Effects of dietary protein supply, weaning age and experimental enterotoxigenic Escherichia coli infection on newly weaned pigs: performance

An experiment was conducted to investigate the effects of post-weaning dietary protein supply and weaning age on the performance of pigs in the absence of in-feed antimicrobial growth promoters (AGP) when artificially challenged with enterotoxigenic Escherichia coli (ETEC), a pathogen associated with post-weaning colibacillosis (PWC). The experiment consisted of a complete 2 × 2 × 2 factorial combination of two weaning ages (4 v. 6 week), two levels of dietary protein (H, 230 g CP/kg v. L, 130 g CP/kg) and challenge with ETEC (+ v. -). An additional four treatments were added to test for the effects of protein source (DSMP, dried skimmed milk powder v. SOYA, soybean meal) and AGP inclusion (yes v. no) on challenged pigs of both weaning ages. At weaning (day 0), pigs were assigned to one of the experimental treatments for 2 weeks. On day 14 post-weaning, the same standard grower ration was fed to all animals until 10 weeks of age. On day 3 post weaning, challenged pigs were administered per os with 109 cfu ETEC. The ETEC challenge had a detrimental short-term effect on performance, decreasing average daily gain (ADG) (days 3 to 6; P = 0.014) in both 4- and 6-week weaned animals. Compared with their non-infected counterparts, challenged 4-week weaned pigs on the H diet demonstrated a larger decrease in ADG immediately post infection than those on the L diet, -42% and -25%, respectively (P = 0.088). This effect was smaller in the 6-week weaned pigs, -26% and -19% for the H and L diets, respectively. Pigs fed SOYA had lower (P < 0.001) daily intake and ADG (day 0 to 14) than those fed DSMP, with 6-week weaned pigs being affected to a greater extent than 4-week weaned pigs. In the absence of AGP, increasing weaning age and decreasing dietary protein level, especially in earlier weaned pigs, may help to minimise the effects of PWC on performance, particularly in sub-optimal environments.     

Impact of early inoculation of probiotics to suckling piglets on postweaning diarrhoea – A challenge study with Enterotoxigenic E. Coli F18

Postweaning diarrhoea caused by Enterotoxigenic Escherichia coli (ETEC) is a threat to the pig industry. With an intensified focus on finding alternatives to the use of medical zinc oxide and antibiotics in newly weaned pigs, the objective of this study was to investigate the effect of early inoculation of probiotics to suckling piglets on subsequently ETEC faecal shedding and immune parameters in ETEC F18-challenged weaned piglets. Sixty pigs weaned on day 28 of age were assigned to three treatment groups: (i) Negative Control (non-challenged), (ii) Positive Control (challenged) and (iii) Probiotic (challenged and inoculated with a multi-species probiotic product during suckling). On days 1 and 2 postweaning, pigs in the Positive Control and Probiotic groups were challenged with 5 × 10⁸ colony-forming unit ETEC F18/pig/day, whereas pigs in the Negative Control group were provided with NaCl. Growth and diarrhoea incidence were not significantly affected by ETEC challenge or probiotic administration. ETEC F18 shedding and C-reactive protein concentration in plasma were significantly lower in the Negative Control group, confirming a successful challenge model. Pigs in the Probiotic group had a significantly reduced number of pigs shedding ETEC F18 and STb toxin in faeces compared with the Positive Control group. Probiotic application did not significantly impact the concentration of C-reactive protein, haptoglobin and cytokines in plasma nor haematology numbers. In conclusion, weaned pigs administered with a multi-species probiotic product early in life had a more rapid response towards the pathogen challenge and a faster clearance of ETEC compared with the Positive Control group. Administration of a multi-species probiotic to newborn piglets may thus promote resilience in the newly weaned pig. However, further studies with pigs subjected to a more severe pathogen challenge are needed to confirm these results and to investigate the mechanism of action of the probiotic intervention.     

Myocardial Injury after Surgery Is a Risk Factor for Weaning Failure from Mechanical Ventilation in Critical Patients Undergoing Major Abdominal Surgery

Background

Myocardial injury after noncardiac surgery (MINS) is a newly proposed concept that is common among adults undergoing noncardiac surgery and associated with substantial mortality. We analyzed whether MINS was a risk factor for weaning failure in critical patients who underwent major abdominal surgery.

Methods

This retrospective study was conducted in the Department of Critical Care Medicine of Peking University People''s Hospital. The subjects were all critically ill patients who underwent major abdominal surgery between January 2011 and December 2013. Clinical and laboratory parameters during the perioperative period were investigated. Backward stepwise regression analysis was performed to evaluate MINS relative to the rate of weaning failure. Age, hypertension, chronic renal disease, left ventricular ejection fraction before surgery, Acute Physiologic and Chronic Health Evaluation II score, pleural effusion, pneumonia, acute kidney injury, duration of mechanical ventilation before weaning and the level of albumin after surgery were treated as independent variables.

Results

This study included 381 patients, of whom 274 were successfully weaned. MINS was observed in 42.0% of the patients. The MINS incidence was significantly higher in patients who failed to be weaned compared to patients who were successfully weaned (56.1% versus 36.5%; P<0.001). Independent predictive factors of weaning failure were MINS, age, lower left ventricular ejection fraction before surgery and lower serum albumin level after surgery. The MINS odds ratio was 4.098 (95% confidence interval, 1.07 to 15.6; P = 0.04). The patients who were successfully weaned had shorter hospital stay lengths and a higher survival rate than those who failed to be weaned.

Conclusion

MINS is a risk factor for weaning failure from mechanical ventilation in critical patients who have undergone major abdominal surgery, independent of age, lower left ventricular ejection fraction before surgery and lower serum albumin levels after surgery.     

Alterations in Ileal Mucosa Bacteria Related to Diet Complexity and Growth Performance in Young Pigs

Background

Evaluation of the prolonged impact of weaning diet on ileal mucosa bacteria and during periods of reduced and improved growth was conducted using 454 pyrosequencing.

Methodology/Principal Findings

Weaned pigs were fed HIGH or LOW complexity diets, with or without antibiotics, for 6 weeks, followed by a common grower diet. Pigs were killed at 2 (n = 4 or 5) and 8 (n = 6) weeks post-weaning (periods of reduced and improved growth, respectively). Mucosal bacteria were removed; DNA was extracted and amplified using the V1–V3 region of the 16S rRNA gene. Mucosal bacteria clustered more closely by week post-weaning than diet but 44% of bacterial species did not change from week 2 to 8. There was no effect of diet complexity or antibiotic inclusion on indices of bacterial diversity. Firmicutes made up 91 and 96% of total reads at week 2 and 8, respectively. The proportion of Clostridium paraputrificum increased (P = 0.003) from week 2 to 8 in pigs fed LOW but didn’t change in pigs fed HIGH; whereas Clostridium leptum decreased (P = 0.02) from week 2 to 8 in pigs fed LOW but didn’t change in pigs fed HIGH. The proportion of Sarcina genus was 3-fold higher in pigs fed A+ compared to A− at week 2 and 5-fold higher at week 8 despite the lack of in-feed antibiotics at that time.

Conclusions/Significance

Shifts in mucosal bacteria populations may be related to dietary induced changes in growth performance during reduced and improved growth but further studies are required to confirm causative relationship. Weaning diet results in species specific prolonged alterations in mucosal bacteria, particularly where high levels of in-feed antibiotics are used. A considerable portion of ileal mucosal bacteria colonize early and remain stable over time despite changes in diet.     

Differential Effects of Early Weaning for HIV-Free Survival of Children Born to HIV-Infected Mothers by Severity of Maternal Disease

Background

We previously reported no benefit of early weaning for HIV-free survival of children born to HIV-infected mothers in intent-to-treat analyses. Since early weaning was poorly accepted, we conducted a secondary analysis to investigate whether beneficial effects may have been hidden.

Methods

958 HIV-infected women in Lusaka, Zambia, were randomized to abrupt weaning at 4 months (intervention) or to continued breastfeeding (control). Children were followed to 24 months with regular HIV PCR tests and examinations to determine HIV infection or death. Detailed behavioral data were collected on when all breastfeeding ended. Most participants were recruited before antiretroviral treatment (ART) became available. We compared outcomes among mother-child pairs who weaned earlier or later than intended by study design adjusting for potential confounders.

Results

Of infants alive, uninfected and still breastfeeding at 4 months in the intervention group, 16.1% who weaned as instructed acquired HIV or died by 24 months compared to 16.0% who did not comply (p = 0.98). Children of women with less severe disease during pregnancy (not eligible for ART) had worse outcomes if their mothers weaned as instructed (RH = 2.60 95% CI: 1.06–6.36) compared to those who continued breastfeeding. Conversely, children of mothers with more severe disease (eligible for ART but did not receive it) who weaned early had better outcomes (p-value interaction = 0.002). In the control group, weaning before 15 months was associated with 3.94-fold (95% CI: 1.65–9.39) increase in HIV infection or death among infants of mothers with less severe disease.

Conclusion

Incomplete adherence did not mask a benefit of early weaning. On the contrary, for women with less severe disease, early weaning was harmful and continued breastfeeding resulted in better outcomes. For women with more advanced disease, ART should be given during pregnancy for maternal health and to reduce transmission, including through breastfeeding.

Trial Registration

Clinical trials.gov {"type":"clinical-trial","attrs":{"text":"NCT00310726","term_id":"NCT00310726"}}NCT00310726     

Effects of dietary protein supply, weaning age and experimental enterotoxigenic Escherichia coli infection on newly weaned pigs: health

Weaning is often associated with post-weaning colibacillosis (PWC), caused by enterotoxigenic Escherichia coli (ETEC). The objective was to investigate the effects of manipulating dietary protein supply and increasing weaning age on enteric health and ETEC shedding of newly weaned pigs exposed to an experimental ETEC challenge. The experiment consisted of a complete 2 × 2 × 2 factorial combination of weaning age (4 v. 6 weeks), dietary protein content (H, 230 g crude protein (CP)/kg v. L, 130 g CP/kg) and experimental ETEC challenge (+ v. -); all foods were free from in-feed antimicrobial growth promoters (AGP). An additional four treatments were added to allow the effect of protein source (DSMP, dried skimmed milk powder v. SOYA, soybean meal) and AGP inclusion (yes v. no) to be investigated in challenged pigs of both weaning ages. On day 3 post-weaning challenged pigs were administered per os with 109 cfu ETEC O149. A subset of pigs was euthanased on days 0 and 6 post weaning to assess enteric health and small intestine morphology. Both weaning age and dietary protein content affected the consequences of ETEC challenge. ETEC excretion persisted longer in the 4-week-weaned pigs than those weaned at 6 weeks. Although not significant, the numbers of ETEC shed in the faeces post infection (days 4 to 14) were higher on the H than L diet, especially in the 4-week-weaned pigs (P = 0.093). Lowering CP level led to significantly firmer faeces post challenge (days 3 to 6) and decreased colonic digesta pH. Protein level had no effect on small intestine villous heights or crypt depths. There was no significant effect of protein source on ETEC excretion or enteric health. Results suggest that increasing weaning age and decreasing the level of dietary protein, especially in earlier weaned pigs, may help to maintain enteric health and minimise the effects of PWC.     

Escherichia coli Expressing EAST1 Toxin Did Not Cause an Increase of cAMP or cGMP Levels in Cells, and No Diarrhea in 5-Day Old Gnotobiotic Pigs

Background

Enterotoxigenic Escherichia coli (ETEC) strains are the leading bacterial cause of diarrhea to humans and farm animals. These ETEC strains produce heat-labile toxin (LT) and/or heat-stable toxins that include type I (STa), type II (STb), and enteroaggregative heat-stable toxin 1 (EAST1). LT, STa, and STb (in pigs) are proven the virulence determinants in ETEC diarrhea. However, significance of EAST1 in ETEC-associated diarrheal has not been determined, even though EAST1 is highly prevalent among ETEC strains.

Methodology/Principal Findings

In this study, we constructed E. coli strains to express EAST1 toxin as the only toxin and studied them in cell lines and five-day old gnotobiotic piglets to determine significance of EAST1 toxin. Data from in vitro studies indicated that EAST1 did not stimulate an increase of intracellular cyclic AMP or GMP levels in T-84 cells or porcine cell line IPEC-J2, nor did it enhance LT or STa toxin of ETEC strains in stimulation of cAMP or cGMP in T-84 cells. In addition, 5-day old gnotobiotic pigs challenged with E. coli strains expressing EAST1 as the only toxin did not developed diarrhea or signs of clinical disease during 72 h post-inoculation.

Conclusion/Significance

Results from this study indicated that EAST1 alone is not sufficient to cause diarrhea in five-day old gnotobiotic pigs, and suggest that EAST1 likely is not a virulence determinant in ETEC-associated diarrhea.     

Antibiotics Increase Gut Metabolism and Antioxidant Proteins and Decrease Acute Phase Response and Necrotizing Enterocolitis in Preterm Neonates

Background

The appropriate use of antibiotics for preterm infants, which are highly susceptible to develop necrotizing enterocolitis (NEC), is not clear. While antibiotic therapy is commonly used in neonates with NEC symptoms and sepsis, it remains unknown how antibiotics may affect the intestine and NEC sensitivity. We hypothesized that broad-spectrum antibiotics, given immediately after preterm birth, would reduce NEC sensitivity and support intestinal protective mechanisms.

Methodology/Principal Findings

Preterm pigs were treated with antibiotics for 5 d (oral and systemic doses of gentamycin, ampicillin and metrodinazole; AB group) and compared with untreated pigs. Only the untreated pigs showed evidence of NEC lesions and reduced digestive function, as indicated by lowered villus height and activity of brush border enzymes. In addition, 53 intestinal and 22 plasma proteins differed in expression between AB and untreated pigs. AB treatment increased the abundance of intestinal proteins related to carbohydrate and protein metabolism, actin filaments, iron homeostasis and antioxidants. Further, heat shock proteins and the complement system were affected suggesting that all these proteins were involved in the colonization-dependent early onset of NEC. In plasma, acute phase proteins (haptoglobin, complement proteins) decreased, while albumin, cleaved C3, ficolin and transferrin increased.

Conclusions/Significance

Depressed bacterial colonization following AB treatment increases mucosal integrity and reduces bacteria-associated inflammatory responses in preterm neonates. The plasma proteins C3, ficolin, and transferrin are potential biomarkers of the colonization-dependent NEC progression in preterm neonates.     

Histomorphometric characteristics of immune cells in small intestine of pigs perorally immunized with vaccine candidate F18ac+ nonenterotoxigenic E. coli strain

Colidiarrhea and colienterotoxemia caused by F4⁺ and/or F18⁺ enterotoxigenic E. coli (ETEC) strains are the most prevalent infections of suckling and weaned pigs. Here we tested the immunogenicity and protective effectiveness of attenuated F18ac⁺ non-ETEC vaccine candidate strain against challenge infection with F4ac⁺ ETEC strain by quantitative phenotypic analysis of small intestinal leukocyte subsets in weaned pigs.We also evaluated levamisole as an immune response modifier (IRM) and its adjuvanticity when given in the combination with the experimental vaccine. The pigs were parenterally immunized with either levamisole (at days -2, -1 and 0) or with levamisole and perorally given F18ac⁺ non-ETEC strain (at day 0), and challenged with F4ac⁺ ETEC strain 7 days later.At day 13 the pigs were euthanatized and sampled for immunohistological/histomorphometrical analyses. Lymphoid CD3⁺, CD45RA⁺, CD45RC⁺, CD21⁺, IgA⁺ and myeloid SWC3⁺ cell subsets were identified in jejunal and ileal epithelium, lamina propria and Peyer’s patches using the avidin-biotin complex method, and their numbers were determined by computer-assisted histomorphometry. Quantitative immunophenotypic analyses showed that levamisole treated pigs had highly increased numbers of jejunal CD3⁺, CD45RC⁺ and SWC3⁺ cells (p<0.05) as compared to those recorded in nontreated control pigs.In the ileum of these pigs we have recorded that only CD21⁺ cells were significantly increased (p<0.01). The pigs that were treated with levamisole adjuvanted experimental vaccine had significantly increased numbers of all tested cell subsets in both segments of the small intestine. It was concluded that levamisole adjuvanted F18ac⁺ non-ETEC vaccine was a requirement for the elicitation of protective gut immunity in this model; nonspecific immunization with levamisole was less effective, but confirmed its potential as an IRM.Key words: nonspecific/specific immunization, E. coli, gut immune cells, pigs.Porcine colidiarrhea and colienterotoxemia induced with F4⁺ and/or F18⁺ enterotoxigenic Escherichia coli (ETEC) strains are economically the most significant diseases of swine which account for moderate to high mortality rates and growth retardation, causing death of 5 million pigs per year in the World. Protection from ETEC is a constant challenge due to high genetic flexibility of this widespread bacterial organism.The virulence characteristics of ETEC are strongly dependent on the production of fimbrial adhesins and enterotoxins (Nagy and Fekete, 1999).The ability of adhesion of ETEC to intestinal wall is mainly due to the production of fimbriae. Enterotoxins produced by adherent ETEC strains act locally on enterocytes and stimulate increased water and electrolyte secretion and decreased fluid absorption. Several types of porcine ETEC are known today, including ETEC strain producing F18 fimbriae with their variants “ab” and “ac” (Bretschinger et al., 1990; Nagy and Fekete, 1999; Zang et al., 2007). The ETEC strains causing diarrhea mostly express F4 or F18 adhesins (Fairbrother et al., 2005; Zang et al., 2007). Nearly all known E. coli enterotoxin genes are produced by ETEC strains expressing either F4 or F18 fimbria. Zhang et al. (2007) have conclude that the dominant pathotypes causing diarrhea in weaned pigs are porcine ETEC strains expressing either F4 fimbria and heat-labile (LT) / heat stable (STb) toxins or LT/STb/EAST1 toxins, or F18 fimbria and STa/STb/Stx2e toxins. However, F18ab is more frequently associated with Shiga like toxin 2e, whereas F18ac is more frequently associated with enterotoxin STI (Cheng et al., 2005). Olasz et al. (2005) showed that the 200-kb plasmid, called pF18, contained the genes responsible for F18 fimbriae production.The curing of F18⁺ ETEC strain, i.e. loss of plasmid carrying the heat stable toxin genes in bacterial mutants has been performed by the plasmid transformation and conjugation following co-culturing of donor and recipient strains as reported earlier (Olasz et al., 2005). New vaccination strategies include the oral immunization of pigs with live avirulent nontoxigenic E. coli strains carrying the fimbrial adhesins F4 and/or F18 (Fairbrother et al., 2005).Receptors for F18ab and F18ac variants are increasingly produced up to the weaning age and the fimbriae F18ac seem to have more receptors around the ileal Peyer''s patches (Nagy et al., 1992). The colonization of the small intestine by an F18⁺ ETEC strain causes enterotoxemia. The typical clinical symptoms of the disease are neurological signs such as ataxia, convulsions and paralysis (Vögeli et al., 1996). It is well known that enterotoxic colibacillosis produces significant losses in two different age groups of pigs: first among newborn pigs and later at the postweaning age (Nagy and Fekete, 1999). The disease usually starts a few days after lacteal protection completely ceases (within the first 2 weeks after weaning), especially when weaning occurs at 3–4 weeks of age.Thus, the success of a vaccine against porcine colidiarrhea and colienterotoxemia depends upon applying it in the most efficient form at the optimal time and matching the right protective antigens with the type of virulence factors of ETEC present in the given animal population (Nagy and Fekete, 2005).The gut mucosal immune system contains specialized lymphoid tissues where environmental antigens are presented inducing B- and T-cell responses (Stokes et al., 1994). These responses are regulated by T cells and cytokines and they lead to plasma cell differentiation and the secretion of IgA antibodies onto intestinal mucosal surfaces.The aggregated lymphoid tissue such as Peyer’s patches and solitary lymphoid cells in the lamina propria both play important roles in the induction and regulation of immune responses in the gut associated lymphoid tissues (GALT) (Lacković et al.,1997b). Such organization of the GALT may provide immune protection at mucosal surfaces where the infection actually occurs (McGhee et al., 1992). Bertschinger et al. (2000) demonstrated the protective effects of a live oral vaccine containing F18 fimbria against porcine postweaning diarrhea and oedema disease.In this study we have examined the distribution and quantitative patterns of the subsets of T and B cells as well as of macrophages and secretory IgA⁺ plasma cells within GALT compartments of 4 weeks old pigs perorally immunized with an attenuated F18ac⁺ non-ETEC vaccine candidate strain against porcine colienterotoxemia. Additionally, we have evaluated adjuvanticity of levamisole in the combination with the experimental vaccine and its immunostimulatory effect when applied as an immune response modifier (IRM). Levamisole (2,3,5,6-tetrahydro-6-phenylimidazole thiazole), was originally described as a highly effective anti-helminthic compound (Thienpont et al., 1966). Subsequent studies have established its ability to restore and enhance depressed immune responses in domestic food animals and to act as an effective adjuvant for parenteral and oral vaccines (Mulcahy and Quinn, 1986; Jenkins and Hurdle, 1989; Božić et al., 2002).     

The level of protein in milk formula modifies ileal sensitivity to LPS later in life in a piglet model

Background

Milk formulas have higher protein contents than human milk. This high protein level could modify the development of intestinal microbiota, epithelial barrier and immune functions and have long-term consequences.

Methodology/Principal findings

We investigated the effect of a high protein formula on ileal microbiota and physiology during the neonatal period and later in life. Piglets were fed from 2 to 28 days of age either a normoprotein (NP, equivalent to sow milk) or a high protein formula (HP, +40% protein). Then, they received the same solid diet until 160 days. During the formula feeding period ileal microbiota implantation was accelerated in HP piglets with greater concentrations of ileal bacteria at d7 in HP than NP piglets. Epithelial barrier function was altered with a higher permeability to small and large probes in Ussing chambers in HP compared to NP piglets without difference in bacterial translocation. Infiltration of T cells was increased in HP piglets at d28. IL-1β and NF-κB sub-units mRNA levels were reduced in HP piglets at d7 and d28 respectively; plasma haptoglobin also tended to be reduced at d7. Later in life, pro-inflammatory cytokines secretion in response to high doses of LPS in explants culture was reduced in HP compared to NP piglets. Levels of mRNA coding the NF-κB pathway sub-units were increased by the challenge with LPS in NP piglets, but not HP ones.

Conclusions/Significance

A high protein level in formula affects the postnatal development of ileal microbiota, epithelial barrier and immune function in piglets and alters ileal response to inflammatory mediators later in life.     

Hemoglobin Levels and Weaning Outcome of Mechanical Ventilation in Difficult-To-Wean Patients: A Retrospective Cohort Study

Introduction

The effect of hemoglobin levels on the weaning outcomes of mechanically ventilated patients remains under debate, particularly for the patients with difficult weaning. This study aims to evaluate the effect of hemoglobin levels on weaning outcomes in difficult-to-wean patients.

Methods

This retrospective cohort study was conducted in a university-affiliated teaching hospital in Taiwan. Patients who fulfilled the criteria of difficult weaning were enrolled. Medical records were reviewed to obtain data on hemograms, biochemistry tests, transfusion records, comorbidities and weaning outcome. The association between hemoglobin levels and 30-day weaning outcomes was evaluated using a logistic regression model.

Results

A total of 751 patients received mechanical ventilation during the study period, 138 of whom fulfilled the criteria of difficult weaning. Compared with the patients whose hemoglobin was <8 g/dL, those with higher hemoglobin levels were more likely to be successfully weaned (odds ratio [OR], 3.69; 95% CI, 1.22–11.15 for hemoglobin 8–10 g/dL and OR, 4.16, 95% CI, 1.30–13.29 for hemoglobin >10 g/dL). Multivariate analysis showed that the odds ratio for weaning success remained significant for hemoglobin levels of 8–10 g/dL (adjusted OR, 3.3; 95% CI, 1.07–10.15) with borderline significance for hemoglobin level > 10 g/dL (adjusted OR, 2.95, 95% CI, 0.88–9.96).

Conclusions

Hemoglobin level is independently associated with weaning outcome in difficult-to-wean patients. Further studies are needed to evaluate whether a restrictive transfusion trigger for acute critical illness is also appropriate for such patients.     

The mycotoxin deoxynivalenol potentiates intestinal inflammation by Salmonella typhimurium in porcine ileal loops

Background and Aims

Both deoxynivalenol (DON) and nontyphoidal salmonellosis are emerging threats with possible hazardous effects on both human and animal health. The objective of this study was to examine whether DON at low but relevant concentrations interacts with the intestinal inflammation induced by Salmonella Typhimurium.

Methodology

By using a porcine intestinal ileal loop model, we investigated whether intake of low concentrations of DON interacts with the early intestinal inflammatory response induced by Salmonella Typhimurium.

Results

A significant higher expression of IL-12 and TNFα and a clear potentiation of the expression of IL-1β, IL-8, MCP-1 and IL-6 was seen in loops co-exposed to 1 µg/mL of DON and Salmonella Typhimurium compared to loops exposed to Salmonella Typhimurium alone. This potentiation coincided with a significantly enhanced Salmonella invasion in and translocation over the intestinal epithelial IPEC-J2 cells, exposed to non-cytotoxic concentrations of DON for 24 h. Exposure of Salmonella Typhimurium to 0.250 µg/mL of DON affected the bacterial gene expression level of a limited number of genes, however none of these expression changes seemed to give an explanation for the increased invasion and translocation of Salmonella Typhimurium and the potentiated inflammatory response in combination with DON.

Conclusion

These data imply that the intake of low and relevant concentrations of DON renders the intestinal epithelium more susceptible to Salmonella Typhimurium with a subsequent potentiation of the inflammatory response in the gut.     

Directed evaluation of enterotoxigenic Escherichia coli autotransporter proteins as putative vaccine candidates

Background

Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in developing countries, where it accounts for millions of infections and hundreds of thousands of deaths annually. While vaccine development to prevent diarrheal illness due to ETEC is feasible, extensive effort is needed to identify conserved antigenic targets. Pathogenic Escherichia coli, including ETEC, use the autotransporter (AT) secretion mechanism to export virulence factors. AT proteins are comprised of a highly conserved carboxy terminal outer membrane beta barrel and a surface-exposed amino terminal passenger domain. Recent immunoproteomic studies suggesting that multiple autotransporter passenger domains are recognized during ETEC infection prompted the present studies.

Methodology

Available ETEC genomes were examined to identify AT coding sequences present in pathogenic isolates, but not in the commensal E. coli HS strain. Passenger domains of the corresponding autotransporters were cloned and expressed as recombinant antigens, and the immune response to these proteins was then examined using convalescent sera from patients and experimentally infected mice.

Principal Findings

Potential AT genes shared by ETEC strains, but absent in the E. coli commensal HS strain were identified. Recombinant passenger domains derived from autotransporters, including Ag43 and an AT designated pAT, were recognized by antibodies from mice following intestinal challenge with {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407, and both Ag43 and pAT were identified on the surface of ETEC by flow cytometry. Likewise, convalescent sera from patients with ETEC diarrhea recognized Ag43 and pAT, suggesting that these proteins are expressed during both experimental and naturally occurring ETEC infections and that they are immunogenic. Vaccination of mice with recombinant passenger domains from either pAT or Ag43 afforded protection against intestinal colonization with ETEC.

Conclusions

Passenger domains of conserved autotransporter proteins could contribute to protective immune responses that develop following infection with ETEC, and these antigens consequently represent potential targets to explore in vaccine development.     

Comprehensive postmortem analyses of intestinal microbiota changes and bacterial translocation in human flora associated mice

Background

Postmortem microbiological examinations are performed in forensic and medical pathology for defining uncertain causes of deaths and for screening of deceased tissue donors. Interpretation of bacteriological data, however, is hampered by false-positive results due to agonal spread of microorganisms, postmortem bacterial translocation, and environmental contamination.

Methodology/Principal Findings

We performed a kinetic survey of naturally occurring postmortem gut flora changes in the small and large intestines of conventional and gnotobiotic mice associated with a human microbiota (hfa) applying cultural and molecular methods. Sacrificed mice were kept under ambient conditions for up to 72 hours postmortem. Intestinal microbiota changes were most pronounced in the ileal lumen where enterobacteria and enterococci increased by 3–5 orders of magnitude in conventional and hfa mice. Interestingly, comparable intestinal overgrowth was shown in acute and chronic intestinal inflammation in mice and men. In hfa mice, ileal overgrowth with enterococci and enterobacteria started 3 and 24 hours postmortem, respectively. Strikingly, intestinal bacteria translocated to extra-intestinal compartments such as mesenteric lymphnodes, spleen, liver, kidney, and cardiac blood as early as 5 min after death. Furthermore, intestinal tissue destruction was characterized by increased numbers of apoptotic cells and neutrophils within 3 hours postmortem, whereas counts of proliferative cells as well as T- and B-lymphocytes and regulatory T-cells decreased between 3 and 12 hours postmortem.

Conclusions/Significance

We conclude that kinetics of ileal overgrowth with enterobacteria and enterococci in hfa mice can be used as an indicator for compromized intestinal functionality and for more precisely defining the time point of death under defined ambient conditions. The rapid translocation of intestinal bacteria starting within a few minutes after death will help to distinguish between relevant bacteria and secondary contaminants thus providing important informations for routine applications and future studies in applied microbiology, forensic pathology, and criminal medicine.     

Effects of Administration of Live or Inactivated Virulent Rhodococccus equi and Age on the Fecal Microbiome of Neonatal Foals

Background

Rhodococcus equi is an important pathogen of foals. Enteral administration of live, virulent R. equi during early life has been documented to protect against subsequent intrabronchial challenge with R. equi, indicating that enteral mucosal immunization may be protective. Evidence exists that mucosal immune responses develop against both live and inactivated micro-organisms. The extent to which live or inactivated R. equi might alter the intestinal microbiome of foals is unknown. This is an important question because the intestinal microbiome of neonates of other species is known to change over time and to influence host development. To our knowledge, changes in the intestinal microbiome of foals during early life have not been reported. Thus, the purpose of this study was to determine whether age (during the first month of life) or administration of either live virulent R. equi (at a dose reported to protect foals against subsequent intrabronchial challenge, viz., 1×10¹⁰ colony forming units [CFU]) or inactivated virulent R. equi (at higher doses, viz., 2×10¹⁰ and 1×10¹¹ [CFU]) altered the fecal microbiome of foals.

Methodology/Principal Findings

Fecal swab samples from 42 healthy foals after vaccination with low-dose inactivated R. equi (n = 9), high-dose inactivated R. equi (n = 10), live R. equi (n = 6), control with cholera toxin B (CTB, n = 9), and control without CTB (n = 8) were evaluated by 454-pyrosequencing of the 16S rRNA gene and by qPCR. No impact of treatment was observed among vaccinated foals; however, marked and significant differences in microbial communities and diversity were observed between foals at 30 days of age relative to 2 days of age.

Conclusions

The results suggest age-related changes in the fecal microbial population of healthy foals do occur, however, mucosal vaccination does not result in major changes of the fecal microbiome in foals.     

Pre- and Postnatal Nutritional Histories Influence Reproductive Maturation and Ovarian Function in the Rat

Background

While prepubertal nutritional influences appear to play a role in sexual maturation, there is a need to clarify the potential contributions of maternal and childhood influences in setting the tempo of reproductive maturation. In the present study we employed an established model of nutritional programming to evaluate the relative influences of prenatal and postnatal nutrition on growth and ovarian function in female offspring.

Methods

Pregnant Wistar rats were fed either a calorie-restricted diet, a high fat diet, or a control diet during pregnancy and/or lactation. Offspring then were fed either a control or a high fat diet from the time of weaning to adulthood. Pubertal age was monitored and blood samples collected in adulthood for endocrine analyses.

Results

We report that in the female rat, pubertal timing and subsequent ovarian function is influenced by the animal''s nutritional status in utero, with both maternal caloric restriction and maternal high fat nutrition resulting in early pubertal onset. Depending on the offspring''s nutritional history during the prenatal and lactational periods, subsequent nutrition and body weight gain did not further influence offspring reproductive tempo, which was dominated by the effect of prenatal nutrition. Whereas maternal calorie restriction leads to early pubertal onset, it also leads to a reduction in adult progesterone levels later in life. In contrast, we found that maternal high fat feeding which also induces early maturation in offspring was associated with elevated progesterone concentrations.

Conclusions

These observations are suggestive of two distinct developmental pathways leading to the acceleration of pubertal timing but with different consequences for ovarian function. We suggest different adaptive explanations for these pathways and for their relationship to altered metabolic homeostasis.     

Early hypothalamic FTO overexpression in response to maternal obesity--potential contribution to postweaning hyperphagia

Background

Intrauterine and postnatal overnutrition program hyperphagia, adiposity and glucose intolerance in offspring. Single-nucleotide polymorphisms (SNPs) of the fat mass and obesity associated (FTO) gene have been linked to increased risk of obesity. FTO is highly expressed in hypothalamic regions critical for energy balance and hyperphagic phenotypes were linked with FTO SNPs. As nutrition during fetal development can influence the expression of genes involved in metabolic function, we investigated the impact of maternal obesity on FTO.

Methods

Female Sprague Dawley rats were exposed to chow or high fat diet (HFD) for 5 weeks before mating, throughout gestation and lactation. On postnatal day 1 (PND1), some litters were adjusted to 3 pups (vs. 12 control) to induce postnatal overnutrition. At PND20, rats were weaned onto chow or HFD for 15 weeks. FTO mRNA expression in the hypothalamus and liver, as well as hepatic markers of lipid metabolism were measured.

Results

At weaning, hypothalamic FTO mRNA expression was increased significantly in offspring of obese mothers and FTO was correlated with both visceral and epididymal fat mass (P<0.05); body weight approached significance (P = 0.07). Hepatic FTO and Fatty Acid Synthase mRNA expression were decreased by maternal obesity. At 18 weeks, FTO mRNA expression did not differ between groups; however body weight was significantly correlated with hypothalamic FTO. Postnatal HFD feeding significantly reduced hepatic Carnitine Palmitoyltransferase-1a but did not affect the expression of other hepatic markers investigated. FTO was not affected by chronic HFD feeding.

Significance

Maternal obesity significantly impacted FTO expression in both hypothalamus and liver at weaning. Early overexpression of hypothalamic FTO correlated with increased adiposity and later food intake of siblings exposed to HFD suggesting upregulation of FTO may contribute to subsequent hyperphagia, in line with some human data. No effect of maternal obesity was observed on FTO in adulthood.     

Dynamics of Japanese Encephalitis Virus Transmission among Pigs in Northwest Bangladesh and the Potential Impact of Pig Vaccination

Background

Japanese encephalitis (JE) virus infection can cause severe disease in humans, resulting in death or permanent neurologic deficits among survivors. Studies indicate that the incidence of JE is high in northwestern Bangladesh. Pigs are amplifying hosts for JE virus (JEV) and a potentially important source of virus in the environment. The objectives of this study were to describe the transmission dynamics of JEV among pigs in northwestern Bangladesh and estimate the potential impact of vaccination to reduce incidence among pigs.

Methodology/Principal Findings

We conducted a comprehensive census of pigs in three JE endemic districts and tested a sample of them for evidence of previous JEV infection. We built a compartmental model to describe JEV transmission dynamics in this region and to estimate the potential impact of pig vaccination. We identified 11,364 pigs in the study area. Previous JEV infection was identified in 30% of pigs with no spatial differences in the proportion of pigs that were seropositive across the study area. We estimated that JEV infects 20% of susceptible pigs each year and the basic reproductive number among pigs was 1.2. The model suggest that vaccinating 50% of pigs each year resulted in an estimated 82% reduction in annual incidence in pigs.

Conclusions/Significance

The widespread distribution of historic JEV infection in pigs suggests they may play an important role in virus transmission in this area. Future studies are required to understand the contribution of pig infections to JE risk in humans and the potential impact of pig vaccination on human disease.