Differences in enteric pathogens and intestinal microbiota between diarrheic weaned piglets and healthy penmates.

Postweaning diarrhea (PWD) is a multifactorial disease caused by different aetiological agents, like viruses or bacteria and where the role of the microbiota remains unclear. The aim of this study was to assess differences between healthy and diarrheic weaned pigs concerning the prevalence of pathogens and changes in the intestinal microbiota. Eighteen farms with PWD were selected and 277 fecal samples were collected (152 diarrheic vs 125 healthy). Presence of Rotavirus A (RVA), B (RVB), C (RVC) and Porcine Epidemic Diarrhea Virus (PEDV), virulence factors of Escherichia coli and Clostridioides difficile were analyzed by PCR. Finally, the microbiota composition was also study by 16 S rRNA sequencing on 148 samples (102 diarrheic vs 46 healthy). RVA (53.95 % vs 36 %, p=0.04) and RVB (49.67 % vs 28.8 %, p<0.001) were more frequent in diarrheic animals. Furthermore, RVA viral load was higher in diseased animals. VT2 toxin was significantly associated with diarrhea, whereas other virulence factors were not. Presence of C. difficile and PEDV was almost negligible. Regarding microbiota changes, Fusobacteriota phylum was more frequent in diarrheic samples and Ruminococcaceae family in healthy penmates. During the first week postweaning, Enterobacteriace and Campylobacteria were enriched in animals presenting diarrhea. Furthermore, Lactobacillus was detected in those individuals with no RVA infection. In conclusion, RVA seems to play a primary role in PWD. Classic E. coli virulence factors were not associated with diarrhea, indicating the need for revising their implication in disease. Moreover, Lactobacillus was found frequently in animals negative for RVA, suggesting some protective effect.

Effect of an organic acid blend as an antibiotic alternative on growth performance, antioxidant capacity, intestinal barrier function, and fecal microbiota in weaned piglets.

This study was conducted to evaluate the effects of dietary organic acid blend on growth performance, antioxidant capacity, intestinal barrier function, and fecal microbiota in weaned piglets compared to antibiotic growth promoters (AGP). A total of 90 weaned crossbred barrows (24 ± 1 days of age) with an initial body weight of 7.40 kg were allocated into 3 experimental treatments. Each treatment consisted of 6 replicate pens, with 5 piglets housed in each pen. The dietary treatments included the basal diet (NC), the basal diet supplemented with antibiotics (PC), and the basal diet supplemented with organic acid blend (OA). On day 42, one piglet per pen was randomly selected for plasma and small intestinal sample collection. The results showed that dietary AGP significantly improved growth performance and reduced diarrhea incidence compared to the NC group (P < 0.05). Dietary OA tended to increase body weight on day 42 (P = 0.07) and average daily gain from day 0 to 42 (P = 0.06) and reduce diarrhea incidence (P = 0.05). Dietary OA significantly increased plasma catalase (CAT) activity and decreased the plasma concentration of malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin (IL)-8, and IL-6, which were accompanied by upregulated the relative mRNA abundance of superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1) and nuclear factor erythroid 2-related factor 2 (NRF2) in comparison to that in the NC group (P < 0.05). Moreover, pigs fed the OA diet significantly increased the ratio of villus height to crypt depth and upregulated the relative expression of zonula occludens-1 (ZO-1) and Claudin1 gene in the jejunum compared to the NC group (P < 0.05). Interestingly, dietary AGP or OA did not affect the fecal microbiota structure or volatile fatty acid content (P > 0.05). In conclusion, our results suggested that dietary OA supplementation could improve growth performance and antioxidant capacity and protect the intestinal barrier of weaned piglets, therefore it has the potential to be consideredas an alternative to AGP in the pig industry.

Effects of increasing dietary arginine supply during the three first weeks after weaning on pig growth performance, plasma amino acid concentrations, and health status.

A total of 425 weaned pigs (Exp. 1: 225 pigs [5.8 ±â€…0.9 kg]; Exp. 2: 200 pigs [6.1 ±â€…1.2 kg]) were used to determine the optimal dietary standardized ileal digestible (SID) arginine (Arg) level in early nursery diets based on growth and health responses. The basal diet in Exp.1 was formulated to meet SID Arg recommendation (0.66%; NRC, 2012) and in Exp. 2, SID Arg was set to simulate current industry practices for feeding nursery pigs (1.15 %). Basal diets were supplemented with 0.3%, 0.6%, 0.9%, and 1.2% of l-arginine to provide five levels of dietary SID Arg. Experimental diets were fed during phases I (days 0 to 7) and II (days 8 to 21) with common diets until market. Feed disappearance and body weight (BW) were measured on days 7, 14, 21, and 43. Final BW was recorded at first removal of pigs for market. Pen fecal score was assigned daily from days 0 to 21. Plasma immunoglobulin A (IgA) was determined on days 0, 7, and 14 and amino acids (AAs) concentration and plasma urea nitrogen (PUN) on days 0 and 14. Orthogonal polynomial contrasts were used to determine the linear and quadratic effects of dietary Arg. Optimal SID Arg was determined by fitting the data with piecewise regression, using growth performance as the primary response variable. In Exp. 1, dietary Arg linearly increased (P < 0.1) BW, average daily gain (ADG), and gain to feed ratio (G:F) ratio on day 21, as well as reduced (χ2 = 0.004) the percentage of pigs that lost weight (PLW) in week 1 by 29%. Dietary Arg resulted in linear improvement (P = 0.082) of ADG for the overall nursery period and quadratic improvement (P < 0.1) of final BW at marketing. In Exp. 2, dietary Arg linearly increased (P < 0.05) ADG and average daily feed intake (ADFI) in week 1, BW and ADFI (P < 0.1) on day 14, as well as reduced (χ2 ≤ 0.001) PLW in week 1. From days 0 to 21, G:F was improved quadratically (P < 0.1). Dietary Arg linearly increased (P < 0.1) ADG and BW on day 43. Dietary Arg supplementation decreased the incidence (χ2 < 0.05) of soft and watery feces during the first weeks after weaning and lower concentration of plasma IgA on days 7 and 14. Dietary Arg linearly and/or quadratically influenced plasma AA concentrations (P < 0.05), including an increase in Arg, Leu, Phe, Val, citrulline, ornithine, and PUN concentrations. Overall, weaned pigs exhibit optimal nursery growth performance and health when provided with dietary SID Arg ranging from 1.5% to 1.9%. This dietary range contributes to a reduction in the occurrence of fall-back pigs and improvements in final BW at marketing.

Characterization of Antimicrobial Resistance in Escherichia coli Isolated from Diarrheic and Healthy Weaned Pigs in Catalonia.

Postweaning diarrhea (PWD) is a multifactorial concern in the swine industry that leads to high antibiotic consumption, usually without testing susceptibility, increasing the risk of the selection of Escherichia coli-resistant strains. In this study, 251 E. coli strains isolated from fecal samples of diarrheic (n = 148) and apparently healthy piglets (n = 103) in farms in Catalonia were tested against their susceptibility to fourteen different antimicrobials. The phenotypic antimicrobial resistance (AMR) testing revealed high levels of AMR, with 41.4% of the isolates presenting a multidrug-resistant (MDR) profile. More specifically, resistance to class D (prudence) antimicrobials such as erythromycin (99.6%), amoxicillin (95.2%), streptomycin (91.6%), tetracycline (88.8%), lincospectin (64.5%), and sulfamethoxazole/trimethoprim (60%) was very high, as well as to class C (caution) antimicrobials such as florfenicol (45%). A special concern was observed for antimicrobial category B (restrict), like quinolones and colistin, that both presented a high rate of resistance. Colistin use was substantially reduced in Spain, but resistance is still present in weaned pigs, presenting a MIC90 of 4 µg/mL. This suggests that reducing antibiotic use is not enough to eliminate this AMR. Finally, it was found that piglets suffering diarrhea were more commonly carriers of MDR strains than the healthy ones (49.3% vs. 35%, p = 0.031). Therefore, given the high rates of resistance to the most commonly used antimicrobials, especially in diseased pigs, a new non-antibiotic-based approach should be implemented for the management of PWD.

Effect of different ratios of phytogenic feed additives on growth performance, nutrient digestibility, intestinal barrier integrity, and immune response in weaned pigs challenged with a pathogenic Escherichia coli.

This study was conducted to investigate the effects of supplementing different ratios of phytogenic feed additives (PFA) to weaned pigs challenged with pathogenic Escherichia coli on growth performance, nutrient digestibility, intestinal barrier integrity, and immune response, and to determine the optimal mixing ratio for post-weaning diarrhea (PWD) prevention. A total of 48 4-wk-old weaned pigs with initial body weight of 8.01 ± 0.39 kg were placed in individual metabolic cages, and then randomly assigned to eight treatment groups. The eight treatments were as follows: a basal diet without E. coli challenge (negative control, NC), a basal diet with E. coli challenge (positive control, PC), PC with supplementing 0.1% mixture of 20% bitter citrus extract (BCE), 10% microencapsulated blend of thymol and carvacrol (MEO), and 70% excipient (T1), PC with supplementing 0.1% mixture of 10% MEO, 20% premixture of grape seed and grape marc extract, green tea, and hops (PGE), and 60% excipient (T2), PC with supplementing 0.1% mixture of 10% BCE, 10% MEO, 10% PGE, and 70% excipient (T3), PC with supplementing 0.1% mixture of 20% BCE, 20% MEO, and 60% excipient (T4), PC with supplementing 0.1% mixture of 20% MEO, 20% PGE, and 60% excipient (T5), and PC with supplementing 0.1% mixture of 10% BCE, 20% MEO, 10% PGE, and 60% excipient (T6). The experiments progressed in 16 days, including 5 days before and 11 days after the first E. coli challenge (day 0). In the E. coli challenge treatments, all pigs were orally inoculated by dividing a total of 10 mL of E. coli F 18 for three consecutive days from day 0 postinoculation (PI). Compared with the PC group, the PFA2 and PFA6 groups significantly increased (P < 0.05) feed efficiency and decreased (P < 0.05) diarrhea during the entire period. At day 11 PI, the PFA6 group significantly improved (P < 0.05) gross energy digestibility compared to the PFA1 group. The PFA6 group significantly decreased (P < 0.05) tumor necrosis factor α (TNF-α) and interleukin-6 in serum and increased (P < 0.05) the villus height to crypt depth ratio (VH:CD). The PFA2 significantly decreased (P < 0.05) the relative protein expression of calprotectin in the ileum. In conclusion, improvements in growth performance, diarrhea reduction, and immunity enhancement are demonstrated when 10% BCE, 20% MEO, 10% PGE, and 60% excipient are mixed.

Phytogenic feed additives (PFA) include various herbs and spices, such as essential oils and polyphenols. Flavonoids and polyphenols contained in PFA are generally known to have antioxidant and antibacterial actions and based on this, PFA is considered an alternative to antibiotics in the swine industry. Pathogenic Escherichia coli infection is one of the most important causes of post-weaning diarrhea (PWD) in pigs. PWD causes intestinal damage, which leads to severe diarrhea, reduced growth performance, and mortality in weaned pigs, resulting in significant financial loss to the swine industry. Therefore, this study was conducted to investigate the effects of supplementing different ratios of PFA to weaned pigs challenged with E. coli and determine the optimal mixing ratio for PWD prevention. Our study results showed that growth performance was improved when supplementing a mixture of 10% bitter citrus extract (BCE), 20% microencapsulated blend of thymol and carvacrol (MEO), 10% premixture of grape seed and grape marc extract, green tea, and hops (PGE), and 60% excipient. Also, the effect of improving the immune response and intestinal morphology was shown. In conclusion, a mixture of 10% BCE, 20% MEO, 10% PGE, and 60% excipients is considered the optimal mixing ratio.

Oral administration of heat-inactivated Escherichia coli during suckling alleviated Salmonella typhimurium-derived intestinal injury after rat weaning.

Introduction: Newly weaned animals are susceptible to a wide range of microbial infections taking a high risk of developing post-weaning diarrhea. Trained immunity is the capacity of the innate immune system to produce a stronger and non-specific response against a secondary infection after the inflammatory response caused by previous stimulus has returned to normal state. The objective of this study was to evaluate if the heat-inactivated Escherichia coli (IEC) as an immunostimulant on suckling pups elicits a protective effect on the intestine of post-weaning rats challenged with Salmonella Typhimurium (S.Typhimurium). We adapted a newborn rat model for this purpose. Methods: Sixty newborn pups were randomly separated into two groups: IEC group (n =30) orally administrated IEC during suckling, while the CON group received orally the same dose of saline. Both of the two group challenged with various doses of S.Typhimurium after experiencing a 4-week resting period. Twelve of individuals were selected to detect the survival rate, and ten of the rest were necropsied 48 hours post-challenge. Results and Discussion: The results showed that oral administration of IEC during suckling alleviated the injury in ileal morphology induced by post-weaning S.Typhimurium infection via increasing the levels of two tight junction proteins [zonula occluden-1 (ZO-1) and Occludin-1] and several secreted proteins (Lysozyme, Mucin-2, and SIgA) in the intestinal mucosa. Furthermore, the pre-stimulation with IEC significantly increased cytokines tumor necrosis factor-alpha (TNF- α) and interleukin-1 beta (IL-1 ß) expressions in an enhanced secondary reaction way after experiencing a 4-week resting period. This implicated the possible involvement of trained immunity. The 16S rDNA sequence results showed that pre-stimulation with IEC decreased the abundance of Clostridia, Prevotella, Christensenellaceae_R-7_group and Parabacteroides after intestinal infection of S.Typhimurium. Our results confirmed that the previous oral administration of IEC had a protective effect on S.Typhimurium-induced intestinal injury in weaned rats by inducing a robust immune response. The present study suggested a new strategy for preventing intestinal infection of newborn animals.

Lactoferrin Decreases Enterotoxigenic Escherichia coli-Induced Fluid Secretion and Bacterial Adhesion in the Porcine Small Intestine.

Enterotoxigenic Escherichia coli (ETEC) infections are one of the most prevalent causes of post-weaning diarrhea in piglets, resulting in morbidity, mortality and elevated use of antibiotics. The emergence and further spread of antimicrobial resistance together with the growing demand for high quality animal protein requires the identification of novel alternatives for antimicrobials. A promising alternative is lactoferrin, as we previously showed that it can both inhibit the growth and degrade bacterial virulence factors of porcine ETEC strains in vitro. Aiming to confirm these findings in vivo, we performed a small intestinal segment perfusion experiment in piglets. Here, we showed that lactoferrin could not only decrease ETEC-induced fluid secretion, but also their ability to colonize the small intestinal epithelium. Furthermore, while ETEC infection induced pro-inflammatory cytokine mRNA expression in this experiment, lactoferrin was not able to counteract these responses. In addition, a bacterial motility assay showed that lactoferrin can reduce the motility of ETEC. Our findings further support the use of lactoferrin as an alternative for antimicrobials and also show its potential for the prevention of ETEC infections in pigs.

The Role of Probiotics in Alleviating Postweaning Diarrhea in Piglets From the Perspective of Intestinal Barriers.

Early weaning of piglets is an important strategy for improving the production efficiency of sows in modern intensive farming systems. However, due to multiple stressors such as physiological, environmental and social challenges, postweaning syndrome in piglets often occurs during early weaning period, and postweaning diarrhea (PWD) is a serious threat to piglet health, resulting in high mortality. Early weaning disrupts the intestinal barrier function of piglets, disturbs the homeostasis of gut microbiota, and destroys the intestinal chemical, mechanical and immunological barriers, which is one of the main causes of PWD in piglets. The traditional method of preventing PWD is to supplement piglet diet with antibiotics. However, the long-term overuse of antibiotics led to bacterial resistance, and antibiotics residues in animal products, threatening human health while causing dysbiosis of gut microbiota and superinfection of piglets. Antibiotic supplementation in livestock diets is prohibited in many countries and regions. Regarding this context, finding antibiotic alternatives to maintain piglet health at the critical weaning period becomes a real emergency. More and more studies showed that probiotics can prevent and treat PWD by regulating the intestinal barriers in recent years. Here, we review the research status of PWD-preventing and treating probiotics and discuss its potential mechanisms from the perspective of intestinal barriers (the intestinal microbial barrier, the intestinal chemical barrier, the intestinal mechanical barrier and the intestinal immunological barrier) in piglets.

Interaction of CP levels in maternal and nursery diets, and its effect on performance, protein digestibility, and serum urea levels in piglets.

Reduced protein levels in nursery diets have been associated with a lower risk of postweaning diarrhea, but the interaction with CP levels in maternal diet on the performance of the offspring remains unclear. The objective of this study was to determine the effect of protein content in sow gestation and piglet nursery diets on the performance of the piglets until slaughter. This was studied in a 2 × 2 factorial trial (35 sows, 209 piglets), with higher or lower (H or L) dietary CP in sow diets (168 vs 122 g CP/kg) during late gestation. A standard lactation feed was provided for all sows (160 g CP/kg). For both sow treatments, half of the litters received a higher or lower CP in the piglet nursery diet (210 vs 166 g CP/kg). This resulted in four possible treatment combinations: HH, HL, LH and LL, with sow treatment as first and piglet treatment as second letter. For each phase, all diets were iso-energetic and had a similar level of essential amino acids. Ps*p is the p-value for the interaction effect between sow and piglet treatment. In the nursery phase (3.5-9 weeks of age), a tendency toward interaction between piglet and sow treatments with feed efficiency (Ps*p = 0.08) was observed with HH having the highest gain:feed ratio (G:F) (0.74 ± 0.01), LH the lowest (0.70 ± 0.01) and the other two groups intermediate. In the growing-finishing phase, an interaction was observed between the piglet and sow diets with decreased G:F for LH (Ps*p = 0.04) and a tendency toward interaction with increased daily feed intake for LH (Ps*p = 0.07). The sow diet showed a tendency toward a long-lasting effect on the dressing percentage and meat thickness of the offspring, which was higher for the progeny of H sows (Ps < 0.01 and Ps = 0.02, respectively). At 23 weeks, serum urea concentrations tended to be lower for the HH and LL groups (Ps*p = 0.07). Fecal consistency scores were higher at day 10-day 14 after weaning for piglets from L sows (Ps = 0.03 and Ps < 0.01, respectively). At day 7 after weaning, fecal consistency score was higher for piglets fed the higher protein diet (Pp < 0.01). At 8 weeks of age, the apparent total tract digestibility of CP (ATTDCP) interacted between piglet and sow diet (Ps*p = 0.02), with HH showing the highest digestibility values. In conclusion, the protein levels in sow late-gestation and piglet nursery diets interacted with feed efficiency, ATTDCP and serum urea concentrations in the nursery phase.

Development of piglet gut microbiota at the time of weaning influences development of postweaning diarrhea - A field study.

Postweaning diarrhea is a common issue in pig production which is currently controlled by feed supplementation with zinc oxide. However, new alternatives are being sought due to an expected ban on zinc oxide in feed supplementation from 2022 in the EU. One possible alternative is to use novel types of probiotics consisting of microbiota characteristic for healthy weaned piglets. In this study, we therefore collected rectal swabs of piglets 3 days before weaning and 4 days after weaning in a commercial farm considering all risks of field trial like the use of antibiotics, classified the piglets as predisposed, healthy or sick and using 16S rRNA sequencing, we determined and compared the microbiota composition. Increased Actinobacteria before weaning was a marker of piglets predisposed for diarrhea. Increased Chlamydia or Helicobacter before weaning was surprisingly a marker of healthy and resistant piglets after weaning. After weaning, unclassified Clostridiales, Deltaproteobacteria, Selenomonadales, Fusobacterium, Akkermansia or Anaerovibrio increased in microbiota of piglets with postweaning diarrhea while an increase in Prevotella and Faecalibacterium was characteristic for healthy, weaned piglets. Both changes in individual microbiota members and also correct timing of microbiota reshaping around weaning and the increase of mainly Prevotella species just after weaning are equally important for resistance to postweaning diarrhea in piglets under field conditions.

Porcine and Bovine Forms of Lactoferrin Inhibit Growth of Porcine Enterotoxigenic Escherichia coli and Degrade Its Virulence Factors.

Postweaning diarrhea (PWD) is an economically important, multifactorial disease affecting pigs within the first 2 weeks after weaning. The most common agent associated with PWD is enterotoxigenic Escherichia coli (ETEC). Currently, antibiotics are used to control PWD, and this has most likely contributed to an increased prevalence of antibiotic-resistant strains. This puts pressure on veterinarians and farmers to decrease or even abandon the use of antibiotics, but these measures need to be supported by alternative strategies for controlling these infections. Naturally derived molecules, such as lactoferrin, could be potential candidates due to their antibacterial or immune-modulating activities. Here, we analyzed the ability of bovine lactoferrin (bLF), porcine lactoferrin (pLF), and ovotransferrin (ovoTF) to inhibit ETEC growth, degrade ETEC virulence factors, and inhibit adherence of these pathogens to porcine intestinal epithelial cells. Our results revealed that bLF and pLF, but not ovoTF, inhibit the growth of ETEC. Furthermore, bLF and pLF can degrade several virulence factors produced by ETEC strains, more specifically F4 fimbriae, F18 fimbriae, and flagellin. On the other hand, ovoTF degrades F18 fimbriae and flagellin but not F4 fimbriae. An in vitro adhesion assay showed that bLF, ovoTF, and pLF can decrease the number of bacteria adherent to epithelial cells. Our findings demonstrate that lactoferrin can directly affect porcine ETEC strains, which could allow lactoferrin to serve as an alternative to antimicrobials for the prevention of ETEC infections in piglets.IMPORTANCE Currently, postweaning F4+ and F18+Escherichia coli infections in piglets are controlled by the use of antibiotics and zinc oxide, but the use of these antimicrobial agents most likely contributes to an increase in antibiotic resistance. Our work demonstrates that bovine and porcine lactoferrin can inhibit the growth of porcine enterotoxigenic E. coli strains. In addition, we also show that lactoferrin can reduce the adherence of these strains to small intestinal epithelial cells, even at a concentration that does not inhibit bacterial growth. This research could allow us to develop lactoferrin as an alternative strategy to prevent enterotoxigenic E. coli (ETEC) infections in piglets.

Application of a Novel Epitope- and Structure-Based Vaccinology-Assisted Fimbria-Toxin Multiepitope Fusion Antigen of Enterotoxigenic Escherichia coli for Development of Multivalent Vaccines against Porcine Postweaning Diarrhea.

Enterotoxigenic Escherichia coli (ETEC) strains producing K88 (F4) or F18 fimbriae and enterotoxins are the predominant cause of pig postweaning diarrhea (PWD). We recently identified neutralizing epitopes of fimbriae K88 and F18, heat-labile toxin (LT), heat-stable toxins type I (STa) and type II (STb), and Shiga toxin 2e (Stx2e). In this study, we explored a novel epitope- and structure-based vaccinology platform, multiepitope fusion antigen (MEFA), for PWD vaccine development. By using an epitope substitution LT toxoid, which lacks enterotoxicity but retains immunogenicity, as the backbone to present neutralizing epitopes of two ETEC fimbriae and four toxins, we generated PWD fimbria-toxin MEFA to mimic epitope native antigenicity. We then examined MEFA protein immunogenicity and evaluated MEFA application in PWD vaccine development. Mice subcutaneously immunized with PWD MEFA protein developed strong IgG responses to K88, F18, LT, and STb and moderate responses to the toxins Stx2e and STa. Importantly, MEFA-induced antibodies inhibited adherence of K88 or F18 fimbrial bacteria to pig intestinal cells and also neutralized LT, STa, STb, and Stx2e toxicity. These results indicated that PWD fimbria-toxin MEFA induced neutralizing antibodies against an unprecedent two fimbriae and four toxins and strongly suggested a potential application of this MEFA protein in developing a broadly protective PWD vaccine.IMPORTANCE ETEC-associated postweaning diarrhea (PWD) causes significant economic losses to swine producers worldwide. Currently, there is no effective prevention against PWD. A vaccine that blocks ETEC fimbriae (K88 and F18) from attaching to host receptors and prevents enterotoxins from stimulating water hypersecretion in pig small intestinal epithelial cells can effectively protect against PWD and significantly improves pig health and well-being. The fimbria-toxin MEFA generated from this study induced neutralizing antibodies against both ETEC fimbriae and all four ETEC toxins, suggesting a great potential of this fimbria-toxin MEFA in PWD vaccine development and further supporting the general application of this novel MEFA vaccinology platform for multivalent vaccine development.

Mapping the Neutralizing Epitopes of Enterotoxigenic Escherichia coli K88 (F4) Fimbrial Adhesin and Major Subunit FaeG.

Enterotoxigenic Escherichia coli (ETEC) strains that produce immunologically heterogeneous fimbriae and enterotoxins are the primary cause of neonatal diarrhea and postweaning diarrhea in young pigs. A multivalent vaccine inducing protective immunity against ideally all ETEC fimbriae and enterotoxins could be effective against diarrhea in young pigs. However, developing a vaccine to broadly protect against various ETEC virulence determinants has proven challenging. Recently developed structure- and epitope-based multiepitope fusion antigen (MEFA) technology that presents neutralizing epitopes of various virulence determinants at a backbone immunogen and that mimics epitope native immunogenicity suggests the feasibility of developing multivalent vaccines. With neutralizing epitopes from ETEC fimbria F18 and enterotoxins being identified, it becomes urgent to identify protective epitopes of K88 (F4) fimbriae, which play a major role in pig neonatal and postweaning diarrhea. In this study, we identified B-cell immunodominant epitopes in silico from the K88ac fimbrial major subunit (also adhesin) FaeG and embedded each epitope in a heterogeneous carrier for epitope fusions. We then immunized mice with each epitope fusion protein and examined epitope antigenicity and also neutralizing activities of epitope-induced antibodies. Data showed that while all nine FaeG epitope fusions induced antibodies to K88ac fimbria, anti-K88 IgG antibodies derived from epitopes MTGDFNGSVD (ep1), LNDLTNGGTK (ep2), GRTKEAFATP (ep3), ELRKPDGGTN (ep4), PMKNAGGTKVGAVKVN (ep5), and RENMEYTDGT (ep8) significantly inhibited adherence of K88ac fimbrial bacteria to porcine intestinal cell line IPEC-J2, indicating that these peptides were the neutralizing epitopes of K88ac fimbrial major subunit FaeG and suggesting the future application of FaeG epitopes in ETEC vaccine development.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains producing K88ac fimbriae and enterotoxins are a major cause of porcine neonatal diarrhea and postweaning diarrhea in the United States. Currently, there is no vaccine to induce broadly protective antiadhesin and antitoxin immunity against ETEC-associated diarrhea. To develop a broadly effective ETEC vaccine, we need to target the most important if not all ETEC virulence determinants. While conventional vaccinology approaches encounter difficulties at integrating or including heterogeneous ETEC fimbria and toxin antigens into a vaccine product, multiepitope fusion antigen (MEFA) structural vaccinology provides a new platform to combine neutralizing antigenic elements or epitopes from various heterogeneous virulence factors for broad immunity and protection. Identification of the neutralizing epitopes of K88ac fimbria from this study added the last antigens to an MEFA-based multivalent vaccine against ETEC-associated diarrhea in pigs. An effective vaccine against pig diarrhea can significantly improve swine health and well-being and reduce economic losses to the swine industry worldwide.

Intestinal challenge with enterotoxigenic Escherichia coli in pigs, and nutritional intervention to prevent postweaning diarrhea.

Gut health of nursery pigs immediately after weaning is tightly associated with their growth performance and economic values. Postweaning diarrhea (PWD) is one of the major concerns related to gut health of nursery pigs which often is caused by infections of enterotoxigenic Escherichia coli (ETEC), mainly including F4 (K88)+ and F18+E. coli. The main virulence factors of ETEC are adhesins (fimbriae or pili) and enterotoxins. The common types of fimbriae on ETEC from PWD pigs are F18+ and F4+. Typically, PWD in pigs is associated with both F18+ and F4+ ETEC infections whereas pre-weaning diarrhea in pigs is associated with F4+ ETEC infection. Enterotoxins including heat-labile enterotoxins (LT) and heat-stable peptide toxins (ST) are associated with causing diarrhea in pigs. At least 109 to 1010 ETEC are required to induce diarrhea in nursery pigs typically lasting 1 to 5 days after ETEC infection. Antibiotics used to be the most effective way to prevent PWD, however, with the increased bacterial resistance to antibiotics, alternatives to the use of antibiotics are urgently needed to prevent PWD. Immunopropylaxis and nutritional intervention of antimicrobial minerals (such as zinc oxide and copper sulfate), organic acids, functional feedstuffs (such as blood plasma and egg yolk antibodies), direct fed microbials, phytobiotics, and bacteriophage can potentially prevent PWD associated with ETEC. Some other feed additives such as nucleotides, feed enzymes, prebiotic oligosaccharides, and clay minerals can enhance intestinal health and thus indirectly help with preventing PWD. Numerous papers show that nutritional intervention using selected feed additives can effectively prevent PWD.

Feeding a diet containing resistant potato starch influences gastrointestinal tract traits and growth performance of weaned pigs.

The aim was to evaluate the effects of feeding resistant potato starch (RPS) as a natural source of resistant starch to weaned pigs for 28 d immediately after weaning. Sixty piglets (Yorkshire-Landrace × Duroc) weaned at 21 ± 2 d (1:1 male:female) with an initial BW of 7.2 ± 0.78 kg were assigned in a completely randomized design to 1 of 5 dietary treatments to give 6 observations per treatment and 2 pigs per pen. Dietary treatments consisted of a negative control corn-soybean meal-wheat-wheat middlings-based diet (NC; no antimicrobial agents added) or the NC supplemented with RPS either as powder or in capsules and each included at 0.5 or 1.0% as a top-dressing on each day. Diets were formulated to meet 1998 NRC specifications. Pigs were offered the experimental diets on an ad libitum basis for 28 d and water was available at all times. The ADG, ADFI, and G:F were determined weekly. Fecal score was determined daily for 14 d after weaning. At the conclusion of study, 1 pig from each pen was randomly selected and euthanized (n = 6 per treatment) to determine visceral organ weight, digesta pH, VFA, and ammonia N (NH3-N) concentrations. Resistant potato starch supplementation improved (P < 0.001) fecal score, and pigs offered 1.0% RPS had more solid feces (P < 0.05) than those offered 0.5% RPS during the first 14 d after weaning, independent of the form of RPS. Resistant potato starch supplementation decreased (P < 0.05) ileal and cecal digesta pH regardless of the levels of RPS or mode of delivery. The total VFA concentrations in cecal digesta were greater (P < 0.05) but the molar proportion of branched-chain fatty acids were lower (P < 0.05) for pigs fed the RPS-containing diets compared with those fed the NC, irrespective of the RPS levels or the form of RPS. However, there were no differences (P > 0.10) in visceral organ weights, growth performance, and digestibilities of DM, CP, Ca, and P among treatments. The results of this experiment indicate that supplementing a weaner pig diet with at least 0.5% RPS independent of mode of delivery has the potential to enhance outcomes characteristic of a functional gut in weaned pigs without adverse effects on growth.

Prevalence of the paa gene (porcine attaching and effacing associated) in porcine enteropathogenic Escherichia coli (PEPEC) associated with postweaning diarrhea in south Brazil.

Paa (porcine attaching and effacing associated) may be an important virulence factor E. coli of piglets with diarrhea. This study showed for the first time in Brazil the prevalence of the paa gene (22%) in E. coli strains isolated from piglets and these isolates also harboured genes for other adhesins and toxins LT II, STa and STb.

Prevalence of the paa gene (porcine attaching and effacing associated) in porcine enteropathogenic Escherichia coli (PEPEC) associated with postweaning diarrhea in south Brazil

Paa (porcine attaching and effacing associated) may be an important virulence factor E. coli of piglets with diarrhea. This study showed for the first time in Brazil the prevalence of the paa gene (22%) in E. coli strains isolated from piglets and these isolates also harboured genes for other adhesins and toxins LT II, STa and STb.

Caracterización genotipica de aislamientos de Escherichia coli obtenidos de cerdos con diarrea posdestete y enfermedad de los edemas / Genotypic characterization of toxigenic Escherichia coli isolated from pigs with postweaning diarrhea (PWD) and edema disease (ED)

El objetivo del trabajo fue caracterizar mediante PCR 47 aislamientos de Escheríchia coli recuperados de 32 cerdos con diagnóstico clínico de diarrea posdestete (DPD) y de 3 cerdos con enfermedad de los edemas (ED). Sobre 44 aislamientos provenientes de cerdos con DPD, 42 (95,5 %) fueron caracterizados como E. coli enterotoxigénicos (ETEC) y 2 (4,5 %) como E. coli productores de toxina Shiga (STEC). Catorce aislamientos de ETEC (33,3 %) fueron positivos para los genes estl/estlI/fedA. El genotipo más complejo fue eltA/estll/east1/faeG/aidA. Los aislamientos provenientes de cerdos con ED se clasificaron como STEC porcinos y fueron portadores de stxJaidA. Once aislamientos (25 %) fueron portadores del gen que codifica la expresión de la adhesina AIDA-I. Sin embargo, en ningún aislamiento se detectaron los genes que codifican la expresión de las adhesinas F5, F6, F41, de intimina y de "Paa". La prevención de la DPD y de la ED podría realizarse mediante el desarrollo de vacunas que generen anticuerpos contra las adhesinas de las cepas de E. coli prevalentes en la Argentina.

The purpose of this work was to characterize 47 Escherichia coli strains isolated from 32 pigs diagnosed with postweaning diarrhea and tree pigs with edema disease by PCR. Forty two (95.5 %) of the strains isolated from diarrheic pigs were characterized as enterotoxigenic E. coli (ETEC) and 2 (4.5 %) as Shiga toxin-producing E. coli (STEC). Fourteen (33.3 %) ETEC strains were positive for est/estll/fedA genes. The most complex genotype was eltA/estl/faeG/aidA. Strains isolated from pigs with ED were classified as porcine STEC and were stxjaidA carriers. Eleven (25 %) strains carried the gene encoding adhesln protein AIDA-I. However, genes coding for F5, F6, F41, intimin and Paa were not detected. The development of vaccines generating antibodies against prevalent E. coli adhesins in Argentina could be useful for the prevention of PWD and ED.

Caracterización genotipica de aislamientos de Escherichia coli obtenidos de cerdos con diarrea posdestete y enfermedad de los edemas / Genotypic characterization of toxigenic Escherichia coli isolated from pigs with postweaning diarrhea (PWD) and edema disease (ED)

El objetivo del trabajo fue caracterizar mediante PCR 47 aislamientos de Escheríchia coli recuperados de 32 cerdos con diagnóstico clínico de diarrea posdestete (DPD) y de 3 cerdos con enfermedad de los edemas (ED). Sobre 44 aislamientos provenientes de cerdos con DPD, 42 (95,5 %) fueron caracterizados como E. coli enterotoxigénicos (ETEC) y 2 (4,5 %) como E. coli productores de toxina Shiga (STEC). Catorce aislamientos de ETEC (33,3 %) fueron positivos para los genes estl/estlI/fedA. El genotipo más complejo fue eltA/estll/east1/faeG/aidA. Los aislamientos provenientes de cerdos con ED se clasificaron como STEC porcinos y fueron portadores de stxJaidA. Once aislamientos (25 %) fueron portadores del gen que codifica la expresión de la adhesina AIDA-I. Sin embargo, en ningún aislamiento se detectaron los genes que codifican la expresión de las adhesinas F5, F6, F41, de intimina y de "Paa". La prevención de la DPD y de la ED podría realizarse mediante el desarrollo de vacunas que generen anticuerpos contra las adhesinas de las cepas de E. coli prevalentes en la Argentina.(AU)

The purpose of this work was to characterize 47 Escherichia coli strains isolated from 32 pigs diagnosed with postweaning diarrhea and tree pigs with edema disease by PCR. Forty two (95.5 %) of the strains isolated from diarrheic pigs were characterized as enterotoxigenic E. coli (ETEC) and 2 (4.5 %) as Shiga toxin-producing E. coli (STEC). Fourteen (33.3 %) ETEC strains were positive for est/estll/fedA genes. The most complex genotype was eltA/estl/faeG/aidA. Strains isolated from pigs with ED were classified as porcine STEC and were stxjaidA carriers. Eleven (25 %) strains carried the gene encoding adhesln protein AIDA-I. However, genes coding for F5, F6, F41, intimin and Paa were not detected. The development of vaccines generating antibodies against prevalent E. coli adhesins in Argentina could be useful for the prevention of PWD and ED.(AU)

Frequency of virulence genes in Escherichia coli strains isolated from piglets with diarrhea in the North Parana State, Brazil / Frequência de genes de virulência em Escherichia coli isolada de suínos com diarréia no norte do Estado do Paraná, Brasil

Identification of Escherichia coli causing porcine postweaning diarrhea requires knowledge regarding the prevalent pathotypes within a given region. A total of 100 Escherichia coli isolates from piglets with diarrhea in Londrina city, Parana State, South Brazil, were screened for the presence of genes for F4, F5, F6, F18, F41 fimbrial antigens by specific probes and for enterotoxins (STa, STb, LT and STx2e) by polymerase chain reaction (PCR). The results showed that 60% of the isolates were positive for one or more of the fimbrial antigens and 92% were positive at least for one of the virulence factors examined. Virulence factor genesdetected were F4 (44%), F18 (38%), F5 (30%), F41 (32%), F6 (25%), LTp-I (71%), STa (40%), STb (47%) andSTx2e (3%). Twenty four patterns of virulence factor according to the different virulence genes form werefound and the most frequent virulence gene pattern was F4, F18, F41, STa, STb and LT. Most of the isolates that carried genes for adhesins also harboured genes for toxins.

A identificação de amostras de Escherichia coli responsáveis por diarréia pós-desmame em suínos requerconhecimento dos patotipos prevalentes dentro de uma dada região. Cem amostras de Escherichia coli isoladas de leitões com diarréia no Estado do Paraná, Brasil, foram testadas para apresença dos genes que codificam antígenos fimbriais F4, F5, F6, F18, F41 e para a produção de enterotoxinas (STa, STb, LT and STx2e), através de sondas e da técnica da PCR (polymerasechain reaction). Os resultados mostraram que 60% dos isolados foram positivos para um ou mais antígenos fimbriais e 92% foram positivos para pelo menos um dos fatores de virulência examinados. Os genes de virulência detectados foram F4 (44%), F18 (38%), F5 (30%), F41 (32%), F6 (25%), LTp-I (71%), STa(40%), STb (47%) e STx2e (3%). Vinte e quatro padrões de virulência, de acordo com as diferentes combinações dos genes de virulência, foram encontrados e o mais prevalente foi F4,F18, F41, STa, STb e LT. A maioria das amostras que carreiam genes para adesinas também transportam genes para produção de toxinas.