An integrated transcriptomics and metabolomics study of the immune response of newly hatched chicks to the cytosine-phosphate-guanine oligonucleotide stimulation.

The immunological immaturity of the innate immune system during the first-week post-hatch enables pathogens to infect chickens, leading to the death of the animals. Current preventive solutions to improve the resistance of chicks to infections include vaccination, breeding, and sanitation. Other prophylactic solutions have been investigated, such as the stimulation of animal health with immunostimulants. Recent studies showed that administration of immune-modulators to one-day-old chicks, or in ovo, significantly reduces mortality in experimental bacterial or viral infection challenge models. Owing to a lack of molecular biomarkers required to evaluate chicken immune responses and assess the efficacy of vaccines or immune-modulators, challenge models are still used. One way to reduce challenge experiments is to define molecular signatures through omics approaches, resulting in new methodologies to rapidly screen candidate molecules or vaccines. This study aims at identifying a dual transcriptomics and metabolomics blood signature after administration of CpG-ODN (cytosine-phosphate-guanine oligodeoxynucleotides), a reference immune-stimulatory molecule. A clinical study was conducted with chicks and transcriptomics and metabolomics analyses were performed on whole-blood and plasma samples, respectively. Differentially expressed genes and metabolites with different abundance were identified in chicks treated with CpG-ODN. The results showed that CpG-ODN activated the innate immune system, within hours after administration, and its effect lasted over time, as metabolomics and transcriptomics profiles still varied 6 D after administration. In conclusion, through an integrated clinical omics approach, we deciphered in part the mode of action of CpG-ODN in post-hatch chicks.

Caecal microbiota compositions from 7-day-old chicks reared in high-performance and low-performance industrial farms and systematic culturomics to select strains with anti-Campylobacter activity.

There is growing interest in exploring the chickens' intestinal microbiota and understanding its interactions with the host. The objective is to optimize this parameter in order to increase the productivity of farm animals. With the goal to isolate candidate probiotic strains, specific culturomic methods were used in our study to culture commensal bacteria from 7-days old chicks raised in two farms presenting long history of high performance. A total of 347 isolates were cultured, corresponding to at least 64 species. Among the isolates affiliated to the Firmicutes, 26 had less than 97% identity of their partial 16S sequence with that of the closest described species, while one presented less than 93% identity, thus revealing a significant potential for new species in this ecosystem. In parallel, and in order to better understand the differences between the microbiota of high-performing and low-performing animals, caecal contents of animals collected from these two farms and from a third farm with long history of low performance were collected and sequenced. This compositional analysis revealed an enrichment of Faecalibacterium-and Campylobacter-related sequences in lower-performing animals whereas there was a higher abundance of enterobacteria-related sequences in high-performing animals. We then investigated antibiosis activity against C. jejuni ATCC 700819 and C. jejuni field isolate as a first phenotypic trait to select probiotic candidates. Antibiosis was found to be limited to a few strains, including several lactic acid bacteria, a strain of Bacillus horneckiae and a strain of Escherichia coli. The antagonist activity depended on test conditions that mimicked the evolution of the intestinal environment of the chicken during its lifetime, i.e. temperature (37°C or 42°C) and oxygen levels (aerobic or anaerobic conditions). This should be taken into account according to the stage of development of the animal at which administration of the active strain is envisaged.

Multivariate analysis of the immune response to a vaccine as an alternative to the repetition of animal challenge studies for vaccines with demonstrated efficacy.

The assessment of vaccine combinations, or the evaluation of the impact of minor modifications of one component in well-established vaccines, requires animal challenges in the absence of previously validated correlates of protection. As an alternative, we propose conducting a multivariate analysis of the specific immune response to the vaccine. This approach is consistent with the principles of the 3Rs (Refinement, Reduction and Replacement) and avoids repeating efficacy studies based on infectious challenges in vivo. To validate this approach, a set of nine immunological parameters was selected in order to characterize B and T lymphocyte responses against canine rabies virus and to evaluate the compatibility between two canine vaccines, an inactivated rabies vaccine (RABISIN®) and a combined vaccine (EURICAN® DAPPi-Lmulti) injected at two different sites in the same animals. The analysis was focused on the magnitude and quality of the immune response. The multi-dimensional picture given by this 'immune fingerprint' was used to assess the impact of the concomitant injection of the combined vaccine on the immunogenicity of the rabies vaccine. A principal component analysis fully discriminated the control group from the groups vaccinated with RABISIN® alone or RABISIN®+EURICAN® DAPPi-Lmulti and confirmed the compatibility between the rabies vaccines. This study suggests that determining the immune fingerprint, combined with a multivariate statistical analysis, is a promising approach to characterizing the immunogenicity of a vaccine with an established record of efficacy. It may also avoid the need to repeat efficacy studies involving challenge infection in case of minor modifications of the vaccine or for compatibility studies.

Effect of sow vaccination against porcine circovirus type 2 (PCV2) on virological profiles in herds with or without PCV2 systemic disease.

We investigated porcine circovirus type 2 (PCV2) virological profiles in herds affected (PCVAD-AH, n = 5) or non-affected (PCVAD-NAH, n = 4) by PCV2-associated diseases (PCVAD), before and after 1 y of PCV2 gilt and sow vaccination. Fresh feces from the floor (5 pens/age/farm) and 5 blood samples (1/pen) were collected at 3, 9, 15, 21 wk. Individual feces and blood samples were collected from 5 gilts and 15 sows. Sampling was repeated 1 y after vaccination. Quantitative PCR on feces, PCV2 antibodies in blood serum and cell-mediated immunity were investigated. Before vaccination, pigs of PCVAD-AH had higher viral load in feces (9 and 15 wk), lower IgG and higher IgM (3 wk) and lower lymphocyte counts (9 and 15 wk) suggesting immunosuppression. Vaccination reduced viral load in growers, increased IgG (3 wk) suggesting improved maternal immunity, reduced IgM (3 wk), increased total antibody titers in sows and increased CD79a cells in the pigs.

Effet de la vaccination des truies contre le circovirus porcin de type 2 (PCV2) sur les profils virologiques des troupeaux atteints ou non de la maladie systémique PCV2. Nous avons fait une enquête sur les profils virologiques du circovirus porcin de type 2 (PCV2) dans les troupeaux affectés (PCVAD-AH, n = 5) ou non affectés (PCVAD-NAH, n = 4) par les maladies associées au PCV2 (MAPCV), 1 an avant et 1 an après la vaccination des cochettes et des truies contre le PCV2. Des fèces fraîches sur le plancher (5 enclos/âge/ferme) et 5 échantillons de sang (1/enclos) ont été prélevés à 3, 9, 15 et 21 semaines. Des fèces individuelles et des échantillons sanguins ont été préIevés auprès de 5 cochettes et de 15 truies. L'échantillonnage a été répété 1 an après la vaccination. La RCP quantitative sur les fèces, les anticorps de PCV2 dans le sérum sanguin et l'immunité à médiation cellulaire ont fait l'objet d'une enquête. Avant la vaccination, les porcs de PCVAD-AH présentaient une charge virale supérieure dans les fèces (à 9 et à 15 semaines), une IgG inférieure et une IgM supérieure (à 3 semaines) ainsi qu'une numération inférieure des lymphocytes (à 9 et à 15 semaines) suggérant l'immunosuppression. La vaccination a réduit la charge virale chez les porcs en croissance, a augmenté les IgG (à 3 semaines) suggérant une immunité maternelle améliorée, a réduit les IgM (à 3 semaines), a augmenté le total des titres d'anticorps chez les truies et a augmenté les cellules CD79a chez les porcs.(Traduit par Isabelle Vallières).

Intradermal vaccination with un-adjuvanted sub-unit vaccines triggers skin innate immunity and confers protective respiratory immunity in domestic swine.

Intradermal (ID) vaccination constitutes a promising approach to induce anti-infectious immunity. This route of immunization has mostly been studied with influenza split-virion vaccines. However, the efficacy of ID vaccination for sub-unit vaccines in relation to underlying skin innate immunity remains to be explored for wider application in humans. Relevant animal models that more closely mimic human skin immunity than the widely used mouse models are therefore necessary. Here, we show in domestic swine, which shares striking anatomic and functional properties with human skin, that a single ID delivery of pseudorabies virus (PRV) glycoproteins without added adjuvant is sufficient to trigger adaptive cellular and humoral immune responses, and to confer protection from a lethal respiratory infection with PRV. Analysis of early events at the skin injection site revealed up-regulation of pro-inflammatory cytokine and chemokine genes, recruitment of neutrophils and monocytes and accumulation of inflammatory DC. We further show that the sustained induction of pro-inflammatory cytokine genes results from the combined effects of skin puncture, liquid injection in the dermis and viral antigens. These data highlight that immune protection against respiratory infection can be induced by ID vaccination with a subunit vaccine and reveal that adjuvant requirements are circumvented by the mechanical and antigenic stress caused by ID injection, which triggers innate immunity and mobilization of inflammatory DC at the immunization site. ID vaccination with sub-unit vaccines may thus represent a safe and efficient solution for protection against respiratory infections in swine and possibly also in humans, given the similarity of skin structure and function in both species.

DNA vaccination of neonate piglets in the face of maternal immunity induces humoral memory and protection against a virulent pseudorabies virus challenge.

DNA vaccination represents a unique opportunity to overcome the limitations of conventional vaccine strategy in early life in the face of maternal-derived immunity. We used the model of pseudorabies virus (PRV) infection in pigs to further explore the potential of DNA vaccination in piglets born to sows repeatedly vaccinated with a PRV inactivated vaccine. A single immunisation of 8-week-old piglets with a DNA vaccine expressing secreted forms of PRV gB, gC, and gD, triggered an active serological response, confirming that DNA vaccination can over-ride significant residual maternal-derived immunity. A clear anamnestic response was evidenced when a secondary DNA vaccination was performed at 11 weeks of age, suggesting that DNA vaccination, performed in the face of passive immunity, elicited a strong humoral memory. We subsequently explored the potential of DNA vaccination in neonate piglets (5-6 days of age) in the face of very high titres of maternal antibodies and demonstrated that very high titres of passive antibodies selectively inhibited serological responses but not the establishment of potent memory responses. Finally, we demonstrated that DNA vaccination provided protection against an infectious PRV challenge at the end of the fattening period (i.e. at approximately 5 months of age). Collectively, our results pave the way for a new flexible vaccination program, which could ensure uninterrupted protection of fattening pigs over their entire economical life under field conditions.