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.
Highlights
Eimeria species are parasitic protozoa that cause coccidiosis, an intestinal disease commonly characterised by malabsorption, diarrhoea and haemorrhage that is important in chickens
Transfected E. tenella sporozoites expressed foreign antigens under the regulation of two different promoter regions The functionality of promoters 5’Et-Actin and 5’Et-Translation initiation factor (TIF) was confirmed by detection of red fluorescence in sporozoites transfected with constructs containing mCherry cloned downstream of each promoter (Table 1, Fig. 2a)
Quantification of transgene copy number per parasite genome and the level of transgene transcription was not carried out in these short-term sporozoite cultures because non-integrated plasmid was detected, evidenced by a number of mCitrine copies per genome determined by quantitative Polymerase Chain Reaction (PCR) (qPCR) to exceed 35,000
Summary
Eimeria species are parasitic protozoa that cause coccidiosis, an intestinal disease commonly characterised by malabsorption, diarrhoea and haemorrhage that is 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. Eimeria genomes are much larger than those of viral vectors, in consequence they can tolerate the insertion and expression of several foreign antigens They induce a broad range of potent immune responses following oral administration [12] having the potential to be exploited as a flexible oral vaccine vector for intracellular and extracellular pathogens
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