Innate responses and biomarkers of resistance to Eimeria infection in the chicken
Item statusRestricted Access
Embargo end date30/06/2019
Coccidiosis is an intestinal disease caused by the protozoan parasite Eimeria, of which E. tenella and E. maxima are a common cause of disease in the poultry industry, causing weight gain loss, decreased feed efficiency and mortality in poultry. Coccidiosis is usually controlled by the application of anti-coccidial drugs or by vaccination, but drug resistance in Eimeria has been reported and vaccines require the passage of live Eimeria oocysts through the birds and are therefore expensive and difficult to produce. Alternative solutions are to develop subunit vaccines and to breed chickens for resistance to Eimeria by identifying resistance biomarkers, both of which require characterisation of the chicken immune response to Eimeria. To characterise immune responses to Eimeria, this study aimed to investigate the response of antigen presenting cells (APC) to Eimeria and determine which chicken pathogen recognition receptors (PRRs) recognise Eimeria vaccine candidates using in vitro techniques. The role of T helper (Th) 17 cells during E. maxima and E. tenella infection was also investigated in vivo through infection of a commercial broiler line. This study also aimed to identify biomarkers of Eimeria resistance by characterising the immune response to E. maxima and E. tenella in chicken lines which exhibit differential resistance and susceptibility to both these Eimeria spp. The development of chicken bone marrow-derived macrophage (BMM) and dendritic cell (BMDC) cultures provides an opportunity to study the responses of host-derived APC to Eimeria antigens and potential vaccine candidates in vitro. Here, both BMM and BMDC responded to an E. tenella oocyst crude lysate by upregulating mRNA expression of proinflammatory mediators (IL1B, IL6 and NOS2), BMM appeared more regulatory in nature (upregulated IL10 mRNA expression) and BMDC appeared more Th1-promoting (upregulated IFNG mRNA expression). Immune mapped protein 1 (IMP1) and apical membrane antigen 1 (AMA1) are two Eimeria vaccine candidates that have been shown to elicit protective immunity to Eimeria. In response to vaccine candidates IMP1 and AMA1, BMM responded in an inflammatory fashion through increased expression of IL6 and NOS2 mRNA. These results indicate that chicken macrophages and dendritic cells can recognise Eimeria and Eimeria vaccine candidates and facilitate inflammation though production of proinflammatory cytokines, but also have roles in promoting Th1 responses and in immune regulation. In order to trigger innate immune responses, pathogen associated molecular patterns (PAMPs) must be recognised by host PRRs, present on the surface of APC. Currently it is not known which Eimeria PAMPs are detected by which chicken PRRs. Use of a reporter gene assay identified that recombinant IMP1 and AMA1 are recognised by Toll-like receptor (TLR)1LB/2A heterodimers however further investigation is needed to determine other Eimeria PAMPs that are recognised by other chicken PRRs. Th1 responses are known to be important for the resolution of Eimeria infection however Th17 responses during Eimeria infection are less well characterised. Thought to contribute to immunopathology during Eimeria infection, Th17 responses represent a potential target in improving the outcome of Eimeria infection in chickens. Surprisingly, RT-qPCR analysis revealed no changes in the mRNA expression of Th17-associated cytokines in the gut of E. maxima- or E. tenella-infected Ross 308 broilers with the exception of IL21, indicating that IL-21 is acting in another capacity than as a Th17 effector during Eimeria infection. IL-21 is a highly pluripotent cytokine and further study would be required to characterise the role of IL-21 during Eimeria infection. In order to breed chickens for resistance, biomarkers of Eimeria resistance must first be identified. Line 15I and C.B12 chickens display inverse resistance and susceptibility to E. maxima and E. tenella. To identify biomarkers of resistance to E. maxima and E. tenella, the immune response of these lines to both Eimeria spp. was phenotyped. A higher increase in serum IL-10 was observed in E. maxima-infected susceptible line 15I than line C.B12 supporting a previous report that IL-10 is involved in susceptibility to E. maxima. RT-qPCR analysis revealed earlier increases of IFNG, IL10 and IL21 mRNA in the gut of resistant line C.B12 birds following E. maxima infection, indicating that a prompt immune response is a factor in resistance to E. maxima. No biomarkers of resistance to primary E. tenella infection were identified and further interrogation of the immune responses of these lines is required, particularly in response to secondary E. tenella infection. The results of this study have furthered our understanding of the role of APCs during Eimeria infection and following vaccination with IMP1 and AMA1 and support IMP1 and AMA1 as suitable vaccine candidates. IL-21 was identified as an important cytokine during Eimeria infection and further study is required to assess if IL-21 is beneficial or damaging to clearance of the parasite and to evaluate its potential as a therapeutic target. This study also confirmed previous findings that IL- 10 is involved in susceptibility to Eimeria and identified that a rapid response is important for resistance to E. maxima, providing a basis for further study to identify biomarkers of Eimeria resistance.