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.

Intrinsic alterations in peripheral neutrophils from cystic fibrosis newborn piglets.

BACKGROUND: The hallmark of the cystic fibrosis (CF) lung disease is a neutrophil dominated lung environment that is associated to chronic lung tissue destruction and ultimately the patient's death. It is unclear whether the exacerbated neutrophil response is primary related to a defective CFTR or rather secondary to chronic bacterial colonization and inflammation. Here, we hypothesized that CF peripheral blood neutrophils present intrinsic alteration at birth before the start of an inflammatory process. METHODS: Peripheral blood neutrophils were isolated from newborn CFTR+/+ and CFTR-/- piglets. Neutrophils immunophenotype was evaluated by flow cytometry. Lipidomic and proteomic profile were characterized by liquid chromatography/tandem mass spectrometry (LC-MS/MS), intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) followed by top-down high-resolution mass spectrometry (HRMS), respectively. The ability of CF neutrophils to kill pseudomonas aeruginosa was also evaluated. RESULTS: Polyunsaturated fatty acid metabolites analysis did not show any difference between CFTR+/+ and CFTR-/- neutrophils. On the other hand, a predictive mathematical model based on the ICM-MS proteomic profile was able to discriminate between both genotypes. Top-down proteomic analysis identified 19 m/z differentially abundant masses that corresponded mainly to proteins related to the antimicrobial response and the generation of reactive oxygen species (ROS). However, no alteration in the ability of CFTR-/- neutrophils to kill pseudomonas aeruginosa in vitro was observed. CONCLUSIONS: ICM-MS demonstrated that CFTR-/- neutrophils present intrinsic alterations already at birth, before the presence of any infection or inflammation.

Late weaning is associated with increased microbial diversity and Faecalibacterium prausnitzii abundance in the fecal microbiota of piglets.

BACKGROUND: In pig production systems, weaning is a crucial period characterized by nutritional, environmental, and social stresses. Piglets transition from a milk-based diet to a solid, more complex plant-based diet, and their gut physiology must adapt accordingly. It is well established that piglets weaned later display improved health, better wean-to-finish growth performance, and lower mortality rates. The aim of this study was to evaluate the impact of weaning age on fecal microbiota diversity and composition in piglets. Forty-eight Large White piglets were divided into 4 groups of 12 animals that were weaned at different ages: 14 days (early weaning), 21 days (a common weaning age in intensive pig farming), 28 days (idem), and 42 days (late weaning). Microbiota composition was assessed in each group by sequencing the 16S rRNA gene using fecal samples taken on the day of weaning, 7 days later, and at 60 days of age. RESULTS: In each group, there were significant differences in fecal microbiota composition before and after weaning (p < 0.05), confirming that weaning can drastically change the gut microbiota. Microbiota diversity was positively correlated with weaning age: microbial alpha diversity and richness were higher in piglets weaned at 42 days of age both on the day of weaning and 7 days later. The abundance of Faecalibacterium prausnitzii operational taxonomic units (OTUs) was also higher in piglets weaned at 42 days of age. CONCLUSIONS: Overall, these results show that late weaning increased gut microbiota diversity and the abundance of F. prausnitzii, a microorganism with positive effects in humans. Piglets might thus derive a competitive advantage from later weaning because they have more time to accumulate a higher diversity of potentially beneficial microbes prior to the stressful and risky weaning period.

IPEC-1 variable immune response to different serovars of Salmonella enterica subsp. enterica.

Salmonella is a genus of Gram-negative bacteria in the Enterobacteriaceae family causing various illnesses. The ability of the different serovars of Salmonella enterica subsp. enterica to infect a host and to induce pathology relies in part on their cellular and molecular interactions with the intestinal epithelium. In the current study, an in vitro approach using non-polarized or polarized IPEC-1 porcine intestinal epithelial cells were used in order to assess the relation between adhesion, invasion, and induction of the immune response as a function of the serotype of Salmonella. Five serovars, Choleraesuis (host-adapted), Typhimurium (ubiquitous), Typhisuis (host-restricted), which are relevant for pig infection, and Dublin and Gallinarum, which are host-restricted or host-adapted, were studied. A strong variation was observed in the percentages of adhesion and invasion amongst the S. enterica serovars used to interact with the non-polarized and polarized cells. Subsequently, differences were identified between serovars in terms of immune response induced. Serovars Typhimurium and Typhisuis induced a strong innate immune response four and half hours after the beginning of cell stimulation while Choleraesuis, Gallinarum, and Dublin did not. A strong inflammatory response could limit the spread of the porcine serovars to the gut while, with a weak response, bacteria may not be constrained by the immune response enabling severe systemic diseases. Different repertoires of adhesion factors and of secreted protein effectors between Salmonella serovars interacting with IPEC-1 cells probably explains the differences in their early pathogenic behaviours.

CFTR-deficient pigs display alterations of bone microarchitecture and composition at birth.

BACKGROUND: The lack of cystic fibrosis transmembrane conductance regulator (CFTR) function causes cystic fibrosis (CF), predisposing to severe lung disease, reduced growth and osteopenia. Both reduced bone content and strength are increasingly recognized in infants with CF before the onset of significant lung disease, suggesting a developmental origin and a possible role in bone disease pathogenesis. The role of CFTR in bone metabolism is unclear and studies on humans are not feasible. Deletion of CFTR in pigs (CFTR -/- pigs) displays at birth severe malformations similar to humans in the intestine, respiratory tract, pancreas, liver, and male reproductive tract. METHODS: We compared bone parameters of CFTR -/- male and female pigs with those of their wild-type (WT) littermates at birth. Morphological and microstructural properties of femoral cortical and trabecular bone were evaluated using micro-computed tomography (µCT), and their chemical compositions were examined using Raman microspectroscopy. RESULTS: The integrity of the CFTR -/- bone was altered due to changes in its microstructure and chemical composition in both sexes. Low cortical thickness and high cortical porosity were found in CFTR -/- pigs compared to sex-matched WT littermates. Moreover, an increased chemical composition heterogeneity associated with higher carbonate/phosphate ratio and higher mineral crystallinity was found in CFTR -/- trabecular bone, but not in CFTR -/- cortical bone. CONCLUSIONS: The loss of CFTR directly alters the bone composition and metabolism of newborn pigs. Based on these findings, we speculate that bone defects in patients with CF could be a primary, rather than a secondary consequence of inflammation and infection.

First molecular and serological evidence of Coxiella burnetti infection among sheep and goats of Jammu province of India.

The epidemiology and prevalence of Q fever in India is largely unknown. There are very few serologic and molecular reports of Q fever in India and these are old reports. The objective of this study was to investigate, for the first time, the presence of Coxiella burnetii infection in sheep and goat flocks of Jammu province of Jammu and Kashmir, India. A total of 148 milk (110 sheep and 38 goats) samples, 282 sera (170 sheep and 112 goats), and 152 vaginal swabs (123 sheep and 29 goats) were collected from farms with incidences of repeated abortion. The LSI Q fever ruminant serum/milk ELISA kit was used to identify anti-C. burnetii antibodies and nested PCR was employed to detect DNA in vaginal swabs. Overall, 42 (38.2%; 95% CI: 29.2-47.9) sheep and 9 (23.7%; 95% CI: 12.0-40.6) goat milk samples, and 21 (12.4%; 95% CI: 8.0-18.5) sheep and 11 (9.8%; 95% CI: 5.2-17.3) goat sera were ELISA positive. In addition, nine (7.3%; 95% CI: 3.6-13.8) vaginal swabs from sheep tested positive by nested PCR; however, C. burnetii could not be found in any of the vaginal swabs from goat. These results indicate that sheep seem to be a more important reservoir of C. burnetii than goats posing a risk for human infection in this area.

Immunomodulating effect of a seaweed extract from Ulva armoricana in pig: Specific IgG and total IgA in colostrum, milk, and blood.

The transfer of passive immunity from sows to piglets can be improved through the administration of immuno-stimulating products before farrowing. This study evaluated the immuno-stimulating effect of an algal sulfated polysaccharide extract (MSP extract) from the green algae Ulva armoricana when administrated orally to sows at the end of gestation. Four diets were tested: Control (no MSP extract), MSP1 (2 g/day of MSP extract), MSP2 (8 g/day), and MSP3 (16 g/day). The experimental diets were provided in two periods: before the last atrophic rhinitis vaccine booster, and a week before farrowing. Anti-Bordetella IgG antibodies were recorded in blood, colostrum, and milk, and total IgA were measured in colostrum and milk. Titer kinetics between the blood sampled before farrowing and colostrum displayed an increase in specific IgG for MSP3. Moreover, the MSP2 diet increased the level of total IgA in milk compared to the control group. Although the immuno-stimulating effect of MSP extract on piglet performance was not concurrent across the different supplementation levels, the present study supports the use of natural algae extract (MSP) as an immunomodulating solution in swine production.

Limited shedding of an S-InDel strain of porcine epidemic diarrhea virus (PEDV) in semen and questions regarding the infectivity of the detected virus.

PEDV is mainly transmitted by the oro-fecal route although PEDV shedding in semen has already been shown for an S-non-InDel PEDV strain infection. The aim of this study was to determine if PEDV can be shed in semen from SPF (specific pathogens free) boars infected by a French S-InDel PEDV strain (PEDV/FR/001/2014) and in case of positive semen to determine the infectivity of that semen. Both infected boars had diarrhea after inoculation and shed virus in feces. PEDV genome was also detected by RT-qPCR in the sperm-rich fraction of semen (6.94 × 103 and 4.73 × 103 genomic copies/mL) from the two boars infected with the S-InDel PEDV strain but only once at 7DPI. In addition, PEDV RNA in Peyer's patches and in mesenteric lymph nodes was also present for the two inoculated boars. The PEDV positive semen (S-non-InDel and S-InDel) sampled during a previous trial and in this boar trial were inoculated to six SPF weaned pigs. The inoculated piglets did not seroconvert and did not shed virus throughout the duration of the study except for one pig at 18 DPI. But, PEDV could be detected in intestinal tissues such as duodenum, jejunum and jejunum Peyer's patches by RT-qPCR except for one pig. Even if PEDV genome has been detected in semen, experimental infection of piglets with positive semen failed to conclude to the infectivity of the detected PEDV.

Lessons learnt from a porcine epidemic diarrhea (PED) case in France in 2014: Descriptive epidemiology and control measures implemented.

An acute epidemic of porcine epidemic diarrhea (PED) has affected the USA since 2013 and spread all around the world. In France, the immune status of the pig population against PED virus (PEDV) was expected to be low due to the absence of circulation of the virus since the 80's and a compulsory notification of PED was set up in 2014. Here, we reported the first case of a PED outbreak in December 2014 in the North of France after a long absence of the disease, the monitoring of the excretion and the control measure implementation. The isolated strain in France in December 2014 was a PEDV "S-InDel" strain which was close to the "S-InDel" German PEDV strain isolated in May 2014. The individual shedding duration of PEDV in feces was estimated around 20 days for pigs of different ages. Biosecurity measures implemented allowed the limitation of PEDV spread to fattening and farrowing rooms without dissemination to the nursery block. Using strict biosecurity measures, direct shipment of infected fatteners to the slaughterhouse, strict decontamination protocols with a quarantine of 6 weeks for replacement gilts without voluntary contamination helped PEDV fade out within the herd and avoided the spread to other herds. PEDV presence in manure was investigated as well as the inactivation treatment of the virus present in the liquid manure. An increase to a pH 12 of liquid manure by liming led to the absence of PEDV detection by RT-PCR after seven days.

Better horizontal transmission of a US non-InDel strain compared with a French InDel strain of porcine epidemic diarrhoea virus.

From the severe porcine epidemic diarrhoea (PED) epidemics that struck in 2013 in the United States of America and other countries of North and South America, two types of porcine epidemic diarrhoea virus (PEDV) were isolated, namely the InDel and the non-InDel strains. They are differentiated by insertions/deletions in the S1 nucleotide sequence of the S gene, and differences in virulence were observed from the clinical cases. In 2014, a PED outbreak occurred in a pig farm in France, from which an InDel strain was isolated. This study aimed at comparing, under experimental conditions, the pathogenicity and the direct and indirect transmissions between a non-InDel strain isolated from a PED-affected piglet in 2014 in the USA and the French InDel strain. All infected pigs showed clinical signs with the non-InDel strain although only the inoculated and direct contact pigs showed clinical signs in the InDel strain group. Although viral RNA was detected in air samples with both strains, the indirect contact pigs remained free from infection with the InDel strain in contrast to the non-InDel group in which airborne transmission occurred in the indirect contact pigs. All infected pigs shed virus in faeces regardless of PEDV strain with 9 of 30 pigs showing intermittent faecal shedding. The transmission rate by direct contact was found to be 2.17-fold higher than the non-InDel strain compared with the InDel. In conclusion, the InDel strain was less pathogenic than the non-InDel strain in our experimental conditions. The transmission route differed between the two strains. Direct contact was the main transmission route for the InDel strain, although the non-InDel strain was transmitted through direct contact and indirectly through the air.

Evidence of porcine epidemic diarrhea virus (PEDV) shedding in semen from infected specific pathogen-free boars.

In 2013, PED emerged for the first time in the United States (US). The porcine epidemic diarrhea virus (PEDV) spread quickly throughout North America. Infection with PEDV causes watery diarrhea and up to 100% mortality in piglets, particularly for highly pathogenic non-InDel strains circulating in the US. PEDV is mainly transmitted by the fecal-oral route. Transmission via the venereal route has been suspected but not previously investigated. The aim of the study was to determine if PEDV could be detected in semen from infected specific pathogen-free (SPF) boars inoculated with a PEDV US non-InDel strain suggesting venereal transmission may occur. Two boars orally inoculated with PEDV showed clinical signs and virus shedding in feces. Transient presence of the PEDV genome was detected by RT-qPCR in the seminal (5.06 × 102 to 2.44 × 103 genomic copies/mL) and sperm-rich fraction of semen (5.64 × 102 to 3.40 × 104 genomic copies/mL) and a longer duration of viral shedding was observed in the sperm-rich fraction. The evidence of PEDV shedding in semen raises new questions in term of disease spread within the pig population with the use of potentially contaminated semen.

The Pig: A Relevant Model for Evaluating the Neutrophil Serine Protease Activities during Acute Pseudomonas aeruginosa Lung Infection.

The main features of lung infection and inflammation are a massive recruitment of neutrophils and the subsequent release of neutrophil serine proteases (NSPs). Anti-infectious and/or anti-inflammatory treatments must be tested on a suitable animal model. Mice models do not replicate several aspects of human lung disease. This is particularly true for cystic fibrosis (CF), which has led the scientific community to a search for new animal models. We have shown that mice are not appropriate for characterizing drugs targeting neutrophil-dependent inflammation and that pig neutrophils and their NSPs are similar to their human homologues. We induced acute neutrophilic inflammatory responses in pig lungs using Pseudomonas aeruginosa, an opportunistic respiratory pathogen. Blood samples, nasal swabs and bronchoalveolar lavage fluids (BALFs) were collected at 0, 3, 6 and 24 h post-insfection (p.i.) and biochemical parameters, serum and BAL cytokines, bacterial cultures and neutrophil activity were evaluated. The release of proinflammatory mediators, biochemical and hematological blood parameters, cell recruitment and bronchial reactivity, peaked at 6h p.i.. We also used synthetic substrates specific for human neutrophil proteases to show that the activity of pig NSPs in BALFs increased. These proteases were also detected at the surface of lung neutrophils using anti-human NSP antibodies. Pseudomonas aeruginosa-induced lung infection in pigs results in a neutrophilic response similar to that described for cystic fibrosis and ventilator-associated pneumonia in humans. Altogether, this indicates that the pig is an appropriate model for testing anti-infectious and/or anti-inflammatory drugs to combat adverse proteolytic effects of neutrophil in human lung diseases.

Mycoplasma hyopneumoniae does not affect the interferon-related anti-viral response but predisposes the pig to a higher level of inflammation following swine influenza virus infection.

In pigs, influenza A viruses and Mycoplasma hyopneumoniae (Mhp) are major contributors to the porcine respiratory disease complex. Pre-infection with Mhp was previously shown experimentally to exacerbate the clinical outcomes of H1N1 infection during the first week after virus inoculation. In order to better understand the interactions between these pathogens, we aimed to assess very early responses (at 5, 24 and 48 h) after H1N1 infection in pigs pre-infected or not with Mhp. Clinical signs and macroscopic lung lesions were similar in both infected groups at early times post-H1N1 infection; and Mhp pre-infection affected neither the influenza virus replication nor the IFN-induced antiviral responses in the lung. However, it predisposed the animals to a higher inflammatory response to H1N1 infection, as revealed by the massive infiltration of neutrophils and macrophages into the lungs and the increased production of pro-inflammatory cytokines (IL-6, IL-1ß and TNF-α). Thus, it seems it is this marked inflammatory state that would play a role in exacerbating the clinical signs subsequent to H1N1 infection.

Phylogenetic network analysis applied to pig gut microbiota identifies an ecosystem structure linked with growth traits.

The ecological interactions within the gut microbial communities are complex and far from being fully understood. Here we report the first study that aims at defining the interaction network of the gut microbiota in pigs and comparing it with the enterotype-like clustering analysis. Fecal microbiota of 518 healthy piglets was characterized by 16S ribosomal RNA gene sequencing. Two networks were constructed at the genus and operational taxonomic unit levels. Within-network interactions mirrored the human gut microbiota relationships, with a strong co-exclusion between Prevotella and Ruminococcus genera, and were consistent with the two enterotype-like clusters identified in the pig microbiota. Remarkably, the cluster classification of the individuals was significantly associated with the body weight at 60 days of age (P=0.005) and average daily gain (P=0.027). To the best of our knowledge, this is the first study to provide an integrated overview of the porcine gut microbiota that suggests a conservation of the ecological community interactions and functional architecture between humans and pig. Moreover, we show that the microbial ecosystems and porcine growth traits are linked, which allows us to foresee that the enterotype concept may have an important role in the animal production industry.

Computed Tomography (CT) Scanning Facilitates Early Identification of Neonatal Cystic Fibrosis Piglets.

BACKGROUND: Cystic Fibrosis (CF) is the most prevalent autosomal recessive disease in the Caucasian population. A cystic fibrosis transmembrane conductance regulator knockout (CFTR-/-) pig that displays most of the features of the human CF disease has been recently developed. However, CFTR-/- pigs presents a 100% prevalence of meconium ileus that leads to death in the first hours after birth, requiring a rapid diagnosis and surgical intervention to relieve intestinal obstruction. Identification of CFTR-/- piglets is usually performed by PCR genotyping, a procedure that lasts between 4 to 6 h. Here, we aimed to develop a procedure for rapid identification of CFTR-/- piglets that will allow placing them under intensive care soon after birth and immediately proceeding with the surgical correction. METHODS AND PRINCIPAL FINDINGS: Male and female CFTR+/- pigs were crossed and the progeny was examined by computed tomography (CT) scan to detect the presence of meconium ileus and facilitate a rapid post-natal surgical intervention. Genotype was confirmed by PCR. CT scan presented a 94.4% sensitivity to diagnose CFTR-/- piglets. Diagnosis by CT scan reduced the birth-to-surgery time from a minimum of 10 h down to a minimum of 2.5 h and increased the survival of CFTR-/- piglets to a maximum of 13 days post-surgery as opposed to just 66 h after later surgery. CONCLUSION: CT scan imaging of meconium ileus is an accurate method for rapid identification of CFTR-/- piglets. Early CT detection of meconium ileus may help to extend the lifespan of CFTR-/- piglets and, thus, improve experimental research on CF, still an incurable disease.

Early-life establishment of the swine gut microbiome and impact on host phenotypes.

Early bacterial colonization and succession within the gastrointestinal tract has been suggested to be crucial in the establishment of specific microbiota composition and the shaping of host phenotype. Here, the composition and dynamics of faecal microbiomes were studied for 31 healthy piglets across five age strata (days 14, 36, 48, 60 and 70 after birth) together with their mothers. Faecal microbiome composition was assessed by 16S rRNA gene 454-pyrosequencing. Bacteroidetes and Firmicutes were the predominant phyla present at each age. For all piglets, luminal secretory IgA concentration was measured at day 70, and body weight was recorded until day 70. The microbiota of suckling piglets was mainly represented by Bacteroides, Oscillibacter, Escherichia/Shigella, Lactobacillus and unclassified Ruminococcaceae genera. This pattern contrasted with that of Acetivibrio, Dialister, Oribacterium, Succinivibrio and Prevotella genera, which appeared increased after weaning. Lactobacillus fermentum might be vertically transferred via breast milk or faeces. The microbiota composition coevolved with their hosts towards two different clusters after weaning, primarily distinguished by unclassified Ruminococcaceae and Prevotella abundances. Prevotella was positively correlated with luminal secretory IgA concentrations, and body weight. Our study opens up new possibilities for health and feed efficiency manipulation via genetic selection and nutrition in the agricultural domain.

Pattern recognition receptors in the gut: analysis of their expression along the intestinal tract and the crypt/villus axis.

Pattern recognition receptors (PRRs) play a critical role in the detection of microorganisms and the induction of inflammatory and immune responses. Using PCR and Western-blot analysis, this study investigated the differential expression in the intestine of 14 PRRs and nine associated cytokines. Thirty-two pigs were used to determine the expression of these markers (1) along the proximal/distal axis of the small intestine (duodenum, jejunum, and ileum) and (2) between the intestinal segments and their respective lymphoid organs (Peyer's patches [PP] and mesenteric lymph nodes [MLN]). Six additional animals were used to quantify the expression of these genes along the crypt/villus axis of jejunum, using microdissected samples. Most genes showed increased expression (1) in the distal than in the proximal parts of the small intestine (TLR3, 5, RIG-I, IL-1ß, IL-8, and IFN-γ); (2) in lymphoid organs (TLR1, 2, 6, 9, 10, IL-10, TNF-α), especially the MLN (TLR4, 7, 8, NOD1, NOD2, NALP3, IFN-α, IL-6, IL-12, and TGF-ß), than in intestinal segments. The analysis along the crypt/villus identified: (1) genes with higher expression in lamina propria (TLR1, 2, 4, 9, NOD1, NOD2, IL-1ß, IL-10, TGF-ß, TNF-α) and (2) genes with higher expression in the villus (TLR3, 5, 6, RIG-I, IL-6). These results highlight the differential expression of PRRs and cytokines along the proximal/distal and the crypt/villus axis of the intestine, contributing to a fine analysis of the complex functional architecture of the small intestine and should be related to the gut microbiota.

Innate immune response to a H3N2 subtype swine influenza virus in newborn porcine trachea cells, alveolar macrophages, and precision-cut lung slices.

Viral respiratory diseases remain of major importance in swine breeding units. Swine influenza virus (SIV) is one of the main known contributors to infectious respiratory diseases. The innate immune response to swine influenza viruses has been assessed in many previous studies. However most of these studies were carried out in a single-cell population or directly in the live animal, in all its complexity. In the current study we report the use of a trachea epithelial cell line (newborn pig trachea cells - NPTr) in comparison with alveolar macrophages and lung slices for the characterization of innate immune response to an infection by a European SIV of the H3N2 subtype. The expression pattern of transcripts involved in the recognition of the virus, interferon type I and III responses, and the host-response regulation were assessed by quantitative PCR in response to infection. Some significant differences were observed between the three systems, notably in the expression of type III interferon mRNA. Then, results show a clear induction of JAK/STAT and MAPK signaling pathways in infected NPTr cells. Conversely, PI3K/Akt signaling pathways was not activated. The inhibition of the JAK/STAT pathway clearly reduced interferon type I and III responses and the induction of SOCS1 at the transcript level in infected NPTr cells. Similarly, the inhibition of MAPK pathway reduced viral replication and interferon response. All together, these results contribute to an increased understanding of the innate immune response to H3N2 SIV and may help identify strategies to effectively control SIV infection.

Extensive expression differences along porcine small intestine evidenced by transcriptome sequencing.

The aim of this study was to analyse gene expression along the small intestine (duodenum, jejunum, ileum) and in the ileal Peyer's patches in four young pigs with no clinical signs of disease by transcriptome sequencing. Multidimensional scaling evidenced that samples clustered by tissue type rather than by individual, thus prefiguring a relevant scenario to draw tissue-specific gene expression profiles. Accordingly, 1,349 genes were found differentially expressed between duodenum and jejunum, and up to 3,455 genes between duodenum and ileum. Additionally, a considerable number of differentially expressed genes were found by comparing duodenum (7,027 genes), jejunum (6,122 genes), and ileum (6,991 genes) with ileal Peyer's patches tissue. Functional analyses revealed that most of the significant differentially expressed genes along small intestinal tissues were involved in the regulation of general biological processes such as cell development, signalling, growth and proliferation, death and survival or cell function and maintenance. These results suggest that the intrinsic large turnover of intestinal tissues would have local specificities at duodenum, ileum and jejunum. In addition, in concordance with their biological function, enteric innate immune pathways were overrepresented in ileal Peyer's patches. The reported data provide an expression map of the cell pathway variation in the different small intestinal tissues. Furthermore, expression levels measured in healthy individuals could help to understand changes in gene expression that occur in dysbiosis or pathological states.

CCL28 involvement in mucosal tissues protection as a chemokine and as an antibacterial peptide.

CCL28 chemokine is expressed by epithelial cells of various mucosal tissues. This chemokine binds to CCR3 and CCR10 receptors and plays an essential role in the IgA antibody secreting cells (IgA-ASC) homing to mucosal surfaces and to lactating mammary gland as well. In addition, CCL28 has been shown to exert a potent antimicrobial activity against both Gram-negative and Gram-positive bacteria and fungi. Using the pig model, we investigated the expression of both CCR10 and CCR3 receptors in a large panel of mucosal tissues. RT-PCR analysis revealed the expression of CCR3 and CCR10 mRNA in salivary glands, nasal mucosae, Peyer's patches, small and large intestine, suggesting the presence of leucocytes expressing these receptors within these tissues. CCR10 mRNA was observed in sow mammary gland at late gestation with an increasing level during lactation. Recombinant porcine CCL28 protein was produced and mass spectrometry analysis revealed antimicrobial chemokines features such as a high pI value (10.2) and a C-terminal highly positively-charged region. Using a viable count assay, we showed that CCL28 displayed antimicrobial activity against enteric pathogens and was effective in killing Salmonella serotypes Dublin and Choleraesuis, enteroinvasive Escherichia coli K88 and non-pathogenic E. Coli K12. The potent antimicrobial function of CCL28 combined with its wide distribution in mucosal tissues and secretions suggest that this protein plays an important role in innate immune protection of the epithelial surfaces.