a simple method for isolation of Eimeria spp. from the mixed field samples

Khalafalla, Reda E.; Daugschies, Arwid

doi:10.1007/s00436-010-1840-z

Cited by 15 publications

(7 citation statements)

References 10 publications

(11 reference statements)

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“…Eimeria species sporozoites Sporulated oocysts of E. tenella (strain LE-01 Eten-05/1) were maintained at the Institute of Parasitology, University of Leipzig, Germany, by using a single oocyst infection and subsequent passage every 6 months as described before (Khalafalla and Daugschies 2010;Shirley 1977Shirley , 1995.…”

Section: Methodsmentioning

confidence: 99%

In vivo evaluation of anticoccidial effect of antibody fragments expressed in pea (Pasum sativum) on Eimeria tenella sporozoites

Khalafalla

Daugschies²

2010

Parasitol Res

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Coccidiosis in chicken causes great economic losses. The increasing resistance of Eimeria species to anticoccidials has induced the search for alternative methods of control. In vivo antibody neutralization assay was conducted to study the inhibitory effect of nine antibody fragments (Ab1-Ab9) on Eimeria tenella sporozoites. These anti-E. tenella antibody fragments were expressed in pea plant (Pasum sativum). To assess the inhibitory effect on parasite reproduction, the in vivo antibody neutralization assay was done by retrograde infection of chicken with sporozoites previously incubated with antibody fragments. The pre-incubated sporozoites with the examined antibody fragments displayed a reduced ability to reproduce following intracloacal application to chicken (especially Ab1, Ab3, Ab5, and Ab9). Other antibody fragments (Ab2, Ab4, Ab6, Ab7, and Ab8) were less capable to reduce sporozoite infectivity and reproduction.

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Section: Methodsmentioning

confidence: 99%

In vivo evaluation of anticoccidial effect of antibody fragments expressed in pea (Pasum sativum) on Eimeria tenella sporozoites

Khalafalla

Daugschies²

2010

Parasitol Res

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“…The Yangzhou E. necatrix strain used in the present study was originally isolated from chickens that died from E. necatrix infection in 2009 (Yangzhou, Jiangsu, China). The identity of the strain was determined using the single-oocyst method described previously [ 23 ] and confirmed by microscopic examination and sequence analysis of the ribosomal RNA (rRNA) gene internal transcribed spacer region [ 24 ]. All animal experiments were approved by and conducted in strict accordance with the guidelines of the Animal Care and Use Committee of the College of Veterinary Medicine, Yangzhou University.…”

Section: Methodsmentioning

confidence: 99%

Full-length transcriptome analysis and identification of transcript structures in Eimeria necatrix from different developmental stages by single-molecule real-time sequencing

et al. 2021

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Background Eimeria necatrix is one of the most pathogenic parasites, causing high mortality in chickens. Although its genome sequence has been published, the sequences and complete structures of its mRNA transcripts remain unclear, limiting exploration of novel biomarkers, drug targets and genetic functions in E. necatrix. Methods Second-generation merozoites (MZ-2) of E. necatrix were collected using Percoll density gradients, and high-quality RNA was extracted from them. Single-molecule real-time (SMRT) sequencing and Illumina sequencing were combined to generate the transcripts of MZ-2. Combined with the SMRT sequencing data of sporozoites (SZ) collected in our previous study, the transcriptome and transcript structures of E. necatrix were studied. Results SMRT sequencing yielded 21,923 consensus isoforms in MZ-2. A total of 17,151 novel isoforms of known genes and 3918 isoforms of novel genes were successfully identified. We also identified 2752 (SZ) and 3255 (MZ-2) alternative splicing (AS) events, 1705 (SZ) and 1874 (MZ-2) genes with alternative polyadenylation (APA) sites, 4019 (SZ) and 2588 (MZ-2) fusion transcripts, 159 (SZ) and 84 (MZ-2) putative transcription factors (TFs) and 3581 (SZ) and 2039 (MZ-2) long non-coding RNAs (lncRNAs). To validate fusion transcripts, reverse transcription-PCR was performed on 16 candidates, with an accuracy reaching up to 87.5%. Sanger sequencing of the PCR products further confirmed the authenticity of chimeric transcripts. Comparative analysis of transcript structures revealed a total of 3710 consensus isoforms, 815 AS events, 1139 genes with APA sites, 20 putative TFs and 352 lncRNAs in both SZ and MZ-2. Conclusions We obtained many long-read isoforms in E. necatrix SZ and MZ-2, from which a series of lncRNAs, AS events, APA events and fusion transcripts were identified. Information on TFs will improve understanding of transcriptional regulation, and fusion event data will greatly improve draft versions of gene models in E. necatrix. This information offers insights into the mechanisms governing the development of E. necatrix and will aid in the development of novel strategies for coccidiosis control. Graphical Abstract

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“…Four wild strains (D2, D4, D5, and D8) of E . tenella were isolated from the field using the single oocyst method (Khalafalla and Daugschies ) and confirmed to be resistant to diclazuril using in vivo drug sensitivity tests in chickens. These four strains were propagated, and the SO were collected and purified.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, Et STP transcripts in SO of DZR, MRR, and DS strains were assayed using the same method. Field DZR strains (D2, D4, D5, and D8) were obtained from the wild using the single oocyst method (Khalafalla and Daugschies ), and Et STP mRNA transcripts of SO of the field DZR strains were analyzed. The SO of DZR strains resistant to different concentrations of diclazuril (0.2, 0.5, 0.8, and 1.0 ppm) and MRR strains resistant to different concentrations of maduramicin (3 and 5 ppm) were compared with DS strains.…”

Section: Methodsmentioning

confidence: 99%

Molecular characterization of serine/threonine protein phosphatase of Eimeria tenella

Zhao

Zhu

et al. 2020

J Eukaryotic Microbiology

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Avian coccidiosis is a widespread and economically significant poultry disease caused by several Eimeria species, including Eimeria tenella. Previously, E. tenella serine/threonine protein phosphatase (EtSTP) was found to be differentially expressed in drug‐sensitive (DS) and drug‐resistant strains using RNA‐seq. In the present study, we found that transcription and translation levels of EtSTP were higher in diclazuril‐resistant (DZR) strains and maduramicin‐resistant (MRR) strains than in DS strains using quantitative real‐time PCR (qPCR) and Western blotting. Enzyme activity results indicated that the catalytic activity of EtSTP was higher in the two drug‐resistant strains than in DS strains. Western blot and qPCR analysis also showed that expression levels of EtSTP were higher in unsporulated oocysts (UO) and second‐generation merozoites (SM). Indirect immunofluorescence localization showed that EtSTP was located in most areas of the parasite with the exception of refractile bodies, and fluorescence intensity was enhanced during development. In vitro inhibition experiments showed that the ability of sporozoites (SZ) to invade cells was significantly decreased after treatment with anti‐rEtSTP antibody. These results indicated that EtSTP acted mainly during the developmental and reproductive stages of the parasite and may be related to the resistance of coccidia to external drug pressure.

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Single oocyst infection: a simple method for isolation of Eimeria spp. from the mixed field samples

Abstract: The study described a simple method for single oocyst infection which is usually used to maintain the Eimeria spp. as pure strains in the laboratory and to isolate a single species from the mixed field samples.

Cited by 15 publications

References 10 publications

In vivo evaluation of anticoccidial effect of antibody fragments expressed in pea (Pasum sativum) on Eimeria tenella sporozoites

In vivo evaluation of anticoccidial effect of antibody fragments expressed in pea (Pasum sativum) on Eimeria tenella sporozoites

Full-length transcriptome analysis and identification of transcript structures in Eimeria necatrix from different developmental stages by single-molecule real-time sequencing

Molecular characterization of serine/threonine protein phosphatase of Eimeria tenella

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