Search

SUMMARY Pentameric ligand-gated ion-channels rapidly transduce synaptic neurotransmitter signals to an electrical response in post-synaptic neuronal or muscle cells and control the neuromusculature of a majority of multicellular animals. A wide range of pharmaceuticals target these receptors including ethanol, nicotine, anti-depressants and other mood regulating drugs, compounds that control pain and mobility and are targeted by a majority of anthelmintic drugs used to control parasitic infection of humans and livestock. Major advances have been made in recent years to our understanding of the structure, function, activity and the profile of compounds that can activate specific receptors. It is becoming clear that these anthelmintic drug targets are not fixed, but differ in significant details from one nematode species to another. Here we review what is known about the evolution of the pentameric ligand-gated ion-channels, paying particular attention to the nematodes, how we can infer the origins of such receptors and understand the factors that determine how they change both over time and from one species to another. Using this knowledge provides a biological framework in which to understand these important drug targets and avenues to identify new receptors and aid the search for new anthelmintic drugs.

Chlamydia (C.) pecorum, an obligate intracellular bacterium, may cause severe diseases in ruminants, swine and koalas, although asymptomatic infections are the norm. Recently, we identified genetic polymorphisms in the ompA, incA and ORF663 genes that potentially differentiate between high-virulence C. pecorum isolates from diseased animals and low-virulence isolates from asymptomatic animals. Here, we expand these findings by including additional ruminant, swine, and koala strains. Coding tandem repeats (CTRs) at the incA locus encoded a variable number of repeats of APA or AGA amino acid motifs. Addition of any non-APA/AGA repeat motif, such as APEVPA, APAVPA, APE, or APAPE, associated with low virulence (P<10-4), as did a high number of amino acids in all incA CTRs (P = 0.0028). In ORF663, high numbers of 15-mer CTRs correlated with low virulence (P = 0.0001). Correction for ompA phylogram position in ORF663 and incA abolished the correlation between genetic changes and virulence, demonstrating co-evolution of ompA, incA, and ORF663 towards low virulence. Pairwise divergence of ompA, incA, and ORF663 among isolates from healthy animals was significantly higher than among strains isolated from diseased animals (P≤10-5), confirming the longer evolutionary path traversed by low-virulence strains. All three markers combined identified 43 unique strains and 4 pairs of identical strains among all 57 isolates tested, demonstrating the suitability of these markers for epidemiological investigations.

Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement and are targets of important "classical" anthelmintics like levamisole and pyrantel, as well as "novel" anthelmintics like tribendimidine and derquantel. Four biophysical subtypes of nAChR have been observed electrophysiologically in body muscle of the nematode parasite Oesophagostomum dentatum, but their molecular basis was not understood. Additionally, loss of one of these subtypes (G 35 pS) was found to be associated with levamisole resistance. In the present study, we identified and expressed in Xenopus oocytes, four O. dentatum nAChR subunit genes, Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, to explore the origin of the receptor diversity. When different combinations of subunits were injected in Xenopus oocytes, we reconstituted and characterized four pharmacologically different types of nAChRs with different sensitivities to the cholinergic anthelmintics. Moreover, we demonstrate that the receptor diversity may be affected by the stoichiometric arrangement of the subunits. We show, for the first time, different combinations of subunits from a parasitic nematode that make up receptors sensitive to tribendimidine and derquantel. In addition, we report that the recombinant levamisole-sensitive receptor made up of Ode-UNC-29, Ode-UNC-63, Ode-UNC-38 and Ode-ACR-8 subunits has the same single-channel conductance, 35 pS and 2.4 ms mean open-time properties, as the levamisole-AChR (G35) subtype previously identified in vivo. These data highlight the flexible arrangements of the receptor subunits and their effects on sensitivity and resistance to the cholinergic anthelmintics; pyrantel, tribendimidine and/or derquantel may still be effective on levamisole-resistant worms.

In order to improve the identification of avian pathogenic Escherichia coli (APEC) strains, an extensive characterization of 1,491 E. coli isolates was conducted, based on serotyping, virulence genotyping, and experimental pathogenicity for chickens. The isolates originated from lesions of avian colibacillosis (n = 1,307) or from the intestines of healthy animals (n = 184) from France, Spain, and Belgium. A subset (460 isolates) of this collection was defined according to their virulence for chicks. Six serogroups (O1, O2, O5, O8, O18, and O78) accounted for 56.5% of the APEC isolates and 22.5% of the nonpathogenic isolates. Thirteen virulence genes were more frequently present in APEC isolates than in nonpathogenic isolates but, individually, none of them could allow the identification of an isolate as an APEC strain. In order to take into account the diversity of APEC strains, a statistical analysis based on a tree-modeling method was therefore conducted on the sample of 460 pathogenic and nonpathogenic isolates. This resulted in the identification of four different associations of virulence genes that enables the identification of 70.2% of the pathogenic strains. Pathogenic strains were identified with an error margin of 4.3%. The reliability of the link between these four virulence patterns and pathogenicity for chickens was validated on a sample of 395 E. coli isolates from the collection. The genotyping method described here allowed the identification of more APEC isolates with greater reliability than the classical serotyping methods currently used in veterinary laboratories.

The Salmonella outer membrane protein Rck mediates a Zipper-like entry mechanism controlled by Rac, the Arp2/3 complex, and actin polymerization. However, little is known about the early steps leading to Rac activation and Rck-mediated internalization. The use of pharmacological inhibitors or PI 3-kinase dominant-negative mutant induced more than 80% less invasion without affecting attachment. Moreover, Rck-mediated internalization caused an increase in the association of p85 with at least one tyrosine-phosphorylated protein, indicating that class I PI 3-kinase activity was stimulated. We also report that this PI 3-kinase activity is essential for Rac1 activation. However, Rac recruitment at the Rck-mediated entry site was independent of its activation. Using a pharmacological approach or Akt-knockout cells, we also demonstrated that Akt was phosphorylated in response to Rck-mediated internalization as demonstrated by immunoblotting analysis and that all three Akt isoforms were required during this process. Overall, our results describe a signaling pathway involving tyrosine phosphorylation, class I PI 3-kinase, Akt activation, and Rac activation, leading to Rck-dependent Zipper entry. The specificity of this signaling pathway with regard to that of the type 3 secretion system, which is the other invasion process of Salmonella, is discussed.

The transcriptional activator RamA is involved in multidrug resistance (MDR) by increasing expression of the AcrAB-TolC RND-type efflux system in several pathogenic Enterobacteriaceae. In Salmonella enterica serovar Typhimurium (S. Typhimurium), ramA expression is negatively regulated at the local level by RamR, a transcriptional repressor of the TetR family. We here studied the DNA-binding activity of the RamR repressor with the ramA promoter (P(ramA)). As determined by high-resolution footprinting, the 28-bp-long RamR binding site covers essential features of P(ramA), including the -10 conserved region, the transcriptional start site of ramA, and two 7-bp inverted repeats. Based on the RamR footprint and on electrophoretic mobility shift assays (EMSAs), we propose that RamR interacts with P(ramA) as a dimer of dimers, in a fashion that is structurally similar to the QacR-DNA binding model. Surface plasmon resonance (SPR) measurements indicated that RamR has a 3-fold-lower affinity (K(D) [equilibrium dissociation constant] = 191 nM) for the 2-bp-deleted P(ramA) of an MDR S. Typhimurium clinical isolate than for the wild-type P(ramA) (K(D) = 66 nM). These results confirm the direct regulatory role of RamR in the repression of ramA transcription and precisely define how an alteration of its binding site can give rise to an MDR phenotype.

Dendritic cells (DCs) are well known as professional antigen-presenting cells (APC) able to initiate specific T-cell responses to pathogens in lymph nodes (LN) draining the site of infection. However, the respective contribution of migratory and LN-resident DCs in this process remains unclear. As DC subsets represent important targets for vaccination strategies, more precise knowledge of DC subsets able to present vaccine antigens to T cells efficiently is required. To investigate the capacities of DCs migrating in the lymph (L-DCs) to initiate a specific T-cell response, we used physiologically generated DCs collected from a pseudoafferent lymphatic cannulation model in sheep. The CD1b+ L-DCs were assessed for presenting antigens from the vaccine attenuated strain of Salmonella enterica serovar Abortusovis. CD1b+ L-DCs were able to phagocytose, process and to present efficiently Salmonella antigens to effector/memory T cells in vitro. They were shown to be efficient APC for the priming of allogeneic naive T cells associated with inducing both IFN-γ and IL-4 responses. They were also efficient in presenting Salmonella antigens to autologous naive T cells associated with inducing both IFN-γ and IL-10 responses. The capacities of L-DCs to process and present Salmonella antigens to T cells were investigated in vivo after conjunctival inoculation of Salmonella. The CD1b+ L-DCs collected after inoculation were able to induce the proliferative response of CD4+ T cells suggesting the in vivo capture of Salmonella antigens by the CD1b+ L-DCs, and their potential to present them directly to CD4+ T cells. In this study, CD1b+ L-DCs present potential characteristics of APC to initiate by themselves T cell priming in the LN. They could be used as target cells for driving immune activation in vaccinal strategies.

A deletion of ∼20 amino acids in the stalk of neuraminidase is frequently observed upon transmission of influenza A viruses from waterfowl to domestic poultry. A pair of recombinant H7N1 viruses bearing either a short- or long-stalk neuraminidase was genetically engineered. Inoculation of the long-stalk-neuraminidase virus resulted in a higher cloacal excretion in ducks and led conversely to lower-level oropharyngeal excretion in chickens, associated with a higher-level local immune response and better survival. Therefore, a short-stalk neuraminidase is a determinant of viral adaptation and virulence in chickens but is detrimental to virus replication and shedding in ducks.

Salmonella enterica serotype Senftenberg (S. Senftenberg) has recently become more frequent in poultry flocks. Moreover some strains have been implicated in severe clinical cases. To explain the causes of this emergence in farm animals, 134 S. Senftenberg isolates from hatcheries, poultry farms and human clinical cases were analyzed. Persistent and non-persistent strains were identified in chicks. The non-persistent strains disappeared from ceca a few weeks post inoculation. This lack of persistence could be related to the disappearance of this serotype from poultry farms in the past. In contrast, persistent S. Senftenberg strains induced an intestinal asymptomatic carrier state in chicks similar to S. Enteritidis, but a weaker systemic infection than S. Enteritidis in chicks and mice. An in vitro analysis showed that the low infectivity of S. Senftenberg is in part related to its low capacity to invade enterocytes and thus to translocate the intestinal barrier. The higher capacity of persistent than non-persistent strains to colonize and persist in the ceca of chickens could explain the increased persistence of S. Senftenberg in poultry flocks. This trait might thus present a human health risk as these bacteria could be present in animals before slaughter and during food processing.

The Suppressor Of Cytokine Signaling (SOCS) proteins are key physiological regulators of the immune system. Little is known about tissue expression of SOCS and data in pigs are extremely scarce. In order to further study SOCS in pigs, preliminary data must be collected. In the current report, we first identified the three most suitable reference genes in ten porcine tissues. The beta-2-microglobulin (B2MI) reference gene was most often particularly suitable in our conditions. Then, using three reference genes we determined the mRNA expression of SOCS1-7 and CIS in every selected tissue. Constitutive mRNA expression was identified for all the members of the SOCS family in the ten tissues. Interestingly, the constitutive mRNA expression of SOCS1, SOCS3, SOCS7 and CIS was rather heterogeneous between tissues while for SOCS2, SOCS4, SOCS5 and SOCS6 differences of expression were less obvious. Highest CIS and SOCS mRNA expressions were observed in large intestine (SOCS1, SOCS3, SOCS4, SOCS6, and CIS), small intestine (SOCS1, SOCS4, SOCS6, and CIS), spleen (SOCS2, SOCS3, SOCS5, SOCS7, and CIS), trachea (SOCS3) and thymus (SOCS1, SOCS2, SOCS4, SOCS7, and CIS). These data will help for further studies about the role of SOCS proteins in the control of porcine innate and adaptive responses., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Interleukin-17A (IL-17A) and IL-17F have been shown to mediate a crucial crosstalk between the immune system and various epithelial tissues, stimulating various defensive mechanisms to bacterial infections. A number of studies have characterized the response to IL-17A and IL-17F of epithelial cells from airways, intestine, and skin, but not from the mammary gland. To evaluate the potential contribution of IL-17 to the immune defense of the mammary gland, we analyzed the effects of recombinant bovine IL-17A and IL-17F on primary bovine mammary epithelial cells (MEC) by quantitative PCR and ELISA. We found expression (mRNA) of the two components of the IL-17 receptor complex, IL-17RA and IL-17RC, in mammary tissue and MEC in vitro. The expression of a number of genes encoding cytokines, chemokines and proteins endowed with antibacterial activities was increased by IL-17A, and to a lesser extent by IL-17F, but the magnitude of responses was modest. As expected, responses were augmented by the combination of IL-17A or IL-17F with TNF-α. Interestingly, responses of a few of the tested genes, such as IL8, CCL20, iNOS, and CfB, were augmented by the combination of IL-17A with staphylococcal lipoteichoic acid or muramyl dipeptide, bacterial agonists of the innate immune system. This can be interpreted as indicating that IL-17A and IL-17F are tailored to exert their full potential in a septic environment. MEC responses were characterized by the expression of chemokines targeting not only neutrophils (CXCL3 and CXCL8) but also mononuclear leucocytes (CCL2, CCL20). Production of IL-6 was low and the inflammatory cytokines TNF-α and IL-1β were expressed (mRNA) but proteins were not secreted. Altogether, our results suggest that IL-17A and IL-17F have a potential to modulate the mammary gland immune response to mastitis-causing pathogens., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

Cotyledons (n=170) from dairy cattle were analysed for Coxiella burnetii by real-time (rt) PCR targeting the IS1111a and icd genes. Positive cases (n=90) and a random selection of negative cases (n=20) were examined by histology, immunohistochemistry and, if infection level was high, by fluorescence in situ hybridisation. PCR results were compared to bulk tank milk (BTM) antibody levels. Placental infection was detected in cows from herds at all BTM antibody levels. However the likelihood of placental infection was generally higher in herds with intermediate or high BMT antibody levels than in herds with low antibody levels. Histological examination revealed a range of mostly mild cotyledonary changes; C. burnetii infection was only rarely associated with inflammation. This may explain why bovine Q fever is usually not clinically apparent. Nevertheless, infected cattle will shed C. burnetii at calving and this can occur even in herds without BTM antibodies., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Toll-like receptor 4 (TLR4), which recognizes lipopolysaccharide from Gram-negative bacteria, plays a major role in resistance of mice and humans to Salmonella infection. In chickens, Salmonella may establish a carrier state whereby bacteria are able to persist in the host organism without triggering clinical signs. Based on cellular morphological parameters, we developed a method, without using antibodies, to separate three cecal cell subpopulations: lymphocytes, enterocytes, and a population encompassing multiple cell types. We analyzed the mRNA expression of TLR4, interleukin-1β (IL-1β), IL-8, IL-12, and lipopolysaccharide-induced tumor necrosis factor alpha factor (LITAF) in cecal subpopulations of chicks from inbred lines resistant or susceptible to the carrier state infected with Salmonella enterica serovar Enteritidis. The results showed that resistance to the carrier state in chicks is associated with a larger percentage of lymphocytes and with higher levels of expression of TLR4 and IL-8 at homeostasis in the three cell subpopulations, as well as with a higher level of expression of LITAF in lymphocytes during the carrier state. In contrast to the early phase of infection, the carrier state is characterized by no major cell recruitment differences between infected and noninfected animals and no significant modification in terms of TLR4, IL-1β, IL-8, IL-12, and LITAF expression in all cell subpopulations measured. However, TLR4 expression increased in the lymphocytes of chicks from the susceptible line, reaching the same level as that in infected chicks from the resistant line. These observations suggest that the carrier state is characterized by a lack of immune activation and highlight the interest of working at the level of the cell population rather than that of the organ.

A gene cluster involved in the metabolism of prebiotic short-chain fructooligosaccharides (scFOS) has recently been identified in the extra-intestinal avian pathogenic Escherichia coli strain BEN2908. This gene cluster, called the fos locus, plays a major role in the initiation stage of chicken intestinal colonization. This locus is composed of six genes organized as an operon encoding a sugar transporter and enzymes involved in scFOS metabolism, and of a divergently transcribed gene encoding a transcriptional regulator, FosR, belonging to the LacI/GalR family. To decipher the regulation of scFOS metabolism, we monitored the fos operon promoter activity using a luciferase reporter gene assay. We demonstrated that the expression of fos genes is repressed by FosR, controlled by catabolite repression and induced in the presence of scFOS. Using electrophoretic mobility shift assays and surface plasmon resonance experiments, we showed that FosR binds to two operator sequences of the fos operon promoter region. This binding to DNA was inhibited in the presence of scFOS, especially by GF2. We then propose a model of scFOS metabolism regulation in a pathogenic bacterium, which will help to identify the environmental conditions required for fos gene expression and to understand the role of this locus in intestinal colonization., (© 2011 Blackwell Publishing Ltd.)

Avian influenza virus (AIV) infections of the chicken occur via the respiratory route. Unlike ducks which are considered as a natural AIV reservoir, chickens are highly susceptible to AIV infections and do not possess the RIG-I pattern recognition receptor involved in triggering the antiviral interferon response. To study the chicken innate immune response to AIV in the respiratory tract, we established an epithelial cell line (CLEC213) from lung explants of white leghorn chickens. CLEC213 cells exhibited a polyhedral morphology and formed cohesive clusters bound through tight junctions as assessed by electron microscopy. Expression of E-cadherin but not vimentin could be detected as expected for cells of epithelial origin. In addition, CLEC213 cells showed characteristics similar to those of mammalian type II pneumocytes, including the presence of intracytoplasmic vacuoles filled with a mucopolysaccharide material, alkaline phosphatase activity, transcription of chicken lung collectins genes (cLL and SPA), and some intracytoplasmic lamellar-like bodies. CLEC213 cells showed a constitutive expression level of TLR3 and TLR4 and were responsive to stimulation with the respective agonists, poly (I:C) and LPS: between 4h and 24h after treatment, a strong increase in the expression of IFN-α, IFN-β and IL-8 genes could be detected. Furthermore, CLEC213 cells supported efficient growth of the low pathogenicity avian influenza virus H6N2 (A/duck/France/05057a/2005) in the presence or the absence of trypsin in the culture media. At 4h post-infection, the H6N2 virus induced highly elevated levels of expression of IFN-α and IL-8, moderately elevated levels of LITAF, TGF-β4 and CCL5. However, an increase of IFN-β gene expression could not be detected in response to AIV infection. In conclusion, like mammalian type II pneumocytes, CLEC213 are able to mount a robust cytokine and chemokine immune response to microbial patterns and viral infection. We hypothesize that they could derive from lung atrial granular cells. The involvement of such type of lung epithelial cells in the respiratory tract defence of the chicken can thus be further studied., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Probiotic yeasts may provide protection against intestinal inflammation induced by enteric pathogens. In piglets, infection with F4+ enterotoxigenic Escherichia coli (ETEC) leads to inflammation, diarrhea and intestinal damage. In this study, we investigated whether the yeast strains Saccharomyces cerevisiae (Sc, strain CNCM I-3856) and S. cerevisiae variety boulardii (Sb, strain CNCM I-3799) decreased the expression of pro-inflammatory cytokines and chemokines in intestinal epithelial IPI-2I cells cultured with F4+ ETEC. Results showed that viable Sc inhibited the ETEC-induced TNF-α gene expression whereas Sb did not. In contrast, killed Sc failed to inhibit the expression of pro-inflammatory genes. This inhibition was dependent on secreted soluble factors. Sc culture supernatant decreased the TNF-α, IL-1α, IL-6, IL-8, CXCL2 and CCL20 ETEC-induced mRNA. Furthermore, Sc culture supernatant filtrated fraction < 10 kDa displayed the same effects excepted for TNF-α. Thus, our results extended to Sc (strain CNCM I-3856) the inhibitory effects of some probiotic yeast strains onto inflammation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Mammary epithelial cells (MEC) participate in the first line of defense of the mammary gland to invading pathogens. In vitro culture of MEC is widely used as a model to study the capacity of these cells to sense and respond to mastitis-causing bacteria. Analysis of gene expression by quantitative PCR (qPCR) following exposure to bacteria or bacterial constituents is a powerful tool to assess responses of MEC to pathogens. Although internal standards such as reference genes are required for qPCR to yield valid data, the validation of proper genes to quantify mRNA transcripts in MEC exposed to pro-inflammatory stimuli has never been reported. In this study, 10 commonly used reference genes belonging to different functional classes (ACTB, ATP5B, EIF2B2, GAPDH, PPIA, SDHA, SUZ12, UXT, YWHAZ, and 18s rRNA) were analyzed by qPCR to determine the most stable in bovine MEC unstimulated and stimulated with mastitis pathogens (Staphylococcus aureus or Escherichia coli), microbial agonists of the innate immune system (lipoteichoic acid and muramyl dipeptide, or lipopolysaccharide), or proinflammatory cytokines (IL-17A and tumor necrosis factor-α). An M value was used as a measure of gene stability as determined using the geNorm application. This study demonstrated that the expression of the 10 reference genes was stable under the different experimental conditions. These data will be useful for bovine mastitis research in selecting reference genes and validating reverse transcription-qPCR data., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Salmonella causes a wide range of diseases from acute gastroenteritis to systemic typhoid fever, depending on the host. To invade non-phagocytic cells, Salmonella has developed different mechanisms. The main invasion system requires a type III secretion system (T3SS) known as T3SS-1, which promotes a Trigger entry mechanism. However, other invasion factors have recently been described in Salmonella, including Rck and PagN, which were not expressed under our bacterial culture conditions. Based on these observations, we used adhesion and invasion assays to analyse the respective roles of Salmonella Enteritidis T3SS-1-dependent and -independent invasion processes at different times of infection. Diverse cell lines and cell types were tested, including endothelial, epithelial and fibroblast cells. We demonstrated that cell susceptibility to the T3SS-1-independent entry differs by a factor of nine between the most and the least permissive cell lines tested. In addition, using scanning electron and confocal microscopy, we showed that T3SS-1-independent entry into cells was characterized by a Trigger-like alteration, as for the T3SS-1-dependent entry, and also by Zipper-like cellular alteration. Our results demonstrate for what is believed to be the first time that Salmonella can induce Trigger-like entry independently of T3SS-1 and can induce Zipper-like entry independently of Rck. Overall, these data open new avenues for discovering new invasion mechanisms in Salmonella.

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.

Extra-intestinal pathogenic Escherichia coli (ExPEC) strains are responsible for a wide range of diseases in humans and animals. Using in vitro invasion assays and transmission electron microscopy, we showed that BEN2908, an ExPEC strain of avian origin (also termed APEC for Avian Pathogenic E. coli), is able to usurp cellular endocytic pathways to invade A549 human type II pneumocytes and LMH avian hepatocytes where it is able to survive over several days. Although type 1 fimbriae are the major adhesin of BEN2908, proportions of adherent fimbriated or afimbriated bacteria that entered cells were comparable. Internalization of BEN2908 into human pneumocytes reinforces previous studies indicating that APEC strains could represent a zoonotic risk., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: The Salmonella genomic island 1 (SGI1) is a Salmonella enterica-derived integrative mobilizable element (IME) containing various complex multiple resistance integrons identified in several S. enterica serovars and in Proteus mirabilis. Previous studies have shown that SGI1 transfers horizontally by in trans mobilization in the presence of the IncA/C conjugative helper plasmid pR55., Methodology/principal Findings: Here, we report the ability of different prevalent multidrug resistance (MDR) plasmids including extended-spectrum β-lactamase (ESBL) gene-carrying plasmids to mobilize the multidrug resistance genomic island SGI1. Through conjugation experiments, none of the 24 conjugative plasmids tested of the IncFI, FII, HI2, I1, L/M, N, P incompatibility groups were able to mobilize SGI1 at a detectable level (transfer frequency <10(-9)). In our collection, ESBL gene-carrying plasmids were mainly from the IncHI2 and I1 groups and thus were unable to mobilize SGI1. However, the horizontal transfer of SGI1 was shown to be specifically mediated by conjugative helper plasmids of the broad-host-range IncA/C incompatibility group. Several conjugative IncA/C MDR plasmids as well as the sequenced IncA/C reference plasmid pRA1 of 143,963 bp were shown to mobilize in trans SGI1 from a S. enterica donor to the Escherichia coli recipient strain. Depending on the IncA/C plasmid used, the conjugative transfer of SGI1 occurred at frequencies ranging from 10(-3) to 10(-6) transconjugants per donor. Of particular concern, some large IncA/C MDR plasmids carrying the extended-spectrum cephalosporinase bla(CMY-2) gene were shown to mobilize in trans SGI1., Conclusions/significance: The ability of the IncA/C MDR plasmid family to mobilize SGI1 could contribute to its spread by horizontal transfer among enteric pathogens. Moreover, the increasing prevalence of IncA/C plasmids in MDR S. enterica isolates worldwide has potential implications for the epidemic success of the antibiotic resistance genomic island SGI1 and its close derivatives.

The genus Listeria consists of eight species but only two are pathogenic. Human listeriosis due to Listeria monocytogenes is a foodborne disease. L. monocytogenes is widespread in the environment living as a saprophyte, but is also capable of making the transition into a pathogen following its ingestion by susceptible humans or animals. It is now known that many distinct strains of L. monocytogenes differ in their virulence and epidemic potential. Unfortunately, there is currently no standard definition of virulence levels and no complete comprehensive overview of the evolution of Listeria species and L. monocytogenes strains taking into account the presence of both epidemic and low-virulence strains. This article focuses on the methods and genes allowing us to determine the pathogenic potential of Listeria strains, and the evolution of Listeria virulence. The presence of variable levels of virulence within L. monocytogenes has important consequences on detection of Listeria strains and risk analysis but also on our comprehension of how certain pathogens will behave in a population over evolutionary time.

The range of leucocytes susceptible to the leucotoxin LukM/F', a two-component pore-forming toxin of Staphylococcus aureus causing mastitis in ruminants, had not been defined. We used fluorescent-labeled LukM to investigate the binding of this toxin to bovine cells and to identify its cellular targets among bovine, human and murine leucocytes. LukM bound to bovine blood neutrophils from all the individuals tested with similar affinity, with an apparent dissociation constant (Kd) of 1.81 ± 0.14 nM and 13 3100 ± 506 binding sites. The amount of LukM bound to bovine neutrophils did not depend on the presence of the complementary component LukF', suggesting that the binding of LukM to its ligand does not depend on the formation of pore-forming oligomers, and that the number of bound LukM molecules corresponds to the number of available cell membrane ligands. Other staphylococcal class S components of bipartite leucotoxins (HlgA, HlgC, LukE, LukS-PV) were inefficient competitors of LukM for the binding to bovine neutrophils, indicating that LukM has a distinct ligand on target cells. Bovine blood neutrophils bound slightly more LukM than did milk neutrophils, and much more than did ovine and caprine blood neutrophils. Bovine monocytes and milk macrophages readily bound LukM, whereas blood lymphocytes did not. Human neutrophils bound little LukM and were resistant to LukM/F' at the highest tested concentration (40 nM). Murine neutrophils bound LukM and were susceptible to the toxicity of LukM/F', exhibiting flattening and nucleus alteration beginning at 0.3 nM concentration. Among murine peritoneal exudate cells, T lymphocytes (CD3+) and monocytes/macrophages (F4/80+) bound LukM, whereas binding to B lymphocytes (CD19+) was not detected. These results indicate that cells of the myeloid lineage are the main targets of LukM/F' in dairy ruminants, and that resident or inflammatory migrated phagocytes are susceptible to this toxin., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The control of gastro-intestinal nematodes remains largely based on anthelminthic treatments, however spreading of anthelmintic resistance has reduced their efficacy. The genes involved in the transition to parasitic lifestyle could constitute targets of interest to develop alternative control strategies. In the trichostrongylid nematode Haemonchus contortus, we have used a SSH (Suppressive Subtractive Hybridization) based approach to generate two distinct subtracted cDNA libraries specifically enriched in cDNA expressed during the early parasitic fourth stage larvae L4 (5 days post-infection). A total of 200 clones were subjected to dot-blot experiments and 46 clones were selected for further characterization. The 46 corresponding expressed sequence tags (EST) were found to cluster into nine contigs. The corresponding full-length cDNA was obtained for all candidates. The genes encoding potentially secreted proteins were investigated in more detail. RT-PCR experiments confirmed their specific expression or over expression from the early L4 larvae to the adult stages and search for homologs in the trichostrongylid species T. circumcincta was performed in order to investigate whether they may be novel cross-specific targets., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Staphylococcus aureus, a major pathogen for the mammary gland of dairy ruminants, elicits the recruitment of neutrophils into milk during mastitis, but the mechanisms are incompletely understood. We investigated the response of the bovine mammary gland to muramyl dipeptide (MDP), an elementary constituent of the bacterial peptidoglycan, alone or in combination with lipoteichoic acid (LTA), another staphylococcal microbial-associated molecular pattern (MAMP). MDP induced a prompt and marked influx of neutrophils in milk, and its combination with LTA elicited a more intense and prolonged influx than the responses to either stimulus alone. The concentrations of several chemoattractants for neutrophils (CXCL1, CXCL2, CXCL3, CXCL8, and C5a) increased in milk after challenge, and the highest increases followed challenge with the combination of MDP and LTA. MDP and LTA were also synergistic in inducing in vitro chemokine production by bovine mammary epithelial cells (bMEpC). Nucleotide-binding oligomerization domain 2 (NOD2), a major sensor of MDP, was expressed (mRNA) in bovine mammary tissue and by bMEpC in culture. The production of interleukin-8 (IL-8) following the stimulation of bMEpC by LTA and MDP was dependent on the activation of NF-κB. LTA-induced IL-8 production did not depend on platelet-activating factor receptor (PAFR), as the PAFR antagonist WEB2086 was without effect. In contrast, bMEpC and mammary tissue are known to express Toll-like receptor 2 (TLR2) and to respond to TLR2 agonists. Although the levels of expression of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1β were increased by LTA and MDP at the mRNA level, no protein could be detected in the bMEpC culture supernatant. The level of induction of IL-6 was low at both the mRNA and protein levels. These results indicate that MDP and LTA exert synergistic effects to induce neutrophilic inflammation in the mammary gland. These results also show that bMEpC could contribute to the inflammatory response by recognizing LTA and MDP and secreting chemokines but not proinflammatory cytokines. Overall, this study indicates that the TLR2 and NOD2 pathways could cooperate to trigger an innate immune response to S. aureus mastitis.

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.

In pigs raised for meat production, weaning is a critical period because of related physiological perturbations and negative consequences on performance. Previous studies have shown that early weaning could either impair development of mucosal barrier function or boost intestinal immunologic parameters. In order to obtain further knowledge about the impact of ultra-early weaning on the porcine immune system development, three groups of piglets were weaned at different ages and compared to the unweaned control group. Lower IgA concentrations in ultra-early and early weaned piglets than in other piglets were identified in serum. In the mesenteric lymph node (MLN), significant differences in the mRNA expression of IL17a, TGF beta and FOXP3 were found between specific groups. Indeed, IL17a mRNA was mainly detected in ultra-early weaned piglets while FOXP3 and TGF beta mRNA were associated to both ultra-early weaned and suckling piglets. Reduced serum IgA concentration and MLN induction of a Th17 cytokine in ultra-early weaned piglets could be related to alterations of the mucosal barrier functions consecutive to the milk deprivation. All together, our findings suggest a crucial role for endogenous milk factors onto the onset of IgA synthesis., ((c) 2010 Elsevier B.V. All rights reserved.)

We report the dissemination of a conjugative IncI1 plasmid carrying bla(CTX-M-1), conferring resistance to extended-spectrum cephalosporins, in Salmonella enterica isolates from poultry and humans in France from 2003 to 2008. By IncI1 plasmid subtyping, this plasmid was shown to be genetically related to that found in Escherichia coli isolates from healthy poultry in France.

Chlamydophila pecorum strains are commonly found in the intestine and vaginal mucus of asymptomatic ruminants and may therefore induce a positive serological response when the animals are tested for C. abortus. They have also been associated with different pathological diseases in ruminants, swine and koala. The aim of this study was to identify specific C. pecorum immunodominant antigens which could be used in ELISA tests allowing to distinguish between animals infected with C. pecorum and those infected with other chlamydial species. A gene encoding 35-kDa inclusion membrane protein incA of C. pecorum was isolated by immunoscreening of the C. pecorum DNA library using ovine anti-C. pecorum antibodies. The recombinant IncA protein did not react with a murine serum directed against C. abortus but did react with a specific monoclonal antibody of C. pecorum and toward several ovine serum samples obtained after experimental infection with different C. pecorum strains. This protein could be a good candidate for specific diagnosis of C. pecorum infection., ((c) 2009 Elsevier B.V. All rights reserved.)

Salmonella can invade non-phagocytic cells through its type III secretion system (T3SS-1), which induces a Trigger entry process. This study showed that Salmonella enterica, subspecies enterica serovar Enteritidis can also invade cells via the Rck outer membrane protein. Rck was necessary and sufficient to enable non-invasive E. coli and Rck-coated beads to adhere to and invade different cells. Internalization analysis of latex beads coated with different Rck peptides showed that the peptide containing amino acids 140-150 promoted adhesion, whereas amino acids between 150 and 159 modulated invasion. Expression of dominant-negative derivatives and use of specific inhibitors demonstrated the crucial role of small GTPases Rac1 and Cdc42 in activating the Arp2/3 complex to trigger formation of actin-rich accumulation, leading to Rck-dependent internalization. Finally, scanning and transmission electron microscopy with Rck-coated beads and E. coli expressing Rck revealed microvillus-like extensions that formed a Zipper-like structure, engulfing the adherent beads and bacteria. Overall, our results provide new insights into the Salmonella T3SS-independent invasion mechanisms and strongly suggest that Rck induces a Zipper-like entry mechanism. Consequently, Salmonella seems to be the first bacterium found to be able to induce both Zipper and Trigger mechanisms to invade host cells.

Suppressor of cytokine signaling (SOCS) proteins are key physiological regulators of both innate and adaptive immunity. These proteins belong to the three major classes of modulators of cytokines signaling. In the following article, we used porcine polarized intestinal cells to study early response to the protozoan, Entamoeba histolytica, and we identified by rapid amplification of cDNA ends (RACE) PCR porcine SOCS1, SOCS4, SOCS5 and SOCS6 encoding sequences. With more than 92% identity predicted porcine SOCS proteins are very similar to their human counterparts. Among SOCS transcripts, only SOCS2 mRNA was significantly induced in epithelial intestinal cells in response to the cytolytic activity of the parasite. The transcriptomic profile obtained after 3h of co-culture of polarized intestinal cells with E. histolytica was clearly oriented toward inflammation and the recruitment of neutrophils. These transcriptomic data have been normalized with accuracy by the utilisation of multiple validated reference genes. The analysis offers a first set of reference genes useful for future studies in porcine intestinal cells. Our data shed light on the understanding of the early response of polarized intestinal cells to E. histolytica and identified a potential involvement of SOCS2 in the parasite regulation of the host response., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Concentrations of the chemoattractants CXCL1, CXCL2, CXCL3, CXCL8, and C5a in milk were reduced by the preparation of milk whey by high-speed centrifugation or with rennet. About half of the chemoattractants (35 to 65%) were associated with the casein micelle sediment, except when whey was prepared by acidification. Consequently, quantification of chemoattractants should be carried out preferentially with skimmed milk samples or, whenever whey is needed, with acidic whey samples. The interference of milk or milk whey with the enzyme-linked immunosorbent assays (ELISAs) used to quantify the chemoattractants was moderate, as long as tetramethylbenzidine (TMB), not ABTS [2,2'-azino-bis-(3-ethylbenzthiazoline-sulfonate)], was used as the substrate of peroxidase. These considerations will help to assess more precisely a component of the immune response of the mammary gland to infection.

Chlamydophila pecorum found in the intestine and vaginal mucus of asymptomatic ruminants has also been associated with different pathological conditions in ruminants, swine and koalas. Some endangered species such as water buffalos and bandicoots have also been found to be infected by C. pecorum. The persistence of C. pecorum strains in the intestine and vaginal mucus of ruminants could cause long-term sub-clinical infection affecting the animal's health. C. pecorum strains present many genetic and antigenic variations, but coding tandem repeats have recently been found in some C. pecorum genes, allowing C. pecorum strains isolated from sick animals to be differentiated from those isolated from asymptomatic animals. This review provides an update on C. pecorum infections in different animal hosts and the implications for animal health. The taxonomy, typing and genetic aspects of C. pecorum are also reviewed., (INRA, EDP Sciences, 2010.)

The cDNA-AFLP (cDNA-amplified fragment length polymorphism) method comparing transcripts from levamisole-resistant and susceptible Haemonchus contortus isolates has led to the successful identification of a number of potentially useful levamisole-resistance markers. In the present study, we report the characterization of the transcript-derived fragment (TDF) named HAX which was confirmed to be specifically expressed in three levamisole-resistant isolates by RT-PCR experiments. Cloning and sequencing of the full-length cDNA sequence of HAX revealed high similarity to the Caenorhabditis elegans acr-8 gene and its putative H. contortus orthologue encoding a nicotinic acetylcholine receptor subunit. This Hco-acr-8b short isoform corresponded to a spliced variant of Hco-acr-8 mRNA containing the two first exons and a part of intron 2. As nicotinic acetylcholine receptors constitute the pharmacological target of levamisole, Hco-acr-8b may potentially be involved in the molecular mechanisms leading to levamisole-resistance acquisition in H. contortus.

A Gram-negative, non-motile, non-spore-forming coccoid bacterium (strain BO1(T)) was isolated recently from a breast implant infection of a 71-year-old female patient with clinical signs of brucellosis. Affiliation of strain BO1(T) to the genus Brucella was confirmed by means of polyamine pattern, polar lipid profile, fatty acid profile, quinone system, DNA-DNA hybridization studies and by insertion sequence 711 (IS711)-specific PCR. Strain BO1(T) harboured four to five copies of the Brucella-specific insertion element IS 711, displaying a unique banding pattern, and exhibited a unique 16S rRNA gene sequence and also grouped separately in multilocus sequence typing analysis. Strain BO1(T) reacted with Brucella M-monospecific antiserum. Incomplete lysis was detected with bacteriophages Tb (Tbilisi), F1 and F25. Biochemical profiling revealed a high degree of enzymic activity and metabolic capabilities. In multilocus VNTR (variable-number tandem-repeat) analysis, strain BO1(T) showed a very distinctive profile and clustered with the other 'exotic' Brucella strains, including strains isolated from marine mammals, and Brucella microti, Brucella suis biovar 5 and Brucella neotomae. Comparative omp2a and omp2b gene sequence analysis revealed the most divergent omp2 sequences identified to date for a Brucella strain. The recA gene sequence of strain BO1(T) differed in seven nucleotides from the Brucella recA consensus sequence. Using the Brucella species-specific multiplex PCR assay, strain BO1(T) displayed a unique banding pattern not observed in other Brucella species. From the phenotypic and molecular analysis it became evident that strain BO1( T) was clearly different from all other Brucella species, and therefore represents a novel species within the genus Brucella. Because of its unexpected isolation, the name Brucella inopinata with the type strain BO1(T) (=BCCN 09-01(T)=CPAM 6436(T)) is proposed.

In many species such as sheep and pig, there are two types of Peyer's patches (PP): several discrete patches in the jejunum and a long and continuous patch in the ileum. Most of the immunoglobulin A in the gut is generated by B-cells in the PP germinal centers. Moreover, swine like ovine ileal PP might be important for antigen independent B-cell repertoire diversification. We examined, by quantitative real-time PCR, the expression of 36 transcripts of antimicrobial peptides, chemokines, interleukines, Toll-like receptors and transcription factors from both PP and we highlighted the differences by a principal component analysis. Ileal PP was characterized by a higher mRNA expression of CCL28, IL5, IL10, TLR2 and TLR4 while jejunal PP showed higher mRNA expression of antimicrobial peptides, CCL25, FOXP3, IL4, T-Bet, TSLP and SOCS2. Then, we analyzed some VDJ rearrangements to assess immunoglobulin repertoire diversity in jejunal and ileal PP from weaned piglets. The IgA and IgM repertoires were more diverse in ileal than in jejunal piglet PP. All these results could be related to the rarefaction of interfollicular T-cell zone and the presence in ileal versus jejunal lumen of a more diversified microflora. These findings shed a light on the functional differences between both PP.

The purpose of this article is to present the diseases induced in humans and animals by the different species of Chlamydophila, after providing an overview on the history of these infectious agents and their taxonomy. The route of transmission and the available methods for prevention and control in the different animal species are reviewed., (Copyright 2009 Elsevier B.V. All rights reserved.)

Natural killer (NK) cells have a key role in the innate immune response against pathogens because of their cytotoxic properties and production of interferon-gamma (IFN-gamma). Some insight into ruminant NK cell biology has been gained through the characterization of bovine NK cells as NKp46(+)/CD3(-) cells. However, ovine NK cells have been little studied because of the lack of specific antibodies. Most NK cells in humans and cattle express CD16. We found that an antibody against human CD16 that cross-reacts with bovine NK cells also recognizes cell populations in ovine peripheral blood mononuclear cells. Using double labelling with CD14 revealed the same profile as described in other species, and we identified a putative NK cell population. We therefore sorted this ovine CD16(+)/CD14(-) cell population and tested it for NK cell characteristics. More than 80% of sorted CD16(+)/CD14(-) cells expressed perforin. After a week of culture in the presence of IL-2 and IL-15, ovine CD16(+)/CD14(-) cells had become large cells with intra-cytoplasmic granules containing perforin, and the vast majority displayed an activated CD2(-/low)/CD25(+)/CD8(+) phenotype, as observed for bovine NKp46(+)/CD3(-) cells. Moreover, these cells expressed transcripts for the NKp46 receptor, and were cytotoxic in a CD16-mediated redirected lysis assay against a murine cell line, P815, and in a direct lysis assay against the ovine cell line, IDO5. Finally, ovine CD16(+)/CD14(-) cells having expanded for 7 days in culture secreted IFN-gamma in response to IL-12 in a dosedependent manner. Taken together, these findings led us to conclude that the ovine CD16(+)/CD14(-) lymphocyte sub-population displays the phenotype and functional characteristics of NK cells.

This is the first report of benzimidazole (BZ) resistance in the nematode Trichostrongylus axei in sheep. Trichostrongylus axei infects several species of herbivores including sheep, cattle and horses, and the emergence of anthelmintic resistance could lead to significant problems in its control. Benzimidazole resistance in two sheep flocks in central France was detected by post-treatment worm counts. The sequencing of a central region of the isotype 1 beta-tubulin gene from adult T. axei recovered post-mortem revealed only one, non-synonymous single nucleotide polymorphism at position 200 (Phe200Tyr), which had already been reported for other nematodes. Seven years after BZ treatment ceased, T. axei helminths present were still resistant to BZ suggesting these parasites do not revert to susceptibility to this anthelmintic, even when the selection pressure had been removed for many years. The findings also highlight major changes in the make-up of the nematode burden in sheep flocks that accompanies the emergence of BZ resistance., (Copyright 2008 Elsevier Ltd. All rights reserved.)

Background: The development of mucosal vaccines is crucial to efficiently control infectious agents for which mucosae are the primary site of entry. Major drawbacks of these protective strategies are the lack of effective mucosal adjuvant. Synthetic oligodeoxynucleotides that contain several unmethylated cytosine-guanine dinucleotide (CpG-ODN) motifs are now recognized as promising adjuvants displaying mucosal adjuvant activity through direct activation of TLR9-expressing cells. However, little is known about the efficacy of these molecules in stimulating the intestinal immune system in neonates., Methodology/principal Findings: First, newborn mice received CpG-ODN orally, and the intestinal cytokine and chemokine response was measured. We observed that oral administration of CpG-ODN induces CXC and CC chemokine responses and a cellular infiltration in the intestine of neonates as detected by immunohistochemistry. We next compared the efficiency of the oral route to intraperitoneal administration in stimulating the intestinal immune responses of both adults and neonates. Neonates were more responsive to TLR9-stimulation than adults whatever the CpG-ODN administration route. Their intestinal epithelial cells (IECs) indirectly responded to TLR9 stimulation and contributed to the CXC chemokine response, whereas other TLR9-bearing cells of the lamina-propria produced CC chemokines and Th1-type cytokines. Moreover, we showed that the intestine of adult exhibited a significantly higher level of IL10 at homeostasis than neonates, which might be responsible for the unresponsiveness to TLR9-stimulation, as confirmed by our findings in IL10-deficient mice., Conclusions/significance: This is the first report that deciphers the role played by CpG-ODN in the intestine of neonates. This work clearly demonstrates that an intraperitoneal administration of CpG-ODN is more efficient in neonates than in adults to stimulate an intestinal chemokine response due to their lower IL-10 intestinal level. In addition we report the efficiency of the oral route at inducing intestinal chemokine responses in neonate that might be taken into consideration for further vaccine development against neonatal diseases.