Innate responses and biomarkers of resistance to Eimeria infection in the chicken

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

Evaluation of glycosylated FlpA and SodB as subunit vaccines against campylobacter jejuni colonisation in chickens

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide and the handling or consumption of contaminated poultry meat is the key source of infection. C. jejuni proteins FlpA and SodB and glycoconjugates containing the C. jejuni N-glycan have been separately reported to be partially protective vaccines in chickens. In this study, two novel glycoproteins generated by protein glycan coupling technology-G-FlpA and G-SodB (with two and three N-glycosylation sites, respectively)-were evaluated for efficacy against intestinal colonisation of chickens by C. jejuni strain M1 relative to their unglycosylated variants. Two independent trials of the same design were performed with either a high challenge dose of 107 colony-forming units (CFU) or a minimum challenge dose of 102 CFU of C. jejuni M1. While antigen-specific serum IgY was detected in both trials, no reduction in caecal colonisation by C. jejuni M1 was observed and glycosylation of vaccine antigens had no effect on the outcome. Our data highlight inconsistencies in the outcome of C. jejuni vaccination trials that may reflect antigen-, challenge strain-, vaccine administration-, adjuvant- and chicken line-specific differences from previously published studies. Refinement of glycoconjugate vaccines by increasing glycosylation levels or using highly immunogenic protein carriers could improve their efficacy

Multivalent poultry vaccine development using Protein Glycan Coupling Technology.

BackgroundPoultry is the world's most popular animal-based food and global production has tripled in the past 20 years alone. Low-cost vaccines that can be combined to protect poultry against multiple infections are a current global imperative. Glycoconjugate vaccines, which consist of an immunogenic protein covalently coupled to glycan antigens of the targeted pathogen, have a proven track record in human vaccinology, but have yet to be used for livestock due to prohibitively high manufacturing costs. To overcome this, we use Protein Glycan Coupling Technology (PGCT), which enables the production of glycoconjugates in bacterial cells at considerably reduced costs, to generate a candidate glycan-based live vaccine intended to simultaneously protect against Campylobacter jejuni, avian pathogenic Escherichia coli (APEC) and Clostridium perfringens. Campylobacter is the most common cause of food poisoning, whereas colibacillosis and necrotic enteritis are widespread and devastating infectious diseases in poultry.ResultsWe demonstrate the functional transfer of C. jejuni protein glycosylation (pgl) locus into the genome of APEC χ7122 serotype O78:H9. The integration caused mild attenuation of the χ7122 strain following oral inoculation of chickens without impairing its ability to colonise the respiratory tract. We exploit the χ7122 pgl integrant as bacterial vectors delivering a glycoprotein decorated with the C. jejuni heptasaccharide glycan antigen. To this end we engineered χ7122 pgl to express glycosylated NetB toxoid from C. perfringens and tested its ability to reduce caecal colonisation of chickens by C. jejuni and protect against intra-air sac challenge with the homologous APEC strain.ConclusionsWe generated a candidate glycan-based multivalent live vaccine with the potential to induce protection against key avian and zoonotic pathogens (C. jejuni, APEC, C. perfringens). The live vaccine failed to significantly reduce Campylobacter colonisation under the conditions tested but was protective against homologous APEC challenge. Nevertheless, we present a strategy towards the production of low-cost "live-attenuated multivalent vaccine factories" with the ability to express glycoconjugates in poultry

Investigating the effect of providing monetary incentives to participants on completion rates of referred co-respondents: an embedded randomized controlled trial. Study within a trial (SWAT) protocol

Background Parent-report questionnaires are a common method of generating data on child outcomes in mental health studies. A second report from another person who knows the child (co-respondent) is implemented to reduce bias and increase objectivity. The success of this approach is dependent on the engagement of co-respondents, which can be difficult. Financial incentives are used to increase data return in clinical trials, and to promote referral rates in online marketing. This protocol describes the use of an embedded randomised controlled trial (RCT) to investigate the effect of financial incentives on rates of co-respondent data completion. In the host RCT (of an online intervention designed to reduce the impact of a parent's anxiety on their child) index participants (i.e. parents) are asked to invite a co-respondent to complete measures on the index child. This study will test the hypothesis that providing monetary incentives to index participants will increase the outcome measure completion rate of co-respondents. Methods Embedded RCT of two parallel groups. Participants in the intervention arm will be sent a £10 voucher if their chosen co-respondent completes online baseline measures. Participants in the control arm will not be offered payment regardless of their chosen co-respondent's behaviour. 1754 participants will take part. Analysis will compare co-respondent outcome measure completion rates between the two arms at baseline and follow-up. Conclusion Findings from this study will provide evidence on the impact of offering payment to index participants on return rates of co-respondent data. This will inform resource allocation within future clinical trials

Quantitative trait loci and transcriptome signatures associated with avian heritable resistance to Campylobacter.

Funder: BiotechnologyCampylobacter is the leading cause of bacterial foodborne gastroenteritis worldwide. Handling or consumption of contaminated poultry meat is a key risk factor for human campylobacteriosis. One potential control strategy is to select poultry with increased resistance to Campylobacter. We associated high-density genome-wide genotypes (600K single nucleotide polymorphisms) of 3000 commercial broilers with Campylobacter load in their caeca. Trait heritability was modest but significant (h2 = 0.11 ± 0.03). Results confirmed quantitative trait loci (QTL) on chromosomes 14 and 16 previously identified in inbred chicken lines, and detected two additional QTLs on chromosomes 19 and 26. RNA-Seq analysis of broilers at the extremes of colonisation phenotype identified differentially transcribed genes within the QTL on chromosome 16 and proximal to the major histocompatibility complex (MHC) locus. We identified strong cis-QTLs located within MHC suggesting the presence of cis-acting variation in MHC class I and II and BG genes. Pathway and network analyses implicated cooperative functional pathways and networks in colonisation, including those related to antigen presentation, innate and adaptive immune responses, calcium, and renin-angiotensin signalling. While co-selection for enhanced resistance and other breeding goals is feasible, the frequency of resistance-associated alleles was high in the population studied and non-genetic factors significantly influenced Campylobacter colonisation

Role of caecal microbiota in the differential resistance of inbred chicken lines to colonization by Campylobacter jejuni

Campylobacter is the leading foodborne bacterial diarrhoeal illness in many countries, with up to 80 % of human cases attributed to the avian reservoir. The only control strategies currently available are stringent on-farm biosecurity and carcass treatments. Heritable differences in the resistance of chicken lines to Campylobacter colonisation have been reported and resistance-associated quantitative trait loci are emerging, albeit their impact on colonization appears modest. Recent studies indicated a protective role of the microbiota against colonization by Campylobacter in chickens. Furthermore, in murine models, differences in resistance to bacterial infections can be partially transferred between lines by transplantation of gut microbiota. In this study, we investigated whether heritable differences in colonization of inbred chicken lines by Campylobacter jejuni are associated with differences in caecal microbiota. We performed homologous and heterologous caecal microbiota transplants between line 61 (resistant) and line N (susceptible), by orally administering caecal contents collected from 3-week-old donors to day-of-hatch chicks. Recipient birds were challenged (day 21) with C. jejuni 11168H. In birds given homologous microbiota, the differential resistance of lines to C. jejuni colonization was reproduced. Contrary to our hypothesis, transfer of caecal microbiota from line 61 to line N significantly increased C. jejuni colonization. No significant difference in the overall composition of the caecal microbial communities of the two lines was identified, albeit line-specific differences for specific operational taxonomic units were identified. Our data suggest that while heritable differences in avian resistance to Campylobacter colonization exist, these are not explained by significant variation in the caecal microbiota