Weaning transition, but not the administration of probiotic candidate Kazachstania slooffiae, shaped the gastrointestinal bacterial and fungal communities in nursery piglets.

As in-feed antibiotics are phased out of swine production, producers are seeking alternatives to facilitate improvements in growth typically seen from this previously common feed additive. Kazachstania slooffiae is a prominent commensal fungus in the swine gut that peaks in relative abundance shortly after weaning and has beneficial interactions with other bacteriome members important for piglet health. In this study, piglets were supplemented with K. slooffiae to characterize responses in piglet health as well as fungal and bacterial components of the microbiome both spatially (along the entire gastrointestinal tract and feces) and temporally (before, during, and after weaning). Litters were assigned to one of four treatments: no K. slooffiae (CONT); one dose of K. slooffiae 7 days before weaning (day 14; PRE); one dose of K. slooffiae at weaning (day 21; POST); or one dose of K. slooffiae 7 days before weaning and one dose at weaning (PREPOST). The bacteriome and mycobiome were analyzed from fecal samples collected from all piglets at day 14, day 21, and day 49, and from organ samples along the gastrointestinal (GI) tract at day 21 and day 49. Blood samples were taken at day 14 and day 49 for cytokine analysis, and fecal samples were assayed for antimicrobial resistance. While some regional shifts were seen in response to K. slooffiae administration in the mycobiome of the GI tract, no remarkable changes in weight gain or health of the animals were observed, and changes were more likely due to sow and the environment. Ultimately, the combined microbiome changed most considerably following the transition from suckling to nursery diets. This work describes the mycobiome along the piglet GI tract through the weaning transition for the first time. Based on these findings, K. slooffiae administered at this concentration may not be an effective tool to hasten colonization of K. slooffiae in the piglet GI tract around the weaning transition nor support piglet growth, microbial gut health, or immunity. However, diet and environment greatly influence microbial community development.

Transcriptional Reprogramming in Rumen Epithelium during the Developmental Transition of Pre-Ruminant to the Ruminant in Cattle.

We present an analysis of transcriptomic dynamics in rumen epithelium of 18 Holstein calves during the transition from pre-rumination to rumination in cattle-fed hay or concentrated diets at weaning. Three calves each were euthanized at 14 and 42 d of age to exemplify preweaning, and six calves each were provided diets of either milk replacer and grass hay or calf starter to introduce weaning. The two distinct phases of rumen development and function in cattle are tightly regulated by a series of signaling events and clusters of effectors on critical pathways. The dietary shift from liquid to solid feeds prompted the shifting of gene activity. The number of differentially expressed genes increased significantly after weaning. Bioinformatic analysis revealed gene activity shifts underline the functional transitions in the ruminal epithelium and signify the transcriptomic reprogramming. Gene ontogeny (GO) term enrichment shows extensively activated biological functions of differentially expressed genes in the ruminal epithelium after weaning were predominant metabolic functions. The transcriptomic reprogramming signifies a correlation between gene activity and changes in metabolism and energy production in the rumen epithelium, which occur at weaning when transitioning from glucose use to VFA use by epithelium during the weaning.

Thermophile Lytic Enzyme Fusion Proteins that Target Clostridium perfringens.

Clostridium perfringens is a bacterial pathogen that causes necrotic enteritis in poultry and livestock, and is a source of food poisoning and gas gangrene in humans. As the agriculture industry eliminates the use of antibiotics in animal feed, alternatives to antibiotics will be needed. Bacteriophage endolysins are enzymes used by the virus to burst their bacterial host, releasing bacteriophage particles. This type of enzyme represents a potential replacement for antibiotics controlling C. perfringens. As animal feed is often heat-treated during production of feed pellets, thermostable enzymes would be preferred for use in feed. To create thermostable endolysins that target C. perfringens, thermophile endolysin catalytic domains were fused to cell wall binding domains from different C. perfringens prophage endolysins. Three thermostable catalytic domains were used, two from prophage endolysins from two Geobacillus strains, and a third endolysin from the deep-sea thermophilic bacteriophage Geobacillus virus E2 (GVE2). These domains harbor predicted L-alanine-amidase, glucosaminidase, and L-alanine-amidase activities, respectively and degrade the peptidoglycan of the bacterial cell wall. The cell wall binding domains were from C. perfringens prophage endolysins (Phage LYtic enzymes; Ply): PlyCP18, PlyCP10, PlyCP33, PlyCP41, and PlyCP26F. The resulting fifteen chimeric proteins were more thermostable than the native C. perfringens endolysins, and killed swine and poultry disease-associated strains of C. perfringens.

Yeasts of Burden: Exploring the Mycobiome-Bacteriome of the Piglet GI Tract.

Interactions between the bacteria and fungi in the gut microbiome can result in altered nutrition, pathogenicity of infection, and host development, making them a crucial component in host health. Associations between the mycobiome and bacteriome in the piglet gut, in the context of weaning, remain unknown. Weaning is a time of significant stress, dietary changes, microbial alterations, and a predisposition to infection. The loss of animal health and growth makes potential microbial interventions of interest to the swine industry. Recent studies have demonstrated the diversity and development of the microbiome in the gastrointestinal (GI) tract of piglets during weaning, resulting from the dietary and physiological changes. Despite these advances, the role of the mycobiota in piglet health and its contribution to overall microbiome development remains mostly unknown. In this study we investigated the bacteriome and the mycobiome after weaning in the GI tract organs and feces from 35-day old piglets. Following weaning, the α-diversity and amplicon sequence variants (ASVs) counts of the bacteriome increased, proximally to distally, from the stomach to the feces along the GI tract, while the mycobiome α-diversity and ASV counts were highest in the porcine stomach. ß-diversity analyses show distinct clusters based on organ type in the bacteriome and mycobiome, but dispersion remained relatively constant in the mycobiome between organ/fecal sites. Bacteroidetes, Firmicutes, and Epsilonbacteraeota were the most abundant bacterial phyla present in the GI tract and feces based on mean taxonomic composition with high variation of composition found in the stomach. In the mycobiome, the dominant phyla were Ascomycota and Basidiomycota, and the stomach mycobiome did not demonstrate the same high level of variation observed in the bacteriome. Potential interactions between genera were found in the lower piglet GI bacteriome and mycobiome with positive correlations found between the fungus, Kazachstania, and several bacterial species, including Lactobacillus. Aspergillus demonstrated negative correlations with the short chain fatty acid-producing bacteria Butyricoccus, Subdoligranulum, and Fusicatenibacter. This study demonstrates the distinct colonization dynamics between fungi and bacteria in the GI tract and feces of piglets directly following weaning and the potential interactions of these microbes in the porcine gut ecosystem.

The piglet mycobiome during the weaning transition: a pilot study1.

The importance of the microbiota in the gastrointestinal tract of animals is recognized as a critical player in host health. Recently, the significance of the mycobiome has been recognized, but culture-independent studies are limited, especially in swine. Weaning is a time of stress, dietary changes, and a predisposition to infections, making it a time point of interest to industry. In this pilot study, we sought to assess and characterize the mycobiome in the feces of swine from birth through the critical weaning transition to investigate the mycobiome population and its temporal dynamics in piglet feces. Cultured fecal samples demonstrate a significant increase in fungal burden following weaning that does not differ from adult levels, suggesting stable colonization. Culturable fungi were not found in any environmental samples tested, including water, food, sow milk or colostrum. To determine the fungal diversity present and to address the problem of unculturable fungi, we performed a pilot study utilizing ITS and 16S rRNA focused primers for high-throughput sequencing of fungal and bacterial species, respectively. Bacterial populations increase in diversity over the experimental timeline (days 1 to 35 postbirth), but the fungal populations do not demonstrate the same temporal trend. Following weaning, there is a dynamic shift in the feces to a Saccharomycetaceae-dominated population. The shift in fungal population was because of the dominance of Kazachstania slooffiae, a poorly characterized colonizer of animal gastrointestinal tracts. This study provides insights into the early colonization and subsequent establishment of fungi during the weaning transition in piglets. Future studies will investigate the effect of the mycobiome on piglet growth and health during the weaning transition.

Metabolomic analysis of longissimus from underperforming piglets relative to piglets with normal preweaning growth.

BACKGROUND: Recent increases in intra-litter variability in weaning weight have raised swine production costs. A contributor to this variability is the normal birth weight pig that grows at a slower rate than littermates of similar birth weight. The goal of this study was to interrogate biochemical profiles manifested in skeletal muscle originating from slow growing (SG) and faster growing littermates (control), with the aim of identifying differences in metabolic pathway utilization between skeletal muscle of the SG pig relative to its littermates. Samples of longissimus muscle from littermate pairs of pigs were collected at 21 d of age for metabolomic analysis (Metabolon, Inc., Durham, NC). RESULTS: Birth weights did not differ between littermate pairs of SG and Control pigs (P > 0.05). Weaning weights differed by 1.51 ± 0.19 kg (P < 0.001). Random forest (RF) analysis was effective at segregating the metabolome of muscle samples by growth rate, resulting in a predictive accuracy of 81% versus random segregation (50%). Decreases in sugars in the pentose phosphate pathway (PPP) in the longissimus of SG pigs were detected (P < 0.05). Decreases were also apparent in glycolytic intermediates (glycerol-3-phosphate and lactate) and key glycolysis-derived intermediates (glucose-6-phosphate and fructose-6-phosphate; P < 0.05). SG pigs had increased levels of phospholipids, lysolipids, diacylglycerols, and sphingolipids (P < 0.05). Pathway analysis identified a cluster of molecules associated with muscle and collagen/extracellular matrix breakdown that are increased in the SG pig (glutamate, 3-methylhistidine and hydroxylated proline moieties; P < 0.05). Nicotinate metabolism was altered in SG pigs, resulting in a 78% decrease in the nicotinamide adenine dinucleotide pool (P < 0.05). CONCLUSIONS: These metabolomic data provide the first evidence for biochemical mechanisms that should be investigated to determine if they have a potential role in the slow growth in some normal birth weight piglets that contribute to increased intra-litter variability in weaning weights and provides essential information and potential targets for the development of nutritional intervention strategies.

Regulation of cytokine gene expression by orosomucoid in neonatal swine adipose tissue.

BACKGROUND: Porcine adipose tissue expresses orosomucoid (ORM1) mRNA, a protein with anti-inflammatory and immunomodulatory properties. Previous research has demonstrated that porcine ORM1 can reduce insulin stimulated glucose metabolism in porcine adipose tissue in vitro. The present study was designed to examine the preweaning ontogeny of ORM1 mRNA abundance in porcine subcutaneous adipose and to determine if ORM1 can regulate mRNA abundance of inflammatory cytokines that contribute to insulin resistance in primary cultures derived from neonatal porcine subcutaneous adipose tissue. Cultures were differentiated in vitro and subsequently the adipocyte containing cultures were incubated for 24 h with 0-5000 ng porcine ORM1/mL medium. Cultures were then harvested, total RNA extracted for use in reverse transcription and the mRNA abundance of cytokine mRNA quantified by real-time PCR. RESULTS: ORM1 mRNA abundance within neonatal adipose tissue does not change from d 1 to d 21 of age and is a very small fraction relative to liver mRNA abundance. The ORM1 mRNA level in porcine adipocytes and stromal-vascular cells are similar (P > 0.05). Treatment with ORM1 did not affect TNFα (tumor necrosis factor α) mRNA level (P > 0.05), while interleukin 6 (IL6) mRNA abundance was reduced 32 % at 1,000 ng ORM1/mL (P < 0.01). However, TNFα protein content in the cell culture media was reduced by ORM1 treatment (5,000 ng/mL, P < 0.05), whereas ORM1 had no detectable effect on the media content of IL6 (P > 0.05). The reduction of macrophage migration inhibitory factor (MIF) mRNA abundance by ORM1 was dose dependent (P < 0.01). Monocyte chemotactic protein (MCP) mRNA level was reduced 27 % by 1,000 ng ORM1/mL (P < 0.05). CONCLUSIONS: The data suggest that ORM1 has limited effects TNFα, IL6, MIF or MCP expression at the concentrations tested. Secondly, these cytokines do not appear to contribute to the reported insulin resistance induced by ORM1 in porcine adipose tissue in vitro as an increase in the abundance of these inflammatory cytokines would be predicted during an insulin resistant state.

Identification of alpha-1 acid glycoprotein (AGP) as a potential marker of impaired growth in the newborn piglet.

Two studies were conducted to investigate the relationship between circulating levels of haptoglobin and α-1 acid glycoprotein (AGP) and growth in neonatal pigs. Circulating serum AGP, but not haptoglobin, was higher (P<0.001) in newborn runts than average-sized littermates. At 1 and 3 weeks, AGP and haptoglobin were similar among control and runt piglets. To determine the possible association between AGP and growth rate, blood was collected between the first and second day after birth in piglets from 10 average litters. Birthweight was positively correlated with growth rate through 21 days (linear regression correlation coefficient (CC), 0.43 (P<0.006); 0.299 (P<0.003) in males and females, respectively). Plasma AGP at birth was negatively correlated with growth (CC, -0.429 (P<0.006); -0.351 (P<0.01) in males and females, respectively). When AGP was calculated on a per kg birthweight basis, the CC with growth improved by 25 and 34% in males and females, respectively, compared with birthweight alone. Haptoglobin in blood was not correlated with growth. These data suggest that AGP at birth is reflective of growth conditions in utero or fetal maturation and may serve as an early predictive biomarker for pre-weaning growth rate.

Identification of protein carbonyls in serum of the fetal and neonatal pig.

Oxidation of serum proteins leads to non-reversible carbonyl formation which alters their function and is associated with stress-related disease processes. The primary objective of this study was to quantify and identify oxidized serum proteins in fetal and newborn piglets. Protein carbonyls were converted to hydrazones with dinitrophenyl hydrazine and quantified spectrophotometrically. For identification, serum protein carbonyls were derivatized with biotin hydrazide, separated by 2D PAGE and stained with FITC-avidin. Biotin-labeled proteins were excised from gels and identified by mass spectrometry. At birth, carbonyls were determined to be approximately 600 pmole/mg serum protein. Fetuses at 50 and 100 days of gestation had similar levels of protein carbonyls as newborns. Carbonyl levels were also similar for control and runt (<1 kg at birth) piglets between 1 and 21 days of age; however, distribution of many proteins varied by age and was also influenced by birth weight. Major oxidized proteins identified in fetal (f) and newborn (n) pigs included; albumin (f, n), transferrin (f, n), fetuin-A (f, n) alpha fetoprotein (f, n), plasminogen (f, n), fetuin-B (f), alpha-1-antitrypsin (f, n) alpha-1-acid glycoprotein (f) and immunoglobulins (n). While abundance and distribution of oxidized proteins changed over time, these changes appear to primarily reflect relative amounts of those proteins in serum.

Porcine leptin alters isolated adipocyte glucose and fatty acid metabolism.

This study examined if leptin can acutely affect glucose or fatty acid metabolism in pig adipocytes and whether leptin's actions on lipogenesis are manifested through interaction with insulin or growth hormone. Subcutaneous adipose tissue was obtained from approximately 55 kg crossbred barrows at the USDA abattoir. Isolated adipocytes were prepared using a collagenase procedure. Experiments assessed U-14C-glucose or 1-14C-palmitate metabolism in isolated adipocytes exposed to: basal medium (control), 100 nM insulin, 100 ng/ml porcine growth hormone, 100 ng/ml recombinant porcine leptin, and combinations of these hormones. Treatments were performed in triplicate and the experiment was repeated with adipocytes isolated from five different animals. Cell aliquots (250 microl) were added to 1 ml of incubation medium, then incubated for 2h at 37 degrees C for measurement of glucose and palmitate oxidation or incorporation into lipid. Incubation of isolated adipocytes with insulin increased glucose oxidation rate by 18% (P<0.05), while neither growth hormone nor leptin affected glucose oxidation (P>0.5). Total lipid synthesis from glucose was increased by approximately 25% by 100 nM insulin or insulin+growth hormone (P<0.05). Insulin+leptin reduced the insulin response by 37% (P<0.05). The combination of all three hormones increased total lipid synthesis by 35%, relative to controls (P<0.05), a rate similar to insulin alone. Fatty acid synthesis was elevated by insulin (32%, P<0.05) or growth hormone (13%, P<0.05). Leptin had no effect on fatty acid synthesis (P>0.05). Leptin reduced the esterification rate by 10% (P<0.05). Growth hormone and insulin could overcome leptin's inhibition of palmitate esterification (P>0.05).