Effects of a novel, non-invasive pre-hatch application of probiotic for broilers on development of cecum microbiota and production performance.

BACKGROUND: Probiotics are used in the broiler industry to increase production performance. Most often a probiotic is applied by mixing it in the feed, but studies have shown that earlier application may be advantageous. Therefore, in ovo application where the probiotic is administrated into the egg before hatch has been investigated as an alternative application method. However, in ovo application may impact hatchability negatively and may not be feasible at all hatcheries. The purpose of this study was to investigate the effect of a novel non-invasive method for mass application before hatch. The probiotic (E. faecium 669) was applied as a single dose by spray on the unhatched eggs and production performance and development of the cecal microbiota until slaughter was compared with a control flock. Through 16S rRNA sequencing of cecal samples from 25 broilers at day 7, 21 and 37 we compared the microbiota composition and richness for each group. The study was repeated for additional recording of production performance and re-isolation of the probiotic E. faecium from the intestine. RESULTS: In both trials the probiotic E. faecium could be re-isolated from the yolk sac and intestine at hatch and at day 7. Broilers in the probiotic treated groups had a higher performance in terms of bodyweight at day 34 and European production efficiency factor. Finally, a significant reduction of first-week and overall mortality was observed in the probiotic group in the first trial. Based on 16S rRNA profiling, significant differences in alpha diversity were found exclusively at day 37. Estimation of beta diversities, however, identified significant differences in microbiota composition between the control and probiotic group at day 7, 21 and 37. CONCLUSION: The probiotic E. faecium strain successfully colonized broilers before/during hatch after a single spray application at day 18 of incubation. Positive effects of the probiotic were observed in multiple production parameters, including reduced mortality in trial 1, and microbiota analyses indicate significantly different microbiota compositions throughout the experimental phase. Taken together, the novel low-tech mass administration of E. faecium (669) may be considered a feasible strategy for improvements of production parameters in broiler production.

Isolation and characterization of multidrug resistant Gallibacterium anatis biovar haemolytica strains from Polish geese and hens.

Gallibacterium anatis biovar haemolytica is a bacterium that is frequently associated with infections of the reproductive tract and respiratory system in poultry. To assess the current prevalence and resistance profile of these bacteria in Poland, we collected and investigated 63 strains of Gallibacterium from diseased domestic poultry flocks including geese, laying hens, breeding hens and an ornamental hen. Detailed characterization of the isolates included the analysis of phenotypic antimicrobial resistance profiles and biofilm formation ability. Furthermore, the genetic background of 40 selected isolates regarding the presence of virulence and antimicrobial resistance genes and mobile genetic elements was determined. All investigated isolates were multidrug resistant, most prominently to ß-lactams, fluoroquinolones, sulfonamides and macrolides. A total of 48 different resistance profiles were detected. Of all isolates, 50.8% formed a strong biofilm, where strains isolated from geese appeared to be better at biofilm formation than strains isolated from laying and breeding hens. Single-nucleotide polymorphism genotyping revealed that G. anatis bv. haemolytica strains are restricted in host and geographical distribution, and the geese isolates showed greater phylogenetic similarity. Whole genome sequencing enabled identification of 25 different antimicrobial resistance determinants. The most common resistance genes were tetB, blaROB-1, and blaTEM-1 which may be located on mobile genetic elements. All isolates possessed the toxin gene gtxA, and the fimbrial gene flfA was identified in 95% of strains. Our results indicated that all G. anatis bv. haemolytica isolates showed multidrug resistant phenotypes. Strains isolated from geese were characterized by the highest percentage of isolates resistant to selected antimicrobials, probably reflecting host-related adaptations.

Genomic Analysis of Escherichia coli Longitudinally Isolated from Broiler Breeder Flocks after the Application of an Autogenous Vaccine.

Escherichia coli is the main bacterial cause of major economic losses and animal welfare issues in poultry production. In this study, we investigate the effect of an autogenous vaccine on E. coli strains longitudinally isolated from broiler breeder flocks on two farms. In total, 115 E. coli isolates were sequenced using Illumina technologies, and compared based on a single-nucleotide polymorphism (SNP) analysis of the core-genome and antimicrobial resistance (AMR) genes they carried. The results showed that SNP-based phylogeny corresponds to a previous multilocus-sequence typing (MLST)-based phylogeny. Highly virulent sequence types (STs), including ST117-F, ST95-B2, ST131-B2 and ST390-B2, showed a higher level of homogeneity. On the other hand, less frequent STs, such as ST1485, ST3232, ST7013 and ST8573, were phylogenetically more distant and carried a higher number of antimicrobial resistance genes in most cases. In total, 25 antimicrobial genes were detected, of which the most prevalent were mdf(A) (100%), sitABCD (71.3%) and tet(A) (13.91%). The frequency of AMR genes showed a decreasing trend over time in both farms. The highest prevalence was detected in strains belonging to the B1 phylogenetic group, confirming the previous notion that commensal strains act as reservoirs and carry more resistance genes than pathogenic strains that are mostly associated with virulence genes.

A multi-omics approach unravels metagenomic and metabolic alterations of a probiotic and synbiotic additive in rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Animal protein production is increasingly looking towards microbiome-associated services such as the design of new and better probiotic solutions to further improve gut health and production sustainability. Here, we investigate the functional effects of bacteria-based pro- and synbiotic feed additives on microbiome-associated functions in relation to growth performance in the commercially important rainbow trout (Oncorhynchus mykiss). We combine complementary insights from multiple omics datasets from gut content samples, including 16S bacterial profiling, whole metagenomes, and untargeted metabolomics, to investigate bacterial metagenome-assembled genomes (MAGs) and their molecular interactions with host metabolism. RESULTS: Our findings reveal that (I) feed additives changed the microbiome and that rainbow trout reared with feed additives had a significantly reduced relative abundance of the salmonid related Candidatus Mycoplasma salmoninae in both the mid and distal gut content, (II) genome resolved metagenomics revealed that alterations of microbial arginine biosynthesis and terpenoid backbone synthesis pathways were directly associated with the presence of Candidatus Mycoplasma salmoninae, and (III) differences in the composition of intestinal microbiota among feed types were directly associated with significant changes of the metabolomic landscape, including lipids and lipid-like metabolites, amino acids, bile acids, and steroid-related metabolites. CONCLUSION: Our results demonstrate how the use of multi-omics to investigate complex host-microbiome interactions enable us to better evaluate the functional potential of probiotics compared to studies that only measure overall growth performance or that only characterise the microbial composition in intestinal environments. Video Abstract.

Integrative analyses of probiotics, pathogenic infections and host immune response highlight the importance of gut microbiota in understanding disease recovery in rainbow trout (Oncorhynchus mykiss).

AIMS: Given the pivotal role played by the gut microbiota in regulating the host immune system, great interest has arisen in the possibility of controlling fish health by modulating the gut microbiota. Hence, there is a need to better understand of the host-microbiota interactions after disease responses to optimize the use of probiotics to strengthen disease resilience and recovery. METHODS AND RESULTS: We tested the effects of a probiotic feed additive in rainbow trout and challenged the fish with the causative agent for enteric red mouth disease, Yersinia ruckeri. We evaluated the survival, host immune gene expression and the gut microbiota composition. Results revealed that provision of probiotics and exposure to Y. ruckeri induced immune gene expression in the host, which were associated with changes in the gut microbiota. Subsequently, infection with Y. ruckeri had very little effect on microbiota composition when probiotics were applied, indicating that probiotics increased stabilisation of the microbiota. Our analysis revealed potential biomarkers for monitoring infection status and fish health. Finally, we used modelling approaches to decipher interactions between gut bacteria and the host immune gene responses, indicating removal of endogenous bacteria elicited by non-specific immune responses. CONCLUSIONS: We discuss the relevance of these results emphasizing the importance of host-microbiota interactions, including the protective potential of the gut microbiota in disease responses. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results highlight the functional consequences of probiotic-induced changes in the gut microbiota post infection and the resulting host immune response.

Bacterial Outer Membrane Vesicles as a Versatile Tool in Vaccine Research and the Fight against Antimicrobial Resistance.

Gram-negative bacteria include a number of pathogens that cause disease in humans and animals. Although antibiotics are still effective in treating a considerable range of infections caused by Gram-negative bacteria, the alarming increase of antimicrobial resistance (AMR) induced by excessive use of antibiotics has raised global concerns. Therefore, alternative strategies must be developed to prevent and treat bacterial infections and prevent the advent of a postantibiotic era. Vaccines, one of the greatest achievements in the history of medical science, hold extraordinary potential to prevent bacterial infections and thereby reduce the need for antibiotics. Novel bacterial vaccines are urgently needed, however, and outer membrane vesicles (OMVs), naturally produced by Gram-negative bacteria, represent a promising and versatile tool that can be employed as adjuvants, antigens, and delivery platforms in the development of vaccines against Gram-negative bacteria. Here, we provide an overview of the many roles OMVs can play in vaccine development and the mechanisms behind these applications. Methods to improve OMV yields and a comparison of different strategies for OMV isolation aiming at cost-effective production of OMV-based vaccines are also reviewed.

Synbiotic feed supplementation significantly improves lipid utilization and shows discrete effects on disease resistance in rainbow trout (Oncorhynchus mykiss).

Enteric redmouth disease caused by the bacterial pathogen Yersinia ruckeri is the main reason for antimicrobial prescription, and a cause of substantial economic losses and decreased animal welfare in aquaculture. Given the importance of the intestinal microbiota in digestion and disease, our aim was to investigate whether synbiotic feed supplementation strategies could improve feed performance and disease resistance. Four experimental synbiotic feeds formulated with pre- and probiotics were tested against a commercially available probiotic control feed. Each experimental feed was evaluated for feed performance, effects on gross as well as intestinal morphometrics, and finally their effect on resistance against a waterborne experimental infection with Yersinia ruckeri serotype O1, biotype 2. While co-supplementing Pediococcus acidilactici with citrus flavonoids or bacterial paraprobiotics significantly improved utilization of feed lipid content relative to the control group, a decrease in lipid utilization was observed for feeds that combined P. acidilactici with yeast paraprobiotics. No significant improvements on disease resistance were observed. Still, synbiotic formulations including P. acidilactici led to reduced risks relative to that of the control group, while an increased relative risk was observed for a Bacillus-based formulation. In conclusion, two of the synbiotic supplements significantly improved lipid utilization and contributed to minor increases in disease resistance.

Injection Vaccines Formulated with Nucleotide, Liposomal or Mineral Oil Adjuvants Induce Distinct Differences in Immunogenicity in Rainbow Trout.

Protection facilitated by the widespread use of mineral oil adjuvanted injection vaccines in salmonid fish comes with adverse effects of varying severity. In this study, we characterized the immunological profiles of two alternative vaccine formulations, both with proven efficacy and an improved safety profile in rainbow trout. Experimental injection vaccines were prepared on an identical whole-cell Aeromonas salmonicida bacterin platform and were formulated with CpG oligodeoxynucleotides, a liposomal (CAF01) or a benchmark mineral oil adjuvant, respectively. A naïve group, as well as bacterin and saline-injected groups were also included. Following administration, antigen-specific serum antibody titers, the tissue distribution of immune cell markers, and the expression of immune-relevant genes following the in vitro antigenic restimulation of anterior kidney leukocytes was investigated. Immunohistochemical staining suggested prolonged antigen presentation for the particulate formulations and increased mucosal presence of antigen-presenting cells in all immunized fish. Unlike the other immunized groups, the CAF01 group only displayed a transient elevation in specific antibody titers and immunohistochemical observations, and the transcription data suggest an increased role of cell-mediated immunity for this group. Finally, the transcription profile of the CpG formulation approached that of a TH1 profile. When compared to the benchmark formulation, CAF01 and CpG adjuvants induce slight, but distinct differences in the resulting protective immune responses. This is important, as it allows a broader immunological approach for the future development of safer vaccines.

Alternatives to mineral oil adjuvants in vaccines against Aeromonas salmonicida subsp. salmonicida in rainbow trout offer reductions in adverse effects.

In an effort to reduce the frequency and severity of adverse reactions seen from the use of mineral oil adjuvants in salmonid fish, the effects of two alternative adjuvants were assessed, focusing on the induction of adverse effects as well as protection. Using rainbow trout (Oncorhynchus mykiss) as recipients, injection vaccines based on formalin-inactivated Aeromonas salmonicida subspecies salmonicida were formulated with CpG oligodeoxynucleotides, the liposomal cationic adjuvant formulation 01 (CAF01) or with Freund's incomplete adjuvant and administered intraperitoneally. Control groups of unvaccinated, Tris-buffered saline-injected or bacterin-injected individuals were included, and each group included in the study held a total number of 240 individuals. Subsequently, individuals from each group were examined for differences in Fulton's condition factor, macro- and microscopic pathological changes, as well as protection against experimental infection with A. salmonicida. While adverse effects were not eliminated, reductions in microscopic and macroscopic adverse effects, in particular, were seen for both the nucleotide- and liposome-based vaccine formulations. Furthermore, the induced protection appears similar to that of the benchmark formulation, thus introducing viable, potential alternative types of adjuvants for use in future fish vaccines.

Global 3D imaging of Yersinia ruckeri bacterin uptake in rainbow trout fry.

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM) in rainbow trout, and the first commercially available fish vaccine was an immersion vaccine against ERM consisting of Y. ruckeri bacterin. The ERM immersion vaccine has been successfully used in aquaculture farming of salmonids for more than 35 years. The gills and the gastrointestinal (GI) tract are believed to be the portals of antigen uptake during waterborne vaccination against ERM; however, the actual sites of bacterin uptake are only partly understood. In order to obtain insight into bacterin uptake during waterborne vaccination, optical projection tomography (OPT) together with immunohistochemistry (IHC) was applied to visualize bacterin uptake and processing in whole rainbow trout fry. Visualization by OPT revealed that the bacterin was initially taken up via gill lamellae from within 30 seconds post vaccination. Later, bacterin uptake was detected on other mucosal surfaces such as skin and olfactory bulb from 5 to 30 minutes post vaccination. The GI tract was found to be filled with a complex of bacterin and mucus at 3 hours post vaccination and the bacterin remained in the GI tract for at least 24 hours. Large amounts of bacterin were present in the blood, and an accumulation of bacterin was found in filtering lymphoid organs such as spleen and trunk kidney where the bacterin accumulates 24 hours post vaccination as demonstrated by OPT and IHC. These results suggest that bacterin is taken up via the gill epithelium in the earliest phases of the bath exposure and from the GI tract in the later phase. The bacterin then enters the blood circulatory system, after which it is filtered by spleen and trunk kidney, before finally accumulating in lymphoid organs where adaptive immunity against ERM is likely to develop.

Adverse and long-term protective effects following oil-adjuvanted vaccination against Aeromonas salmonicida in rainbow trout.

Prophylactic measures against Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis, have been an active field of research for decades, with studies mainly focused on Atlantic salmon (Salmo salar). In the present study we have examined the protective and adverse effects of mineral oil-adjuvanted injection vaccines on rainbow trout (Oncorhynchus mykiss). A commercial vaccine and an experimental auto vaccine, as well as their respective adjuvant formulations alone were used to evaluate their individual effects, both prior to and during an experimental waterborne infection challenge. Macro- and microscopic examination revealed signs of vaccine-induced adverse effects from 10 weeks to 14 months post vaccination. Both vaccines induced statistically significant protection during the experimental challenge (P=0.018 for both vaccines), as well as significantly elevated levels of specific circulating antibodies prior to and during the experimental challenge when compared to an unvaccinated control group. During the early, critical time points of the infection, both vaccines appeared to protect against pathological changes to the liver and spleen, which provides a probable explanation for the reduced mortality seen in the vaccinated groups. A significant correlation was found between the level of A. salmonicida-specific antibodies measured prior to challenge and the endpoint survival of each group after the experimental infection, and furthermore, the levels of these antibodies remained elevated for at least 14 months post vaccination.

Oral and anal vaccination confers full protection against enteric redmouth disease (ERM) in rainbow trout.

The effect of oral vaccines against bacterial fish diseases has been a topic for debate for decades. Recently both M-like cells and dendritic cells have been discovered in the intestine of rainbow trout. It is therefore likely that antigens reaching the intestine can be taken up and thereby induce immunity in orally vaccinated fish. The objective of this project was to investigate whether oral and anal vaccination of rainbow trout induces protection against an experimental waterborne infection with the pathogenic enterobacteria Yersinia ruckeri O1 biotype 1 the causative agent of enteric redmouth disease (ERM). Rainbow trout were orally vaccinated with AquaVac ERM Oral (MERCK Animal Health) or an experimental vaccine bacterin of Y. ruckeri O1. Both vaccines were tested with and without a booster vaccination four months post the primary vaccination. Furthermore, two groups of positive controls were included, one group receiving the experimental oral vaccine in a 50 times higher dose, and the other group receiving a single dose administered anally in order to bypass the stomach. Each group was bath challenged with 6.3 × 10(8) CFU/ml Y. ruckeri, six months post the primary vaccination. The challenge induced significant mortality in all the infected groups except for the groups vaccinated anally with a single dose or orally with the high dose of bacterin. Both of these groups had 100% survival. These results show that a low dose of Y. ruckeri bacterin induces full protection when the bacterin is administered anally. Oral vaccination also induces full protection, however, at a dose 50 times higher than if the fish were to be vaccinated anally. This indicates that much of the orally fed antigen is digested in the stomach before it reaches the second segment of the intestine where it can be taken up as immunogenic antigens and presented to lymphocytes.

3D visualization of the initial Yersinia ruckeri infection route in rainbow trout (Oncorhynchus mykiss) by optical projection tomography.

Despite the fact that enteric redmouth disease (ERM) in farmed rainbow trout is one of the most devastating disease problems, little is known about the initial route of infection and pathogenicity of the aetiological agent, Yersinia ruckeri. In order to determine the initially infected organs, optical projection tomography (OPT), a novel three-dimensional (3D) bio-imaging technique, was applied. OPT not only enables the visualization of Y. ruckeri on mucosal surfaces but also the 3D spatial distribution in whole organs, without sectioning. Rainbow trout were infected by bath challenge exposure to 1 × 10(8) CFU/ml of Y. ruckeri O1 for 1 hour. Three fish were sampled for OPT and immunohistochemistry (IHC) 1, 10 and 30 minutes, 1, 3, 6, 12 and 24 hours, as well as 2, 3, 7 and 21 days after the start of the infection period. Y. ruckeri was re-isolated from the blood of infected fish as early as 1 minute post infection. Both OPT and IHC analysis confirmed that the secondary gill lamellae were the only tissues infected at this early time point, indicating that Y. ruckeri initially infects gill epithelial cells. The experimentally induced infection caused septicemia, and Y. ruckeri was found in all examined organs 7 days post infection including the brain, which correlated with the peak in mortality. To the best of our knowledge this is the first description of Y. ruckeri infection in the brain, which is likely to cause encephalitis. This in part could explain the lethality of ERM in rainbow trout. Using OPT scanning it was possible to visualize the initial route of entry, as well as secondary infection routes along with the proliferation and spread of Y. ruckeri, ultimately causing significant mortality in the exposed rainbow trout. These results demonstrate that OPT is a state-of-the-art technique capable of visualizing pathogenesis at high resolution.

Long-lasting protection induced by bath vaccination against Aeromonas salmonicida subsp. salmonicida in rainbow trout.

For decades Aeromonas salmonicida subsp. salmonicida (from here referred to as A. salmonicida) has been recognized as the causative agent of typical furunculosis. This disease has had a major impact on aquaculture worldwide, making it a target for international research, particularly within the field of immunoprohylaxis. Initial studies attempted vaccination via oral route and immersion. However, these vaccination methods proved insufficient when compared to intraperitoneally (i.p.) injected vaccines. The focus of vaccine research regarding A. salmonicida shifted towards the i.p.-injected vaccines during the 1980's and -90's, resulting in oil-adjuvanted vaccines providing high levels of protection over longer periods of time. The majority of this research has been conducted using salmon, while rainbow trout, which is also a commercially important species, has played a much less central role. In this study, we have examined the effect of a bath vaccination using an experimental A. salmonicida bacterin. Rainbow trout were vaccinated by a 5 min bath in a formalin-inactivated bacterin. Half of these fish was booster vaccinated using 50% of the initial vaccine dose 10 weeks post primary immunization. Along with an un-vaccinated control group, the fish were challenged by waterborne infection 24 weeks post primary immunization. Both vaccinated groups showed a significantly increased survival (>93% survival) compared to a 70% survival in the un-vaccinated control group (P = 0.005 and P = 0.019 for single and dual immunizations, respectively). When comparing the survival of the single and dual immunization groups, there was no significant difference (P = 0.531). ELISA showed no significant induction of specific circulating antibodies in either vaccinated group. These results are interesting with regard to the protective mechanisms, seen in the light of previous results obtained using bath as well as i.p. vaccination against furunculosis in salmonid fishes.

Acute cholesterol depletion leads to net loss of the organic osmolyte taurine in Ehrlich Lettré tumor cells.

In mammalian cells, the organic osmolyte taurine is accumulated by the Na-dependent taurine transporter TauT and released though the volume- and DIDS-sensitive organic anion channel. Incubating Ehrlich Lettré tumor cells with methyl-ß-cyclodextrin (5 mM, 1 h) reduces the total cholesterol pool to 60±5% of the control value. Electron spin resonance data indicate a concomitant disruption of cholesterol-rich micro-domains. Active taurine uptake, cellular taurine content, and cell volume are reduced by 50, 20 and 20% compared to control values, respectively, whereas the passive taurine release is increased 4.5-fold under isotonic conditions following cholesterol depletion. However, taurine release under isotonic conditions is insensitive to DIDS and inhibitors of the volume-regulated anion channel. Uptake and release of meAIB are similarly affected following cholesterol depletion. Kinetic analysis reveals that cholesterol depletion increases TauT's affinity toward taurine but reduces its maximal transport capacity. Cholesterol depletion has no impact on TauT regulation by protein kinases A and C. Phospholipase A2 activity, which is required for the activation of volume-sensitive organic anion channel (VSOAC), is increased under isotonic and hypotonic conditions following cholesterol depletion, whereas taurine release under hypotonic conditions is reduced following cholesterol depletion. Hence, acute cholesterol depletion of Ehrlich Lettré cells leads to reduced TauT and VSOAC activities and at the same time increases the release of organic osmolytes via a leak pathway different from the volume-sensitive pathways for amino acids and anions.