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

Abstract

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.

Eimeria tenellaROPkinaseEtROP1induces G0/G1 cell cycle arrest and inhibits host cell apoptosis

“…Moreover, EtROP1 deletion in Eimeria by transgenesis would be relevant to confirm its function in vivo. Unfortunately, genetic manipulation in Eimeria has been limited to knock‐in strain production so far (Clark et al, ; Clark et al, ; Marugan‐Hernandez et al, ; Rieux et al, ). The CRISPR‐Cas9‐mediated genome editing approach should soon keep its promises and allow the generation of knockout virulence‐attenuated strains that may be valuable for vaccination.…”

Eimeria tenella protein trafficking: differential regulation of secretion versus surface tethering during the life cycle

“…Oocysts were harvested one week later, sporulated and used for a subsequent in vivo passage after enrichment of the population for fluorescent parasites by FACS (BD FACS Aria TM III 4 ).…”

Laboratory Growth and Genetic Manipulation of Eimeria tenella

“…A number of 5ʹ and 3ʹ endogenous regulatory regions have been successfully identified from Eimeria genomes and used to drive expression of different exogenous genes (normally fluorescent reporters), most commonly in E. tenella . These include promoters from constitutively expressed genes—such as actin, beta tubulin, and histone H4—but also other regulatory regions from genes expressed during specific stages of the parasite life cycle such as those encoding microneme proteins (MIC) 1, 2, 3, and 5; the surface antigen (SAG) 13 from the zoite stages; and the Gam56 protein from the sexual stages (Clark et al., ; Hanig, Entzeroth, & Kurth, ; Hao, Liu, Zhou, Li, & Suo, ; Kelleher & Tomley, ; Kurth & Entzeroth, ; Marugán‐Hernandez et al., , ; Tang et al., ). Thus, the choice of promoter can determine whether the transgene is expressed throughout the entire parasite life cycle or if it is restricted to specific life cycle stages.…”

“…This approach could streamline commercial vaccine formulations from up to eight Eimeria species/strains, due to the lack of cross‐protective immunity, to just a few lines expressing relevant antigens from all other species (Blake & Tomley, ). The toolbox would also allow expression of additional antigens directed against other pathogens which affect poultry, even modifying their trafficking within the parasite to target antigen exposure to the host immune system (Clark et al., ; Marugán‐Hernandez et al., , ; Pastor‐Fernandez et al., ; Tang et al., ).…”