The use of prebiotics and probiotics in pigs

A review

Author: Dr Louise Mar Agricultural Research Council Livestock usiness Division Ani!al "roduction

Contents
#$ The probiotic and prebiotic concept %$ Aspects relevant to the use of probiotics in pigs %$# Rearing of pigs %$% The porcine digestive tract %$%$# &to!ach %$%$% &!all intestine %$%$' Large intestine %$' Lactic acid bacteria indigenous to pigs %$( Detection and identification of lactic acid bacteria in the porcine gastro) intestinal tract %$* +!!unolog, '$ -se of prebiotics and probiotics ($ .fficac, and !ode of action of probiotics and prebiotics *$ &election of potential probiotic strains *$# The use of gastro)intestinal !odels to screen cultures for probiotic properties *$% The safet, of probiotic bacteria /$ &ituation in &outh Africa 0$ 1eneral discussion

#$ The probiotic and prebiotic concept The concept of probiotics evolved at the beginning of the 20 th century from a hypothesis first proposed by the Nobel Prize winning Russian scientist Elie Metchni off! "e suggested that the long and healthy life span of #ulgarian peasants was due to the consumption of fermented mil products $Metchni off% &'0()! *uring the last few decades% research on probiotics has e+panded beyond bacteria isolated from fermented dairy products to normal microbiota of the intestinal tract $,anders and "uis in-t .eld% &''')! .anbelle et al! $&''0) defined probiotics as natural intestinal bacteria that% after oral administration in effective doses% are able to colonize the animal digestive tract% thus eeping or increasing the natural flora% preventing colonization of pathogenic organisms and securing optimal utility of the feed! Prebiotics are defined as non/digestible food ingredients that affect the host beneficially by selectively stimulating the growth and0or activity of bacteria in the colon $1ibson and Roberfroid% &''2)! This definition was recently amended to 34 prebiotic is a selectively fermented ingredient that allows specific changes% both in the composition and0or activity in the gastrointestinal microbiota that confers benefits upon host well/being and health!- 5n practice% the beneficial bacteria that serve as targets for prebiotics are mostly lactobacilli and bifidobacteria $1ibson et al!% &'''6 #ouhni et al!% 2007)! 8nli e probiotics were allochthonous microorganisms are introduced in the gut% and have to compete against established colonic communities% an advantage of using prebiotics to modify gut function is that the target bacteria are already commensal to the large intestine $Macfarlane et al!% 200()! "owever% if for any reason li e disease% ageing% antibiotic or drug therapy% the appropriate health/promoting bacteria are not present in the bowel% prebiotics are not li ely to be effective! 9ombinations of prebiotics and probiotics are referred to as synbiotics! 9ommercial probiotic products often do not meet e+pected standards in that the composition and viability of the strains may differ from information on the label $"amilton/ Miller et al!% &'''6 "amilton/Miller and ,hah et al!% 20026 :eese% 20026 ;asoli et al!% 200<)! 4nother ma=or issue in relation to the application of probiotics is the poor evidence for efficacy based on clinical trials $>laenhammer and >ullen% &''')! Three issues interfere with the identification of specific health effects of probiotics $>laenhammer and >ullen% &''')! ;irstly% the comple+ity and variability of the gastro/intestinal environment in relation to

gastro/intestinal diseases ma e it difficult to determine the effect probiotics have on health and disease! ,econdly% confusion as to the identity% viability and properties of probiotics lead to strains being incorrectly identified! ?astly% single probiotic strains induce a multitude of effects among different hosts in a test population! 4 mono/strain probiotic is defined as containing one strain of a certain species whereas multi/strain probiotics contain more than one strain of the same species or genus! The term multi/species probiotics is used for preparations containing strains that belong to one or preferably more genera $Timmerman et al.% 2007)! Multi/species preparations have an advantage when compared to mono/ and multi/ strain probiotics $Timmerman et al!% 2007)! Multi/species probiotics benefit from a certain amount of synergism due to the combination of characteristics from different species! The concept of probiotics plays an important role in animal health! Pig rearing has become an intensive commercial industry! Economic losses due to decreased health and performance brought about by intensive farming practices focused on increased production and low costs% are very important! Ma=or efforts have been made to find different ways to improve the rearing of pigs! 4ntibiotics have been used successfully for more than 20 years to enhance growth performance and control the spread of disease $1ustafson and #owen% &''@)! 4ntibiotic resistance is as ancient as antibiotics% protecting antibiotic producing organisms from their own products $Phillips et al!% 2007)! 4ntibiotic resistant variants and species that are inherently resistant can dominate and populate host animals! 5ncreased concern e+ists about the potential of antibiotics in animal feed and their contribution to the growing list of antibiotic/resistant human pathogens $9orpet% &''A6 :illiams and "eymann% &''()! 4lthough the use of antibiotics for growth promotion is still allowed in certain countries% including the 8nited ,tates% 4ustralia and ,outh 4frica% several European countries have implemented strict legislation to prevent the incorporation of antibiotics in animal feed $Ratcliff% 2000)! 5n &'(A ,weden was one of the first countries to ban the incorporation of low/dose antibiotics into animal feed! The Buestion remains% does the use of antibiotics in production animals pose a ris to human healthC 5n a recent review% it was stated that the actual danger appears small and the low dosages used for growth promotion $generally below 0!2D per ton feed) could not be regarded as a hazard $Phillips et al!% 2007)! 4ntibiotics are used in animals and humans% and most of the resistance problem in humans arises from medicinal use! Resistance may develop in bacterial populations present in production animals% and resistant bacteria can contaminate

animal/derived food% but adeBuate coo ing destroys most bacteria! 1rowth/promoting antibiotics predominantly active against 1ram/positive bacteria have very little or no effect on the antibiotic resistance of salmonellae and conseBuently on infections caused by salmonellae! 5n some parts of the world% antibiotics used to treat animals and added to feed as growth promoters may have adverse effects when associated resistance is ta en into account! The same antibiotics are often used to treat humans $Phillips et al!% 2007)! 5n contrast% Piddoc $2002) could not find clear evidence that antibiotic/resistant bacteria isolated from animals% cause infections in humans% for e+ample Buinolone/resistant strains of Salmonella serovar Typhimurium *T&07 are not transmitted through production animals! The flouroBuinolones used therapeutically in animals appear to pose little threat to human health! ;louroBuinolone resistance was recorded in bacteria isolated from humans% in countries where the use of this growth promoter is banned such as ,weden% ;inland and 9anada $Rautelin et al!% &''<6 ,=Egren et al!% &''<6 1audreau and 1ilbert% &''()! ;aecal flora isolated from a healthy person may contain antibiotic resistant enterococci% but most enterococci isolated from animals do not colonize the human intestine $*upont and ,teele% &'(@6 ,94N% &''A% &''(6 #ezoen et al!% &'''6 #utaye et al!% &'''6 4car et al!% 2000)! E. coli resistance is more li ely to be driven by antibiotic use in humans% although an animal origin for at least some clinical isolates cannot be e+cluded $1ulliver et al!% &''')! The banning of antibiotic usage in animal feed remains a controversial issue especially in the way that it affects farming with production animals! Many natural substances have been investigated as alternatives to conventional chemotherapeutic agents $Turner et al!% 2002)! Probiotics are one approach used to improve piglet health and deal with intestinal problems encountered during rearing $.anbelle et al!% &''0)! Fther approaches include acidification of feed or water $9hapman% &'(()% altering dietary formulations for small piglets% the development of feeds with lower protein content $?awrence% &'(<)% and vaccination with attenuated pathogens or with strains genetically modified $1reenwood and Tzipori% &'(@6 Trevallyn/Gones% &'(@)! The administration of growth hormones% somatostatin immunization and enzyme supplementation were also considered as alternatives to antibiotic treatment $Thac er% &'(()! Treatment with psychopharmacological drugs $#=Er et al!% &'(@)% utilization of the lacto/pero+idase system $Reiter% &'(2) and stimulation of hormone/li e proteins $anti/secretory factors) capable of reversing intestinal hyper secretion to reduce symptoms of diarrhoea $?Ennroth et al!% &'(() were proposed! ,ome esoteric substances such as zeolite have reduced diarrhoea in piglets

and increased feed efficiency $Mumpton and ;ishman% &'@@)! Natural substances that enhance growth performance and immune function in pigs include plant products such as seaweed% saponins e+tracted from certain desert plants% spices and herbs $Turner et al!% 2002)! Probiotic preparations may be incorporated in prophylactic agents and it is important to now the mode of action to anticipate the dosage levels $Gonsson and 9onway% &''2)! The use of probiotics should not e+clude other alternatives and a combination of treatments may be complimentary and more effective! %$ Aspects relevant to the use of probiotics in pigs To understand the effect probiotics have on piglets% a thorough understanding of aspects affecting the rearing of pigs and their digestive tract is needed! %$# Rearing of pigs Fne of the ma=or problems in the rearing of pigs is the high mortality rate $ ca! 20D) up to weaning age $#Hc strEm% &'@<)! 5n piggeries pre/weaning mortality is caused by diarrhoea% overlay% splay leg% anaemia% bacterial septicaemia% necrotic enteritis% cold e+posure and0or congenital defects $;ahy et al!% &'(@)! Piglets in a piggery are often born immature% which renders them more vulnerable to infections! Neonatal diarrhoea often manifests 7( h after birth and is largely attributed to the enteroto+ic E. coli strains >(( $most freBuent)% >''% '(@P or ;7& $*e 1raaf and Mooi% &'(A)! Salmonella spp!% Campylobacter spp!% Cryptosporidium% transmissible gastroenteritis virus% rotavirus% porcine adenovirus and coronavirus may also cause diarrhoea $Tzipori% &'(26 ;ahy et al!% &'(@)! The disease manifests by hypersecretion of fluids across the gut wall and into the lumen% triggering the host-s immune system through the various to+ins produced! Piglets are particularly susceptible to diarrhoea during the first three wee s after birth and at weaning age $2&/ to 2(/ days/old)! *uring the first days% the piglet is protected by maternal immunoglobulins in the colostrum $Porter% &'A')! Post/weaning diarrhoea occurs appro+imately 7 to &0 days after weaning!

Enteropathogenic E. coli is the ma=or pathogen $;ahy et al!% &'(@)! Many theories have been proposed as to why disease occurs at weaning! Fne hypothesis is the sudden deprivation of maternal antibodies and other protective factors in the sow-s mil ! 4nother possibility is

sudden changes in diet and0or a compromised metabolism $;ahy et al!% &'(@) that may lead to particles being metabolized by pathogens% which results in an increase of cell numbers! 9hanges in temperature% humidity and other environmental conditions may also affect the animal-s immune system $9arghill% &'(2)% leading to diarrhoea $#=Er et al!% &'(7)! Traditionally% pigs have been weaned after @ to &0 wee s% but piglets are now weaned after < to 7 wee s! 4t this young age the intestinal tract is not able to digest the diets developed for older pigs $9ranwell and Moughan% &'(')! The correct feed formulation is thus of critical importance! ;eed should contain easily digestible components! *uring the fattening stage $si+ months and older) swine dysentery is a problem and feed should be adapted to achieve desirable performances! %$% The porcine digestive tract The length of the gastro/intestinal tract $15T) in the newborn pig is only two meters compared to 20 meters in a mature animal $,lade% 2007)! Probiotics need to resist low p" and proteolytic enzymes in the digestive tract! The retention time% mi+ing of the ingested material with gastric =uices and previous digesta% influences the survival of the administered strains! 5n the anterior part of the small intestine% the most important defense is the fast flow rate that prevents microbial overgrowth% provided the microorganisms do not attach to the epithelium! The presence of bile in this region also represses survival and activity of the microorganisms! 5n the caecum and large intestine probiotics have to compete with a stable indigenous microflora in the healthy host animal% but the passage rate is slower and the microorganisms establish easier $Gonsson and 9onway% &''2)! %$%$# &to!ach The entrance of the stomach has the same type of eratinized sBuamous non/secreting epithelium as the esophagus $Noa es% &'@&)! 5n this region epithelial cells are released continuously and are covered with intestinal bacterial cells including lactobacilli $?ip in% &'(@)! Released sBuamous cells colonized by these bacteria may help to regulate the composition of the digestive microflora by ensuring dominance of the lactic acid bacteria $;uller et al!% &'@(6 #arrow et al!% &'(0)! 5n the stomach% gastric =uices containing mucus% "9l% proteolytic enzymes and low p" are factors influenced by the age of the animal! The stomach p" may be as low as 2!0 in an adult pig% but as high as 2!0 in mil /fed piglets $,lade%

2007)! The intestinal p" of pigs at different ages is listed in Table &! The degree of mi+ing and the rate at which contents pass through the stomach influence the effectiveness of the digestion process! Mi+ing of the digesta depends on dry matter content and particle size! ?iBuid feed and finely ground feed are mi+ed more easily than drier or coarsely ground cereal diets $Ma+well et al!% &'@0)! Table # 4ge Neonatal Pre/weaned :eaned 4dult 9ompiled from ,mith p" .alues in the digestive tract of pigs ,tomach 7!0 / 2!' <!0 I 7!7 2!A I 7!' 2!< I 7!2 and Gones ,mall intestine 9aecum 9olon 4nterior Posterior A!7 I A!( A!< I A!@ A!@ I @!@ A!A I @!2 A!0 I A!' A!0 I A!( A!( I @!2 A!2 I @!7 7!@ I @!< A!< I @!' A!& I @!@ A!A I @!@ <!2 I A!2 A!0 I A!@ 2!( I A!7 2!( I A!( $&'A<)% ,mith $&'A2)% #oucourt and ?y $&'@2)% 9lemens et

al! $&'@2)% #raude et al! $&'@A)% 9ranwell et al! $&'@A)% #arrow et al! $&'@@)% ,chulze $&'@@)% ,chulze and #ath e $&'@@)! %$%$% &!all intestine The acidified portions of digesta entering the duodenum are mi+ed with bile% pancreatic =uice% enzymes and other substances! The p" increases in the small intestine% but variations are less than encountered in the stomach! The difference between piglets and adult pigs is less pronounced $>idder and Manners% &'@()! .ariations are large in the duodenum $p" 2!0 to A!0) and progressively smaller towards the ileum $p" @!0 to @!2)! The activity of microflora in the distal part of the small intestine lowers the p" in this region $;riend et al!% &'A<)! 5t normally ta es 2!2 h for a food particle to pass through the small intestine $>idder and Manners% &'@()! 4t this flow rate% it is difficult for bacteria to multiply fast enough to prevent being washed out and probiotics should be administered in sufficient dosages! 4ttachment to epithelial cells is a prereBuisite for bacteria to colonize the small intestine! .olumes measured for the small intestine can be as much as 0!&% 0!A and 20 ? for very young% weaned and adult pigs% respectively $.odovar et al!% &'A7)!

%$%$' Large intestine

The large intestine consists of the caecum% spiral colon and the distal colon! The rate of passage is slower compared to the small intestine% leading to the establishment of a dense and comple+ anaerobic microflora! The first part of a meal reaches the anus after &0 to 27 h% but the mean retention time is much more variable and can be two to four days $>idder and Manners% &'@()! The large intestine can hold volumes up to 0!07% &!0 and 22!0 ? for very young% weaned and adult pigs% respectively $>idder and Manners% &'@()! The p" of the large intestine remains at appro+imately A!0 $>idder and Manners% &'@()! %$' Lactic acid bacteria indigenous to pigs The pig is a monogastric animal in which the foregut $stomach and small intestine) is colonized by a relatively large variety of microflora! #acteria in the small intestine survive low p" conditions better and bacterial numbers are generally high $&0 @ to &0' cfu0ml) in this section of the 15T $9onway% &'(')! ?actic acid bacteria $?4#)% mostly Lactobacillus and Streptococcus spp! dominate the small intestine $;uller et al!% &'@()! ?4# in the foregut helps the young pig to decrease the stomach p" by the production of lactic acid and other organic acids% mainly from lactose $9ranwell et al!% &'@A6 #arrow et al!% &'@@)! ?4# may regulate the microflora of the small intestine by migrating with the digesta passing down the 15T $;uller et al!% &'@()! 1ram/negative bacteria dominate the caecum $Robinson et al!% &'(&) and 1ram/ positive species the colon $,alinatro et al!% &'@@)! ,pecies often found in the porcine digestive tract are Lactobacillus acidophilus% Lactobacillus delbreuckii% Lactobacillus fermentum% Lactobacillus reuteri% Lactobacillus salivarius% Enterococcus bovis% Enterococcus durans% Enterococcus faecalis% Enterococcus faecium% Streptococcus intestinalis% Streptococcus porcinus, Streptococcus salivarius% Bifidobacterium adolescentis and Bifidobacterium suis $Raibaud et al!% &'A&6 Jani et al!% &'@76 #arrow et al!% &'@@6 ;uller et al!% &'@(6 9ollins et al!% &'(76 Robinson et al!% &'(76 Robinson et al!% &'(()! The selection and establishment of the indigenous ?4# in the neonatal pig develops progressively from birth $,in ovics and Guhasz% &'@76 ,chulze% &'@@)! 4 succession of Lactobacillus spp! occurs in the small intestine $Tannoc et al!% &''0) L. reuteri colonize animals on the first day of birth% with the L. acidophilus group appearing one wee after birth $Naito et al!% &''2)! ?ysozyme in sow-s mil has a significant effect on bacterial colonization of the pre/weaned piglet $,chulze and MKller% &'(0)! 9olostrum from the sow-s mil provides a protective effect against pathogen/ induced diarrhoea $*ucluzeau% &'(2)! 4dverse conditions may lead to changes in the intestinal flora! Mar edly lower numbers of lactobacilli and bifidobacteria were detected in

the foregut of piglets deprived of water and food for @2 h% while numbers of E. coli increased $Morishita and Fgata% &'@0)! %$( Detection and identification of lactic acid bacteria in the porcine gastro)intestinal tract 8nderstanding of the comple+ natural bacterial communities that colonize the 15T of monogastric mammals such as pigs and humans is far from complete! The identification of faecal flora by time/consuming methods where intestinal bacteria had to be isolated and cultured% revealed considerable species diversity $Moore and "oldeman% &'@76 ,alinatro et al!% &'@@6 Moore et al!% &'(@)! Fver the past decade% molecular methods have been developed that may be used to study the diversity of the gut microflora $:ilson and #litchington% &''A)! Molecular biology plays an important role in the field of probiotics% where it is used as a ta+onomic tool! 9urrent techniBues li e genetic fingerprinting% gene seBuencing% oligonucleotide probing and specific primer selection% discriminate closely related bacteria with varying degrees of success $Mc9artney% 2002)! 4dditional methods that are used include *11E% temperature gradient gel electrophoresis $T11E) and fluorescent/ in-situhybridization $;5,")! ;5," can be used to great effect in the identification of intestinal microorganisms! #y applying fluorescently labeled oligonucleotides% individual whole fi+ed cells can be identified in situ $*elong et al!% &'('6 4mann et al!% &''0)! ;5," can be implemented in the detection of probiotic bacteria since r*N4 targeted specific oligonucleotide probes can be designed for the probiotic strains administered% that would enable detection of the cells in the mucus! The addition of fermentable carbohydrates supports the growth of lactobacilli in the ileum and colon of weaning piglets! ;uture molecular biology studies on probiotics and gut flora will lead to a better understanding of the activity and function of microflora $Mc9artney% 2002)! The Buest will be to demonstrate the role of probiotic bacteria in vivo! %$* +!!unolog, 5n the healthy adult pig% immunoglobulins are released into the digestive tract and contribute to the host-s defense against infection! This immune defense starts to function soon after birth and continues up to about < wee s of age $Gonsson and 9onway% &''2)! 4fter < wee s% 5g4 is secreted and provides immune protection $Porter% &'A')! ,ow mil

immunoglobulins inhibit the growth of E. coli $:ilson and ,vendsen% &'@&)% adhesion to enterocytes $Nagy et al!% &'@') and neutralizes to+ins $#randenburg and :ilson% &'@<)! 4t weaning% the piglet is suddenly deprived of mil antibodies and some non/immunological factors such as lactoferrin% transferrin% vitamin #&2/binding protein and the bifidus factor $9ranwell and Moughan% &'(')! 4lthough the immune system of piglets is fully functional at the time of weaning% it may need to be stimulated to prevent diarrhoea! Probiotics may stimulate the immune system $PerdigLn et al!% &'(@6 ,hahani et al!% &'(')! "ypersensitivity responses in the early/weaned piglet may be induced by dietary components! The inta e of small amounts of certain proteins before weaning% particularly soy% sensitizes the immune system $Newby et al!% &'(7)! Mild diarrhoea and some intestinal disturbances may result% leading to increased susceptibility to pathogenic infections! '$ -se of probiotics and prebiotics The interest in probiotics increased during the &'70s% followed by a decline! "owever% interest is escalating again as can be seen from the number of recent publications! Emphasis has shifted from using mil fermented with microbes to selecting for indigenous bacteria! The species used in probiotic products for pigs include L. acidophilus% Lactococcus lactis% L. reuteri% combinations of Lactobacillus spp!% E. faecalis% E. faecium% Bacillus licheniformis% Bacillus subtilis% Bacillus subtilis var! toyoi% Bifidobacterium bifidum% Bifidobacterium pseudolongum% Bifidobacterium thermophilus% Clostridium butyricum% Saccharomyces spp! and other yeasts! Mi+ed combinations of organisms used% include Pediococcus acidilactici% Lactobacillus plantarum% Lactobacillus casei% L. fermentum% Lactobacillus brevis% Lactobacillus delbreuckii subsp! bulgaricus, L. casei% Streptococcus salivarius subsp! thermophilus% L. plantarum% L. acidophilus and E. faecium $Gonsson and 9onway% &''2)! ?actobacilli are strong acid producers and seldom pathogenic $,harpe et al!% &'@<6 ,harpe% &'(&)! 9ertain strains of E. faecalis and E. faecium are pathogenic $"ardie% &'(A6 Mundt% &'(A)! "owever% some non/pathogenic enterococci are incorporated in probiotic products $,trompfovM et al!% 2007)! Many enterococci produce antimicrobial substances $enterocins) and have an effect on spoilage organisms $9intas et al!% &''@6 ,abia et al!% 2002)! Enterococci can be used as probiotic organisms because of high growth rate% adhesion ability and production of enterocins $Maia et al!% 200&)! Non/pathogenic strains of certain E. coli can be administered to prevent subseBuent colonization of other pathogenic bacteria in the 15T

$*uval/5flah et al!% &'(<)! Fne of the best e+amples of probiotic E! coli is strain Nissle &'&@ $EcN)% serotype FAN>2N"& $#lum et al!% &''2)! This strain lac s typical virulence genes and prevents the invasion of ersinia enterocolitica% Shigella fle!neri% Legionella pneumophila and Listeria monocytogenes $4ltenhoefer et al!% 2007)! Probiotic preparations should be administered soon after birth% when disease is anticipated $preventive or curative)! 4dministration could be orally $although this could be very stressful to the animals)% or dispensed in water or feed $pelleted or ground)! Probiotic bacteria can be given as viable organisms in wet% frozen or freeze/dried preparations or pastes $Tournut% &'(')% or as fermented products $Pollman et al!% &'(7)! Pelleting involves high temperatures and pressures that may be lethal to microorganisms $Gonsson and 9onway% &''2)! ,ome streptococci and Bacillus spp! are less affected by heat and may survive% but lactobacilli are more sensitive! 1rowth conditions of the bacteria% harvesting methods and e+posure conditions prior to freeze/drying also influence survival of the cells! Normal indigenous microflora of a healthy pig may not establish in the 15T when piglets are moved directly after birth into a scrupulously clean environment% or after antibiotic treatment! Preparations of ?4# can be administered at these times to initiate the natural seBuential colonization of the digestive tract $9ranwell et al!% &'@A)! :ith normal pig rearing the piglets stay in close contact with the sow for the first wee s% and will be colonized by ?4#! Fn farms with a high incidence of diarrhoeal disease% it may be appropriate to introduce a probiotic strain as early as possible to colonize the digestive tract with probiotic strains that inhibit pathogens! The characteristics and mechanisms of action of the specific strains used will determine whether a single or continuous dosage is preferable! Therapeutic doses are &0' to &0&2 viable organisms per animal per day or &0 A to &0@ added to feed $.anbelle et al! &''0)! The number of organisms given should be sufficient to elicit a beneficial response in the host% but should not induce digestive disorders $Gonsson and 9onway% &''2)! The issue whether administered probiotic microorganisms are transient or adhere in the 15T% influences the dosage reBuired! Transient strains need to be administered at higher levels than strains adhering to and multiplying in the 15T $9onway% &'(')! Prebiotics are often administered in con=unction with probiotics! The dominant prebiotics used are fructo/oligosaccharides $;F,)% oligofructose and inulin trans/galacto/

oligosaccharides $TF,)% gluco/oligosaccharides% glyco/oligosaccharides% lactulose% lactitol% malto/oligosaccharides% +ylo/oligosaccharides% stachyose and raffinose $Monsan and Paul% &''26 Frban et al!% &''@6 Patterson et al!% &''@6 9ollins and 1ibson% &'''6 Patterson and #ur holder% 200<)! 4lthough mannan oligosaccharides $MF,) have been used as prebiotics% they do not enrich probiotic bacterial populations% but act by binding and removing pathogens from the intestinal tract and by stimulating the immune system $,pring et al!% 2000)! The oligomers% galacto/oligosaccharides $1F,)% soybean oligosaccharides% lactosucrose% isomalto/oligosaccharides and palatinose revealed prebiotic potential $Manning et al!% 2007)! The vast ma=ority of studies on prebiotics focused on inulin% ;F,% 1F, and TF,! The latter group of carbohydrates has a history of safe commercial use $Macfarlane et al!% 200()! ($ .fficac, and !ode of action of probiotics and prebiotics 4dministration of probiotic products often gives inconclusive or conflicting results in host animals and determination of the mode of action becomes more difficult $Gonsson% &'(26 Tuschy% &'(A6 9onway% &'(')! Fne important factor to consider is that host susceptibility varies from one animal to the other! Evaluations of probiotic use should include the effect on microflora in the digestive tract! Performance and health can be evaluated by growth rates% feed utilization% number of deaths and occurrence of diarrhoea $Gonsson and 9onway% &''2)! The clinical conditions in which efficiency of probiotics have been reported range from infectious% allergic and inflammatory to neoplastic% suggesting that a single mechanism of action is unli ely $Marteau and ,hanahan% 200<)! .arious hypotheses e+ist to e+plain the mode of action of probiotics% but these remain speculatory $.anbelle et al!% &''0)! "ealth promoting advantages of probiotic preparations include production of antimicrobial substances% organic acids% and prevention of adhesion of pathogenic bacteria in the digestive tract! Fther possible modes of action include production of metabolites able to neutralize bacterial to+ins in situ or inhibition of their production! 4n increase in feed conversion by secretion of enzymes from the microflora% stimulation of the immune system% and proliferation in the 15T were also suggested as possible modes of action! ,a ata et al! $200<) suggested that probiotics modify the metabolism in the microbial ecosystem of the large intestine by increasing the production of short chain fatty acids $,9;4)! This leads to an increase in sodium and water absorption and a decrease in colonic activity! The ,9;4 act as modulators for reBuired functions to ensure a healthy 15T! Fne study assessed the body

weight% wee ly feed inta e and feacal consistency after probiotic supplementation Fne advantage recorded for probiotic supplementation in this study included reduction of weaning diarrhea in piglets $Taras et al!% 200@)! The e+act mechanism of action of probiotics remains largely un nown! Probiotics may contribute to host defense by reinforcing non/ immunological defenses and stimulating both specific and non/specific host immune responses $1ill% 200<)! ?ittle is nown about the relative importance of the probiotic/ stimulated mechanisms in host protection! Prebiotics have been shown to possess some immunomodulatory properties! To assess the effects of prebiotics such as ;F, and 1F, on the immune system% a large number of immunological parameters0mar ers needed to be assessed $Macfarlane et al., 200()! Measurements of these mar ers had to ta e into account the fact that they can be affected by gender and age% and that they might vary because of e+ternal factors such as stress% smo ing and alcohol inta e% which necessitates careful selection of control sub=ects! The gut contains lymphoid tissue that forms a ma=or part of the body-s immune system! E+perimental data obtained suggest that immunomodulation can occur through the use of functional foods such as prebiotics $Macfarlane et al., 200()! Prebiotics li e raffinose have been shown to reduce allergic reactions in children $Nagura et al!% 2002) and results obtained to date with prebiotics in relation to osteoporosis offer some promise $Macfarlane et al!% 200() but studies are limited! ?actulose% ;F, and 1F, have la+ative effects with lactulose well established in the treatment of constipation $*e ,chryver et al!% 2002)! ?atest studies indicate that there might be potential use of prebiotics on their own% or in combination with probiotics% to immunomodulate the diseases li e rheumatoid arthritis and cancer $Macfarlane et al!% 200() but these studies are however in a very early stage! Research data demonstrating the efficacy of prebiotic application in pigs are scarce compared to human studies $Mountzouris et al!% 200A)! Most prebiotic oligosaccharides incorporated into swine diets at levels ranging from 2 to 70 g0 g have resulted in mi+ed but generally not significant effects regarding beneficial modulation of microbial populations determined in various intestinal segments and faeces of pigs $;lic inger et al!% 200<6 Mi elsen et al., 200<)! Prebiotic inclusion levels in feeds at higher levels have resulted in significantly increased levels of bifidobacteria and lactobacilli in the porcine gut% but at the higher cost of nutrient digestibility $,miric y/T=ardes et al!% 200<)! "owever% depression of nutrient digestibility is lin ed to animal performance and health% therefore careful assessment

is reBuired! 5t was concluded that in terms of microflora metabolic activity% the substantially higher numerical trends seen in TF, and ;F, treatments regarding total volatile fatty acid% acetate concentrations and glycolytic activities% it could be postulated that TF, and ;F, promoted saccharolytic activities in the pig colon $Mountzouris et al!% 200A)! Fverall% effects of prebiotics on porcine gut health have often been variable and inconsistent!

*$ &election of potential probiotic strains Probiotic strains are selected based on resistance to lytic enzymes in saliva $lysozyme) and digestive enzymes% growth at low p" and bile salts% and their ability to prevent colonization of pathogenic bacteria! ,timulation of the immune system by the probiotic strains is reBuired to increase cell/mediated immune response! Technological resistance and stability at high temperatures during pelleting% spraying etc! will ensure viability of the probiotic strains after dosage! 9ell adhesion is one of the selection criteria that remain controversial! This aspect was derived from the concept of virulence factors in pathogenic bacteria! 4dherence promotes certain virulence activities li e production of to+ins $Edwards and Puente% &''(6 >lemm and ,chembri% 2000)! ,imilar interactions could be beneficial for probiotic organisms such as lactobacilli! ?actobacilli adhere to mucosal surfaces and thereby limit the adherence of pathogenic bacteria $>otars i et al!% &''@6 >ir=avainen et al!% &''()! ,ome lactobacilli lac the ability to bind mucus in vitro $Gonsson et al!% 200&)! ,ince many of these non/binders were isolated from mucosal surfaces it may be assumed that the growth environment affects the adhesion properties of bacteria $Gonsson et al!% 200&)! The adhesion property of probiotic ?4# is species/specific $#arrow et al!% &'(0)! "ost specificity is a desirable property for probiotic bacteria and is one of the selection criteria $,alminen et al!% &'((6 ,aarela et al!% 2000)! 4dhesion of ?4# in relation to host specificity in human% canine% possum% bird and fish mucus were investigated in vitro $Rin inen et al!% 200<)! Results indicated that the adhesion trait was not host specific but rather characteristic of the species! This suggests that animal models in probiotic adhesion assays may be more applicable to other host species than earlier thought and highlights the fact that the selection criteria for a probiotic may vary according to the application of the probiotic $Rin inen et al!% 200<)! Numerous papers have been published on the isolation and selection of potential probiotic strains $Nemcova et al!% &''@6 9hang et al!% 200&6 1usils et al!% 2002)! Results obtained with

in vivo feeding trials were variable because of the comple+ity of the intestine and variation between individual animals $,imon et al!% 200<)! 9ompetitive e+clusion products containing undefined cultures were effective in pigs $;edor a/9ray et al!% &'''6 1enovese et al!% 2000)% but the possibility that these products may contain pathogens remains $1illian et al!% 2007)! 5ndividual probiotic strains need to be identified before inclusion in a probiotic product $1illian et al!% 2007)! ,election characteristics for prebiotics differ from those proposed for probiotics! Prebiotics should not be hydrolyzed by digestive enzymes or absorbed by mammalian tissues! ,ubstances used as prebiotics must selectively enrich beneficial bacteria $,immering and #laut% 200&)! *$# The use of gastro)intestinal !odels to screen cultures for probiotic properties To select suitable probiotics% the strains have to be studied in the environment where they function! The intestinal tract of humans and animals is not readily available for research purposes! This lead to the development of various models simulating the gastro/intestinal tract $Miller and :olin% &'(&6 .eilleu+ and Rowland% &'(&6 Edwards et al!% &'(26 1ibson et al!% &'((6 Mac;arlane et al!% &'('6 Molly et al!% &''<6 .eenstra et al!% &''<)! 4 uniBue 15T model was developed at the TNF Nutrition and ;ood Research Frganization% based in the Netherlands! 5t was the first in vitro model that included features li e peristaltic movements% physiological transit characteristics% nutrient absorption and water retention $.eenstra et al!% &''<)! These uniBue features made the TNF model very e+pensive to develop and operate! Potential applications included research on the digestibility of carbohydrates and other food ingredients% interactions of fats and proteins% stability of fat and sugar replacers% availability of minerals and survival of bacteria used in fermented foods and probiotics $.eenstra et al!% &''<)! The TNF model could be used in both animal nutrition and pharmaceutical research! ?atest research included studies of the absorption of mycoto+ins in the 15T of pigs $4vantaggiato et al!% 2007) and mechanistic studies on the intragastric formation of nitrosamines% resulting in valuable information being obtained regarding the human cancer ris from the combined inta e of codfish and nitrate/containing vegetables $>rul et al!% 2007)! These in vitro models proved a popular tool for research concerning bacterial populations in the 15T and probiotic bacteria administered to animals and humans! 4dvantages in the use of in vitro models compared to in vivo animal trials and e+periments include cost/effectiveness%

rapid results% reproducibility and most importantly% no ethical constraints $.eenstra et al!% &''<)! *$% The safet, of probiotic bacteria Theoretically% probiotic bacteria may be responsible for side effects such as systemic infections% deleterious metabolic activities% e+cessive immune stimulation in susceptible individuals and gene transfer $Marteau% 200&)! "owever% only a few cases of side effects in humans have been reported $Marteau% 200&)! ?imited information is available on the adverse effects of probiotics in animals% especially pigs! ;uture studies may focus on the degradation of the intestinal mucus layer by probiotics! No mucus degradation was observed in e+periments with gnotobiotic rats $Ruseler/van Embden et al!% &''2)! 4ntibiotic resistance genes% especially those encoded by plasmids% can be transferred between organisms $Marteau% 200&)! This raises the Buestion whether resistance genes can be transferred by probiotics to endogenous flora or to pathogenic microorganisms! Ris of gene transfer depends on the genetic material transferred% nature of the donor and recipient strains and on selective pressure! Probiotics currently used have been assessed as safe in fermented foods% but safety evaluation in microbial food supplements remains controversial since legislation differs between countries $5solauri et al!% 2007)! The ability of specific probiotic strains to survive gastric conditions and adhere to intestinal mucosa following oral administration may entail the ris of bacterial translocation% bacteraemia and sepsis $Table 2)! 5t was proved that probiotics improve the microflora in the gut and thus the overall health status of the host animal and that probiotics have O1R4,P $generally regarded as safe) status $4nadon et al!% 200A)! /$ &ituation in &outh Africa 4lthough the use of antibiotics as a growth stimulant in pig rearing has not been banned thus far in ,outh 4frica% the legislation might be implemented in the future! Therefore the ?ivestoc #usiness division of the 4gricultural Research 9ouncil $4R9) already has a research programme in place called O4lternatives to antibioticsP! 4t the 4nimal ;eed Manufacturers 4ssociation $4;M4) forum in &''(% an overview of the mechanisms of and role of prebiotics and probiotics in animal feeds was presented by the 4R9! *uring the 4;M4 ;orum in March 200@% it was reported that legislation banning the use of antimicrobial growth promoters

$41P-s) was implemented on the &st of Ganuary 200A in the European 8nion $E8)! The Buestion remained% will the same legislation on 41P-s await ,outh 4frica in the futureC 5t was reported at the 4;M4 forum by Maritz $200A) that varying results were obtained in countries where the use 41P-s were banned! 5n *enmar % the use of antibiotics as therapeutic treatment increased after the banning of 41P-s% reflecting increasing problems with diarrhoea $Maritz% 200A)! The same problem was e+perienced in ,weden and was addressed by changes in farm management% hygiene% sectioning% zinc supplementation of piglet feed and the use of medicated feed in some herds $:egener% 2002)! The cost of production in the pig industry also increased after the banning of 41P-s $Maritz% 200A)! The 4R9/5rene is highly committed to the tas of finding alternatives to antibiotics% and therefore freBuently calls on the livestoc % feed and pharmaceutical industries to assist us with research efforts in this regard! 5t is clear that an outright ban of 41P-s in ,outh 4frica will not be feasible $also emphasized by above findings from countries in the E8)% thereby supporting the approach of the 8nited ,tates! ,pecific 41P-s lin ed directly to human medicinal use will have to be phased out! :e hypothesize that a holistic approach will be reBuired as a single alternative is unli ely to be the answer% rather a combination of alternatives might provide products and strategies that will enable 41P-s to be phased out! "uman health is of prime importance% especially with the high incidence of "5.045*, and other eroding conditions in the population% and the threat of antibiotic resistant bacteria to these individuals as well as to the population as a whole! 0$ 1eneral discussion The large population of ?4# present in the digestive tract of a healthy animal ma es piglets ideal candidates for probiotic dosage! The genetic bac ground% physiological health status and diet of the animal may influence the effectiveness of probiotic preparations $Gonsson and 9onway% &''2)! 5t appears difficult to establish a probiotic permanently in the digestive tract of the host animal! Most studies indicate that the indigenous microflora are very efficient in preventing new organisms from establishing permanently $Gonsson and 9onway% &''2)! Fther general disadvantages of probiotics include their high price and the fact that a high dosage of administration is reBuired $1uerra et al!% 200@)! More basic nowledge of the digestive ecosystem is needed to obtain consistent effects from probiotics!

:ith the introduction of molecular based techniBues such as ;5,"% this might be achieved in the future! Table % Potential clinical targets of probiotic intervention Potential mechanism Reduction in duration of rotavirus shedding% normalization of gut Nutritional management of allergic disease and inflammatory bowel disease permeability and microbiota *egradation0 structural modification of enternal antigens% normalization of properties of indigenous microbiota and gut barrier functions% local and systemic inflammatory response% increase in e+pression Reducing the ris of infectious disease Reducing the ris of allergic0inflammatory disease of mucins 5ncrease in 5g4/secreting cells against rotavirus% the e+pression of mucins Promotion of gut barrier functions% anti/inflammatory potential% regulation of the secretion of inflammatory mediators% promotion of the development of the immune system Modified from 5solauri et al! $2007) Ris related to host and strain characteristics *irecting the microbiota towards other adverse outcomes% directing the immune responder type to other adverse outcomes ,trains with pro/ inflammatory effects% adverse effects on innate immunity% translocation% infection Potential ris s Ris related to host and strain characteristics

Effect Nutritional management of acute diarrhoea

,ome of the problems that remain to be solved include the mode of action of probiotics% dose/response relationship% better nowledge of the importance of adhesion% the chemical nature of the receptor sites of different probiotic strains and retaining viability! ,pecific conditions where probiotics can be incorporated% as alternatives to antibiotics need to be

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Edwards% R!4!% Puente% G!?!% &''(! ;imbrial e+pression in enteric bacteriaN 4 critical step in intestinal pathogenesis! Trends Microbiol! A% 2(2/2(@! ;ahy% .!4!% 9onnaughton% *!% *riesen% ,!G!% ,picer% E!M!% &'(@! Preweaning colibacillosis! 5nN 4P,4 9ommittee $Eds!)% Manipulating Pig Production% 4ustralian Pig ,cience association! :erribee% .ictoria% 4ustralia% pp! &@A/&((! ;asoli% ,!% Marzotto% M!% Rizotti% ?!% Rossi% ;!% *ellaglio% ;!% Torriani% ,!% 200<! #acterial composition of commercial probiotic products as evaluated by P9R/*11E analysis! 5nt! G! ;ood! Microbiol! (2% 2'/@0! ;edor a/9ray% P!G!% #ailey% G!,!% ,tern% N!G!% 9o+% N!4!% ?adely% ,!R!% Musgrove% M!% &'''! Mucosal competitive e+clusion to reduce Salmonella in swine! G! ;ood Prot! A2% &<@A/ &<(0! ;lic inger% E!4!% .an ?oo% G!% ;ahey% 1!9!% 200<! Nutritional responses to the presence of inulin and oligofructose in the diets of domesticated animalsN 4 Review! 9rit! Rev! ;ood ,ci! Nutr! 7<% &'/A0! ;uller% R!% #arrow% P!4!% #roo er% #!E!% &'@(! #acteria associated with the gastric epithelium of neonatal pigs! 4ppl! Environ! Microbiol! <2% 2(2/2'&! 1audreau% 9!% 1ilbert% "!% &''(! 4ntimicrobial resistance of clinical strains of Campylobacter "e"uni subsp! "e"uni isolated from &'(2 to &''@ in Suebec% 9anada! 4ntim! 4gents! 9hemother! 72% 2&0A/2&0(! 1enovese% >!G!% 4nderson% R!9!% "arvey% R!#!% Nisbet% *!G!% 2000! 9ompetitive e+clusion treatment reduces the mortality and faecal shedding associated with enteroto+igenic Escherichia coli infection in nursery/raised neonatal pigs! 9an! G! .et! Res! A7% 207/20@! 1ibson% 1!R!% 9ummings% G!"!% Mac;arlane% 1!T!% &'((! 8se of a three/stage continuous culture system to study the effect of mucin on dissimilatory sulphate reduction and methanogenesis by mi+ed populations of human gut bacteria! 4ppl! Environ Microbiol! <&% <2'/<@2! 1ibson% 1!R!% Roberfroid% M!#!% &''2! *ietary modulation of the human colonic microbiotaN introducing the concept of prebiotics! G! Nutr! &22% &70&/&7&2! 1ibson% 1!R!% Rastall% R!4!% Roberfroid% M!#!% &'''! Prebiotics! 5n Colonic #icrobiota% $utrition and %ealth ed! 1ibson% 1!R! and Roberfroid% M!#! pp! &0&/&27! *oordrechtN >luwer 4cademic Press! 1ill% "!,!% 200<! Probiotics to enhance anti/infective defences in the gastro/intestinal tract! #est Pract! Res! 9l! 1a! &@% @22/@@<!

1illian% E!% 1ardiner% 1!E!% 9asey% P!1!% 9asey% 1!% ?ynch% P!#!% ?awlor% P!1!% "ill% 9!% ;itzgerald% 1!;!% ,tanton% 9!% Ross% R!P!% 2007! Relative ability of orally administered Lactobacillus murinus to predominate and persist in the porcine gastro/intestinal tract! 4ppl! Environ! Microbiol! @0% &('2/&'0A! 1reenwood% P!E!% Tzipori% ,!% &'(@! Protection of suc ling piglets from diarrhoea caused by enterogenic Escherichia coli by vaccination of the pregnant sow with recombinant *N4 derived pilus antigens! 5nN 4P,4 9ommittee $Eds!)% Manipulating Pig Production% 4ustralian Pig ,cience 4ssociation! :erribee% .ictoria% 4ustralia% p!2<0! 1uerra% N!P!% #ernardez% P!;!% Mendez% G!% 9achaldora% P!% 9astro% ?!P!% 200@! Production of potentially probiotic lactic acid bacteria and their evaluation as feed additives for weaned piglets! 4nim! ;eed ,ci! Technol! &<7% ('/&0@! 1ulliver% M!4!% #ennett% M!% #egon% M!% &'''! EnterobacteriaN 4ntibiotic resistance found in wild rodents! Nature 70&% 2<<! 1usils% 9!% #u=azha% M!% 1onzales% ,!% 2002! Preliminary studies to design a probiotic for use in swine feed! 5nterciencia 2@% 70'/7&<! 1ustafson% R!"!% #owen% R!E!% &''@! 4ntibiotic use in animal agriculture! G! 4ppl! Microbiol! (<% 2<&/27&! "amilton/Miller% G!M!% ,hah% ,!% :in ler% G!T!% &'''! Public health issues arising from microbiological and labeling Buality of foods and supplements containing probiotic Microorganisms! Public "ealth Nutr! 2% 22</22'! "amilton/Miller% G!M!% ,hah% ,!% 2002! *eficiencies in microbiological Buality and labeling of probiotic supplements! 5nt! G! ;ood Microbiol! @2% &@2/&@A! "ardie% G!M!% &'(A! 1enus Streptococcus. 5nN P!"!4! ,neath% P!"!4! $Ed!)% #ergey-s Manual of ,ystematic #acteriology% vol! 2! :illiams T :il ins% ?ondon% pp! &07</&07@! 5solauri% E!% ,alminen% ,!% Fuwehand% 4!9!% 2007! Probiotics! #est Pract! Res! 9l! 1a! &(% 2''/<&<! Gonsson% E!% &'(2! Lactobacilli as probiotics to pigs and calves! *issertation% *ept! 4nimal Nutrition Management% ,wed! 8niv! 4gric! ,ci! &7(% pp! &/A2! Gonsson% E!% 9onway% P!% &''2! Probiotics for pigs! 5nN ;uller% R! $Ed!)% ProbioticsN The scientific basis! 9hapman T "all% ?ondon% 8>! pp! 2A0/<&A! Gonsson% "!% ,trEm% E!% Roos% ,!% 200&! 4ddition of mucin to the growth medium triggers mucus/binding activity in different strains of Lactobacillus reuteri in vitro! ;EM, Microbiol! ?ett! 207% &'/22! >idder% *!E!% Manners M!G!% &'@(! *igestion in the Pig% ,cientechnica% #ristol!

>ir=avainen% P!.!% Fuwehand% 4!9!% 5solauri% E!% ,alminen% ,!G!% &''(! The ability of probiotic bacteria to bind to human intestinal mucus! ;EM, Microbiol! ?ett! &A@% &(2/ &('! >laenhammer% T!R!% >ullen% M!G!% &'''! ,election and design of probiotics! 5nt! G! ;ood Microbiol! 20% 72/2@! >lemm% P!% ,chembri% M!4!% 2000! #acterial adhesionsN ;unction and structure! 5nt! G! Microbiol! 2'0% 2@/<2! >otars i% ,!;!% ,avage% *!9!% &''@! Models for study of the specificity by which indigenous lactobacilli adhere to murine gastric epithelia! 5nfect! 5mmun! A<% &A'(/&@02! >rul% 9!4!M!% Jeilma er% M!G!% ,chothorst% R!9!% "avenaar% R!% 2007! 5ntragastric formation and modulation of N/nitrosodimethylamine in a dynamic in vitro gastro/intestinal model under human physiological conditions! ;ood 9hem! To+icol! 72% 2&/A<! ?awrence% T!?!G!% &'(<! *ietary manipulation of the environment within the gastro/intestinal tract of the growing pig and its possible influence on disease controlN ,ome thoughts! Pig .et! ,oc! Proc!% 9ambridge% &0% 70/7'! ?ip in% M!% &'(@% Proliferation and differentiation of normal and diseased gastro/intestinal cells! 5nN Gohnson% ?!R! $Ed!)% Physiology of the 1astro/intestinal Tract% 2 nd ed! Raven Press% New Ror % pp! 222/2(7! ?loyd/Gones% 1!% ?au% P!9!>!% &''(! 4 molecular view of microbial diversity in a dynamic landfill in SuUbec! ;EM, Microbiol! ?ett! &@2% 2&'/22A! ?Ennroth% 5!% Martinsson% >!% ?ange% ,!% &'((! Evidence of protection against diarrhoea in suc ling piglets by a hormone/li e protein in sow/mil ! G! .et! Med! <2A% A2(/A<2! Mac;arlane% 1!T!% "ay% ,!% 1ibson% 1!R!% &'('! 5nfluence of mucin on glycosidase% protease and arylamidase activities of human gut bacteria grown in a </stage continuous culture system! G! 4ppl! #acteriol! AA% 70@/7&@! Macfarlane% 1!T!% ,teed% "!% Macfarlane% ,!% 200(! #acterial metabolism and health/related effects of galacto/oligosaccharides and other prebiotics! G! 4ppl! Microbiol! &07% <02/<77! Maia% F!#!% *uarte% R!% ,ilva% 4!M!% 9ara% *!9!% Nicoli% G!R!% 200&! Evaluation of the components of a commercial probiotic in gnotobiotic mice e+perimentally challenged with Salmonella enterica subsp! Enterica ser! Typhimurium! .et! Microbiol! &'% &(</&('! Manning% T!,!% 1ibson% 1!R!% 2007! Prebiotics! #est Pract! Res! 9l! 1a! &(% 2(@/2'(! Maritz% G!% 200A! 4ntimicrobial growth promoters in animal productionN Possible implications of the removal from animal feed! 4;M4 Matri+% ,eptember% 7 I (!

Marteau% P!% ,hanahan% ;!% 200<! #asic aspects and pharmacology of probioticsN 4n overview of pharmaco inetics% mechanisms of action and side effects! #est Pract! Res! 9l! 1a! &@% @22/@70! Marteau% P!% 200&! ,afety aspects of probiotic products! ,cand! G! Nutr! 72% 22/27! Ma+well% 9!.!% Reimann% E!M!% "oe stra% :!1!% >owalczy % T!% #enevenga% N!G!% 1rummer% R!"!% &'@0! Effect of dietary particle size on lesion development and on the contents of various regions of the swine stomach! G! 4nim! ,ci! <0% '&&/'22! Mc9artney% 4!?!% 2002! 4pplication of molecular biological methods for studying probiotics and the gut flora! #r! G! Nutr! ((% ,2'/,<@! Metchni off% E!% &'0(! Prolongation of ?ife! 1! Putnum-s ,ons! New Ror ! Mi elsen% ?!?% Ga obsen% M!% Gensen% #!#!% 200<! Effects of dietary oligosaccharides on microbial diversity and fructo/oligosaccharide degrading bacteria in faeces of piglets in postweaning! 4nim! ;eed ,ci! Technol! &0'% &<</&20! Miller% T!?!% :olin% M!G!% &'(&! ;ermentation by the human large intestine microbial community in an in vitro semi/continuous culture system! 4ppl! Environ Microbiol 72% 700/70@! Molly% >!% .an de :oestyne% M!% .erstraete% :!% &''<! *evelopment of a 2/step multi/ chamber reactor as a simulation of the human intestinal microbial ecosystem! 4ppl! Microbiol! #iotechnol! <'% 227/22(! Monsan% P!% Paul% ;!% &''2! Fligosaccharide feed additives! 5nN R!G! :allace% R!G! 9hesson% 4! $Eds!)% ."9 #iotechnology in 4nimal ;eeds and 4nimal ;eeding% New Ror ! pp! 2<</ 272! Moore% :!E!% "oldeman% ?!.!% &'@7! "uman faecal floraN The normal flora of 20 Gapanese/ "awaiians! 4ppl! Environ! Microbiol! 2@% 'A&/'@'! Moore% :!E!% Moore% ?!.!"!% 9ato% E!P!% :il ins% T!*!% >ornegay% E!T!% &'(@! Effect of high/fibre and high/oil diets on the faecal flora of swine! 4ppl! Environ! Microbiol! 2<% &A<(/&A77! Morishita% R!% Fgata% M!% &'@0! ,tudies on the alimentary flora of pigs! .! 5nfluence of starvation on the microbial flora! Gapan! G! .et! ,ci! <2% &'/27! Mountzouris% >!9!% #ala as% 9!% ;ava% ;!% Tuohy% >!M!% 1ibson% 1!R!% ;egeros% >!% 200A! Profiling of composition and metabolic activities of the colonic microflora of growing pigs fed diets supplemented with prebiotic oligosaccharides! 4naerobe! &2% &@(/&(2! Mumpton% ;!4!% ;ishman% P!"!% &'@@! The application of natural zeolites in animal science and aBuaculture! G! 4nim! ,ci! 72% &&&(/&20<!

Mundt% G!F!% &'(A! Enterococci! 5nN ,neath% P!"!4! $Ed!)% #ergey-s Manual of ,ystematic #acteriology% vol! 2! :illiams T :il ins% ?ondon% pp! &0A</&0A2! Nagura% T!% "achimura% ,!% "ashiguchi% M!% 8eda% R!% >anno% T!% >i uchi% "!% ,ayama% >!% >aminogawa% ,!% 2002! ,uppressive effect of dietary raffinose on T/helper 2 cell/ mediated activity! #r! G! Nutr! ((% 72&/72@! Nagy% ?!>!% #hugal% #!,!% Mc>enzie% T!% &'@'! *uration of anti/adhesive and bactericidal activities of mil from vaccinated sows on Escherichia coli F&7' in the digestive tract of piglets during the nursing period! Res! .et! ,ci! 2@% 2('/2'A! Naito% ,!% "ayashidani% "!% >ane o% >!% Fgawa% M!% #enno% R!% &''2! *evelopment of intestinal lactobacilli in normal piglets! G! 4ppl! #acteriol! @'% 2<0/2<A! Nemcova% R!% ?au ova% 4!% 1ancarci ova% ,!% >astel R!% &''@! &n vitro studies of porcine lactobacilli for possible probiotic use! #erl! Munch! Tierarztl! :ochenschr% &&0% 7&</7&@! Newby% T!G!% Miller% #!% ,to es% 9!R!% &'(7! ?ocal hypersensitivity response to dietary antigens in early/weaned pigs% 5nN "aresign% :!% 9ole% G!4! $Eds!)% Recent 4dvances in 4nimal Nutrition! #utterworth% ?ondon% pp! 7'/2'! Noa es% *!E!% &'@&! 1astric function in the young pig! Ph!*! Thesis% 8niversity of ?ondon! $9ited from 9ranwell and Moughan% &'(')! Frban% G!5!% Patterson% G!4!% ,utton% 4!?!% Richards% 1!N!% &''@! Effect of sucrose thermal oligosaccharide caramel% dietary vitamin/mineral level% and brooding temperature on growth and intestinal bacterial populations in broiler chic ens! Poult! ,ci! @A% 7(2/7'0! Patterson% G!4!% #ur holder% >!M!% 200<! 4pplication of Prebiotics and Probiotics in poultry production! Poult! ,ci! (2% A2@/A<&! Patterson% G!4!% Frban% G!5!% ,utton% 4!?!% Richards% 1!N!% &''@! ,elective enrichment of bifidobacteria in the intestinal tract of broilers by thermally produced estoses and effect on broiler performance! Poult! ,ci! @A% 7'@/200! PerdigLn% 1!% Nader de Macais% M!E!% 4lvarez% ,!% Fliver% 1!% Pesce de Ruiz "olgado% 4!4!% &'(@! Enhancement of immune response in mice fed with Streptococcus thermophilus and Lactobacillus acidophilus! G! *airy! ,ci! @0% '&'/'2A! Phillips% 5!% 9asewell% M!% 9o+% T!% *e 1root% #!% ;riis% 9!% Gones% R!% Nightingale% 9!% Preston% R!% :addell% G!% 2007! *oes the use of antibiotics in food animals pose a ris to human healthC 4 critical review of published data! G! 4ntimicrob! 9hemother! 2<% 2(/22! Piddoc % ?!G!.!% 2002! ;luoroBuinolone resistance in Salmonella serovars isolated from humans and food animals! ;EM, Microbiol! Rev! 2A% </&A!

Pollmann% *!,!% >ennedy% 1!4!% >och% #!4!% 4llee% 1!?!% &'(7! 5nfluence of nonviable Lactobacillus fermentation product in artificially reared pigs! Nutr! Rep! 5nt! 2'% '@@/'(2! Porter% P!% &'A'! Transfer of immunoglobulins 5g1% 5g4 and 5gM to lacteal secretions in the sow and their absorption by the neonatal piglet! #iochem! #iophys! 4cta! &(% <(&/<'2! Raibaud% P!% 9aulet% M!% 1alpin% G!.!% MocBuot% 1!% &'A&! ,tudies on the bacterial flora of the alimentary tract of pigs! 55! ,treptococciN selective enumeration and differentiation of the dominant group! G! 4ppl! #acteriol! 27% 2(2/<0A! Ratcliff% G!% 2000! 4ntibiotic #ans I 4 European Perspective! Proceedings of the 4;M4 ,ymposium onN 5mproving animal performance through nutrition! Pretoria% ,outh 4frica! Rautelin% "!% Ren onen% F!.!% >osunen% T!8!% &''<! 4zithro/mycin resistance in Campylobacter "e"uni and Campylobacter coli. Eur! G! 9lin! Microbiol! 5nfect! *is! &2% (A7/(A2! Reiter% #!% &'(2! Protective proteins in mil % biological significance and e+ploitation! 5nt! *airy ;ed! #ull! &'&%&/<2! Rin inen% M!% :estermarc % E!% ,alminen% ,!% Fuwehand% 4!9!% 200<! 4bsence of host specificity for in vitro adhesion of probiotic lactic acid bacteria to intestinal mucus! .et! Microbiol! '@% 22/A&! Robinson% 5!M!% 4llison% M!G!% #uc lin% G!4!% &'(&! 9haracterization of the cecal bacteria of normal pigs! 4ppl! Environ! Microbiol! 7&% '20/'22! Robinson% 5!M!% ,tromley% G!M!% .arel% .!"!% 9ato% E!P!% &'((! Streptococcus intestinalis% a new species from the colons and faeces of pigs! 5nt! G! ,yst! #acteriol! <(% 272/27(! Robinson% 5!M!% :hipp% ,!9!% #uc lin% G!4!% 4llison% M!G!% &'(7! 9haracterization of predominant bacteria from the colons of normal and dysenteric pigs! 4ppl! Environ! Microbiol! 7(% 'A7/'A'! Ruseler/van Embden% G!1!"!% .an ?ieshout% ?!M!9!% 1osselin % M!G!% Marteau% P!% &''2! 5nability of Lactobacillus casei strain 11% L. acidophilus% and Bifidobacterium bifidum to degrade intestinal mucus glycoproteinsN 9learing the way for mucosa/protective therapy! ,cand! G! 1astroenterol! ('% &'&A/&'&@! ,aarela% M!% Mogensen% 1!% ;ondUn% R!% MHttE% G!% Mattila/,andholm% T!% 2000! Probiotic bacteriaN ,afety% functional and technological properties! G! #iotechnol! (7% &'@/2&2! ,abia% 9!% Manicardi% 1!% Messi% P!% de Niederhausern% ,!% #ondi% M!% 2002! Enterocin 7&A>5% an antilisterial bacteriocin produced by Enterococcus casseliflavus 5M 7&A>5 isolated from 5talian sausages! 5nt! G! ;ood Microbiol! @2% &A</&@0!

,a ata% T!% >o=ima% T!% ;u=ieda% M!% Ta ahashi% M!% Michibata% T!% 200<! 5nfluences of probiotic bacteria on organic acid production by pig caecal bacteria in vitro! Proc! Nutr! ,oc! A2% @</(0! ,alinatro% G!P!% #la e% 5!1!% Muirhead% P!4!% &'@@! 5solation and identification of faecal bacteria from adult swine! 4ppl! Environ! Microbiol! <<% @'/(7! ,alminen% ,!% *eighton% M!4!% #enno% R!% 1orbach% ,!?!% &'((! ?actic acid bacteria in health and disease! 5nN ,alminen% ,!% von :right% 4! $Eds!)% ?actic 4cid #acteria! Microbiology and ;unctional aspects% 2nd ed! Marcel *e er% New Ror % pp! 2&&/22<! ,anders% M!E!% "uis in-t .eld% G!% &'''! #ringing a probiotic/containing functional food to the mar etN microbiological% product% regulatory and labeling issues! 4nt! van ?eeuwenh! 5nt! G! 1en! Mol! Microbiol! @A% 2'</<&2! ,94N% &''A! Report of the ,cientific 9ommittee for 4nimal Nutrition $,94N) on the possible ris for humans of the use of avoparcin as a feed additive! Fpinion e+pressed 2& May &''A! Fffice for E9 Publications% ?u+embourg! ,94N% &''(! Fpinion of the ,cientific 9ommittee for 4nimal Nutrition $,94N) on the immediate and longer/term ris to the value of streptogramins in human medicine posed by the use of virginiamycin as an animal growth promoter% &0 Guly &''(! Fffice for E9 Publications% ?u+embourg! ,chulze% ;!% &'@@! Suantitiven Magen/*arm/;lora/4nalysen beim ;er el vor und nach dem 4bsetzen unter #erKc sichtigung der Pathogenese der >olienteroto+Hmie! 4rch! E+p! .et! Med!% ?eipzig% <&% 2''/<&A! ,chulze% ;!% #ath e% :!% &'@@! Jur Buantativen Jusammensetzung der Magen/*arm/;lora beim ?Huferschwein! 4rch! E+p! .et! Med!% ?eipzig% <&% &A&/&(2! ,chulze% ;!% MKller% 1!% &'(0! ?ysozym in der ,auenmilch und seine #edeutung fKr die ba terielle #esiedlung des Magen/*arm/>anals beim ,augfer el! 4rch! E+p! .et! Med!% ?eipzig% <7% <&@/72<! ,hahani% >!% ;ernandes% "!% 4mer% .!% &'('! 5mmunologic and therapeutic modulation of gastro/intestinal micro ecology by lactobacilli! Microecol! Ther! &(% &0</&07! ,harpe% M!E!% &'(&! The genus Lactobacillus% 5nN ,tarr% M!P! $Ed!)% The Pro aryotes% a "andboo on "abitats% 5solation and 5dentification of #acteria% vol! 2! ,pringer/.erlag% #erlin% pp! &A2</&A@'! ,harpe% M!E!% "ill% ?!R!% ?apage% ,!P!% &'@<! Pathogenic lactobacilli! G! Med! Microbiol! A% 2(&/2(A!

,immering% R!% #laut% M!% 200&! Pro/ and prebiotics I the tasty guardian angelsC 4ppl! Microbiol! #iotechnol! 22%&'/2(! ,imon% F!% .ah=en% :!% ,chare % ?!% 200<! Microorganisms as feed additives I probiotics! 5nN R! F! #all $Ed!)% Proceedings of the 'th 5nternational ,ymposium on *igestive Physiology in Pigs% vol! &% 8niversity of 4lberta% Edmonton% 4lberta% 9anada! pp! 2'2/<&(! ,in ovics% 1!% Guhasz% #!% &'@7! *evelopment of the intestinal flora in suc ling pigs! 4cta! .et! 4cad! ,cient! "ungar! 27% <@2/<(&! ,=Egren% E!E!% ?indblom% 1!#!% >ai=ser% #!% &''<! Rapid development of resistance to Buinolones in Campylobacter in ,weden! 4cta! 1astro/Enterologica #elgica 7A% ,uppl! &0! ,lade% R!% 2007! ;ood tube! Pig 5nt! <7% 22/27! ,miric y/T=ardes% M!R!% 1rieshop% 9!M!% ;lic inger% E!4!% #auer% ?!?!% ;ahey% 1!9!% 200<! *ietary galactooligosaccharides affect ileal and total/tract nutrient digestability% ileal and fecal bacterial concentrations% and ileal fermentative characteristics of growing pigs! G! 4nim! ,ci! (&% 22<2/2272! ,mith% "!:!% &'A2! The development of the flora of the alimentary tract in young animals! G! Pathol! #acteriol! '0% 7'2/2&<! ,mith% "!:!% Gones% G!E!T!% &'A<! Fbservation on the alimentary tract and its bacterial flora in healthy and diseased pigs! G! Pathol! #acteriol! (A% <(@/7&2! ,pring% P!% :en % 9!% *awson% >!4!% Newman% >!E!% 2000! The effect of dietary mannanoligosaccharides on cecal parameters and the concentrations of enteric bacteria in the ceca of Salmonella/challenged broiler chic s! Poult! ,ci! @'% 202/2&&! ,trompfovM% .!% ?au ova% 4!% Fuwehand% 4!9!% 2007! ,election of enterococci for potential canine probiotic additives! .et! Microbiol! &00% &0@/&&7! Tannoc % 1!:!% &''0! The micro ecology of lactobacilli inhabiting the gastro/intestinal tract% 5nN Marshall% >!9! $Ed!)% 4dvances in Microbial Ecology% vol! &&! Plenum Press% New Ror % pp! &7@/&@&! Taras% *!% .ah=en% :!% ,imon% F!% 200@! Probiotics in pigs I modulation of their intestinal distribution and on their impact on health and performance! ?ivestoc ! &0(% 22'/2<&! Thac er% P!4!% &'((! Novel approaches to growth promotion in the pig% 5nN "aresign :!% 9ole% *!G!4! $Eds!)% Recent 4dvances in 4nimal Nutrition! #utterworth% ?ondon% pp! @</ (7!

Timmerman% "!M!% >oning% 9!G!M!% Mulder% ?!% Rombouts% ;!M!% #eynen% 4!9!% 2007! Monostrain% multistrain and multispecies probiotics I 4 comparison of functionality and efficacy! 5nt! G! ;ood Microbiol! 'A% 2&'/2<<! Tournut% G!% &'('! 4pplications of probiotics to animal husbandry! Rev! ,ci! Tech! Fff! 5nt! Epiz!% (% 22&/2AA! Trevallyn/Gones% G!% &'(@! ;ield trials with recombinant *N4 scours vaccine% 5nN 4P,4 9ommittee $Eds!)% Manipulating Pig Production 4ustralian Pig ,cience! :eribee% .ictoria% 4ustralia% p! 2<&! Turner% G!?!% Pas% ,!% *ritz% ,!% Minton% G!E!% 2002! ReviewN 4lternatives to conventional 4ntimicrobials in ,wine *iets! Prof! 4nim! ,ci! &@% 2&@/22A! Tuschy% *!% &'(A! .erwendung von 3Probioti aP als ?eistungsfErderer in der TierernHhrung! Vbers! TierernHhrg!% &7% &2@/&@(! Tzipori% ,!% &'(2! The relative importance of enteric pathogens affecting neonates of domestic animals! 4dv! .et! ,ci! 9omp! Med! 2'% &0</20A! .anbelle% M!% Teller% E!% ;ocant% M!% &''0! Probiotics in animal nutritionN 4 review! 4rch! 4nim! Nutr! #erlin @% 27</2A@! .eenstra% G!% Mine us% M!% Marteau% P!% "avenaar% R!% &''<! New Type 1astro/intestinal% 4 Tool to Test Physiological 4spects of ;oods! 5nt! ;ood 5ngredients! <% 2/(! .eilleu+% #!% Rowland% 5!R!% &'(&! ,imulation of the rat intestinal ecosystem using a two/ stage continuous culture system! G! 1en! Microbiol! &2<% &0</&&2! .odovar% N!% ;lanzy% G!% ;ranWois% 4!9!% &'A7! 5ntestin grele du porc! 5! I *imensions en fonction de l-age et du poids% etude de la =unction du canal cholUdoBue et du canal pancreatic a celui/ci! 4nn! #iol! 4nim! #iochem! #iophys! 7% 2@/<7! :eese% G!,!% 2002! Microbiological evaluation of commercial probiotics! G! 4m! .et! Med! 4ssoc! 220% @'7/@'@! :egener% "!9!% 2002! 8se of antimicrobial growth promoters in food animalsN the ris s outweigh the benefits! 5nN Proceedings of the 5nternational *ebate 9onference on 4ntimicrobial 1rowth PromotersN :orldwide #an on the "orizonC Noordwi= on Jee% the Netherlands! :illiams% R!G!% "eymann% *!?!% &''(! 9ontainment of antibiotic resistance! ,cience% 2@'% &&2</&&27! :ilson% >!"!% #litchington% R!#!% &''A! "uman colonic biota studied by ribosomal *N4 seBuence analysis! 4ppl! Environ! Microbiol! A2% 22@</22@(!

:ilson% :!R!% ,vendsen% G!% &'@&! 5mmunity to Escherichia coli in pigs! The role of mil in protective immunity to E. coli enteritis! 9an! G! 9omp! Med! <2% 2<0/27<! Jani% 1!% #iavati% #!% 9rociani% ;!% Matteuzzi% *!% &'@7! #ifidobacteria from the faeces of piglets! G! 4ppl! #acteriol! <@% 2<@/27@!