Impact of feed additives and host-related factors on bacterial metabolites, mucosal integrity and immune response in the ileum of broilers.

The present study aimed to investigate the effect of age, breed, and sex of broilers, as well as a probiotic or phytobiotic product on mucosal morphology, bacterial metabolites, and immune traits in the ileum of broilers. A total of 2,880 one-day-old male and female broiler chicks from two breeds (Ross308® and Cobb500®) were randomly assigned to 72 pens. Broilers were offered a wheat-soybean diet without (CO), or with either a probiotic (PO; 2.4 × 109 CFU/kg of Bacillus subtilis DSM32324 and DSM32325 and B. amyloliquefaciens DSM25840) or a phytobiotic (PY; grape extract, 165 ppm procyanidin and 585 ppm polyphenols of the diet) product. The trial was conducted with a 3 × 2 × 2 factorial arrangement of diet, breed, and sex in a completely randomized design (6 replicate-pens per treatment). At day 7, 21, and 35, one chicken per pen was slaughtered for collecting ileal tissue to evaluate of histomorphology and mRNA expression, as well as ileal digesta to measure bacterial metabolites. Data were subjected to ANOVA (the main factors; age, diet, breed, and sex) and Four-Way ANOVA (interactions) using GLM procedure. Overall, the concentration of acetate and total short chain fatty acids reached the peak and lactate decreased to its lowest on day 21, but their concentrations at day 7 and 35 were similar (p > 0.05). Spermine, spermidine, and ammonia decreased after day 7, while putrescine and cadaverine increased after day 21 (p < 0.05). mRNA expression of cytokines, mucin 2 (MUC2) and claudin 5 (CLDN5) was similar; increased from day 7 to 21 and decreased afterward (p < 0.05). Villus height, crypt depth and villus surface area increased with age (p < 0.05). Acidic goblet cells (GC) number and density increased after day 21 (p < 0.05). Ross broilers showed higher D-lactate concentration and IFN-γ expression, while Cobb broilers had greater IL-4, IL-6 and TNF-α expression and higher total GC number (p < 0.05). Female displayed higher villus height and GC number and density (mixed and total GC) than male (p < 0.05). The effect of dietary treatment was not found on any investigated variables (p > 0.05). In conclusion, aging of broilers affected ileal histomorphology, cytokine expression, and barrier integrity, as well as bacterial activity. These observed impacts could be attributed to host-microbiota interaction and the direct effects of bacterial metabolites on intestinal cells and immune system.

Novel strategies to improve chicken performance and welfare by unveiling host-microbiota interactions through hologenomics.

Fast optimisation of farming practices is essential to meet environmental sustainability challenges. Hologenomics, the joint study of the genomic features of animals and the microbial communities associated with them, opens new avenues to obtain in-depth knowledge on how host-microbiota interactions affect animal performance and welfare, and in doing so, improve the quality and sustainability of animal production. Here, we introduce the animal trials conducted with broiler chickens in the H2020 project HoloFood, and our strategy to implement hologenomic analyses in light of the initial results, which despite yielding negligible effects of tested feed additives, provide relevant information to understand how host genomic features, microbiota development dynamics and host-microbiota interactions shape animal welfare and performance. We report the most relevant results, propose hypotheses to explain the observed patterns, and outline how these questions will be addressed through the generation and analysis of animal-microbiota multi-omic data during the HoloFood project.

Alterations in bacterial metabolites, cytokines, and mucosal integrity in the caecum of broilers caused by feed additives and host-related factors.

A total of 2,880 one-day-old male and female broiler chicks from two breeds, Ross308 and Cobb500 were randomly assigned to 72 pens. Broilers were offered three diets: a wheat-soybean diet without (CO), or with either a probiotic (probiotic; 2.4 x 109 CFU/kg diet of Bacillus subtilis DSM32324 and DSM32325 and B. amyloliquefaciens DSM25840) or a phytobiotic (phytobiotic; grape extract with 165 ppm procyanidin and 585 ppm polyphenol) product. The trial was conducted with a 3 × 2 × 2 factorial arrangement of diet, breed and sex in a completely randomized design and consisted of 6 replicate-pens per treatment (40 birds per pen). At day 7, 21, and 35, one chicken per pen was slaughtered for caecal sampling to quantify bacterial metabolites (digesta) as well as evaluate mRNA abundance and histomorphology (tissue). Data were subjected to ANOVA using GLM procedure to evaluate age, diet, breed and sex and their interactions. Spearman's correlation (r) was analyzed between metabolite concentration and mRNA abundance. Overall, the concentration of short chain fatty acids increased with age, while lactate decreased from day 7 to 21 (p < 0.05). The mRNA abundance of IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17α, IL-18, IFN-γ and TGF-ß2 increased with age but IL-1ß and TNF-α increased in abundance from day 7 to 21 and then decreased (p < 0.05). Abundance of MUC2 and CLDN5 increased after day 21 (p < 0.05). Caecal crypt depth increased with age (p < 0.05). Acidic goblet cell (GC) number peaked at day 21 (p < 0.05), while mixed GC number was not affected by age. A few impacts of breed, diet and interactions on the investigated variables showed no meaningful biological pattern. Propionate positively correlated with all cytokines investigated (r = 0.150-0.548), except TNF-α. Lactate negatively correlated with pro-inflammatory cytokines like IL-1ß (r = -0.324). Aging affected caecal histomorphology, bacterial activity and genes responsible for barrier integrity and inflammatory response. This effect could be attributed to the interaction between gut microbiota and immune system as well as the direct effect of metabolites on gut histomorphology and cytokine mRNA abundance.

Effects of Exogenous 6-Phytase (EC 3.1.3.26) Supplementation on Performance, Calcium and Phosphorous Digestibility, and Bone Mineralisation and Density in Weaned Piglets.

Phosphorus (P) is an essential mineral for growing piglets, which is poorly accessible in vegetable feedstuffs as it is stored as phytates. Thus, phytase supplementation is essential to increase P availability. Two experiments were conducted to evaluate a novel 6-phytase (EC 3.1.3.26) in weaned pigs fed low-P diets. In each experiment, one hundred and twenty piglets were fed a positive control (PC; adequate in Ca and P), a negative control (NC; limiting in Ca and P), or NC supplemented with 125, 250, or 500 FTU/kg of phytase (NC125, NC250, and NC500, respectively). P content was lower in diets of Experiment 1 than diets of Experiment 2. In Experiment 1, piglets offered PC or phytase diets had higher growth and efficiency compared with NC diets. In Experiment 2, similar effects were obtained, but the effects were less significant. In both experiments, P and Ca ATTD and bone density were significantly increased with phytase supplementation. Moreover, PC and NC500 had higher P concentrations and lower alkaline phosphatase activity in plasma than NC. To conclude, supplementation with the new 6-phytase at doses up to 500 FTU/kg enhanced P utilization, growth performance, and bone density in piglets fed P-limiting diets.

The Control of Intestinal Inflammation: A Major Objective in the Research of Probiotic Strains as Alternatives to Antibiotic Growth Promoters in Poultry.

The reduction of antimicrobial resistance is a major challenge for the scientific community. In a few decades, infections by resistant bacteria are forecasted to be the main cause of death in the world. The withdrawal of antibiotics as growth promoters and their preventive use in animal production is essential to avoid these resistances, but this may impair productivity and health due to the increase in gut inflammation. This reduction in productivity aggravates the problem of increasing meat demand in developing countries and limits the availability of raw materials. Probiotics are promising products to address this challenge due to their beneficial effects on microbiota composition, mucosal barrier integrity, and immune system to control inflammation. Although many modes of action have been demonstrated, the scientific community is not able to describe the specific effects that a probiotic should induce on the host to maximize both productivity and animal health. First, it may be necessary to define what are the innate immune pathways acting in the gut that optimize productivity and health and to then investigate which probiotic strain is able to induce the specific effect needed. This review describes several gaps in the knowledge of host-microbiota-pathogen interaction and the related mechanisms involved in the inflammatory response not demonstrated yet in poultry.

Evaluation of the interval cancer rate and its determinants on the Girona Health Region's early breast cancer detection program.

BACKGROUND: The main aim of this study is to estimate the rate of false negative and true IC on the Program for the Early Detection of Breast Cancer (PEDBC) run by the Girona Health Region (GHR) and compare the clinicopathological characteristics of these tumors with those detected within the same program. METHODS: A retrospective cohort study including all women participating on the Girona PEDBC between 2000 and 2006, with negative mammography screening. The IC included are those detected between the first and second round of screening and between the second and third round. RESULTS: We identified a total of 43 IC, representing an incidence rate of 0.70 cases per 1,000 screened women. Of the 43 probable IC, we were able to classify a total of 22 (51.2%) cases. Of these 22 cases, 54.5% were classified as true interval tumors, 13.6% false negatives, 18.2% occult tumors and the remaining 13.6% minimal sign.We found significant differences in some clinicopathological characteristics of the IC comparing with the tumors detected within the program during the same period. CONCLUSIONS: The IC rate for the PEDBC is within the expected parameters, with a high proportion of cases of true interval cancers (54.5%) and a low proportion of false negatives (13.6%). The results show that the proportional incidence of IC is within the limits set by European Guidelines. Furthermore, it has been confirmed that IC display more aggressive clinicopathological characteristics than screening breast cancers.

The impact of CSFV on the immune response to control infection.

The severity of the acute form of CSF is responsible for the high mortality rate and has been the subject of many studies. Nevertheless, some animals are likely to develop a mild, chronic, or unapparent form of the disease. Paradoxically, this clinical form of the disease has not been well studied, especially regarding its pathogenesis. In this study, we investigated the infection in domestic pigs that is caused by the CSFV Cat01 strain, which is responsible for the 2001-2002 CSFV outbreak in Catalonia, Spain, and which caused mild and nonspecific clinical signs compared to the infection that is caused by another CSFV strain that is responsible for inducing severe clinical symptoms of disease. We assessed the impact of the CSFV infection in the immune system of domestic pigs, mainly on the kinetics of different cytokines, such as IFN-α (innate immunity) and IFN-γ (adaptive immune response), during the first weeks after infection. In addition, we evaluated the impact on the induction of the humoral response and its relation to the course of infection and the RNA CSFV viral load. The IFN-α levels in the serum samples from the pigs that developed a milder form of the CSF disease (infected with Cat01 strain) were lower than those that were detected in the pig with severe clinical CSF signs (Margarita strain). After infection with Cat01 strain, the IFN-γ levels in response to CSFV were detected in addition to the humoral response. Interestingly, in the serum samples of these animals, we detected the lowest load of CSFV RNA. Similarly, the lowest viral load levels were detected in the tonsils of these pigs. Both the T cells and the humoral response that were generated in most of the pigs that were infected with strain Cat01 may be related to the protection in the symptom progression of CSF against this viral strain. These results explain the antiviral role of IFN-γ in the absence of an antibody response. Likewise, these results corroborate the relevance and relationship that exists between the intensity of the T cell response and the protection against CSFV replication. Additionally, these results also explain how the failure to induce optimal levels of humoral and cellular responses after CSFV infection promotes the spread and persistence of the virus.

Immunization with DNA vaccines containing porcine reproductive and respiratory syndrome virus open reading frames 5, 6, and 7 may be related to the exacerbation of clinical disease after an experimental challenge.

Pigs were immunized with DNA plasmids containing different open reading frames (ORFs) of a porcine reproductive and respiratory syndrome virus (PRRSV) genotype I strain. One group was injected with three inoculations of ORF7, a second group was immunized with three inoculations of plasmids containing ORF5 and ORF6, and a third group was kept as controls. Later, +21 days after the last inoculation, animals were challenged with the homologous strain. After the challenge, PRRSV-specific interferon (IFN)-γ-secreting cells and anti-PRRSV IgG antibodies developed faster in DNA vaccinated pigs (p<0.05). However, DNA-immunized pigs showed an exacerbation of the disease compared to the unvaccinated challenged pigs. The data suggest that previous immunization with DNA vaccines against glycoprotein 5 and/or matrix protein of PRRSV, as well as nucleoprotein but to a lesser degree, could result in an exacerbation of the clinical course in terms of fever upon challenge.

A T-cell epitope on NS3 non-structural protein enhances the B and T cell responses elicited by dendrimeric constructions against CSFV in domestic pigs.

It has been recently reported by our group that dendrimeric constructs combining B- and T-cell epitopes from classical swine fever virus (CSFV) provided partial protection against experimental infection. This research evaluated four newly designed constructions while taking into account our previous work, including the direct implication that a T-cell epitope from the NS3 protein contributes to the generation of the immune response against CSFV. To this end, the dendrimeric constructions, including either this NS3 T-cell epitope alone or two different B-cell epitopes without this T-cell epitope, were used to immunise pigs. Thus, construct 1, containing the NS3 T-cell epitope and four copies of a previously described B-cell epitope, significantly reduced the clinical scores and RNA viral loads after challenge relative to the control group. In three out of six animals in this group, vaccination achieved partial protection and was associated with IFN-gamma producing-cells and neutralising antibodies. In contrast, the pigs immunised with construct 2, again with four copies of the B epitope of construct 1 but lacking the T-cell motif, developed more severe clinical signs. Finally, the additional constructs 3 and 4 included four copies of a B epitope that was different from the epitope used in constructs 1 and 2 with or without the abovementioned NS3 T-cell epitope, respectively. Pigs immunised with these latter constructs developed low levels of peptide-specific antibodies that correlated with equally low levels of cellular responses, an absence of neutralising antibodies and a lack of protection. Even so, the clinical scores in the first week after the challenge were less severe for animals vaccinated with construct 3 than for those given construct 4. Our results confirm the relevant role of the B-cell epitope in residues 694-712 of the glycoprotein E2 (which is used in both constructs 1 and 2) for protection against CSFV, as well as the appropriateness of the newly used NS3 peptide as a specific T-cell epitope in domestic pigs.

Positive selection pressure on the B/C domains of the E2-gene of classical swine fever virus in endemic areas under C-strain vaccination.

In Cuba, classical swine fever (CSF) has become an endemic disease with several outbreaks each year, despite the implemented vaccination program. Interestingly, a trend towards a milder presentation of the disease has been observed among the animals during the last years. This study aimed to assess positive selection pressure acting on partial E2 gene of CSF viruses to gain insights into the mechanisms governing virulence and the driving forces of classical swine fever virus (CSFV) evolution in swine populations under regular vaccination. Selection pressure analysis were performed to detect positive selection acting on a particular lineage as well as among sites of the E2-B/C-domain of CSFV nucleotide sequences, reported in a previous study and in the present work, several models, available in the CODEML module of PAML 4.3, were used. In addition, a representative Cuban CSF isolate was assessed in an experimental infection trial for their clinical virulence in order to expand the knowledge regarding CSF viruses circulating in pig populations. The viral genomes sequenced in this study were grouped in a defined cluster within the genotype 1.2, as it has been reported previously for Cuban CSF viruses. The selection pressure analysis didn't find evidence of positive selection (dN/dS of>1) along any branch. The positive selective pressure analysis estimated six new sites under positive selection on E2 partial gene analysed. Besides, the clinical manifestations of the CSF-disease were related mainly to a mild course of the illness. The high number of positively selected sites suggests that these changes could be associated to viral evasion of the host-immune response. These observations highlight a possible association between escape viral variants and the alterations observed in the virulence and pathogenesis of the virus. Therefore, while the vaccination programs have not led to a genotype change, alterations in virulence were suggested to arise.

Immunomodulatory effect of swine CCL20 chemokine in DNA vaccination against CSFV.

The objective of this work was to explore whether a plasmid expressing CCL20 chemokine could improve the immune response against CSFV in co-administration with a DNA vaccine expressing the E2 protein. The immunization of pigs with the DNA vaccine formulation, that contains swine CCL20 chemokine, resulted in the homogenous induction of detectable levels of CSFV antibodies at 36 days after the first injection. Remarkably, immunized animals with E2 DNA vaccine in co-administration with the plasmid containing swine CCL20 developed high titers of neutralizing antibodies against homologous and heterologous CSFV strains and were totally protected upon a lethal viral challenge (sterilizing protection). Our results confirm the role of CCL20 to increase antibody-mediated responses. At the same time suggest the ability of CCL20 to enhance the T helper cell response associated with the induction of neutralizing antibodies against CSFV in pigs previously reported. Systemic replication of virulent CSFV in vivo during the acute phase of infection induces type I IFN. Lower average values of IFN alpha were detected in the serum of pigs immunized with pE2 and pCCL20 at 3 days after challenge. The levels of IFN-alpha detected in pigs immunized with pE2 and principally in non-vaccinated challenged animals can be related to viral load in serum at 3 and 7 days post infection and the clinical signs observed. Our results emphasized the capacity of swine CCL20 chemokine to enhance cellular, humoral and anti viral response with an adjuvant effect in the immune response elicited by E2-DNA vaccination against CSFV. To our knowledge, this is the first report demonstrating the adjuvant effect of swine CCL20 to effectively enhance the potential of DNA vaccine in the immune induction and protection against virus challenge in swine infection model.

Development and validation of a novel SYBR Green real-time RT-PCR assay for the detection of classical swine fever virus evaluated on different real-time PCR platforms.

Classical swine fever is a highly contagious viral disease that causes significant economic losses in pig production on a global scale. The rapid dissemination of the virus and the variability of the clinical signs merit the development of swift and accurate classical swine fever virus (CSFV) detection methods, which can assist in disease control. The development and evaluation of a novel quantitative real-time RT-PCR assay for CSFV detection, based on SYBR Green coupled to melting curve analysis, is described. The analytical and diagnostic performances of the method using two real-time PCR instruments were compared. The assay was specific and detected the major genotypes of CSFV. The limit of detection in cell culture medium and serum was 0.1 TCID50/reaction, while in tissue homogenate for both platforms, it was 1 TCID50/reaction. The limit of detection was 1, 10 and 10² gene copies/µL when nuclease-free water, serum and tissue homogenate, respectively, were used as sample matrices for both instruments. The analysis of 108 tissue homogenate and serum samples from animals infected with CSFV naturally and experimentally and non-infected animals showed that the assay provided a highly sensitive and specific method for classical swine fever.

Phylogenetic networks to study the origin and evolution of porcine circovirus type 2 (PCV2) in Cuba.

Porcine circovirus type 2 (PCV2) is the essential etiological infectious agent of postweaning multisystemic wasting syndrome (PMWS), which is considered one of the most economically important swine diseases worldwide. In this study, a comparison between methodologies based on classical phylogenetic trees and networks to infer the origin of PCV2 in Cuba was performed. In addition, the mechanisms supporting the genetic variability of Cuban PCV2 populations were investigated. A retrospective study, using pig sera collected in Cuba from 1993 to 2004, to evaluate the presence of PCV2 genome and PCV2-specific antibodies was also conducted and revealed a lack of evidence of PCV2 infection in Cuban swine from years 1993 to 2004. A total of 24 complete Cuban PCV2 sequences collected between 2005 and 2009 from different regions of the country were analyzed. Three classical methods of phylogenetic analysis, namely Neighbour-Joining, Maximum Parsimony and Bayesian Inference, as well as haplotype network construction, were used. Whereas the classical phylogenetic trees suggested different origins for the Cuban PCV2 strains, the haplotype network revealed a direct connection between all the Cuban sequences in agreement with the obtained epidemiological and viral sequence data. Moreover, the importation of pigs carried out in 2005 from the Quebec-Ontario region, Canada, seems to be the most likely origin of PCV2 in Cuba. Likewise, the genetic variability of Cuban PCV2 sequences was supported by geographic segregation and positive selection pressure with estimated rates of nucleotide substitution on the order of 3.12×10(-3) and 6.57×10(-3) substitutions/site/year, which are closer to those reported for RNA viruses.

Partial protection against classical swine fever virus elicited by dendrimeric vaccine-candidate peptides in domestic pigs.

We report the immunogenicity of three dendrimeric peptide vaccine candidates for classical swine fever virus (CSFV). Each dendrimeric construct contained four copies of a B-cell epitope from the E2 glycoprotein of CSFV [construct 1: E2 (694-712); 2: E2 (712-727); 3: E2 (829-842)] joined to a T-cell epitope from the NS3 protein (residues 1446-1460). Intramuscular immunization of domestic pigs with the different constructs significantly reduced the clinical score after lethal challenge with CSFV. In contrast, control pigs developed severe clinical signs of the disease. All pigs vaccinated with construct 1, containing a B-cell epitope from the E2 B-C domain, developed an antibody response that recognized not only the original dendrimeric immunogen but also its constituting E2 epitope in linear form, albeit no neutralizing antibodies were detected prior to viral challenge. Two of these pigs were partially protected, which associated with the induction of IFN-γ producing cells and of neutralizing antibodies upon challenge. Interestingly, the serological response elicited by construct 1 lacked antibodies to E2 A domain, used as infection markers. The dendrimeric approach could therefore provide a basis for the development of CSFV marker (DIVA) vaccines, and contribute to a better understanding of the immune responses against CSFV.

Peptide vaccine candidates against classical swine fever virus: T cell and neutralizing antibody responses of dendrimers displaying E2 and NS2-3 epitopes.

Three peptide-based systems integrating B and T antigenic sites of CSFV and displaying the B epitopes in fourfold presentation have been designed and produced, and shown to bring about significant enhancements in immunogenicity over the peptides in monomeric form. Of the different strategies tested for producing the dendrimeric constructs, stepwise SPPS using 3,6-dioxaoctanoic acid as flexible, PEG-like spacer units at the branching points is clearly advantageous, in particular over ligation in solution. The constructs have been used for immunization of domestic pigs, in order to evaluate the protective response induced by each peptide constructs, and to characterize the B- and T-cell response against CSFV in the natural host.

A multiplex RT-PCR assay for the rapid and differential diagnosis of classical swine fever and other pestivirus infections.

Classical swine fever is a highly contagious viral disease causing severe economic losses in pig production almost worldwide. All pestivirus species can infect pigs, therefore accurate and rapid pestivirus detection and differentiation is of great importance to assure control measures in swine farming. Here we describe the development and evaluation of a novel multiplex, highly sensitive and specific RT-PCR for the simultaneous detection and rapid differentiation between CSFV and other pestivirus infections in swine. The universal and differential detection was based on primers designed to amplify a fragment of the 5' non-coding genome region for the detection of pestiviruses and a fragment of the NS5B gene for the detection of classical swine fever virus. The assay proved to be specific when different pestivirus strains from swine and ruminants were evaluated. The analytical sensitivity was estimated to be as little as 0.89TCID(50). The assay analysis of 30 tissue homogenate samples from naturally infected and non-CSF infected animals and 40 standard serum samples evaluated as part of two European Inter-laboratory Comparison Tests conducted by the European Community Reference Laboratory, Hanover, Germany proved that the multiplex RT-PCR method provides a rapid, highly sensitive, and cost-effective laboratory diagnosis for classical swine fever and other pestivirus infections in swine.