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

Virginia Marugan-Hernandez, Charlotte Cockle, Sarah Macdonald, Elaine Pegg, Colin Crouch, Damer P. Blake, Fiona M. Tomley

Eimeria 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. In 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. E. 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.