Development of transgenic lines of Eimeria tenella expressing M2e-enhanced yellow fluorescent protein (M2e-EYFP)

Liu, Xianyong; Zou, Jun; Yin, Guangwen; Su, Huali; Huang, Xiaoxi; Li, Jianan; Xie, Li; Cao, Yingqiong; Yu-juan, Cui; Suo, Xun

doi:10.1016/j.vetpar.2012.12.019

Cited by 24 publications

(26 citation statements)

References 26 publications

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“…Clark et al [10] expressed Campylobacter jejuni antigen CjaA in E. tenella and whilst birds immunised with the transgenic parasites were protected (~90 % reduction in C. jejuni in the caeca 2 weeks after challenge, compared to controls) evidence of immunological recognition of the antigen by the chicken was inconclusive. Moreover, Liu et al [11] generated parasites expressing the M2 protein of the avian influenza virus, but found no evidence of antibody or cellular responses. The only immune responses reported in chickens raised by transgenic Eimeria has been against a fluorescent reporter protein (EYFP) [23], therefore, this is the first time that antibodies against viral proteins expressed in transgenic Eimeria have been reported.…”

Section: Discussionmentioning

confidence: 99%

“…Specifically, Eimeria cannot be cloned in vitro by serial dilution or similar procedures. Instead, single sporocyst infection of chickens is needed to clone Eimeria , which is a time-consuming and inefficient process [11, 28]. Non-clonal populations that have 100 % transgenic parasites can be obtained by applying drug selection over several in vivo passages to parasites where a drug-resistance cassette has been incorporated in the transfection process [9]; however, inclusion of drug-resistant genes is not appropriate for populations to be administrated as live vaccines in field chicken populations.…”

Section: Discussionmentioning

confidence: 99%

“…[8]. Protocols which allow genetic complementation of Eimeria to express heterologous protein-coding sequences [9] raises the interesting possibility that existing Eimeria vaccines could be engineered to express antigens derived from a wide range of poultry pathogens [10, 11]. Eimeria genomes are much larger than those of viral vectors, in consequence they can tolerate the insertion and expression of several foreign antigens.…”

Section: Introductionmentioning

confidence: 99%

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Viral proteins expressed in the protozoan parasite Eimeria tenella are detected by the chicken immune system

et al. 2016

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BackgroundEimeria species are parasitic protozoa that cause coccidiosis, an intestinal disease commonly characterised by malabsorption, diarrhoea and haemorrhage that is particularly important in chickens. Vaccination against chicken coccidiosis is effective using wild-type or attenuated live parasite lines. The development of protocols to express foreign proteins in Eimeria species has opened up the possibility of using Eimeria live vaccines to deliver heterologous antigens and function as multivalent vaccine vectors that could protect chickens against a range of pathogens.ResultsIn this study, genetic complementation was used to express immunoprotective virus antigens in Eimeria tenella. Infectious bursal disease virus (IBDV) causes Gumboro, an immunosuppressive disease that affects productivity and can interfere with the efficacy of poultry vaccination programmes. Infectious laryngotracheitis virus (ILTV) causes a highly transmissible respiratory disease for which strong cellular immunity and antibody responses are required for effective vaccination. Genes encoding the VP2 protein from a very virulent strain of IBDV (vvVP2) and glycoprotein I from ILTV (gI) were cloned downstream of 5’Et-Actin or 5’Et-TIF promoter regions in plasmids that also contained a mCitrine fluorescent reporter cassette under control of the 5’Et-MIC1 promoter. The plasmids were introduced by nucleofection into E. tenella sporozoites, which were then used to infect chickens. Progeny oocysts were sorted by FACS and passaged several times in vivo until the proportion of fluorescent parasites in each transgenic population reached ~20 % and the number of transgene copies per parasite genome decreased to < 10. All populations were found to transcribe and express the transgene and induced the generation of low titre, transgene-specific antibodies when used to immunise chickens.ConclusionsE. tenella can express antigens of other poultry pathogens that are successfully recognised by the chicken immune system. Nonetheless, further work has to be done in order to improve the levels of expression for its future use as a multivalent vaccine vector.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1756-2) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Viral proteins expressed in the protozoan parasite Eimeria tenella are detected by the chicken immune system

et al. 2016

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“…gondii can be investigated and manipulated easily in established in vitro systems using a variety of host cells [2,3]. As with Eimeria species –the most important apicomplexan pathogens in poultry – biotechnological techniques such as transgenic parasites are standard [4–6]. However, no cell culture system is available for many apicomplexan parasites (e.g.…”

Section: Introductionmentioning

confidence: 99%

Isospora suis in an Epithelial Cell Culture System – An In Vitro Model for Sexual Development in Coccidia

et al. 2013

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Coccidian parasites are of major importance in animal production, public health and food safety. The most frequently used representative in basic research on this group is Toxoplasma gondii. Although this parasite is well investigated there is no adequate in vitro model for its sexual development available and knowledge on this important life cycle phase is therefore scarce. The use of Isospora suis , a sister taxon to T. gondii and the causative agent of piglet coccidiosis, could provide a solution for this. In the present study an in vitro model for neonatal porcine coccidiosis in cells representative for the in vivo situation in the piglet gut was developed and evaluated. The parasite development was investigated by light and transmission electron microscopy and optimum culture conditions were evaluated. Intestinal porcine epithelial cells (IPEC-J2) adequately representing the natural host cells supported the development of all endogenous life cycle stages of I . suis , including gametocytes and oocysts. A concentration of 5% fetal calf serum in the culture medium led to highest gametocyte densities on day 12 post infection. Low infection doses (≤1 sporozoite for 100 host cells) were best for oocyst and gametocyte development. The presented system can also be used for immunostaining with established antibodies developed against T. gondii (in our case, anti-TgIMC3 antibodies directed against the inner membrane complex 3). The complete life cycle of I . suis in a cell line representing the natural host cell type and species provides a unique model among coccidian parasites and can be used to address a wide range of topics, especially with regard to the sexual development of coccidia.

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“…To date, transfection of E. tenella sporozoites has been performed using a broad range of plasmid concentrations (Clark et al., ; Liu et al., ; Yan et al., ). However, these studies have not directly evaluated the effect of DNA starting concentration on transfection efficiency, which has now been found to have a dose‐dependent effect.…”

Section: Strategic Planningmentioning

confidence: 99%

Laboratory Growth and Genetic Manipulation of Eimeria tenella

et al. 2019

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Eimeria is a genus of apicomplexan parasites that contains a large number of species, most of which are absolutely host‐specific. Seven species have been recognized to infect chickens. Infection of susceptible chickens results in an intestinal disease called coccidiosis, characterized by mucoid or hemorrhagic enteritis, which is associated with impaired feed conversion or mortality in severe cases. Intensive farming practices have increased the significance of coccidiosis since parasite transmission is favored by high‐density housing of large numbers of susceptible chickens. Routine chemoprophylaxis and/or vaccination with live parasite vaccines provides effective control of Eimeria, although the emergence of drug resistance and the relative cost and production capacity of current vaccine lines can prove limiting. As pressure to reduce drug use in livestock production intensifies, novel vaccination strategies are needed. Development of effective protocols supporting genetic complementation of Eimeria species has until recently been hampered by their inability to replicate efficiently in vitro. Now, the availability of such protocols has raised the prospect of generating transgenic parasite lines that function as vaccine vectors to express and deliver heterologous antigens. For example, this technology has the potential to streamline the production of live anticoccidial vaccines through the generation of parasite lines that co‐express immunoprotective antigens derived from multiple Eimeria species. In this paper we describe detailed protocols for genetic manipulation, laboratory growth, and in vivo propagation of Eimeria tenella parasites, which will encourage future work from other researchers to expand biological understanding of Eimeria through reverse genetics. © 2019 by John Wiley & Sons, Inc.

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Development of transgenic lines of Eimeria tenella expressing M2e-enhanced yellow fluorescent protein (M2e-EYFP)

Cited by 24 publications

References 26 publications

Viral proteins expressed in the protozoan parasite Eimeria tenella are detected by the chicken immune system

Viral proteins expressed in the protozoan parasite Eimeria tenella are detected by the chicken immune system

Isospora suis in an Epithelial Cell Culture System – An In Vitro Model for Sexual Development in Coccidia

Laboratory Growth and Genetic Manipulation of Eimeria tenella

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