livestock animals are subjected to a huge sort of stress factors , such as environmental conditions , animal management or diets that may disrupt the intestinal ecosystem and increase the risk of pathogenic infections . in the swine industry , transition from the liquid to a solid diet during weaning and post - weaning are critic processes due to an important decrease of feed intake , impairing animal performance , immune function , microbiota and therefore , gut homeostasis .
concerning the immune system , these performance stages lead to the end of transference of passive maternal immunity from the sows to the piglets which still have an immature or developing immune system .
this means that piglets become highly susceptible to intestinal pathogens such as enterotoxigenic e.coli ( etec ) or salmonella enterica ser .
antibiotic growth promoters ( agps ) have been extensively used as dietary additives to minimize the risk of infections , especially during the critical periods of animal production , like weaning . however , after european ban of agps ( ec 1831/2003 ) several studies proposed the use of probiotics and prebiotics as natural alternatives to agps . in that sense , the health benefits of probiotics and prebiotics have been widely accepted as gut microbiota stabilizers because they are believed to avoid pathogenic colonization and therefore improving animal welfare .
our group recently published in vitro results that demonstrated the potential of probiotic saccharomyces cerevisiae var boulardii and prebiotic -galactomannan ( gm ) to prevent intestinal infections by e.coli and salmonella in pigs . here
, we aimed to extend the knowledge about the prebiotic role of gm against salmonella challenge according to its polysaccharide structure .
the porcine small intestine epithelial cell line ipi-2i ( ecacc 93100622 ) was established from the ileum of an adult boar ( slad / d haplotype ) .
ipi-2i cells were maintained in dmem glutamax ( invitrogen , spain ) supplemented with 10% fcs ( invitrogen ) , insulin 10 g / ml ( sigma  aldrich , saint - quentin , france ) . in all experiments
, cells were cultured in 6-well plates ( nunc , labclinics , spain ) to confluence . before the addition of pre / probiotics and/or infection ,
cells were washed three times and the cell culture was replaced with dmem media supplemented with insulin 10 g / ml ( sigma  aldrich ) .
cells were used between passages 30 and 70 and periodically tested to avoid mycoplasma contamination ( mycoalert mycoplasma detection kit , lonza ) . to assess the functionality of mannans according to their structure we compared the prebiotic gm ( salmosan , patent wo2009/144070 a2 , licensed to industrial tcnica pecuaria , itpsa , barcelona , spain ) to another mannanoligosaccharide purified from saccharomyces cerevisiae ( mannan sc , sigma ) and to d - mannose monosaccharide ( sigma - aldrich ) in relation to a negative control ( medium ) .
prebiotic gm , mannan sc and d - mannose were diluted in dmem glutamax ( 1 mg / ml ) , vortexed and incubated 30 min at 37c .
immediately before the salmonella infection , gm , mannan sc and d - mannose were respectively added to each well at 10g / ml .
pathogenic salmonella enterica serovar typhimurium ( salmonella ) with antigenic formulae ( 4,12:i:1,2 ) , resistant to ampicillin , chloramphenicol , streptomycin , sulfonamide and tetracycline was provided by dr .
ignacio badiola from centre recerca en sanitat animal ( cresa , irta - uab , bellaterra , spain ) .
aliquots of salmonella were provided in bacterial cryo - preservers ( technical service consultants , ferrer international , spain ) and stored at -80c until use . before infection iec cells ,
a single salmonella cryo - preserver was added to 20 ml of luria - bertani media ( lb ) and cultured for 34h at 37c with 180 rpm rotational agitation ( multitron ht , infors ) . for infection ,
salmonella was used during the exponential growth phase as determined by absorbance at 600 nm .
after 3 h of host cell - pathogen assay performed as described before , 75g / ml of gentamycin ( sigma - aldrich ) were added to each well to avoid bacterial overgrowth .
cytokine protein determination in the culture supernatant was performed by enzyme - linked immunosorbent assays ( elisas ) .
swine il-6 and cxcl8 duoset elisa kits ( r&d systems , vitro sp ) were used according to manufacturer s recommendations .
all statistical analyses were performed using glm procedure ( proc glm ) by sas software version 9.1.3 ( sas institute ) .
means for protein secretion were considered in a 2 * 3 factorial design ( two infection levels * 3 experimental treatments ) with duncan s post - test for group analysis .
superscript letters are used to designate statistical significance : bars with different superscript letters are significantly different ( p < 0.05 ) and bars with same superscript letters indicate no statistical difference ( n.s ) .
the porcine small intestine epithelial cell line ipi-2i ( ecacc 93100622 ) was established from the ileum of an adult boar ( slad / d haplotype ) .
ipi-2i cells were maintained in dmem glutamax ( invitrogen , spain ) supplemented with 10% fcs ( invitrogen ) , insulin 10 g / ml ( sigma  aldrich , saint - quentin , france ) . in all experiments
, cells were cultured in 6-well plates ( nunc , labclinics , spain ) to confluence . before the addition of pre / probiotics and/or infection ,
cells were washed three times and the cell culture was replaced with dmem media supplemented with insulin 10 g / ml ( sigma  aldrich ) .
cells were used between passages 30 and 70 and periodically tested to avoid mycoplasma contamination ( mycoalert mycoplasma detection kit , lonza ) .
to assess the functionality of mannans according to their structure we compared the prebiotic gm ( salmosan , patent wo2009/144070 a2 , licensed to industrial tcnica pecuaria , itpsa , barcelona , spain ) to another mannanoligosaccharide purified from saccharomyces cerevisiae ( mannan sc , sigma ) and to d - mannose monosaccharide ( sigma - aldrich ) in relation to a negative control ( medium ) .
prebiotic gm , mannan sc and d - mannose were diluted in dmem glutamax ( 1 mg / ml ) , vortexed and incubated 30 min at 37c .
immediately before the salmonella infection , gm , mannan sc and d - mannose were respectively added to each well at 10g / ml .
pathogenic salmonella enterica serovar typhimurium ( salmonella ) with antigenic formulae ( 4,12:i:1,2 ) , resistant to ampicillin , chloramphenicol , streptomycin , sulfonamide and tetracycline was provided by dr .
ignacio badiola from centre recerca en sanitat animal ( cresa , irta - uab , bellaterra , spain ) .
aliquots of salmonella were provided in bacterial cryo - preservers ( technical service consultants , ferrer international , spain ) and stored at -80c until use . before infection iec cells ,
a single salmonella cryo - preserver was added to 20 ml of luria - bertani media ( lb ) and cultured for 34h at 37c with 180 rpm rotational agitation ( multitron ht , infors ) . for infection ,
salmonella was used during the exponential growth phase as determined by absorbance at 600 nm .
after 3 h of host cell - pathogen assay performed as described before , 75g / ml of gentamycin ( sigma - aldrich ) were added to each well to avoid bacterial overgrowth .
cytokine protein determination in the culture supernatant was performed by enzyme - linked immunosorbent assays ( elisas ) .
swine il-6 and cxcl8 duoset elisa kits ( r&d systems , vitro sp ) were used according to manufacturer s recommendations .
all statistical analyses were performed using glm procedure ( proc glm ) by sas software version 9.1.3 ( sas institute ) .
means for protein secretion were considered in a 2 * 3 factorial design ( two infection levels * 3 experimental treatments ) with duncan s post - test for group analysis . the p value  0.05 was considered to be significant .
superscript letters are used to designate statistical significance : bars with different superscript letters are significantly different ( p < 0.05 ) and bars with same superscript letters indicate no statistical difference ( n.s ) .
many intestinal pathogenic strains of salmonella and e.coli share type i fimbriae , although from different evolutionary origin , which have high affinity for mannose residues of glycoproteins present on cell surface of the host .
the recognition of these residues leads to bacterial adherence to enterocyte surface and intestinal colonization by the pathogen .
our recent articles showed how probiotic strain of saccaromyces cerevisiae var boulardii ( scb ) and a recently developed prebiotic -galactomannan ( gm ) , derived from fruit seed of the ceratonia silliqua tree , inhibit in vitro adhesion of salmonella and etec to pig intestinal cell line ipi-2i . indeed , both products decreased the overall proinflammatory profile induced by etec or salmonella , with the least expenditure of resources for innate immune response to deal with each pathogen .
mannan derived products , such as scb and gm , are widely accepted to prevent salmonella colonization by binding to the adhesin fim h of the type i fimbriae .
the interaction of probiotic scb and prebiotic gm was already confirmed in our previous work .
however , enterotoxigenic e.coli k88 , which is one on the most important causes of neonatal and post - weaning diarrhea ( pwd ) in piglets , bears type iv fimbriae or pilli .
the latter has been described as mannose resistant to erythrocyte agglutination test in the presence of d - mannose ( 5% ) . nevertheless , the k88 adhesins ( ab , ac and ad ) bind to -galactose residues of receptors of syalic glycoprotein and to neutral glycosphingolipids expressed on the surface of the intestinal mucosa . the presence of -galactose in scb and gm may mimic host receptor for k88 adhesin and therefore inhibit etec attachement to cell surface .
in addition to the residue composition , prebiotic functionality is determined by the size and molecular structure of the saccharides . here , we evaluated functionality of gm according to their polysaccharide structure .
we compared gm to monosaccharide d - mannose and to another mannan oligosaccharide purified from saccharomyces cerevisiae yeast ( mannan sc ) at a final concentration of 10 g / ml . we observed that in vitro challenge with salmonella induces the secretion of proinflammatory cytokine il6 ( 4-fold increase : 224 vs. 897 pg / ml ; figure  1a ) and chemokine cxcl8 ( 2-fold increase : 2645 vs. 5192 pg / ml ; figure  1b ) compared with control without infection . both ,
gm and mannan sc showed a 40% reduction of salmonella - induced secretion of il6 ( 897 vs. 563 and 585 pg / ml , figure  1a ) .
concerning cxcl8 , gm reduce chemokine secretion by 30% ( 5192 vs 3697 pg / ml ; figure  1b ) .
however , mannan sc shows a trend to reduce cxcl8 secretion induced by salmonella ( fig . 
interestingly , the secretion of proinflammatory il6 and cxcl8 in iecs treated with monosaccharide d - mannose was similar to the control of infection ( fig . 
present data confirms early results shown by oyofo et al . and vil et al . who showed higher efficiency of oligomeric mannoses compared with monomeric d - mannose to inhibit in vivo salmonella infection in other productive species like chickens .
in pigs , the addition of a commercial source of mannanoligosaccharides in the diet increased weight gain during the starter period , improved feed efficiency in vivo together with a reduction of enterobacteria counts in the jejunum ( see ref .
indeed , mannanoligosaccharide additives have been shown to modify the composition of the gastrointestinal microbiota , resulting in increased populations of bacteroidetes spp and lactobacillus spp after salmonella infection in vivo , leading to a greater compensatory weight gain of pigs ( see ref .
cytokine il6 ( a ) and chemokine cxcl8 ( b ) concentration in supernatants from ipi-2i cells ( 1  10 cells / well ) co - cultured for 24 h with salmonella ( moi = 4 ) is decreased by gm and mannan sc ( 10 g / ml ) .
regard , our previous in vitro works showed that scb has a higher anti - inflammatory role in etec infections .
in contrast , gm is a potent prophylactic agent against salmonella infections . therefore , the preference of the use of each probiotic or prebiotic additive as a dietary alternative to agps , should be determined , if possible , according to the specific conditions of each farm .
this means the prevalence of etec or salmonella of each productive facility or region . in that sense , considering that etec infections often occur during weaning , while salmonella usually colonizes post - weaning and finishing growing pigs , to enrich diets with scb at early stages to prevent etec infections and with gm to reduce salmonella prevalence at final growing stages should be considered .
in addition , we hypothesize that the prebiotic gm may enhance probiotic scb survival and colonization of the intestinal tract improving host health .
future studies should determine the suitability to combine gm and scb to become a symbiotic additive to prevent intestinal infections .
nowadays in eu , products such as mannan sc and gm are not authorized as zootechnical feed additives under the commission regulation ( ec ) 1831/2003 . in the future
, both products could be authorized following concepts described in the opinion of efsa ( see ref . 23 ) concerning potential new categories of zootechnical feed additives . in this case
it would be additives which improve product quality as  microbial contamination controllers  . however , today it is also possible to introduce them in the european feed legislation according to the regulation ( ec ) 767/2009 where there is a description of how ingredients can be used for particular purposes . in conclusion
, we demonstrated that oligosaccharide structure is crucial for mannans to develop their prebiotic role to inhibit pathogenic adhesion and therefore to reduce proinflamatory immune response induced by the pathogen .
 prebiotics and probiotics are considered natural alternatives to dietary antibiotics in animal production . 
 plant extracts and yeast cell walls are mannose  rich products that can be used as substrate for adhesion of gram - negative bacteria . 
 we assessed whether the structure of these saccharides is relevant to develop their role as prebiotics and therefore , their suitability to be used as alternatives to antibiotics to prevent intestinal infections in pigs . 
 the prebiotic functionality of -galactomannan ( gm ) , mannanoligosaccharide from yeast saccharomyces cerevisiae ( mannan sc ) and monosaccharide d - mannose were studied in porcine intestinal epithelial cells ( ipi-2i ) challenged with salmonella enterica ser . 
 typhimurium . 
 results showed that in vitro challenge with salmonella induces the secretion of proinflammatory cytokine il6 and chemokine cxcl8 compared with control without infection . 
 both gm and mannan sc , attenuate salmonella - induced secretion of il6 and cxcl8 . 
 interestingly , cells treated with d - mannose showed similar levels of proinflammatory il6 and cxcl8 compared with the control of infection . 
 these data suggest that prebiotic role of gm is related to its oligosaccharide structure . 
Introduction
Methods
Intestinal epithelial cell culture (IECs)
Experimental treatments and prebiotic preparation
Host cell-pathogen assay
Determination of cytokine production
Statistical analysis
Results and Discussion