From intestine to beyond: Salmonella entry factors display distinct transcription pattern upon infection in murine models.

The infectious process of bacteria of the genus Salmonella requires the finely regulated use of various virulence factors. Among them, the type 3 secretion system-1 (T3SS-1) and the Rck and PagN invasins are involved in the internalization of the pathogen within eukaryotic cells, but their precise role in the host and in the pathogenic process is still poorly understood. In this study, we aimed to determine the kinetics of expression of these entry factors in a typhoid fever-like and a gastroenteritis model in mice by in vivo imaging using bioluminescent Salmonella Typhimurium reporter strains carrying chromosomal transcriptional fusions. Only pagN and T3SS-1 transcription has been clearly identified. Independently of the pathological model, the caecum was identified as the main transcription site of both pagN and the T3SS-1-encoding gene both at early and late stages of the infection. An intense transcription of pagN was also observed in deep organs in the typhoid fever-like model, while that of T3SS-1 remained quite sporadic in these organs, and mainly focused on the intestine all along the infection. This work will help to understand the respective role of these entry factors at the cellular level in the pathogenesis of Salmonella in vivo.

Topical reinforcement of the cervical mucus barrier to sperm.

Close to half of the world's pregnancies are still unplanned, reflecting a clear unmet need in contraception. Ideally, a contraceptive would provide the high efficacy of hormonal treatments, without systemic side effects. Here, we studied topical reinforcement of the cervical mucus by chitosan mucoadhesive polymers as a form of female contraceptive. Chitosans larger than 7 kDa effectively cross-linked human ovulatory cervical mucus to prevent sperm penetration in vitro. We then demonstrated in vivo using the ewe as a model that vaginal gels containing chitosan could stop ram sperm at the entrance of the cervical canal and prevent them from reaching the uterus, whereas the same gels without chitosan did not substantially limit sperm migration. Chitosan did not affect sperm motility in vitro or in vivo, suggesting reinforcement of the mucus physical barrier as the primary mechanism of action. The chitosan formulations did not damage or irritate the ewe vaginal epithelium, in contrast to nonoxynol-9 spermicide. The demonstration that cervical mucus can be reinforced topically to create an effective barrier to sperm may therefore form the technological basis for muco-cervical barrier contraceptives with the potential to become an alternative to hormonal contraceptives.

In vivo imaging reveals novel replication sites of a highly oncogenic avian herpesvirus in chickens.

In vivo bioluminescence imaging facilitates the non-invasive visualization of biological processes in living animals. This system has been used to track virus infections mostly in mice and ferrets; however, until now this approach has not been applied to pathogens in avian species. To visualize the infection of an important avian pathogen, we generated Marek's disease virus (MDV) recombinants expressing firefly luciferase during lytic replication. Upon characterization of the recombinant viruses in vitro, chickens were infected and the infection visualized in live animals over the course of 14 days. The luminescence signal was consistent with the known spatiotemporal kinetics of infection and the life cycle of MDV, and correlated well with the viral load measured by qPCR. Intriguingly, this in vivo bioimaging approach revealed two novel sites of MDV replication, the beak and the skin of the feet covered in scales. Feet skin infection was confirmed using a complementary fluorescence bioimaging approach with MDV recombinants expressing mRFP or GFP. Infection was detected in the intermediate epidermal layers of the feet skin that was also shown to produce infectious virus, regardless of the animals' age at and the route of infection. Taken together, this study highlights the value of in vivo whole body bioimaging in avian species by identifying previously overlooked sites of replication and shedding of MDV in the chicken host.

In Vivo Tracking of Bacterial Colonization in Different Murine Models Using Bioluminescence: The Example of Salmonella.

Applications of bioluminescence for the in vivo study of pathogenic microorganisms are numerous, ranging from the quantification of virulence gene expression to measuring the effect of antimicrobial molecules on the colonization of tissues and organs by the pathogen. Most studies are performed in mice, but recent works demonstrate that this technique is applicable to larger animals like fish, guinea pigs, ferrets, and chickens. Here, we describe the construction and the utilization of a constitutively luminescent strain of Salmonella Typhimurium to monitor in vivo and ex vivo the colonization of mice in the gastroenteritis, typhoid fever, and asymptomatic carriage models of Salmonella infection.

Evidence of early increased sialylation of airway mucins and defective mucociliary clearance in CFTR-deficient piglets.

BACKGROUND: Bacterial colonization in cystic fibrosis (CF) lungs has been directly associated to the loss of CFTR function, and/or secondarily linked to repetitive cycles of chronic inflammation/infection. We hypothesized that altered molecular properties of mucins could contribute to this process. METHODS: Newborn CFTR+/+ and CFTR-/- were sacrificed before and 6 h after inoculation with luminescent Pseudomonas aeruginosa into the tracheal carina. Tracheal mucosa and the bronchoalveolar lavage (BAL) fluid were collected to determine the level of mucin O-glycosylation, bacteria binding to mucins and the airways transcriptome. Disturbances in mucociliary transport were determined by ex-vivo imaging of luminescent Pseudomonas aeruginosa. RESULTS: We provide evidence of an increased sialylation of CF airway mucins and impaired mucociliary transport that occur before the onset of inflammation. Hypersialylation of mucins was reproduced on tracheal explants from non CF animals treated with GlyH101, an inhibitor of CFTR channel activity, indicating a causal relationship between the absence of CFTR expression and the sialylation of mucins. This increased sialylation was correlated to an increased adherence of P. aeruginosa to mucins. In vivo infection of newborn CF piglets by live luminescent P. aeruginosa demonstrated an impairment of mucociliary transport of this bacterium, with no evidence of pre-existing inflammation. CONCLUSIONS: Our results document for the first time in a well-defined CF animal model modifications that affect the O-glycan chains of mucins. These alterations precede infection and inflammation of airway tissues, and provide a favorable context for microbial development in CF lung that hallmarks this disease.

Effective Nanoparticle-Based Nasal Vaccine Against Latent and Congenital Toxoplasmosis in Sheep.

Toxoplasma gondii is a parasitic protozoan of worldwide distribution, able to infect all warm-blooded animals, but particularly sheep. Primary infection in pregnant sheep leads to millions of abortions and significant economic losses for the livestock industry. Moreover, infected animals constitute the main parasitic reservoir for humans. Therefore, the development of a One-health vaccine seems the best prevention strategy. Following earlier work, a vaccine constituted of total extract of Toxoplasma gondii proteins (TE) associated with maltodextrin nanoparticles (DGNP) was developed in rodents. In this study we evaluated the ability of this vaccine candidate to protect against latent and congenital toxoplasmosis in sheep. After two immunizations by either intranasal or intradermal route, DGNP/TE vaccine generated specific Th1-cellular immune response, mediated by APC-secretion of IFN-γ and IL-12. Secretion of IL-10 appeared to regulate this Th1 response for intradermally vaccinated sheep but was absent in intranasally-vaccinated animals. Finally, protection against latent toxoplasmosis and transplacental transmission were explored. Intranasal vaccination led to a marked decrease of brain cysts compared with the non-vaccinated group. This DGNP/TE vaccine administered intranasally conferred a high level of protection against latent toxoplasmosis and its transplacental transmission in sheep, highlighting the potential for development of such a vaccine for studies in other species.

Characterization of goat prions demonstrates geographical variation of scrapie strains in Europe and reveals the composite nature of prion strains.

Bovine Spongiform Encephalopathy (BSE) is the only animal prion which has been recognized as a zoonotic agent so far. The identification of BSE in two goats raised the need to reliably identify BSE in small ruminants. However, our understanding of scrapie strain diversity in small ruminants remains ill-defined, thus limiting the accuracy of BSE surveillance and spreading fear that BSE might lurk unrecognized in goats. We investigated prion strain diversity in a large panel of European goats by a novel experimental approach that, instead of assessing the neuropathological profile after serial transmissions in a single animal model, was based on the direct interaction of prion isolates with several recipient rodent models expressing small ruminants or heterologous prion proteins. The findings show that the biological properties of scrapie isolates display different patterns of geographical distribution in Europe and suggest that goat BSE could be reliably discriminated from a wide range of biologically and geographically diverse goat prion isolates. Finally, most field prion isolates showed composite strain features, with discrete strain components or sub-strains being present in different proportions in individual goats or tissues. This has important implications for understanding the nature and evolution of scrapie strains and their transmissibility to other species, including humans.

Four types of scrapie in goats differentiated from each other and bovine spongiform encephalopathy by biochemical methods.

Scrapie in goats has been known since 1942, the archetype of prion diseases in which only prion protein (PrP) in misfolded state (PrPSc) acts as infectious agent with fatal consequence. Emergence of bovine spongiform encephalopathy (BSE) with its zoonotic behaviour and detection in goats enhanced fears that its source was located in small ruminants. However, in goats knowledge on prion strain typing is limited. A European-wide study is presented concerning the biochemical phenotypes of the protease resistant fraction of PrPSc (PrPres) in over thirty brain isolates from transmissible spongiform encephalopathy (TSE) affected goats collected in seven countries. Three different scrapie forms were found: classical scrapie (CS), Nor98/atypical scrapie and one case of CH1641 scrapie. In addition, CS was found in two variants-CS-1 and CS-2 (mainly Italy)-which differed in proteolytic resistance of the PrPres N-terminus. Suitable PrPres markers for discriminating CH1641 from BSE (C-type) appeared to be glycoprofile pattern, presence of two triplets instead of one, and structural (in)stability of its core amino acid region. None of the samples exhibited BSE like features. BSE and these four scrapie types, of which CS-2 is new, can be recognized in goats with combinations of a set of nine biochemical parameters.

Protein delivery by porous cationic maltodextrin-based nanoparticles into nasal mucosal cells: Comparison with cationic or anionic nanoparticles.

Different types of biodegradable nanoparticles (NPs) have been studied as delivery systems for proteins into nasal mucosal cells, especially for vaccine applications. Such a nanocarrier must have the ability to be loaded with proteins and to transport this payload into mucosal cells. However, comparative data on nanoparticles' capacity for protein loading, efficiency of subsequent endocytosis and the quantity of nanocarriers used are either lacking or contradictory, making comparisons and the choice of a best candidate difficult. Here we compared 5 types of nanoparticles with different surface charge (anionic or cationic) and various inner compositions as potential vectors: the NPL (cationic maltodextrin NP with an anionic lipid core), cationic and anionic PLGA (Poly Lactic co-Glycolic Acid) NP, and cationic and anionic liposomes. We first quantified the protein association efficiency and NPL associated the largest amount of ovalbumin, used as a model protein. In vitro, the delivery of fluorescently-labeled ovalbumin into mucosal cells (airway epithelial cells, dendritic cells and macrophages) was assessed by flow cytometry and revealed that the NPL delivered protein to the greatest extent in all 3 different cell lines. Taken together, these data underlined the potential of the porous and cationic maltodextrin-based NPL as efficient protein delivery systems to mucosal cells.

Systemic Administration of Avian Defensin 7: Distribution, Cellular Target, and Antibacterial Potential in Mice.

Defensins are natural antimicrobial peptides. The avian beta-defensin AvBD7 isolated from the chicken bone marrow possess broad antibacterial spectrum and strong resistance to proteolysis. However, its ability to fight systemic infections of major concern for public health, such as salmonellosis, is unknown. As a first approach, fluorescence labeling of AvBD7 allowed to track its systemic distribution after intraperitoneal injection in mice using whole body live imaging. It was associated to peritoneal cells and to deeper organs such as the liver. In the next step, the use of labeled AvBD7 allowed to observe its interaction with murine macrophages in culture. After incubation, it was able to penetrate inside the cells through an endocytosis-like mechanism. Furthermore, natural AvBD7 contributed to the control of intracellular multiplication of a multidrug resistant Salmonella strain, after incubation with infected macrophages. Finally, administration in a model of systemic lethal Salmonella infection in mice led to significant improvement of mouse survival, consistently with significant reduction of the liver bacterial load. In conclusion, the results reveal a hitherto unknown intracellular antibacterial effect of AvBD7 in Salmonella target cells and support AvBD7 as a candidate of interest for the treatment of infectious diseases caused by multidrug-resistant pathogenic Enterobacteriaceae.

Residence time and uptake of porous and cationic maltodextrin-based nanoparticles in the nasal mucosa: Comparison with anionic and cationic nanoparticles.

Different types of biodegradable nanoparticles (NP) have been studied as nasal mucosa cell delivery systems. These nanoparticles need to strongly interact with mucosa cells to deliver their payload. However, only a few simultaneous comparisons have been made and it is therefore difficult to determine the best candidate. Here we compared 5 types of nanoparticles with different surface charge (anionic or cationic) and various inner compositions as potential vectors: cationic and anionic liposomes, cationic and anionic PLGA (Poly Lactic co-Glycolic Acid) NP and porous and cationic maltodextrin NP (cationic surface with an anionic lipid core: NPL). We first quantified their nasal residence time after nasal administration in mice using in vivo live imaging and NPL showed the longest residence time. In vitro endocytosis on mucosal cells (airway epithelial cells, macrophages and dendritic cells) using labeled nanoparticles were performed by flow cytometry and confocal microscopy. Among the 5 nanoparticles, NPL were taken up to the greatest extent by the 3 different cell lines and the endocytosis mechanisms were characterized. Taken together, we observed that the nanoparticles' cationic surface charge is insufficient to improve mucosal residence time and cellular uptake and that the NPL are the best candidates to interact with airway mucosal cells.

Structural and functional diversity arising from intra- and inter-haplotype combinations of duplicated DQA and B loci within the ovine MHC.

In sheep, the A and B loci encoding the α and ß chains of the classical class II MHC molecules are DRA and DRB and DQA and DQB. Previous analyses described the duplication of the DQA and DQB genes. The majority of haplotypes include DQA1 and DQA2 loci, however, in a number of haplotypes, DQA1 appears absent and these haplotypes have been described as DQA1 null. In these haplotypes, the DQA2 locus is found in combination with a second locus which appeared more closely related to DQA2 than DQA1, hence the description of this locus as DQA2-like. Here we combine our previous analysis of the DQA transcripts with an analysis of the associated DQB transcripts in ten haplotypes from MHC homozygous animals. This allows the potential for surface expression of different haplotype combinations of DQA and B genes and the functional significance of DQA2-like and its predicted DQB partner to be determined. Atypical DQB transcripts (DQB2-like) were identified in haplotypes classified as DQA1-null and conserved DQB2-like orthologues were identified in other Bovidae indicating trans-species conservation of the allelic lineage. Functional combinations detected by co-transfection of DQ1, DQ2 and DQ2-like genes demonstrates the potential for a wide range of DQ molecules derived from both intra- and inter-haplotype as well as inter-locus combinations. We provide evidence that DQA2-like and B2-like genes form an evolutionary conserved pair which generates structurally distinct class II molecules that are likely to present a distinct range of peptides to CD4+ T cells.

Synthetic parasites: a successful mucosal nanoparticle vaccine against Toxoplasma congenital infection in mice.

AIM: Development of protein vaccine to prevent congenital infection is a major public health priority. Our goal is the design of mucosal synthetic pathogen inducing protective immune responses against congenital toxoplasmosis. MATERIALS & METHODS: Mice were immunized intranasally, establishing pregnancy and challenging orally. Placental immune response, congenital infection, pup growth, parasitic load rates were studied. RESULTS: Pups born to vaccinated infected dams had significantly fewer brain cysts, no intraocular inflammation and normal growth. Protection was associated with a placental cellular Th1 response downregulated by IL-6 and correlated with persistence of vaccine for few hours in the nose before being totally eliminated. CONCLUSION: Our vaccine conferred high protection against congenital toxoplasmosis. These results provide support for future studies of other congenital vaccine.

EU-approved rapid tests might underestimate bovine spongiform encephalopathy infection in goats.

We report the diagnostic sensitivity of 3 EU-approved rapid tests (ELISAs; 1 from IDEXX and 2 from Bio-Rad) for the detection of transmissible spongiform encephalopathy diseases in goats. Ninety-eight goat brainstem samples were tested. All the rapid tests had 100% specificity and ≥80% sensitivity, with the IDEXX test significantly more sensitive than the 2 Bio-Rad tests. All tests detected 100% of samples from goats with clinical scrapie, but missed 8% (IDEXX) to 33% (Bio-Rad SG) of samples from preclinical goats. Importantly, only IDEXX picked up all samples from clinical bovine spongiform encephalopathy (BSE)-infected goats, whereas the other 2 rapid tests missed 15% (Bio-Rad SG) to 25% (Bio-Rad SAP). These results show that a fraction of preclinical scrapie infections are likely missed by EU surveillance, with sensitivity of detection strongly dependent on the choice of the rapid test. Moreover, a significant proportion of clinical BSE infections are underestimated by using either Bio-Rad test. Assuming that the same sensitivity on preclinical goats would also occur in BSE-infected goats, our data suggest that IDEXX is likely the most sensitive test for detecting preclinical field cases of BSE infection in goats, although with an 8% failure rate. These results raise some concerns about the reliability of current EU surveillance figures on BSE infection in goats.

An ancient interlocus recombination increases class II MHC DQA diversity in sheep and other Bovidae.

Animals with fully characterised major histocompatibility complex (MHC) regions are often used to explore the molecular interactions that control the induction of adaptive immunity. The ovine MHC includes two DQA loci, termed DQA1 and DQA2. However, in a minority of haplotypes the DQA1 locus appears absent (DQA1 null) and is replaced by a second locus termed, DQA2-like. This raises a number of questions regarding the origins and function of the DQA2-like sequences. To address this, we have analysed DQA diversity associated with 10 MHC haplotypes, including two classified as DQA1 null. Pair-wise comparison between full-length DQA transcripts from each haplotype identified unique diversity throughout the DQA2-like sequences. Conserved orthologues of the DQA2-like sequences were identified in cattle and goat, and phylogenetic analysis clustered exons 1 and 2 with DQA2 whereas the remainder of the sequence clustered with DQA1. The DQA2-like allelic lineage appears functional and to have arisen from an ancient interlocus recombination between DQA1 and DQA2 loci which predates Bovidae speciation.

Evidence for zoonotic potential of ovine scrapie prions.

Although Bovine Spongiform Encephalopathy (BSE) is the cause of variant Creutzfeldt Jakob disease (vCJD) in humans, the zoonotic potential of scrapie prions remains unknown. Mice genetically engineered to overexpress the human prion protein (tgHu) have emerged as highly relevant models for gauging the capacity of prions to transmit to humans. These models can propagate human prions without any apparent transmission barrier and have been used used to confirm the zoonotic ability of BSE. Here we show that a panel of sheep scrapie prions transmit to several tgHu mice models with an efficiency comparable to that of cattle BSE. The serial transmission of different scrapie isolates in these mice led to the propagation of prions that are phenotypically identical to those causing sporadic CJD (sCJD) in humans. These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions.

Preclinical detection of variant CJD and BSE prions in blood.

The emergence of variant Creutzfeldt Jakob Disease (vCJD) is considered a likely consequence of human dietary exposure to Bovine Spongiform Encephalopathy (BSE) agent. More recently, secondary vCJD cases were identified in patients transfused with blood products prepared from apparently healthy donors who later went on to develop the disease. As there is no validated assay for detection of vCJD/BSE infected individuals the prevalence of the disease in the population remains uncertain. In that context, the risk of vCJD blood borne transmission is considered as a serious concern by health authorities. In this study, appropriate conditions and substrates for highly efficient and specific in vitro amplification of vCJD/BSE agent using Protein Misfolding Cyclic Amplification (PMCA) were first identified. This showed that whatever the origin (species) of the vCJD/BSE agent, the ovine Q171 PrP substrates provided the best amplification performances. These results indicate that the homology of PrP amino-acid sequence between the seed and the substrate is not the crucial determinant of the vCJD agent propagation in vitro. The ability of this method to detect endogenous vCJD/BSE agent in the blood was then defined. In both sheep and primate models of the disease, the assay enabled the identification of infected individuals in the early preclinical stage of the incubation period. Finally, sample panels that included buffy coat from vCJD affected patients and healthy controls were tested blind. The assay identified three out of the four tested vCJD affected patients and no false positive was observed in 141 healthy controls. The negative results observed in one of the tested vCJD cases concurs with results reported by others using a different vCJD agent blood detection assay and raises the question of the potential absence of prionemia in certain patients.

Copy number variation and differential expression of a protective endogenous retrovirus in sheep.

The Jaagsiekte sheep retrovirus exJSRV and its endogenous counterpart enJSRV co-exist in sheep. exJSRV, a betaretrovirus, is the etiological agent of ovine pulmonary adenocarcinoma, and it has been demonstrated in vitro that an enJSRV Gag variant bearing the R-to-W amino acid change at position 21 was able to block exJSRV budding from the cells, providing a potential protective role for the host. In this work, we developed a fast mutation detection assay based on the oligo ligation assay (OLA) that permits the quantification of the relative proportions of the R21 and W21 Gag variants present in individual genomes and in cDNA obtained from normal and exJSRV-induced lung tumors. We have shown that the W21/R21 ratio is variable within and between breeds. We also describe for the first time that putative protecting enJSRV variants were expressed in alveolar type II cells (AECII), the major target of exJSRV.

Neuroimmune connections in ovine pharyngeal tonsil: potential site for prion neuroinvasion.

Recent studies have established the involvement of nasal-associated lymphoid tissues, mainly the pharyngeal tonsil, in prion pathogenesis. However, the mechanisms of the associated neuroinvasion are still debated. To determine potential sites for prion neuroinvasion inside the ovine pharyngeal tonsil, the topography of heavy (200 kDa) and light (70 kDa) neurofilaments and of glial fibrillar acidic protein has been semi-quantitatively analysed inside the various compartments of the tonsil. The results show that the most innervated areas are the interfollicular area and the connective tissue located beneath the respiratory epithelium. The existence of rare synapses between follicular dendritic cells and nerve fibres inside the germinal centre indicates that this mechanism of neuroinvasion is possible but, since germinal centres of lymphoid follicles are poorly innervated, other routes of neuroinvasion are likely. The host PRNP genotype does not influence the pattern of innervation in these various tonsil compartments, unlike ageing during which an increase of nerve endings occurs in a zone of high trafficking cells beneath the respiratory epithelium. A minimal age-related increase of innervation inside the lymphoid follicles has also been observed. An increase in nerve fibre density around the lymphoid follicles, in an area rich in mobile cells such as macrophages and dendritic cells capable of capturing and conveying pathogen prion protein (PrPd), might ensure more efficient infectivity, not in the early phase but in the advanced phase of lymphoinvasion after the amplification of PrPd; alternatively, this area might even act as a direct site of entry during neuroinvasion.

Prionemia and leukocyte-platelet-associated infectivity in sheep transmissible spongiform encephalopathy models.

The dynamics of the circulation and distribution of transmissible spongiform encephalopathy (TSE) agents in the blood of infected individuals remain largely unknown. This clearly limits the understanding of the role of blood in TSE pathogenesis and the development of a reliable TSE blood detection assay. Using two distinct sheep scrapie models and blood transfusion, this work demonstrates the occurrence of a very early and persistent prionemia. This ability to transmit disease by blood transfusion was correlated with the presence of infectivity in white blood cells (WBC) and peripheral blood mononucleated cells (PBMC) as detected by bioassay in mice overexpressing the ovine prion protein PrP (tg338 mice) and with the identification of abnormal PrP in WBC after using protein misfolding cyclic amplification (PMCA). Platelets and a large variety of leukocyte subpopulations also were shown to be infectious. The use of endpoint titration in tg338 mice indicated that the infectivity in WBC (per ml of blood) was 10(6.5)-fold lower than that in 1 g of posterior brainstem sample. In both WBC and brainstem, infectivity displayed similar resistance to PK digestion. The data strongly support the concept that WBC are an accurate target for reliable TSE detection by PMCA. The presence of infectivity in short-life-span blood cellular elements raises the question of the origin of prionemia.