An isotonic protein solution favorably modulated the porcine intestinal immune response and cellular adhesion markers and reduced PEDV shedding in vivo.

Porcine epidemic diarrhea virus (PEDV) causes immensely large economic losses worldwide in the swine industry. PEDV attacks the intestine, disrupts intestinal epithelium morphology and barrier integrity, and results in profound diarrhea and high mortality. A commercially available isotonic protein solution (IPS) (Tonisity Px) has anecdotally been reported to be effective in supportive treatment of piglets with active PEDV infections. This study evaluated the effects of supplementing (or not) the drinking water of 14 day old PEDV-infected piglets with the IPS on the content of E-cadherin, fibronectin, interferon-alpha (IFN-α), and matrix metalloproteinase 9 (MMP-9) in duodenal tissue. The content of PEDV DNA in feces was also measured. Though both groups had similar PEDV shedding at day 1, IPS piglets had significantly lower PEDV shedding at day 5, 14 and 21. The IPS group also had a shorter duration of PEDV virus shedding. Levels of E-cadherin and fibronectin, both of which are structural proteins in the intestine, remained unchanged from baseline in the IPS group, whereas the same molecules decreased significantly in the control group. IFN-α, an antiviral cytokine, and MMP-9, an enzyme that aids in tissue remodeling, were increased at days 5 and 14 post infection, and then decreased at day 21 post-infection in the IPS group compared to control. Overall, the IPS used in this study enhanced epithelial intercellular adhesion (E-cadherin) and extracellular matrix structure (fibronectin), resulted in significantand favorable changes in MMP-9 activity, and favorably modulated IFN-α production. This is the first report of this panel of biomarkers, especially MMP-9 and IFN-α, in the face of in vivo PEDV infection. This is also the first report to investigate a commercially available swine product that does not need to be administered in solid feed, and that is already registered for use throughout Asia, Europe, South America, and North America. Overall, the results of this study serve to clarify the behavior of 4 key biomarkers in the presence of in vivo PEDV infection. The results also indicate that IPS (Tonisity Px) supplementation is a viable intervention to modulate the porcine intestinal immune response with favorable effects on the intestine.

Early-Life Intake of an Isotonic Protein Drink Improves the Gut Microbial Profile of Piglets.

A healthy microbial community in the gut of piglets is critical to minimize the negative performance consequences associated with dietary and environmental changes that occur at weaning. Tonisity Px, an isotonic protein drink, is a potential alternative to balance the gut microbiota as it contains key ingredients for nourishing the small intestine. In the present study, 16 litters comprising 161 piglets were randomly allocated to a group to which Tonisity Px was provided from days 2 to 8 of age (TPX group) or to a control group, to which no Tonisity Px was provided. The TPX group also received Tonisity Px in the 3 days before and after weaning. At days 9, 17, and 30 of age, fecal and ileum samples were collected from piglets belonging to both groups and analyzed using 16S rRNA gene sequencing, semiquantitative PCR of Rotavirus serogroups, and semiquantitative Escherichia coli culture. Overall, Tonisity Px increased the abundance of beneficial bacterial populations (Lactobacillus and Bacteroides species) and reduced potentially pathogenic bacterial populations (E. coli and Prevotellaceae), in both the pre-weaning and post-weaning periods.

Porcine Feed Efficiency-Associated Intestinal Microbiota and Physiological Traits: Finding Consistent Cross-Locational Biomarkers for Residual Feed Intake.

Optimal feed efficiency (FE) in pigs is important for economic and environmental reasons. Previous research identified FE-associated bacterial taxa within the intestinal microbiota of growing pigs. This study investigated whether FE-associated bacteria and selected FE-associated physiological traits were consistent across geographic locations (Republic of Ireland [ROI] [two batches of pigs, ROI1 and ROI2], Northern Ireland [NI], and Austria [AT]), where differences in genetic, dietary, and management factors were minimized. Pigs (n = 369) were ranked, within litter, on divergence in residual feed intake (RFI), and 100 extremes were selected (50 with high RFI and 50 with low RFI) across geographic locations for intestinal microbiota analysis using 16S rRNA amplicon sequencing and examination of FE-associated physiological parameters. Microbial diversity varied by geographic location and intestinal sampling site but not by RFI rank, except in ROI2, where more-feed-efficient pigs had greater ileal and cecal diversity. Although none of the 188 RFI-associated taxonomic differences found were common to all locations/batches, Lentisphaerae, Ruminococcaceae, RF16, Mucispirillum, Methanobrevibacter, and two uncultured genera were more abundant within the fecal or cecal microbiota of low-RFI pigs in two geographic locations and/or in both ROI batches. These are major contributors to carbohydrate metabolism, which was reflected in functional predictions. Fecal volatile fatty acids and salivary cortisol were the only physiological parameters that differed between RFI ranks. Despite controlling genetics, diet specification, dietary phases, and management practices in each rearing environment, the rearing environment, encompassing maternal influence, herd health status, as well as other factors, appears to impact intestinal microbiota more than FE.IMPORTANCE Interest in the role of intestinal microbiota in determining FE in pigs has increased in recent years. However, it is not known if the same FE-associated bacteria are found across different rearing environments. In this study, geographic location and intestinal sampling site had a greater influence on the pig gut microbiome than FE. This presents challenges when aiming to identify consistent reliable microbial biomarkers for FE. Nonetheless, seven FE-associated microbial taxa were common across two geographic locations and/or two batches within one location, and these indicated a potentially "healthier" and metabolically more capable microbiota in more-feed-efficient pigs. These taxa could potentially be employed as biomarkers for FE, although bacterial consortia, rather than individual taxa, may be more likely to predict FE. They may also merit consideration for use as probiotics or could be targeted by dietary means as a strategy for improving FE in pigs in the future.

Exploring a Possible Link between the Intestinal Microbiota and Feed Efficiency in Pigs.

Feed efficiency (FE) is critical in pig production for both economic and environmental reasons. As the intestinal microbiota plays an important role in energy harvest, it is likely to influence FE. Therefore, our aim was to characterize the intestinal microbiota of pigs ranked as low, medium, and high residual feed intake ([RFI] a metric for FE), where genetic, nutritional, and management effects were minimized, to explore a possible link between the intestinal microbiota and FE. Eighty-one pigs were ranked according to RFI between weaning and day 126 postweaning, and 32 were selected as the extremes in RFI (12 low, 10 medium, and 10 high). Intestinal microbiota diversity, composition, and predicted functionality were assessed by 16S rRNA gene sequencing. Although no differences in microbial diversity were found, some RFI-associated compositional differences were revealed, principally among members of Firmicutes, predominantly in feces at slaughter (albeit mainly for low-abundance taxa). In particular, microbes associated with a leaner and healthier host (e.g., Christensenellaceae, Oscillibacter, and Cellulosilyticum) were enriched in low RFI (more feed-efficient) pigs. Differences were also observed in the ileum of low RFI pigs; most notably, Nocardiaceae (Rhodococcus) were less abundant. Predictive functional analysis suggested improved metabolic capabilities in these animals, especially within the ileal microbiota. Higher ileal isobutyric acid concentrations were also found in low RFI pigs. Overall, the differences observed within the intestinal microbiota of low RFI pigs compared with that of their high RFI counterparts, albeit relatively subtle, suggest a possible link between the intestinal microbiota and FE in pigs.IMPORTANCE This study is one of the first to show that differences in intestinal microbiota composition, albeit subtle, may partly explain improved feed efficiency (FE) in low residual feed intake (RFI) pigs. One of the main findings is that, although microbial diversity did not differ among animals of varying FE, specific intestinal microbes could potentially be linked with porcine FE. However, as the factors impacting FE are still not fully understood, intestinal microbiota composition may not be a major factor determining differences in FE. Nonetheless, this work has provided a potential set of microbial biomarkers for FE in pigs. Although culturability could be a limiting factor and intervention studies are required, these taxa could potentially be targeted in the future to manipulate the intestinal microbiome so as to improve FE in pigs. If successful, this has the potential to reduce both production costs and the environmental impact of pig production.

Sequence-based analysis of the intestinal Microbiota of sows and their offspring fed genetically modified maize expressing a truncated form of Bacillus thuringiensis Cry1Ab protein (Bt Maize).

The aim was to investigate transgenerational effects of feeding genetically modified (GM) maize expressing a truncated form of Bacillus thuringiensis Cry1Ab protein (Bt maize) to sows and their offspring on maternal and offspring intestinal microbiota. Sows were assigned to either non-GM or GM maize dietary treatments during gestation and lactation. At weaning, offspring were assigned within sow treatment to non-GM or GM maize diets for 115 days, as follows: (i) non-GM maize-fed sow/non-GM maize-fed offspring (non-GM/non-GM), (ii) non-GM maize-fed sow/GM maize-fed offspring (non-GM/GM), (iii) GM maize-fed sow/non-GM maize-fed offspring (GM/non-GM), and (iv) GM maize-fed sow/GM maize-fed offspring (GM/GM). Offspring of GM maize-fed sows had higher counts of fecal total anaerobes and Enterobacteriaceae at days 70 and 100 postweaning, respectively. At day 115 postweaning, GM/non-GM offspring had lower ileal Enterobacteriaceae counts than non-GM/non-GM or GM/GM offspring and lower ileal total anaerobes than pigs on the other treatments. GM maize-fed offspring also had higher ileal total anaerobe counts than non-GM maize-fed offspring, and cecal total anaerobes were lower in non-GM/GM and GM/non-GM offspring than in those from the non-GM/non-GM treatment. The only differences observed for major bacterial phyla using 16S rRNA gene sequencing were that fecal Proteobacteria were less abundant in GM maize-fed sows prior to farrowing and in offspring at weaning, with fecal Firmicutes more abundant in offspring. While other differences occurred, they were not observed consistently in offspring, were mostly encountered for low-abundance, low-frequency bacterial taxa, and were not associated with pathology. Therefore, their biological relevance is questionable. This confirms the lack of adverse effects of GM maize on the intestinal microbiota of pigs, even following transgenerational consumption.

Effects of feeding Bt MON810 maize to sows during first gestation and lactation on maternal and offspring health indicators.

A total of twenty-four sows and their offspring were used in a 20-week study to investigate the effects of feeding GM maize on maternal and offspring health. Sows were fed diets containing GM or non-GM maize from service to the end of lactation. GM maize-fed sows were heavier on day 56 of gestation (P< 0·05). Offspring from sows fed GM maize tended to be lighter at weaning (P= 0·08). Sows fed GM maize tended to have decreased serum total protein (P= 0·08), and increased serum creatinine (P< 0·05) and γ-glutamyltransferase activity (P= 0·07) on day 28 of lactation. Serum urea tended to be decreased on day 110 of gestation in GM maize-fed sows (P= 0·10) and in offspring at birth (P= 0·08). Both platelet count (P= 0·07) and mean cell Hb concentration (MCHC; P= 0·05) were decreased on day 110 of gestation in GM maize-fed sows; however, MCHC tended to be increased in offspring at birth (P= 0·08). There was a minimal effect of feeding GM maize to sows during gestation and lactation on maternal and offspring serum biochemistry and haematology at birth and body weight at weaning.

Effects of feeding Bt maize to sows during gestation and lactation on maternal and offspring immunity and fate of transgenic material.

BACKGROUND: We aimed to determine the effect of feeding transgenic maize to sows during gestation and lactation on maternal and offspring immunity and to assess the fate of transgenic material. METHODOLOGY/PRINCIPAL FINDINGS: On the day of insemination, sows were assigned to one of two treatments (n = 12/treatment); 1) non-Bt control maize diet or 2) Bt-MON810 maize diet, which were fed for ~143 days throughout gestation and lactation. Immune function was assessed by leukocyte phenotyping, haematology and Cry1Ab-specific antibody presence in blood on days 0, 28 and 110 of gestation and at the end of lactation. Peripheral-blood mononuclear cell cytokine production was investigated on days 28 and 110 of gestation. Haematological analysis was performed on offspring at birth (n = 12/treatment). Presence of the cry1Ab transgene was assessed in sows' blood and faeces on day 110 of gestation and in blood and tissues of offspring at birth. Cry1Ab protein presence was assessed in sows' blood during gestation and lactation and in tissues of offspring at birth. Blood monocyte count and percentage were higher (P<0.05), while granulocyte percentage was lower (P<0.05) in Bt maize-fed sows on day 110 of gestation. Leukocyte count and granulocyte count and percentage were lower (P<0.05), while lymphocyte percentage was higher (P<0.05) in offspring of Bt maize-fed sows. Bt maize-fed sows had a lower percentage of monocytes on day 28 of lactation and of CD4(+)CD8(+) lymphocytes on day 110 of gestation, day 28 of lactation and overall (P<0.05). Cytokine production was similar between treatments. Transgenic material or Cry1Ab-specific antibodies were not detected in sows or offspring. CONCLUSIONS/SIGNIFICANCE: Treatment differences observed following feeding of Bt maize to sows did not indicate inflammation or allergy and are unlikely to be of major importance. These results provide additional data for Bt maize safety assessment.

The effect of feeding Bt MON810 maize to pigs for 110 days on intestinal microbiota.

OBJECTIVE: To assess the effects of feeding Bt MON810 maize to pigs for 110 days on the intestinal microbiota. METHODOLOGY/PRINCIPAL FINDINGS: Forty male pigs (∼40 days old) were blocked by weight and litter ancestry and assigned to one of four treatments; 1) Isogenic maize-based diet for 110 days (Isogenic); 2) Bt maize-based diet (MON810) for 110 days (Bt); 3) Isogenic maize-based diet for 30 days followed by a Bt maize-based diet for 80 days (Isogenic/Bt); 4) Bt maize-based diet for 30 days followed by an isogenic maize-based diet for 80 days (Bt/Isogenic). Enterobacteriaceae, Lactobacillus and total anaerobes were enumerated in the feces using culture-based methods on days 0, 30, 60 and 100 of the study and in ileal and cecal digesta on day 110. No differences were found between treatments for any of these counts at any time point. The relative abundance of cecal bacteria was also determined using high-throughput 16 S rRNA gene sequencing. No differences were observed in any bacterial taxa between treatments, with the exception of the genus Holdemania which was more abundant in the cecum of pigs fed the isogenic/Bt treatment compared to pigs fed the Bt treatment (0.012 vs 0.003%; P≤0.05). CONCLUSIONS/SIGNIFICANCE: Feeding pigs a Bt maize-based diet for 110 days did not affect counts of any of the culturable bacteria enumerated in the feces, ileum or cecum. Neither did it influence the composition of the cecal microbiota, with the exception of a minor increase in the genus Holdemania. As the role of Holdemania in the intestine is still under investigation and no health abnormalities were observed, this change is not likely to be of clinical significance. These results indicate that feeding Bt maize to pigs in the context of its influence on the porcine intestinal microbiota is safe.

Effects of feeding Bt MON810 maize to pigs for 110 days on peripheral immune response and digestive fate of the cry1Ab gene and truncated Bt toxin.

BACKGROUND: The objective of this study was to evaluate potential long-term (110 days) and age-specific effects of feeding genetically modified Bt maize on peripheral immune response in pigs and to determine the digestive fate of the cry1Ab gene and truncated Bt toxin. METHODOLOGY/PRINCIPAL FINDINGS: Forty day old pigs (n = 40) were fed one of the following treatments: 1) isogenic maize-based diet for 110 days (isogenic); 2) Bt maize-based diet (MON810) for 110 days (Bt); 3) Isogenic maize-based diet for 30 days followed by Bt maize-based diet for 80 days (isogenic/Bt); and 4) Bt maize-based diet (MON810) for 30 days followed by isogenic maize-based diet for 80 days (Bt/isogenic). Blood samples were collected during the study for haematological analysis, measurement of cytokine and Cry1Ab-specific antibody production, immune cell phenotyping and cry1Ab gene and truncated Bt toxin detection. Pigs were sacrificed on day 110 and digesta and organ samples were taken for detection of the cry1Ab gene and the truncated Bt toxin. On day 100, lymphocyte counts were higher (P<0.05) in pigs fed Bt/isogenic than pigs fed Bt or isogenic. Erythrocyte counts on day 100 were lower in pigs fed Bt or isogenic/Bt than pigs fed Bt/isogenic (P<0.05). Neither the truncated Bt toxin nor the cry1Ab gene were detected in the organs or blood of pigs fed Bt maize. The cry1Ab gene was detected in stomach digesta and at low frequency in the ileum but not in the distal gastrointestinal tract (GIT), while the Bt toxin fragments were detected at all sites in the GIT. CONCLUSIONS/SIGNIFICANCE: Perturbations in peripheral immune response were thought not to be age-specific and were not indicative of Th 2 type allergenic or Th 1 type inflammatory responses. There was no evidence of cry1Ab gene or Bt toxin translocation to organs or blood following long-term feeding.

High-throughput sequence-based analysis of the intestinal microbiota of weanling pigs fed genetically modified MON810 maize expressing Bacillus thuringiensis Cry1Ab (Bt maize) for 31 days.

The objective of this study was to investigate if feeding genetically modified (GM) MON810 maize expressing the Bacillus thuringiensis insecticidal protein (Bt maize) had any effects on the porcine intestinal microbiota. Eighteen pigs were weaned at ~28 days and, following a 6-day acclimatization period, were assigned to diets containing either GM (Bt MON810) maize or non-GM isogenic parent line maize for 31 days (n = 9/treatment). Effects on the porcine intestinal microbiota were assessed through culture-dependent and -independent approaches. Fecal, cecal, and ileal counts of total anaerobes, Enterobacteriaceae, and Lactobacillus were not significantly different between pigs fed the isogenic or Bt maize-based diets. Furthermore, high-throughput 16S rRNA gene sequencing revealed few differences in the compositions of the cecal microbiotas. The only differences were that pigs fed the Bt maize diet had higher cecal abundance of Enterococcaceae (0.06 versus 0%; P < 0.05), Erysipelotrichaceae (1.28 versus 1.17%; P < 0.05), and Bifidobacterium (0.04 versus 0%; P < 0.05) and lower abundance of Blautia (0.23 versus 0.40%; P < 0.05) than pigs fed the isogenic maize diet. A lower enzyme-resistant starch content in the Bt maize, which is most likely a result of normal variation and not due to the genetic modification, may account for some of the differences observed within the cecal microbiotas. These results indicate that Bt maize is well tolerated by the porcine intestinal microbiota and provide additional data for safety assessment of Bt maize. Furthermore, these data can potentially be extrapolated to humans, considering the suitability of pigs as a human model.

Effects of short-term feeding of Bt MON810 maize on growth performance, organ morphology and function in pigs.

Male weanling pigs (n 32) with a mean initial body weight of 7·5 kg and a mean weaning age of 28 d were used in a 31 d study to investigate the effects of feeding GM (Bt MON810) maize on growth performance, intestinal histology and organ weight and function. At weaning, the pigs were fed a non-GM starter diet during a 6 d acclimatisation period. The pigs were then blocked by weight and litter ancestry and assigned to diets containing 38·9 % GM (Bt MON810) or non-GM isogenic parent line maize for 31 d. Body weight and feed disappearance were recorded on a weekly basis (n 16/treatment), and the pigs (n 10/treatment) were killed on day 31 for the collection of organ, tissue and blood samples. GM maize-fed pigs consumed more feed than the control pigs during the 31 d study (P < 0·05) and were less efficient at converting feed to gain during days 14-30 (P < 0·01). The kidneys of the pigs fed GM maize tended to be heavier than those of control pigs (P = 0·06); however, no histopathological changes or alterations in blood biochemistry were evident. Small intestinal morphology was not different between treatments. However, duodenal villi of GM maize-fed pigs tended to have fewer goblet cells/µm of villus compared with control pigs (P = 0·10). In conclusion, short-term feeding of Bt MON810 maize to weaned pigs resulted in increased feed consumption, less efficient conversion of feed to gain and a decrease in goblet cells/µm of duodenal villus. There was also a tendency for an increase in kidney weight, but this was not associated with changes in histopathology or blood biochemistry. The biological significance of these findings is currently being clarified in long-term exposure studies in pigs.

Fate of transgenic DNA from orally administered Bt MON810 maize and effects on immune response and growth in pigs.

We assessed the effect of short-term feeding of genetically modified (GM: Bt MON810) maize on immune responses and growth in weanling pigs and determined the fate of the transgenic DNA and protein in-vivo. Pigs were fed a diet containing 38.9% GM or non-GM isogenic parent line maize for 31 days. We observed that IL-12 and IFNγ production from mitogenic stimulated peripheral blood mononuclear cells decreased (P<0.10) following 31 days of GM maize exposure. While Cry1Ab-specific IgG and IgA were not detected in the plasma of GM maize-fed pigs, the detection of the cry1Ab gene and protein was limited to the gastrointestinal digesta and was not found in the kidneys, liver, spleen, muscle, heart or blood. Feeding GM maize to weanling pigs had no effect on growth performance or body weight. IL-6 and IL-4 production from isolated splenocytes were increased (P<0.05) in response to feeding GM maize while the proportion of CD4(+) T cells in the spleen decreased. In the ileum, the proportion of B cells and macrophages decreased while the proportion of CD4(+) T cells increased in GM maize-fed pigs. IL-8 and IL-4 production from isolated intraepithelial and lamina propria lymphocytes were also increased (P<0.05) in response to feeding GM maize. In conclusion, there was no evidence of cry1Ab gene or protein translocation to the organs and blood of weaning pigs. The growth of pigs was not affected by feeding GM maize. Alterations in immune responses were detected; however, their biologic relevance is questionable.