Spray-Dried Plasma Promotes Broiler Chick Growth by Enhancing Immune Surveillance.

Spray-dried plasma (SDP) contain a variety of functional proteins that play an immunomodulatory role. To evaluate the potential of SDP to stimulate the immune system, day-old Ross 708 male broiler chicks (200) were allocated randomly to five dietary treatments. Treatment 1 (CX) comprised chicks fed basal unmedicated corn-soybean meal (SBM) without the addition of SDP. Treatment 2 (MX) includes chicks fed unmedicated corn-SBM basal containing Bacitracin methylene disalicylate (BMD) at 0.055 g/kg diet. Treatments 3 (SDP1), 4 (SDP2), and 5 (SDP3) contained chicks given unmedicated corn-SBM basal, into which SDP was included at 10, 20, and 30 g/kg diet, respectively. On d 7, 14, and 21, chicks' body weight and FCR were calculated. Additionally, leucocyte counts, oxidative status, and IgY concentrations were determined in blood. On d 23, fecal populations of selected indicator bacteria species were determined. Results showed that FCR for SP3 was superior (p < 0.05) to other treatments. Likewise, heterophil numbers decreased in MX and SDP treatments compared to CX. Circulating IgY concentration was higher for SDP dietary treatments (p < 0.05) compared to MX. In conclusion, dietary SDP at 30 g/kg enhanced immune surveillance by increasing circulating IgY levels, maintaining a normal oxidative state, and increasing gut Bifidobacteria, thereby improving chick growth performance.

Attenuated Salmonella enterica Serovar Typhimurium, Strain NC983, Is Immunogenic, and Protective against Virulent Typhimurium Challenges in Mice.

Non-typhoidal Salmonella (NTS) serovars are significant health burden worldwide. Although much effort has been devoted to developing typhoid-based vaccines for humans, currently there is no NTS vaccine available. Presented here is the efficacy of a live attenuated serovar Typhimurium strain (NC983). Oral delivery of strain NC983 was capable of fully protecting C57BL/6 and BALB/c mice against challenge with virulent Typhimurium. Strain NC983 was found to elicit an anti-Typhimurium IgG response following administration of vaccine and boosting doses. Furthermore, in competition experiments with virulent S. Typhimurium (ATCC 14028), NC983 was highly defective in colonization of the murine liver and spleen. Collectively, these results indicate that strain NC983 is a potential live attenuated vaccine strain that warrants further development.

An Attenuated Salmonella enterica Serovar Typhimurium Strain and Galacto-Oligosaccharides Accelerate Clearance of Salmonella Infections in Poultry through Modifications to the Gut Microbiome.

Salmonella is estimated to cause one million foodborne illnesses in the United States every year. Salmonella-contaminated poultry products are one of the major sources of salmonellosis. Given the critical role of the gut microbiota in Salmonella transmission, a manipulation of the chicken intestinal microenvironment could prevent animal colonization by the pathogen. In Salmonella, the global regulator gene fnr (fumarate nitrate reduction) regulates anaerobic metabolism and is essential for adapting to the gut environment. This study tested the hypothesis that an attenuated Fnr mutant of Salmonella enterica serovar Typhimurium (attST) or prebiotic galacto-oligosaccharides (GOS) could improve resistance to wild-type Salmonella via modifications to the structure of the chicken gut microbiome. Intestinal samples from a total of 273 animals were collected weekly for 9 weeks to evaluate the impact of attST or prebiotic supplementation on microbial species of the cecum, duodenum, jejunum, and ileum. We next analyzed changes to the gut microbiome induced by challenging the animals with a wild-type Salmonella serovar 4,[5],12:r:- (Nalr) strain and determined the clearance rate of the virulent strain in the treated and control groups. Both GOS and the attenuated Salmonella strain modified the gut microbiome but elicited alterations of different taxonomic groups. The attST produced significant increases of Alistipes and undefined Lactobacillus, while GOS increased Christensenellaceae and Lactobacillus reuteri The microbiome structural changes induced by both treatments resulted in a faster clearance after a Salmonella challenge.IMPORTANCE With an average annual incidence of 13.1 cases/100,000 individuals, salmonellosis has been deemed a nationally notifiable condition in the United States by the Centers for Disease Control and Prevention (CDC). Earlier studies demonstrated that Salmonella is transmitted by a subset of animals (supershedders). The supershedder phenotype can be induced by antibiotics, ascertaining an essential role for the gut microbiota in Salmonella transmission. Consequently, modulation of the gut microbiota and modification of the intestinal microenvironment could assist in preventing animal colonization by the pathogen. Our study demonstrated that a manipulation of the chicken gut microbiota by the administration of an attenuated Salmonella strain or prebiotic galacto-oligosaccharides (GOS) can promote resistance to Salmonella colonization via increases of beneficial microorganisms that translate into a less hospitable gut microenvironment.

A comparison of sequencing platforms and bioinformatics pipelines for compositional analysis of the gut microbiome.

BACKGROUND: Advancements in Next Generation Sequencing (NGS) technologies regarding throughput, read length and accuracy had a major impact on microbiome research by significantly improving 16S rRNA amplicon sequencing. As rapid improvements in sequencing platforms and new data analysis pipelines are introduced, it is essential to evaluate their capabilities in specific applications. The aim of this study was to assess whether the same project-specific biological conclusions regarding microbiome composition could be reached using different sequencing platforms and bioinformatics pipelines. RESULTS: Chicken cecum microbiome was analyzed by 16S rRNA amplicon sequencing using Illumina MiSeq, Ion Torrent PGM, and Roche 454 GS FLX Titanium platforms, with standard and modified protocols for library preparation. We labeled the bioinformatics pipelines included in our analysis QIIME1 and QIIME2 (de novo OTU picking [not to be confused with QIIME version 2 commonly referred to as QIIME2]), QIIME3 and QIIME4 (open reference OTU picking), UPARSE1 and UPARSE2 (each pair differs only in the use of chimera depletion methods), and DADA2 (for Illumina data only). GS FLX+ yielded the longest reads and highest quality scores, while MiSeq generated the largest number of reads after quality filtering. Declines in quality scores were observed starting at bases 150-199 for GS FLX+ and bases 90-99 for MiSeq. Scores were stable for PGM-generated data. Overall microbiome compositional profiles were comparable between platforms; however, average relative abundance of specific taxa varied depending on sequencing platform, library preparation method, and bioinformatics analysis. Specifically, QIIME with de novo OTU picking yielded the highest number of unique species and alpha diversity was reduced with UPARSE and DADA2 compared to QIIME. CONCLUSIONS: The three platforms compared in this study were capable of discriminating samples by treatment, despite differences in diversity and abundance, leading to similar biological conclusions. Our results demonstrate that while there were differences in depth of coverage and phylogenetic diversity, all workflows revealed comparable treatment effects on microbial diversity. To increase reproducibility and reliability and to retain consistency between similar studies, it is important to consider the impact on data quality and relative abundance of taxa when selecting NGS platforms and analysis tools for microbiome studies.

Oral Administration of Astrovirus Capsid Protein Is Sufficient To Induce Acute Diarrhea In Vivo.

The disease mechanisms associated with the onset of astrovirus diarrhea are unknown. Unlike other enteric virus infections, astrovirus infection is not associated with an inflammatory response or cellular damage. In vitro studies in differentiated Caco-2 cells demonstrated that human astrovirus serotype 1 (HAstV-1) capsid protein alone disrupts the actin cytoskeleton and tight junction complex, leading to increased epithelial barrier permeability. In this study, we show that oral administration of purified recombinant turkey astrovirus 2 (TAstV-2) capsid protein results in acute diarrhea in a dose- and time-dependent manner in turkey poults. Similarly to that induced by infectious virus, TAstV-2 capsid-induced diarrhea was independent of inflammation or histological changes but was associated with increased intestinal barrier permeability, as well as redistribution of sodium hydrogen exchanger 3 (NHE3) from the membrane to the cytoplasm of the intestinal epithelium. Unlike other viral enterotoxins that have been identified, astrovirus capsid induces diarrhea after oral administration, reproducing the natural route of infection and demonstrating that ingestion of intact noninfectious capsid protein may be sufficient to provoke acute diarrhea. Based on these data, we hypothesize that the astrovirus capsid acts like an enterotoxin and induces intestinal epithelial barrier dysfunction. IMPORTANCE: Acute gastroenteritis, with its sequela diarrhea, is one of the most important causes of childhood morbidity and mortality worldwide. A variety of infectious agents cause gastroenteritis, and in many cases, an enterotoxin produced by the agent is involved in disease manifestations. Although we commonly think of bacteria as a source of toxins, at least one enteric virus, rotavirus, produces a protein with enterotoxigenic activity during viral replication. In these studies, we demonstrate that oral administration of the turkey astrovirus 2 (TAstV-2) structural (capsid) protein induces acute diarrhea, increases barrier permeability, and causes relocalization of NHE3 in the small intestine, suggesting that rotavirus may not be alone in possessing enterotoxigenic activity.

Development of the Chick Microbiome: How Early Exposure Influences Future Microbial Diversity.

The concept of improving animal health through improved gut health has existed in food animal production for decades; however, only recently have we had the tools to identify microbes in the intestine associated with improved performance. Currently, little is known about how the avian microbiome develops or the factors that affect its composition. To begin to address this knowledge gap, the present study assessed the development of the cecal microbiome in chicks from hatch to 28 days of age with and without a live Salmonella vaccine and/or probiotic supplement; both are products intended to promote gut health. The microbiome of growing chicks develops rapidly from days 1-3, and the microbiome is primarily Enterobacteriaceae, but Firmicutes increase in abundance and taxonomic diversity starting around day 7. As the microbiome continues to develop, the influence of the treatments becomes stronger. Predicted metagenomic content suggests that, functionally, treatment may stimulate more differences at day 14, despite the strong taxonomic differences at day 28. These results demonstrate that these live microbial treatments do impact the development of the bacterial taxa found in the growing chicks; however, additional experiments are needed to understand the biochemical and functional consequences of these alterations.

Genetic variation within the Mx gene of commercially selected chicken lines reveals multiple haplotypes, recombination and a protein under selection pressure.

The Mx protein is one of the best-characterized interferon-stimulated antiviral mediators. Mx homologs have been identified in most vertebrates examined; however, their location within the cell, their level of activity, and the viruses they inhibit vary widely. Recent studies have demonstrated multiple Mx alleles in chickens and some reports have suggested a specific variant (S631N) within exon 14 confers antiviral activity. In the current study, the complete genome of nine elite egg-layer type lines were sequenced and multiple variants of the Mx gene identified. Within the coding region and upstream putative promoter region 36 SNP variants were identified, producing a total of 12 unique haplotypes. Each elite line contained from one to four haplotypes, with many of these haplotypes being found in only one line. Observation of changes in haplotype frequency over generations, as well as recombination, suggested some unknown selection pressure on the Mx gene. Trait association analysis with either individual SNP or haplotypes showed a significant effect of Mx haplotype on several egg production related traits, and on mortality following Marek's disease virus challenge in some lines. Examination of the location of the various SNP within the protein suggests synonymous SNP tend to be found within structural or enzymatic regions of the protein, while non-synonymous SNP are located in less well defined regions. The putative resistance variant N631 was found in five of the 12 haplotypes with an overall frequency of 47% across the nine lines. Two Mx recombinants were identified within the elite populations, indicating that novel variation can arise and be maintained within intensively selected lines. Collectively, these results suggest the conflicting reports in the literature describing the impact of the different SNP on chicken Mx function may be due to the varying context of haplotypes present in the populations studied.

Characterization of turkey inducible nitric oxide synthase and identification of its expression in the intestinal epithelium following astrovirus infection.

The inducible nitric oxide synthase (iNOS) enzyme has long been recognized as a key mediator of innate immune responses to infectious diseases across the phyla. Its role in killing or inactivating bacterial, parasitic, and viral pathogens has been documented in numerous host systems. iNOS, and its innate immune mediator NO has also been described to have negative consequence on host tissues as well; therefore understanding the pathogenesis of any infectious agent which induces iNOS expression requires a better understanding of the role iNOS and NO play in that disease. Previous studies in our laboratory and others have demonstrated evidence for increased levels of iNOS and activity of its innate immune mediator NO in the intestine of turkeys infected with astrovirus. To begin to characterize the role iNOS plays in the innate immune response to astrovirus infection, we identified, characterized, developed tkiNOS specific reagents, and demonstrated that the intestinal epithelial cells induce expression of iNOS following astrovirus infection. These data are the first to our knowledge to describe the tkiNOS gene, and demonstrate that astrovirus infection induces intestinal epithelial cells to express iNOS, suggesting these cells play a key role in the antiviral response to enteric infections.

Astrovirus infection induces sodium malabsorption and redistributes sodium hydrogen exchanger expression.

Astroviruses are known to be a leading cause of diarrhea in infants and the immunocompromised; however, our understanding of this endemic pathogen is limited. Histological analyses of astrovirus pathogenesis demonstrate clinical disease is not associated with changes to intestinal architecture, inflammation, or cell death. Recent studies in vitro have suggested that astroviruses induce actin rearrangement leading to loss of barrier function. The current study used the type-2 turkey astrovirus (TAstV-2) and turkey poult model of astrovirus disease to examine how astrovirus infection affects the ultrastructure and electrophysiology of the intestinal epithelium. These data demonstrate that infection results in changes to the epithelial ultrastructure, rearrangement of F-actin, decreased absorption of sodium, as well as redistribution of the sodium/hydrogen exchanger 3 (NHE3) from the membrane to the cytoplasm. Collectively, these data suggest astrovirus infection induces sodium malabsorption, possibly through redistribution of specific sodium transporters, which results in the development of an osmotic diarrhea.