Maria C. Walsh

Predominance of a bacteriocin-producing Lactobacillus salivarius component of a five-strain probiotic in the porcine ileum and effects on host immune phenotype.

Relative predominance of each of five probiotic strains was investigated in the ileum of weaned pigs, compared with that in feces, when administered in combination at c. 5 x 10(9) CFU day(-1) for 28 days. Probiotic was excreted at 10(6)-10(9) CFU g(-1) feces, while ileal survival ranged from 10(2) to 10(6) CFU g(-1) digesta. In contrast to the feces, where Lactobacillus murinus DPC6002 predominated, the bacteriocin-producing Lactobacillus salivarus DPC6005 dominated over coadministered strains both in the ileum digesta and in mucosa. Probiotic administration did not alter counts of culturable fecal Lactobacillus or Enterobacteriaceae but higher ileal Enterobacteriaceae were observed in the ileal digesta of probiotic-fed pigs (P<0.05). We observed decreased CD25 induction on T cells and monocytes (P<0.01) and decreased CTLA-4 induction (P<0.05) by the mitogen phytohemagglutinin on CD4 T cells from the probiotic group. Probiotic treatment also increased the proportion of CD4+ CD8+ T cells within the peripheral T-cell population and increased ileal IL-8 mRNA expression (P<0.05). In conclusion, superior ileal survival of L. salivarius compared with the other coadministered probiotics may be due to a competitive advantage conferred by its bacteriocin. The findings also suggest that the five-strain combination may function as a probiotic, at least in part, via immunomodulation.

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.

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.

Controlling Salmonella infection in weanling pigs through water delivery of direct-fed microbials or organic acids. Part I: Effects on growth performance, microbial populations, and immune status1

Pigs (n = 88) weaned at 19 ± 2 d of age were used in a 14-d study to evaluate the effects of water-delivered direct-fed microbials (DFM) or organic acids on growth, immune status, Salmonella infection and shedding, and intestinal microbial populations after intranasal inoculation of Salmonella Typhimurium (10(10) cfu/pig). Pigs were challenged with Salmonella 6 d after commencement of water treatments. Treatments were 1) control diet; 2) control diet + DFM (Enterococcus faecium, Bacillus subtilis, and Bacillus licheniformis) in drinking water at 10(9) cfu/L for each strain of bacteria; 3) control diet + an organic acid-based blend (predominantly propionic, acetic, and benzoic acid) in drinking water at 2.58 mL/L; and 4) control diet + 55 mg/kg of carbadox. Serum samples were taken on d 6, 8, 10, and 14 for determination of tumor necrosis factor α (TNFα) concentrations. Fecal samples were taken on d 0, 5, 7, and 11 for determination of Salmonella shedding and enumeration of coliforms. Pigs were euthanized on d 6, 8, 10, and 14. Intestinal and cecal tissue and digesta and mesenteric lymph nodes were sampled and analyzed for Salmonella. Duodenal, jejunal, and ileal mucosal scrapings were sampled for measurement of mucosal TNFα concentrations. Water delivery of DFM prevented a decline in ADG on d 2 to 6 postchallenge compared with the negative control (P < 0.05). Coliform counts tended to be greater (P = 0.09) in the cecum of the DFM treatment group on d 2 postinfection compared with the negative control and acid treatment groups. However, Salmonella prevalence in the feces, gastrointestinal tract, or lymph nodes was not affected by water delivery of acids or DFM. Serum and mucosal TNFα concentrations were not affected by treatment throughout the study with the exception of ileal concentrations on d 4 postchallenge, which were greater in the negative control group compared with all other treatments (P < 0.05). The in-feed antibiotic was the only treatment that reduced Salmonella prevalence and this was localized to the cecum on d 8 postinfection. In conclusion, the DFM and organic acid treatments used in this study offered little or no benefits to pigs infected with Salmonella and should not be considered under the constraints of this study as viable alternatives to in-feed antibiotics in a pathogen challenge situation.

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

ABSTRACT 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.

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.

Effect of feeding genetically modified Bt MON810 maize to ∼40-day-old pigs for 110 days on growth and health indicators

A total of 72 male weaned pigs were used in a 110-day study to investigate the effect of feeding genetically modified (GM) Bt MON810 maize on selected growth and health indicators. It was hypothesised that in pigs fed Bt maize, growth and health are not impacted compared with pigs fed isogenic maize-based diets. Following a 12-day basal period, pigs (10.7 ± 1.9 kg body weight (BW); ∼40 days old) were blocked by weight and ancestry and randomly assigned to treatments: (1) non-GM maize diet for 110 days (non-GM), (2) GM maize diet for 110 days (GM), (3) non-GM maize diet for 30 days followed by GM maize diet up to day 110 (non-GM/GM) and (4) GM maize diet for 30 days followed by non-GM maize diet up to day 110 (GM/non-GM). BW and daily feed intake were recorded on days 0, 30, 60 and 110 (n = 15). Body composition was determined by dual energy X-ray absorptiometry (n = 10) on day 80. Following slaughter on day 110, organs and intestines were weighed and sampled for histological analysis and urine was collected for biochemical analysis (n = 10). Serum biochemistry analysis was performed on days 0, 30, 60, 100 and 110. Growth performance and serum biochemistry were analysed as repeated measures with time and treatment as main factors. The slice option of SAS was used to determine treatment differences at individual time points. There was no effect of feeding GM maize on overall growth, body composition, organ and intestinal weight and histology or serum biochemistry on days 60 and 100 and on urine biochemistry on day 110. A treatment × time interaction was observed for serum urea (SU; P < 0.05), creatinine (SC; P < 0.05) and aspartate aminotransferase (AST; P < 0.05). On day 30, SU was lower for the non-GM/GM treatment compared with the non-GM, GM and GM/non-GM treatments (P < 0.05). On day 110, SC was higher for the non-GM/GM and GM/non-GM treatments compared with non-GM and GM treatments (P < 0.05). Overall, serum total protein was lower for the GM/non-GM treatment compared with the non-GM/GM treatment (P < 0.05). The magnitude of change observed in some serum biochemical parameters did not indicate organ dysfunction and the changes were not accompanied by histological lesions. Long-term feeding of GM maize to pigs did not adversely affect growth or the selected health indicators investigated.

Transgenerational effects of feeding genetically modified maize to nulliparous sows and offspring on offspring growth and health

This study assessed the effect of feeding genetically modified maize expressing a truncated form of the Cry1Ab protein from Bacillus thuringiensis (Bt MON810 maize) to sows during gestation and lactation and their offspring from weaning to 115 d postweaning on offspring growth and health. After weaning at approximately 28 d of age (d 0), individually penned, mixed sex pigs (approximately 8 kg BW) from sows fed isogenic or Bt maize diets were blocked by sow treatment, sex, and BW and randomly assigned to Bt or isogenic maize diets as follows: i) isogenic maize-fed sow/isogenic maize-fed offspring (iso/iso); ii) isogenic maize-fed sow/Bt maize-fed offspring (iso/Bt); iii) Bt maize-fed sow/isogenic maize-fed offspring (Bt/iso); and iv) Bt maize-fed sow/Bt maize-fed offspring (Bt/Bt). Growth performance was recorded at intervals to harvest at approximately 105 kg BW (n=15/treatment) and blood samples were taken for biochemical analysis on d 0, 30, 70, 100, and 115 postweaning (n=10/treatment). Pigs were harvested on d 115 postweaning (n=10/treatment), and carcass weight, backfat depth, and organ weights (heart, kidney, spleen, and liver) were recorded. Kidney, liver, lymph nodes, and small intestine were collected for histological analysis. Offspring from Bt maize-fed sows were heavier than offspring from isogenic maize-fed sows on d 30 (P<0.05), 100 (P<0.05), and 115 postweaning (P<0.05) and had greater overall ADG (P<0.05). Overall ADFI was greater for offspring from sows fed Bt maize (P<0.05) and for Bt maize-fed pigs (P<0.05). Offspring from Bt maize-fed sows had greater carcass (P<0.05) and lighter spleen (P<0.05) weights. Dressing percentage was greater for Bt maize-fed pigs than isogenic maize-fed pigs (P<0.05), and livers were lighter for pigs in the Bt/Bt group than pigs in the iso/Bt or Bt/iso group (P<0.05). Offspring from Bt maize-fed sows also had greater duodenal crypt depths (P<0.05) and lower villus height/crypt depth ratios (P<0.05). No pathology was observed in the organs, and serum biochemistry values generally remained within normal limits and no overall differences were observed, with the exception of overall γ glutamyltransferase, which was less for pigs on the Bt/Bt treatment than pigs on the iso/Bt and Bt/iso treatments. These results indicate that transgenerational consumption of Bt maize diets is not detrimental to pig growth and health.

Bioavailability of the anti-clostridial bacteriocin thuricin CD in gastrointestinal tract

Thuricin CD is a two component narrow spectrum bacteriocin comprising two peptides with targeted activity against Clostridium difficile. This study examined the bioavailability of thuricin with a view to developing it as an effective antimicrobial against intestinal infection. One of the peptides, Trn-β, was found to be degraded by the gastric enzymes pepsin and α-chymotrypsin both in vitro and in vivo, whereas Trn-α was resistant to digestion by these enzymes and hence was detected in the intestinal porcine digesta following oral ingestion by pigs. In order to determine if spores of the producing organism Bacillus thuringiensis DPC 6431 could be used to deliver the bacteriocin to the gut, spores were fed to 30 mice (approx. 10(8)-2×10(8) per animal) and their germination, growth and production of thuricin in the gastrointestinal tract (GIT) of the animals was monitored. Almost 99 % of the spores delivered to the GIT were excreted in the first 24 h and neither Trn-α nor Trn-β was detected in the gut or faecal samples of the test mice, indicating that ingestion of B. thuringiensis spores may not be a suitable vehicle for the delivery of thuricin CD. When thuricin CD was delivered rectally to mice (n = 40) and C. difficile shedding monitored at 1, 6, 12 and 24 h post-treatment, there was a >95 % (>1.5 log units) reduction of C. difficile 027 in the colon contents of infected mice (n = 10) 1 h post-treatment compared with the control group (n = 10; P<0.001). Furthermore, 6 h post-treatment there was a further 1.5 log reduction in C. difficile numbers (n = 10) relative to the control group (n = 10; P<0.05). These results would suggest that rectal administration of thuricin may be a promising mode of delivery of thuricin CD to the colon.

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.