Genetic diversity and detection of atypical porcine pestivirus infections.

Atypical porcine pestivirus (APPV), an RNA virus member of the Flaviviridae family, has been associated with congenital tremor in newborn piglets. Previously reported quantitative polymerase chain reaction (qPCR)-based assays were unable to detect APPV in novel cases of congenital tremor originated from multiple farms from U.S. Midwest (MW). These assays targeted the viral polyprotein coding genes, which were shown to display substantial variation, with sequence identity ranging from 58.2% to 70.7% among 15 global APPV strains. In contrast, the 5'-untranslated region (5' UTR) was found to have a much higher degree of sequence conservation. In order to obtain the complete 5' UTR of the APPV strains originated from MW, the 5' end of the viral cDNA was obtained by using template switching approach followed by amplification and dideoxy sequencing. Eighty one percent of the 5' UTR was identical across 14 global and 5 MW strains with complete or relatively complete 5' UTR. Notably, some of the most highly conserved 5' UTR segments overlapped with potentially important regions of an internal ribosome entry site (IRES), suggesting their functional role in viral protein translation. A newly designed single qPCR assay, targeting 100% conserved 5' UTR regions across 19 strains, was able to detect APPV in samples of well documented cases of congenital tremor which originated from five MW farm sites (1-18 samples/site). As these fully conserved 5' UTR sequences may have functional importance, we expect that assays targeting this region would broadly detect APPV strains that are diverse in space and time.

Effects of mannan oligosaccharides and Lactobacillus mucosae on growth performance, immune response, and gut health of weanling pigs challenged with Escherichia coli lipopolysaccharides.

Addition of pre- and probiotics may confer growth and health benefits when added to the diet of pigs. To determine the effects of feeding mannan oligosaccharide (MOS) and Lactobacillus mucosae (LM) as prebiotic and probiotic sources in weanling pigs under immune challenge, 96 weaned pigs were randomly allotted to 16 experimental pens within a 2 × 2 factorial arrangement of treatments. Control diets with or without 0.1% yeast-derived MOS were randomly assigned to pens and 109 cfu/pig LM broth or a control broth were top-dressed daily. Pigs were fed one of four dietary treatments (control, MOS, LM, and MOS+LM) in Phases I and II (days 0 to 7 and days 7 to 21 postweaning, respectively) and a common diet during Phase III (days 21 to 35 postweaning). On day 14, all pigs were challenged with 100 µg/kg body weight (BW) Escherichia coli lipopolysaccharide (LPS) via intraperitonial injection. Feed disappearance and pig BW were measured weekly. Blood and fecal samples were collected weekly, and additional blood samples were collected on days 1 and 3 post-LPS challenge. On days 15 and 21, one pig per pen was euthanized for collection of ileal mucosa and duodenal and ileal tissue samples. From days 0 to 14, feeding LM decreased gain-to-feed ratio (G:F; P < 0.05). An interaction between LM and MOS was observed for G:F on days 14 to 21 (P < 0.05); G:F in LM (715 g/kg) was greater compared with MOS+LM (P < 0.05; 600 g/kg) and control (P < 0.10; 615 g/kg), but was not different (P > 0.10) from MOS (674 g/kg). After pigs were fed a common diet (days 21 to 35), G:F was decreased (P < 0.05) in the LM treatment groups. Pigs fed diets that included MOS had increased serum immunoglobulin (Ig) G on days 1 and 3 post-LPS challenge and 2 wk after removal of treatments (P < 0.05) and on days 14 and 21 postweaning (P < 0.10) compared with pigs fed diets without MOS. On day 15, mucosal immunoglobulin G was increased (P < 0.05) in control vs. MOS and LM groups. Circulating IL-1ß in control and MOS+LM pigs increased (P < 0.05) on day 1 post-LPS challenge but did not change (P > 0.10) in MOS and LM groups. On day 15, pigs fed LM had decreased (P < 0.05) ileal crypt depth compared with pigs fed the control diet. On day 21, fecal propionate and butyrate tended to be lower (P < 0.10) in pigs fed MOS vs. control and MOS+LM diet. These preliminary findings suggest that feeding LM alone improved feed efficiency and ileal morphological structure during the first week of LPS challenge; additionally, feeding LM and MOS may have beneficial effects relative to immune biomarkers.

Differential longitudinal establishment of human fecal bacterial communities in germ-free porcine and murine models.

The majority of microbiome studies focused on understanding mechanistic relationships between the host and the microbiota have used mice and other rodents as the model of choice. However, the domestic pig is a relevant model that is currently underutilized for human microbiome investigations. In this study, we performed a direct comparison of the engraftment of fecal bacterial communities from human donors between human microbiota-associated (HMA) piglet and mouse models under identical dietary conditions. Analysis of 16S rRNA genes using amplicon sequence variants (ASVs) revealed that with the exception of early microbiota from infants, the more mature microbiotas tested established better in the HMA piglets compared to HMA mice. Of interest was the greater transplantation success of members belonging to phylum Firmicutes in the HMA piglets compared to the HMA mice. Together, these results provide evidence for the HMA piglet model potentially being more broadly applicable for donors with more mature microbiotas while the HMA mouse model might be more relevant for developing microbiotas such as those of infants. This study also emphasizes the necessity to exercise caution in extrapolating findings from HMA animals to humans, since up to 28% of taxa from some donors failed to colonize either model.

Effect of dietary medium-chain fatty acids on nursery pig growth performance, fecal microbial composition, and mitigation properties against porcine epidemic diarrhea virus following storage.

An experiment was conducted to evaluate the effect of dietary medium-chain fatty acid (MCFA) addition on nursery pig growth performance, fecal microbial composition, and mitigation of porcine epidemic diarrhea virus (PEDV) following storage. A total of 360 pigs (DNA 400 × 200, Columbus, NE; initially 6.7 ± 0.07 kg) were randomized to pens (5 pigs per pen) on the day of weaning (approximately 20 d of age), allowed a 6-d acclimation, blocked by BW, and randomized to dietary treatment (9 pens per treatment). All MCFA (Sigma-Aldrich, St. Louis, MO) were guaranteed ≥98% purity, including hexanoic (C6:0), octanoic (C8:0), and decanoic (C10:0) acids. Treatment diets were formulated in 2 phases (7 to 11 and 11 to 23 kg BW) and formulated to meet or exceed NRC requirement estimates. Treatments (n = 8) were a dose response including 0%, 0.25%, 0.5%, 1.0%, and 1.5% added MCFA blend (1:1:1 ratio C6:0, C8:0, and C10:0), as well as treatments with individual additions of 0.5% C6:0, C8:0, or C10:0. Fecal samples were collected from pigs fed control and 1.5% MCFA blend diets on days 0 and 14 and analyzed using 16s rDNA sequencing. Following feed manufacture, feed was stored in bags at barn temperature and humidity for 40 d before laboratory inoculation with PEDV. Subsamples of retained feed were inoculated with PEDV to achieve a titer of 104 TCID50/g and separate sample bottles were analyzed on 0 and 3 d post-inoculation (dpi). Overall, ADG and ADFI were increased (linear, P ≤ 0.010) and feed efficiency (G:F) improved (linear, P = 0.004) with increasing MCFA blend. Pigs fed 0.5% C8:0 had greater (P = 0.038) ADG compared with pigs fed the control diet, and G:F was improved (P ≤ 0.024) when pigs were fed 0.5% C6:0, 0.5% C8:0, or 0.5% C10:0 compared with control. An inclusion level × day interaction was observed (quadratic, P = 0.023), where PEDV Ct values increased (quadratic, P = 0.001) on 0 dpi with increasing levels of MCFA blend inclusion and also increased on 3 dpi (linear, P < 0.001). Fecal microbial diversity and composition were similar between control and 1.5% MCFA blend. In summary, the use of MCFA in nursery pig diets improves growth performance, provides residual mitigation activity against PEDV, and does not significantly alter fecal microbial composition.

BOARD INVITED REVIEW: The pig microbiota and the potential for harnessing the power of the microbiome to improve growth and health1.

A variety of microorganisms inhabit the gastrointestinal tract of animals including bacteria, archaea, fungi, protozoa, and viruses. Pioneers in gut microbiology have stressed the critical importance of diet:microbe interactions and how these interactions may contribute to health status. As scientists have overcome the limitations of culture-based microbiology, the importance of these interactions has become more clear even to the extent that the gut microbiota has emerged as an important immunologic and metabolic organ. Recent advances in metagenomics and metabolomics have helped scientists to demonstrate that interactions among the diet, the gut microbiota, and the host to have profound effects on animal health and disease. However, although scientists have now accumulated a great deal of data with respect to what organisms comprise the gastrointestinal landscape, there is a need to look more closely at causative effects of the microbiome. The objective of this review is intended to provide: 1) a review of what is currently known with respect to the dynamics of microbial colonization of the porcine gastrointestinal tract; 2) a review of the impact of nutrient:microbe effects on growth and health; 3) examples of the therapeutic potential of prebiotics, probiotics, and synbiotics; and 4) a discussion about what the future holds with respect to microbiome research opportunities and challenges. Taken together, by considering what is currently known in the four aforementioned areas, our overarching goal is to set the stage for narrowing the path towards discovering how the porcine gut microbiota (individually and collectively) may affect specific host phenotypes.

The effects of prebiotics on growth performance and in vitro immune biomarkers in weaned pigs.

The objective of the experiment was to investigate the effects of prebiotics in nursery pigs on growth performance and immune biomarkers. Sixty-four weaned pigs (31 ± 1 d; BW 8 ± 0.1 kg) of mixed gender were housed (4 pigs/pen) in an environmentally controlled nursery with ad libitum access to feed and water over a 35-d study. Pigs were randomly assigned to one of four treatments: control (53% corn, 32% SBM, 7% fishmeal, 8% others), control + 2.5% GroBiotic-S (GS), control + 0.05% chicory (CL), or control + 0.5% chicory (CH). Feeders and pigs were weighed weekly. On day 21, blood samples were obtained from three pigs/treatment for collection of peripheral blood mononuclear cells (PBMC). Isolated PBMC were cultured and subsequently challenged with lipopolysaccharide (LPS; 20 ng/mL). Cell culture supernatants were collected for quantification of the pro- and anti-inflammatory cytokines, interleukin (IL)-8 and IL-10, respectively. Dietary treatment had no effect on BW. At days 28 to 35, pigs fed GS (790 ± 15 g), CL (704 ± 15 g), or CH (692 ± 15 g) had greater (P < 0.05) ADG compared with control (643 ± 15 g) pigs. In addition, overall (days 0-35), pigs fed GS (823 ± 18 g), CL (783 ± 18 g), or CH (782 ± 18 g) had greater (P < 0.05) ADFI compared with control, and ADFI for GS-fed pigs was greater (P < 0.05) than either CL or CH. There was no difference in G:F among treatments. In vitro LPS challenge increased (P < 0.05) IL-8 secretion from PBMC isolated from CL (23,731 ± 3,221 pg/mL) pigs compared with control (10,061 ± 3,221 pg/mL) and CH (12,411 ± 3,221 pg/mL) pigs. Secretion of IL-10 from PBMC isolated from CL (63 ± 9 pg/mL) pigs was greater (P < 0.05) compared with control (22 ± 9 pg/mL) pigs and tended (P < 0.1) to be greater compared with CH (34 ± 9 pg/mL) pigs. Results indicate that inclusion of prebiotics in nursery pig diets has positive effects on growth performance and may have immunomodulatory effects (in vitro) on cells isolated from prebiotic-fed pigs.

Synaptogyrin-2 influences replication of Porcine circovirus 2.

Porcine circovirus 2 (PCV2) is a circular single-stranded DNA virus responsible for a group of diseases collectively known as PCV2 Associated Diseases (PCVAD). Variation in the incidence and severity of PCVAD exists between pigs suggesting a host genetic component involved in pathogenesis. A large-scale genome-wide association study of experimentally infected pigs (n = 974), provided evidence of a host genetic role in PCV2 viremia, immune response and growth during challenge. Host genotype explained 64% of the phenotypic variation for overall viral load, with two major Quantitative Trait Loci (QTL) identified on chromosome 7 (SSC7) near the swine leukocyte antigen complex class II locus and on the proximal end of chromosome 12 (SSC12). The SNP having the strongest association, ALGA0110477 (SSC12), explained 9.3% of the genetic and 6.2% of the phenotypic variance for viral load. Dissection of the SSC12 QTL based on gene annotation, genomic and RNA-sequencing, suggested that a missense mutation in the SYNGR2 (SYNGR2 p.Arg63Cys) gene is potentially responsible for the variation in viremia. This polymorphism, located within a protein domain conserved across mammals, results in an amino acid variant SYNGR2 p.63Cys only observed in swine. PCV2 titer in PK15 cells decreased when the expression of SYNGR2 was silenced by specific-siRNA, indicating a role of SYNGR2 in viral replication. Additionally, a PK15 edited clone generated by CRISPR-Cas9, carrying a partial deletion of the second exon that harbors a key domain and the SYNGR2 p.Arg63Cys, was associated with a lower viral titer compared to wildtype PK15 cells (>24 hpi) and supernatant (>48hpi)(P < 0.05). Identification of a non-conservative substitution in this key domain of SYNGR2 suggests that the SYNGR2 p.Arg63Cys variant may underlie the observed genetic effect on viral load.

Fecal Microbiota Transplantation Is Associated With Reduced Morbidity and Mortality in Porcine Circovirus Associated Disease.

Porcine circovirus associated disease (PCVAD) is a term used to describe the multi-factorial disease syndromes caused by porcine circovirus type 2 (PCV-2), which can be reproduced in an experimental setting through the co-infection of pigs with PCV-2 and porcine reproductive and respiratory syndrome virus (PRRSV). The resulting PCVAD-affected pigs represent a subpopulation within the co-infected group. In co-infection studies, the presence of increased microbiome diversity is linked to a reduction in clinical signs. In this study, fecal microbiota transplantation (FMT) was investigated as a means to prevent PCVAD in pigs co-infected with PRRSV and PCV-2d. The sources of the FMT material were high-parity sows with a documented history of high health status and robust litter characteristics. The analysis of the donated FMT material showed the absence of common pathogens along with the presence of diverse microbial phyla and families. One group of pigs (n = 10) was administered the FMT while a control group (n = 10) was administered a sterile mock-transplant. Over the 42-day post-infection period, the FMT group showed fewer PCVAD-affected pigs, as evidenced by a significant reduction in morbidity and mortality in transplanted pigs, along with increased antibody levels. Overall, this study provides evidence that FMT decreases the severity of clinical signs following co-infection with PRRSV and PCV-2 by reducing the prevalence of PCVAD.

Effects of energy restriction during gilt development on milk nutrient profile, milk oligosaccharides, and progeny biomarkers.

An ongoing study at the University of Nebraska-Lincoln (which included 14 batches of gilts; n = 90 gilts/batch) demonstrated that energy restriction during the developmental period of a gilt increases longevity and may also have beneficial effects on progeny health and growth, particularly, parity 1 progeny. Therefore, we hypothesized that energy restriction during gilt development may affect milk nutrient profile, milk oligosaccharides (OS), and postnatal progeny biomarkers. During the development period, batch 14 gilts (n = 128, 8 gilts/pen) were fed 3 dietary treatments including the following: 1) Control diet formulated to NRC (2012) specifications (CTL); 2) Restricted (20% energy restriction via addition of 40% soy hulls; RESTR); and 3) CTL diet plus addition of crystalline amino acids equivalent to the SID Lys:ME of the RESTR diet (CTL+). All diets were fed ad libitum and applied in a 3-phase feeding regimen during gilt development (days 123 to 230 of age). Average daily feed intake was used to estimate daily metabolizable energy intake (Mcal/d) during each phase (Phase 1: 10.13, 6.97, 9.95; Phase 2: 11.25, 8.05, 10.94; and Phase 3: 9.47, 7.95,11.07) for CTL, RESTR, and CTL+, respectively. After 230 d of age, gilts were bred and fed a common diet. Milk samples were collected from batch 14 gilts (n = 7 per treatment) on days 0 and 14 postfarrowing for compositional analysis of N, CP, dry matter (DM), GE, insulin, and OS. Piglet blood samples (n = 6 piglets/gilt) were obtained on days 1 and 15 postfarrowing for quantification of glucagon-like peptide-2 (GLP-2) and insulin. No effects of developmental diet were observed for milk N, CP, DM, or GE; however, N, CP, DM, and insulin were increased (P < 0.05) on day 1 compared with day 14. A total of 61 different milk OS were identified. Milk OS profile was significantly different for neutral and acidic OS (P < 0.05) on day 0, but there were no significant differences on day 14. For piglet GLP-2, a treatment by day interaction was observed (P < 0.009); specifically, on day 1 GLP concentrations were greater (P < 0.001) in CTL+ compared with RESTR (6.73 vs. 1.21 ng/mL). For serum insulin, a treatment by day interaction was observed (P < 0.01); specifically, insulin in RESTR progeny was greater (P < 0.03) than CTL on day 1. In conclusion, nutritional management of the developing gilt may affect milk nutrient composition, milk OS profile, and piglet serum biomarkers.

Effects of spray-dried porcine plasma on fecal microbiota in nursery pigs.

Spray-dried porcine plasma (SDPP) has been considered as an alternative for in-feed antibiotics to improve pig growth performance; however, the effect of SDPP on gut microbiota is unknown. The objective of this study was to evaluate effects of feeding SDPP on fecal microbial communities of nursery pigs. Ninety-six weaned pigs were assigned to 16 pens, which were allotted to two dietary treatments, including the control or the control + SDPP (5% and 2.5% SDPP inclusion in phase 1 and 2, respectively) diet. Fecal samples were collected at d 0, 7, 14, 21, and 28. Multiplex sequencing of V3 region of the 16S rRNA gene was used to characterize the bacterial community structure of fecal samples. Pearson's correlation tests were performed in Calypso to identify bacterial taxa that were either positively or negatively associated with overall growth performance. Feeding SDPP altered microbial structure at family, genus, and operational taxonomic unit (OTU) classifications; however, fecal microbes shifted with time. At the family level, Clostridiaceae increased (P < 0.001) on d 14, but decreased (P < 0.05) on d 28 in SDPP-fed pigs compared with control pigs. Decreased Veillonellaceae (P < 0.05; d 14) and Lachnospiraceae (P = 0.001; overall) were observed in SDPP-fed pigs compared with control pigs. Feeding SDPP increased lactic acid-producing bacteria (Lactobacillus delbrueckii, d 7) and cellulolytic bacteria (Ruminococcus albus, d 7; Clostridium thermocellum, d 7 and 14; and Clostridium saccharoperbutylacetonicum/beijerinckii, d 14; and Megasphaera elsdenii, d 21). On d 28, feeding SDPP decreased (P < 0.05) Clostridium difficile compared with control pigs. In conclusion, feeding SDPP altered fecal microbial communities in nursery pigs. The results of this study may provide information to help explain the positive effects associated with feeding SDPP on nutrient digestibility and gut health of nursery pigs.

Experimental Evaluation of Host Adaptation of Lactobacillus reuteri to Different Vertebrate Species.

The species Lactobacillus reuteri has diversified into host-specific lineages, implying a long-term association with different vertebrates. Strains from rodent lineages show specific adaptations to mice, but the processes underlying the evolution of L. reuteri in other hosts remain unknown. We administered three standardized inocula composed of strains from different host-confined lineages to mice, pigs, chickens, and humans. The ecological performance of each strain in the gastrointestinal tract of each host was determined by typing random colonies recovered from fecal samples collected over five consecutive days postadministration. Results revealed that rodent strains were predominant in mice, confirming previous findings of host adaptation. In chickens, poultry strains of the lineage VI (poultry VI) and human isolates from the same lineage (human VI) were recovered at the highest and second highest rates, respectively. Interestingly, human VI strains were virtually undetected in human feces. These findings, together with ancestral state reconstructions, indicate poultry VI and human VI strains share an evolutionary history with chickens. Genomic analysis revealed that poultry VI strains possess a large and variable accessory genome, whereas human VI strains display low genetic diversity and possess genes encoding antibiotic resistance and capsular polysaccharide synthesis, which might have allowed temporal colonization of humans. Experiments in pigs and humans did not provide evidence of host adaptation of L. reuteri to these hosts. Overall, our findings demonstrate host adaptation of L. reuteri to rodents and chickens, supporting a joint evolution of this bacterial species with several vertebrate hosts, although questions remain about its natural history in humans and pigs.IMPORTANCE Gut microbes are often hypothesized to have coevolved with their vertebrate hosts. However, the evidence is sparse and the evolutionary mechanisms have not been identified. We developed and applied an experimental approach to determine host adaptation of L. reuteri to different hosts. Our findings confirmed adaptation to rodents and provided evidence of adaptation to poultry, suggesting that L. reuteri evolved via natural selection in different hosts. By complementing phylogenetic analyses with experimental evidence, this study provides novel information about the mechanisms driving host-microbe coevolution with vertebrates and serve as a basis to inform the application of L. reuteri as a probiotic for different host species.

In vivo selection to identify bacterial strains with enhanced ecological performance in synbiotic applications.

One strategy for enhancing the establishment of probiotic bacteria in the human intestinal tract is via the parallel administration of a prebiotic, which is referred to as a synbiotic. Here we present a novel method that allows a rational selection of putative probiotic strains to be used in synbiotic applications: in vivo selection (IVS). This method consists of isolating candidate probiotic strains from fecal samples following enrichment with the respective prebiotic. To test the potential of IVS, we isolated bifidobacteria from human subjects who consumed increasing doses of galactooligosaccharides (GOS) for 9 weeks. A retrospective analysis of the fecal microbiota of one subject revealed an 8-fold enrichment in Bifidobacterium adolescentis strain IVS-1 during GOS administration. The functionality of GOS to support the establishment of IVS-1 in the gastrointestinal tract was then evaluated in rats administered the bacterial strain alone, the prebiotic alone, or the synbiotic combination. Strain-specific quantitative real-time PCR showed that the addition of GOS increased B. adolescentis IVS-1 abundance in the distal intestine by nearly 2 logs compared to rats receiving only the probiotic. Illumina 16S rRNA sequencing not only confirmed the increased establishment of IVS-1 in the intestine but also revealed that the strain was able to outcompete the resident Bifidobacterium population when provided with GOS. In conclusion, this study demonstrated that IVS can be used to successfully formulate a synergistic synbiotic that can substantially enhance the establishment and competitiveness of a putative probiotic strain in the gastrointestinal tract.

Variation in time and magnitude of immune response and viremia in experimental challenges with Porcine circovirus 2b.

BACKGROUND: Porcine circovirus 2 is the primary agent responsible for inducing a group of associated diseases known as Porcine Circovirus Associated Diseases (PCVAD), which can have detrimental effects on production efficiency as well as causing significant mortality. The objective of this study was to evaluate variation in viral replication, immune response and growth across pigs (n = 974) from different crossbred lines. The approach used in this study was experimental infection with a PCV2b strain of pigs at an average of 43 days of age. RESULTS: The sequence of the PCV2b isolate used in the challenge was similar with a cluster of PCV2b isolates known to induce PCVAD and increased mortality rates. The swine leukocyte antigen class II (SLAII) profile of the population was diverse, with nine DQB1 haplotypes being present. Individual viremia and antibody profiles during challenge demonstrate variation in magnitude and time of viral surge and immune response. The correlations between PCV2 specific antibodies and average daily gain (ADG) were relatively low and varied between - 0.14 to 0.08 for IgM and -0.02 and 0.11 for IgG. In contrast, PCV2 viremia was an important driver of ADG decline following infection; a moderate negative correlation was observed between viral load and overall ADG (r = - 0.35, P < 0.001). The pigs with the lowest 10% level of viral load maintained a steady increase in weekly ADG (P < 0.0001) compared to the pigs that had the 10% greatest viral load (P < 0.55). In addition, the highly viremic group expressed higher IgM and IgG starting with d 14 and d 21 respectively, and higher tumor necrosis factor - alpha (TNF-α) at d 21 (P < 0.005), compared to low viremic group. CONCLUSIONS: Molecular sources of the observed differences in viremia and immune response could provide a better understanding of the host factors that influence the development of PCVAD and lead to improved knowledge of swine immunity.