Nathalie K. Chanteloup

Bacterial colonization and in vivo expression of F1 (type 1) fimbrial antigens in chickens experimentally infected with pathogenic Escherichia coli.

Escherichia coli strains that cause septicemia of poultry often possess F1 (type 1) fimbriae (encoded by pil [fim] homologous gene clusters) and/or P fimbriae (encoded by pap homologous gene clusters). These fimbriae are thought to be involved in infection and colonization. To study the dynamics of infection due to E. coli with different virulence determinant profiles and to examine the expression of these fimbriae in vivo, three pathogenic E. coli isolates--O1 (pil+/pap+), O2 (pil+/pap), and O78 (pil+/pap+)--were administered intratracheally to 1.5-week-old chickens. Chickens were euthanatized from 3 to 144 hr after infection. The three isolates caused lesions in 30 to 55% of birds. Colonization rates of the trachea, lungs, internal organs, and pericardial fluid were similar for all three isolates, whereas significant differences among isolates were observed in colonization of the air sacs and blood. Bacteria appeared rapidly in the blood, liver, and spleen, whereas presence in the pericardial fluid generally occurred only after 24 hr postinoculation. The dynamics of colonization of the air sacs varied among isolates. Immunofluorescence of frozen tissue sections demonstrated F1 fimbriae (pil expressed) but not P fimbriae on all three isolates colonizing the trachea and on the O1 and O78 isolates colonizing the air sacs. Results suggest that F1 fimbriae are involved in the early stages of development of colisepticemia by promoting association of pathogenic E. coli with the trachea and air sacs of chickens.

A Metabolic Operon in Extraintestinal Pathogenic Escherichia coli Promotes Fitness under Stressful Conditions and Invasion of Eukaryotic Cells

We identified a carbohydrate metabolic operon (frz) that is highly associated with extraintestinal pathogenic Escherichia coli (ExPEC) strains. The frz operon codes for three subunits of a phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) transporter of the fructose subfamily, for a transcriptional activator of PTSs of the MgA family, for two type II ketose-1,6-bisphosphate aldolases, for a sugar-specific kinase (repressor, open reading frame, kinase family [ROK]), and for a protein of the cupin superfamily. We proved that the frz operon promotes bacterial fitness under stressful conditions, such as oxygen restriction, late stationary phase of growth, or growth in serum or in the intestinal tract. Furthermore, we showed that frz is involved in adherence to and internalization in human type II pneumocytes, human enterocytes, and chicken liver cells by favoring the ON orientation of the fim operon promoter and thus acting on the expression of type 1 fimbriae, which are the major ExPEC adhesins. Both the PTS activator and the metabolic enzymes encoded by the frz operon are involved in these phenotypes.

Broiler lines divergently selected for digestive efficiency also differ in their susceptibility to colibacillosis

Increasing feed efficiency of broiler chickens by selective breeding could lead to decreased feed cost and reduced environmental impact of poultry production. At INRA, two broiler chicken lines (D+/D−) were divergently selected for their digestive efficiency. Strong differences were shown between both lines for the anatomy and histology of the digestive tract, and for the intestinal microbiota composition. In the present study, we investigated whether this selection also had an effect on susceptibility to colibacillosis, which is one of the main causes of economic losses in poultry production. The broiler lines D+/D− were challenged with an avian pathogenic Escherichia coli strain. A first experiment was conducted to assess the 50% lethal dose by subcutaneous infection of hatchlings, whereas a second experiment reproduced colibacillosis by infecting air sacs of 23-day-old chicks. The 50% lethal dose was very low for both lines. However, the line with the higher digestive efficiency (D+) was the less susceptible to colibacillosis. This result is interesting for selection purposes and opens the way to integrative genetic studies of the interactions between digestion efficiency and resistance to colibacillosis.

Unveiling the participation of avian kinin ornithokinin and its receptors in the chicken inflammatory response

Vasoactive peptides are key early mediators of inflammation released through activation of different enzymatic systems. The mammalian kinin-kallikrein (K-KLK) system produces bradykinin (BK) through proteolytic cleavage of a kininogen precursor by enzymes named kallikreins. BK acts through specific ubiquitous G-protein coupled receptors (B1R and B2R) to participate in physiological processes and inflammatory responses, such as activation of mononuclear phagocytes. In chickens, the BK-like nonapeptide ornithokinin (OK) has been shown to promote intracellular calcium increase in embryonic fibroblasts and to be vasodilatory in vivo. Also, one of its receptors (B2R) was already cloned. However, the participation of chicken K-KLK system components in the inflammatory response remains unknown and was therefore investigated. We first showed that B1R, B2R and kininogen 1 (KNG1) are expressed in unstimulated chicken tissues and macrophages. We next showed that chicken B1R and B2R are expressed at transcript and protein levels in chicken macrophages and are upregulated by E. coli LPS or avian pathogenic E. coli (APEC) infection. Interestingly, exogenous OK induced internalization and degradation of OK receptors protein, notably B2R. Also, OK induced intracellular calcium increase and potentiated zymosan-induced ROS production and Dextran-FITC endocytosis by chicken macrophages. Exogenous OK itself did not promote APEC killing and had no pro-inflammatory effect. However, when combined with LPS or APEC, OK upregulated cytokine/chemokine gene expression and NO production by chicken macrophages. This effect was not blocked by canonical non-peptide B1R or B2R receptor antagonists but was GPCR- and PI3K/Akt-dependent. In vivo, pulmonary colibacillosis led to upregulation of OK receptors expression in chicken lungs and liver. Also, colibacillosis led to significant upregulation of OK precursor KNG1 expression in liver and in cultured hepatocytes (LMH). We therefore provide hitherto unknown information on how OK and its receptors are involved in inflammation and infection in chickens.

The role of type I interferons (IFNs) in the regulation of chicken macrophage inflammatory response to bacterial challenge

Abstract Mammalian type I interferons (IFN&agr;/&bgr;) are known to modulate inflammatory processes in addition to their antiviral properties. Indeed, virus‐induced type I interferons regulate the mammalian phagocyte immune response to bacteria during superinfections. However, it remains unresolved whether type I IFNs similarly impact the chicken macrophage immune response. We first evidenced that IFN&agr; and IFN&bgr; act differently in terms of gene expression stimulation and activation of intracellular signaling pathways in chicken macrophages. Next, we showed that priming of chicken macrophages with IFN&agr; increased bacteria uptake, boosted bacterial‐induced ROS/NO production and led to an increased transcriptional expression or production of NOS2/NO, IL1B/IL‐1&bgr; and notably IFNB/IFN&bgr;. Neutralization of IFN&bgr; during bacterial challenge limited IFN&agr;‐induced augmentation of the pro‐inflammatory response. In conclusion, we demonstrated that type I IFNs differently regulate chicken macrophage functions and drive a pro‐inflammatory response to bacterial challenge. These findings shed light on the diverse functions of type I IFNs in chicken macrophages. HighlightsType I IFNs differently regulate intracellular events in chicken macrophages.IFN&agr; priming boosts the macrophage inflammatory response to bacterial challenge.This boost in the inflammatory response is mediated by IFN&bgr;Bacterial uptake is increased if chicken macrophages are primed with IFN&agr;.

Complete Genome Sequences of Two Escherichia coli Phages, vB_EcoM_ ESCO5 and vB_EcoM_ESCO13, Which Are Related to phAPEC8

ABSTRACT We report here the complete genome sequences of two Myoviridae phages that infect various avian-pathogenic Escherichia coli strains and that are closely related to phage phAPEC8.

Characterization of the Phospholipid Platelet-Activating Factor As a Mediator of Inflammation in Chickens

Lipid mediators are known to play important roles in the onset and resolution phases of the inflammatory response in mammals. The phospholipid platelet-activating factor (PAF) is a pro-inflammatory lipid mediator which participates in vascular- and innate immunity-associated processes by increasing vascular permeability, by facilitating leukocyte adhesion to the endothelium, and by contributing to phagocyte activation. PAF exerts its function upon binding to its specific receptor, PAF receptor (PAFR), which is abundantly expressed in leukocytes and endothelial cells (ECs). In chickens, lipid mediators and their functions are still poorly characterized, and the role of PAF as an inflammatory mediator has not yet been investigated. In the present study we demonstrate that primary chicken macrophages express PAFR and lysophosphatidylcholine acyltransferase 2 (LPCAT2), the latter being essential to PAF biosynthesis during inflammation. Also, exogenous PAF treatment induces intracellular calcium increase, reactive oxygen species release, and increased phagocytosis by primary chicken macrophages in a PAFR-dependent manner. We also show that PAF contributes to the Escherichia coli lipopolysaccharide (LPS)-induced pro-inflammatory response and boosts the macrophage response to E. coli LPS via phosphatidylinositol 3-kinase/Akt- and calmodulin kinase II-mediated intracellular signaling pathways. Exogenous PAF treatment also increases avian pathogenic E. coli intracellular killing by chicken macrophages, and PAFR and LPCAT2 are upregulated in chicken lungs and liver during experimental pulmonary colibacillosis. Finally, exogenous PAF treatment increases cell permeability and upregulates the expression of genes coding for proteins involved in leukocyte adhesion to the endothelium in primary chicken endothelial cells (chAEC). In addition to these vascular phenomena, PAF boosts the chAEC inflammatory response to bacteria-associated molecular patterns in a PAFR-dependent manner. In conclusion, we identified PAF as an inflammation amplifier in chicken macrophages and ECs, which suggests that PAF could play important roles in the endothelium-innate immunity interface in birds during major bacterial infectious diseases such as colibacillosis.