Sex-specific transcriptome of the chicken chorioallantoic membrane.

Dimorphism between male and female embryos has been demonstrated in many animal species, including chicken species. Likewise, extraembryonic membranes such as the chorioallantoic membrane (CAM) are likely to exhibit a sex-specific profile. Analysis of the previously published RNA-seq data of the chicken CAM sampled at two incubation times, revealed 783 differentially expressed genes between the CAM of male and female embryos. The expression of some of these genes is sex-dependant only at one or other stage of development, while 415 genes are sex-dependant at both developmental stages. These genes include well-known sex-determining and sex-differentiation genes (DMRT1, HEGM, etc.), and are mainly located on sex chromosomes. This study provides evidence that gene expression of extra-embryonic membranes is differentially regulated between male and female embryos. As such, a better characterisation of associated mechanisms should facilitate the identification of new sex-specific biomarkers.

RNA-seq analysis of the active chick embryo chorioallantoic membrane reveals genes that encode proteins assigned to ion transport and innate immunity.

The chicken chorioallantoic membrane (CAM) is an extraembryonic membrane that is vital for the embryo. It undergoes profound cell differentiation between 11 and 15 days of embryonic incubation (EID), which corresponds to the acquisition of its physiological functions. To gain insight into the functional genes that accompany these biological changes, RNA sequencing of the CAM at EID11 and EID15 was performed. Results showed that CAM maturation coincides with the overexpression of 4225 genes, including many genes encoding proteins involved in mineral metabolism, innate immunity, homeostasis, angiogenesis, reproduction, and regulation of hypoxia. Of these genes, some exhibit variability in expression depending on the chicken breed (broiler versus layer breeds). Besides the interest of these results for the poultry sector, the identification of new functional gene candidates opens additional research avenues in the field of developmental biology.

How Egg Storage Duration Prior to Incubation Impairs Egg Quality and Chicken Embryonic Development: Contribution of Imaging Technologies.

Storing fertilised eggs prior to incubation is a frequent practice in commercial hatcheries to coordinate activities and synchronise hatchings. However, the conditions used to store eggs can have major impacts on egg quality and the subsequent viability of chicken embryos. While storage temperatures of 16-18°C are classically used in hatcheries, the duration of storage varies from three to more than 10 days. We explored the effect of storage duration (zero, three or 10 days; D0, D3 and D10, respectively) at 16°C, 80% relative humidity (RH) on egg quality (Broiler, Ross 308), using computed tomography (CT) and classical measurements (egg weight, eggshell strength, egg white pH, Haugh units, yolk index and colour). The results revealed that a storage duration of up to 10 days negatively affected some egg quality traits (yolk index and volume, air chamber volume and egg white pH). Eggs stored for three or 10 days were further incubated for 11, 13 or 15 days (37.8°C, 55% RH). Eggs were analysed by magnetic resonance imaging (MRI) and CT to assess the development of the embryo and internal egg changes occurring during incubation. First, data showed that the fertility and sex ratio of eggs were not affected by storage duration. However, the mortality of viable eggs was increased in the D10 group compared to the D3 group. Results of non-invasive imaging technologies revealed that the storage of eggs for 10 days impaired embryo growth as early as 11 days of incubation (decrease in brain and embryo volumes). Collectively, these data provide new evidence that the duration of egg storage negatively affects embryonic growth. They further corroborate that this parameter is likely to be crucial to synchronising embryonic stages and maybe reducing the hatching window, hence limiting the time spent by newborn chicks in hatchers. In addition, our results highlight that CT and MRI imaging technologies are useful non-invasive tools to evaluate egg quality prior to incubation and the impact of storage (or incubation) practices on developmental growth of the embryo.

Concomitant Morphological Modifications of the Avian Eggshell, Eggshell Membranes and the Chorioallantoic Membrane During Embryonic Development.

The chicken eggshell (ES) consists of 95% calcium carbonate and 3.5% organic matter, and represents the first physical barrier to protect the developing embryo, while preventing water loss. During the second half of development, calcium ions from the inner ES are progressively solubilized to support mineralization of the embryonic skeleton. This process is mediated by the chorioallantoic membrane (CAM), which is an extraembryonic structure that adheres to the eggshell membranes (ESM) lining the inner ES. The CAM surrounds the embryo and all egg contents by day 11 of incubation (Embryonic Incubation Day 11, EID11) and is fully differentiated and functionally active by day 15 of incubation (Embryonic Incubation Day 15, EID15). In this study, we explored the simultaneous morphological modifications in the ES, ESM and the CAM at EID11 and EID15 by scanning electron microscopy. We observed that the tips of the mammillary knobs of the ES remain tightly attached to the ESM fibers, while their bases become progressively eroded and then detached from the bulk ES. Concomitantly, the CAM undergoes major structural changes that include the progressive differentiation of villous cells whose villi extend to reach the ESM and the ES. These structural data are discussed with respect to the importance of ES decalcification in providing the calcium necessary for mineralization of embryo's skeleton. In parallel, eggshell decalcification and weakening during incubation is likely to impair the ability of the ES to protect the embryo. It is assumed that the CAM could counteract this apparent weakening as an additional layer of physical, cellular and molecular barriers against environmental pressures, including pathogens, dehydration and shocks. However, such hypothesis needs to be further investigated.

Eggshell decalcification and skeletal mineralization during chicken embryonic development: defining candidate genes in the chorioallantoic membrane.

During chicken embryonic development, skeleton calcification mainly relies on the eggshell, whose minerals are progressively solubilized and transported to the embryo via the chorioallantoic membrane (CAM). However, the molecular components involved in this process remain undefined. We assessed eggshell demineralization and calcification of the embryo skeleton after 12 and 16 d of incubation, and analyzed the expression of several candidate genes in the CAM: carbonic anhydrases that are likely involved in secretion of protons for eggshell dissolution (CA2, CA4, CA9), ions transporters and regulators (CALB1, SLC4A1, ATP6V1B2, SGK1, SCGN, PKD2) and vitamin-D binding protein (GC). Our results confirmed that eggshell weight, thickness, and strength decreased during incubation, with a concomitant increase in calcification of embryonic skeletal system. In the CAM, the expression of CA2 increased during incubation while CA4 and CA9 were expressed at similar levels at both stages. SCL4A1 and SCGN were expressed, but not differentially, between the two stages, while the expression of ATP6V1B2 and PKD2 genes decreased. The expression of SGK1 and TRPV6 increased over time, although the expression of the latter gene was barely detectable. In parallel, we analyzed the expression of these candidate genes in the yolk sac (YS), which mediates the transfer of yolk minerals to the embryo during the first half of incubation. In YS, CA2 expression increases during incubation, similar to the CAM, while the expression of the other candidate genes decreases. Moreover, CALB1 and GC genes were found to be expressed during incubation in the YS, in contrast to the CAM where no expression of either was detected. This study demonstrates that the regulation of genes involved in the mobilization of egg minerals during embryonic development is different between the YS and CAM extraembryonic structures. Identification of the full suite of molecular components involved in the transfer of eggshell calcium to the embryo via the CAM should help to better understand the role of this structure in bone mineralization.

Lipopolysaccharide heterogeneity in the atypical group of novel emerging Brucella species.

Recently, novel Brucella strains with phenotypic characteristics that were atypical for strains belonging to the genus Brucella have been reported. Phenotypically many of these strains were initially misidentified as Ochrobactrum spp. Two novel species have been described so far for these strains, i.e., B. microti and B. inopinata, and other strains genetically related to B. inopinata may constitute other novel species as well. In this study, we analyzed the lipopolysaccharides (LPS) (smooth LPS [S-LPS] and rough LPS [R-LPS]) of these atypical strains using different methods and a panel of monoclonal antibodies (MAbs) directed against several epitopes of the Brucella O-polysaccharide (O-PS) and R-LPS. Among the most striking results, Brucella sp. strain BO2, isolated from a patient with chronic destructive pneumonia, showed a completely distinct S-LPS profile in silver stain gels that looked more similar to that of enterobacterial S-LPS. This strain also failed to react with MAbs against Brucella O-PS epitopes and showed weak reactivity with anti-R-LPS MAbs. B. inopinata reference strain BO1 displayed an M-dominant S-LPS type with some heterogeneity relative to the classical M-dominant Brucella S-LPS type. Australian wild rodent strains belonging also to the B. inopinata group showed a classical A-dominant S-LPS but lacked the O-PS common (C) epitopes, as previously reported for B. suis biovar 2 strains. Interestingly, some strains also failed to react with anti-R-LPS MAbs, such as the B. microti reference strain and B. inopinata BO1, suggesting modifications in the core-lipid A moieties of these strains. These results have several implications for serological typing and serological diagnosis and underline the need for novel tools for detection and correct identification of such novel emerging Brucella spp.

Novel IS711 chromosomal location useful for identification of marine mammal Brucella genotype ST27, which is associated with zoonotic infection.

We report a novel IS711 chromosomal location that is specific for the Brucella genotype ST27 previously associated with Pacific marine mammals and human zoonotic infection in New Zealand and Peru. Our data support the previous observation that this peculiar genotype is distinct from those commonly isolated from the Atlantic and currently classified within the species B. ceti and B. pinnipedialis.

Mesenteric lymph node cells from neonates present a prominent IL-12 response to CpG oligodeoxynucleotide via an IL-15 feedback loop of amplification.

At birth, the immune system is still in development making neonates more susceptible to infections. The recognition of microbial ligands is a key step in the initiation of immune responses. It can be mimicked to stimulate the immune system by the use of synthetic ligands recognising pattern recognition receptors. In human and mouse, it has been found that neonatal cytokine responses to toll-like receptor (TLR) ligands differ in many ways from those of adults but the relevant studies have been limited to cord blood and spleen cells. In this study, we compared the responses in neonate and adult sheep to CpG oligodeoxynucleotides (ODN), a TLR9 ligand, in both a mucosal and a systemic organ. We observed that in response to CpG-ODN more IL-12 was produced by neonatal than adult sheep cells from mesenteric lymph nodes (MLN) and spleen. This higher IL-12 response was limited to the first 20 days after birth for MLN cells but persisted for a longer period for spleen cells. The major IL-12-producing cells were identified as CD14+CD11b+. These cells were poor producers of IL-12 in response to direct stimulation with CpG-ODN and required the cooperation of other MLN cells. The difference in response to CpG-ODN between neonates and adults can be attributed to both a higher proportion of CD14+CD11b+ cells in neonate lambs and their higher capacity to produce IL-15. The IL-15 increases IL-12 production by an amplifying feedback loop involving CD40.

Cellular and molecular mechanisms underlying the strong neonatal IL-12 response of lamb mesenteric lymph node cells to R-848.

BACKGROUND: Comparative studies on the response of neonates and adults to TLR stimulation have been almost exclusively limited to comparisons of human neonatal cord blood cells with peripheral blood from adults, and analyses of spleen cell responses in mice. We need to extend these studies and gain further information regarding such responses at mucosal sites. METHODOLOGY/PRINCIPAL FINDINGS: We used sheep as a large animal model to study TLR agonist responses in the lymph nodes draining the intestine, an organ that must adapt to profound changes after birth. In response to the imidazoquinoline compound R-848, neonatal mesenteric lymph node (MLN) and spleen cells produced more IL-12 and, consequently, more IFNγ than their adult counterparts. This difference was age-related for both organs, but the preferential IL-12 response decreased more rapidly in the MLN, with young animals producing similar amounts of this cytokine to adults, from the age of 20 days onwards. Intracellular assays and depletion experiments identified CD14(+)CD11b(+)CD40(+) cells as the main producer of IL-12. These cells accounted for a greater proportion of neonatal than of adult MLN cells, and also produced, in direct response to R-848, more IL-12 after isolation. This strong IL-12 response in neonates occurred despite the production of larger amounts of the regulatory cytokine IL-10 and the stronger upregulation of SOCS-1 and SOCS-3 mRNA levels than in adult cells, and was correlated with an increase in p38/MAPK phosphorylation. CONCLUSIONS/SIGNIFICANCE: This is the first attempt to decipher the mechanism by which neonatal MLN cells produce more IL-12 than adult cells in response to the TLR8 agonist R-848. CD14(+)CD11b(+)CD40(+) IL-12-producing cells were more numerous in neonate than in adult MLN cells and displayed higher intracellular responsiveness upon R-848 stimulation. This work provides relevant information for future vaccination or immunostimulation strategies targeting neonates.

Novel IS711-specific chromosomal locations useful for identification and classification of marine mammal Brucella strains.

We report five new IS711 chromosomal locations that are specific for marine mammal Brucella groups of strains and useful for their identification and classification. Our data support their current classification into two species, Brucella ceti and B. pinnipedialis, with subgroups in each, but also the possibility of additional species.

Neonatal goats display a stronger TH1-type cytokine response to TLR ligands than adults.

In mammals, Toll-like receptors play a critical role in initiating innate immune responses and modulating adaptive immunity, by recognizing conserved microbial molecular patterns. This study was undertaken to identify specific features of the responses to synthetic toll-like receptor (TLR) agonists in goats, for the definition of tailored immunostimulation strategies. We show here, in contrast to what has been shown in mice, that mesenteric lymph nodes (MLNs) cells and splenocytes from neonatal goats produce much higher levels of TH1-type cytokines than adults in response to various TLR agonists. IL-12 was identified as a critical cytokine for IFNgamma production by CD8(+) neonatal cells. The higher level of IL-12 production by neonatal MLN and spleen cells than by adult cells was not correlated with a higher level of TLR expression or lower levels of production of the regulatory cytokine IL-10. In neonates, two cell populations-class II(+) CD8(+) and class II(+) CD8(-) cells-produce IL-12 in response to R848 and Poly I:C, respectively. Thus, goat kids have characteristics that could be exploited to favor development of the TH1-type responses critical for the control of intracellular pathogens.

CCR5 is involved in controlling the early stage of Cryptosporidium parvum infection in neonates but is dispensable for parasite elimination.

Chemokines play a critical role in immune cell trafficking and the transition from an innate to an acquired immune response. We analyzed host response in neonatal mice deficient in chemokine receptor CCR5 following infection with the intracellular protozoan parasite Cryptosporidium parvum. CCR5 neonatal mice had a higher parasite burden at the early stage of infection but eliminated the parasite as efficiently as their wild-type counterparts. The higher sensitivity of neonates at the beginning of infection was not due to an altered IFNgamma response. An increased CCR2-attracting chemokine response associated with the recruitment of CCR2-positive cells in the infected mucosa may have compensated for the absence of CCR5. A lack of CCR5 thus has an impact in the early stage of C. parvum infection in neonates, but this receptor is dispensable for subsequent parasite elimination.

Oral and intraperitoneal administration of phosphorothioate oligodeoxynucleotides leads to control of Cryptosporidium parvum infection in neonatal mice.

BACKGROUND: Neonates are particularly vulnerable to infections, in part because of the incomplete development of their immune system. Recent advances in immunostimulatory treatments based on conserved microbial components led us to assess the potential of oligodeoxynucleotides (ODNs) for decreasing the sensitivity of neonates to Cryptosporidium parvum infection. METHODS: Neonate mice were treated orally or intraperitoneally (ip) with CpG ODNs or non-CpG ODNs 24 h before C. parvum infection, and parasite load and cytokine up-regulation were evaluated. RESULTS: CpG ODN 1668 and non-CpG ODN 1668 administered orally, as well as CpG ODN 1668 administered ip, induced an 80%-95% decrease in intestinal parasite load 6 days after infection. Intraperitoneal and oral pretreatment with CpG ODN 1668 led to a strong initial up-regulation of cytokines and CD69 messenger RNA in the intestine and a decrease in parasite load by a Toll-like receptor 9 (TLR9)-dependent mechanism. By contrast, oral administration of non-CpG ODN 1668 decreased parasite load by a TLR9-independent mechanism. CONCLUSION: The control of neonatal C. parvum infection by ip or oral administration of ODNs is feasible by 2 different mechanisms: (1) the well-known interaction involving CpG/TLR9, leading to the production of cytokines and lymphocyte activation, and (2) a new unknown mechanism that is independent of TLR9 and effective orally.

Similar pattern of iNOS expression, NO production and cytokine response in genetic and vaccination-acquired resistance to Marek's disease.

NO is produced by macrophages through activation of the inducible enzyme NOS and its production is triggered as an antiviral and antitumoral immune mechanism. Replication of Marek's disease herpes virus (MDV) is inhibited by NO in vitro. MDV induces T-lymphomas in the chicken and a genetic resistance to tumor development has been linked to the B21 major histocompatibility complex. During the first initial week of viral replication after inoculation of the highly virulent RB-1B MDV strain, histocompatible B21/B21 chickens developed strong iNOS expression and NO production capacity in the spleen, in parallel with strong systemic NO production in the serum. Comparable NO response was not seen with the vaccinal strain HVT. In contrast, reduction in spleen macrophage number and delay in iNOS gene expression was observed in genetically susceptible B13/B13 chickens after MDV infection, in addition to suppression of IFN-gamma-inducible NO production. However, vaccination with HVT 3 days before RB-1B inoculation restored strong iNOS gene expression in the spleen 1 week later and inducible NO production 3 weeks later. Following the pattern of iNOS gene expression, early strong expression of cytokines with powerful iNOS-inducing activity such as IFN-gamma and CC chemokines from the MIP family (MIP-1beta, K203) was observed in genetic resistance and resistance acquired after vaccination with HVT. In conclusion, resistance to MDV appeared preferentially linked in both types of resistance to the early establishment of cytokine induction characteristic of a Th1 immune response, thus favoring the development of an early and strong NO response.