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Colonisation and succession of the gut microbiotain suckling- and newly weaned piglets
A study on the influence of specific nutritional, probiotic, antibiotic, andgenotypic factors
Ph.D. thesis byAnn-Sofie Riis Poulsen
Section for Immunology and MicrobiologyDepartment of Animal Science
Faculty of Science and TechnologyAarhus University, Foulum
Denmark
September 2016
SupervisorsSenior Scientist Charlotte Lauridsen, Ph.D., Department of Animal Science,Faculty of Science and Technology, Aarhus University, Foulum, Denmark.
Senior Scientist Nuria Canibe, Ph.D., Department of Animal Science, Faculty ofScience and Technology, Aarhus University, Foulum, Denmark.
Assessment committeéProfessor Tine Rask Licht, National Food Institute, Technical University ofDenmark, Denmark.
Senior Research Scientist Claire Rogel-Gaillard, Génétique Animale et BiologieIntégrative (GABI, UMR 1313), INRA, France.
Senior Scientist Stig Purup (chairman), Department of Animal Science, Faculty ofScience and Technology, Aarhus University, Foulum, Denmark.
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PrefaceThis Ph.D. thesis has been submitted to the Faculty of Science and Technology,Aarhus University. It comprises experimental work conducted within the groupof Immunology and Microbiology, Department of Animal Science, in the periodFebruary 2013 to September 2016. Included is a three months stay at Centerfor Microbial Communities, Department of Chemistry and Bioscience, AalborgUniversity, with primary focus on next-generation sequencing. The presentedwork contributes to the pool of data generated in the NEOMUNE consortium(http://neomune.ku.dk).
The Ph.D. project has been supervised by senior scientist Charlotte Lauridsenas main supervisor, and senior scientist Nuria Canibe as co-supervisor; both fromthe research group in Immunology and Microbiology.
The project has been funded partly by the Graduate School of Science andTechnology and partly by the NEOMUNE consortium through Innovation FundDenmark.
Ann-Sofie Riis PoulsenSeptember, 2016Foulum
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AcknowledgementsFirst and foremost I want to thank my supervisors, Nuria Canibe and CharlotteLauridsen, who have been supportive in so many ways. Not only have you educatedme in the difficult art of scientific thinking and writing, you have also always beenthere when moral support and encouragement was needed. It has been a pleasurebeing under your supervision.
Experimental tasks would not have been completed without help from thelaboratory technicians in the group of Immunology and Microbiology. I want tothank Karin Durup, Trine Poulsen, Thomas Rebsdorf and Mona Dinsen. I amgrateful to all of you for teaching me microbiological techniques and for yourinvaluable help during experimental periods. Warm thanks to Helle Handll forhelping me with hundreds of DNA extractions and qPCR tasks during the finalstage of my project. Also, heartfelt thanks to Inger Marie Jepsen, Lene RosborgDal and Hanne Purup for your help during my final animal experiment. Thanksto the staff of SB for taking good care of the piglets and for your help duringweekends and holidays.
I also want to thank Nadieh de Jonge and Jeppe Lund Nielsen for havingme in their molecular laboratory in Aalborg, teaching me about next-generationsequencing and data analysis. Thank you, Nadieh, for putting so much hard workinto my project and for never being further away than an e-mail.
I want to thank everyone in the group of Immunology and Microbiology. Youhave been great colleagues during the last three years and I have enjoyed gettingto know every single one of you. Your kindness and openness has been encouragingand deliberating. A very special thanks to my office mate Samantha Noel. Thankyou for your technical and moral support and our talks about everything andnothing. Also warm thanks to Ditte Søvsø Gundelund Nielsen. I truly appreciateyour friendship, your always kind support and for our many talks about frustratingPh.D. stuff, but also about other more joyful things as our children and families.
Lastly, I want to thank my family for putting up with all the ’pig-talk’, theirunderstanding and support. Especially, I want to thank my husband and daughter,Rasmus and My, for always being there, especially when the Ph.D. life just wasn’tthat fun and for their understanding during busy periods.
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SummaryDenmark produces close to 30 million pigs annually. The production is enabled byintensive production systems, where newborn and newly weaned piglets frequentlyshow clinical symptoms of gastrointestinal disease. Such clinical symptoms oftenrequire antibiotic therapy, and large amounts of antibiotics are used especially inthe post-weaning period. The high consumption of antibiotics is of great concerndue to development of antibiotic resistant bacteria. In addition, a compromised guthealth is associated with reduced animal welfare. Hence, the need for improvinggut health of piglets is immense.
The gut is home to a community of bacteria known as the gut microbiota. Thegut microbiota is a complex and highly diverse community and is nowadays ap-preciated as an additional ’organ’ of the body, serving various functions in relationto host health. Numerous internal and external factors influence the compositionof the gut microbiota, especially during its development. These factors can po-tentially be used for improving gut health of suckling and newly weaned piglets.The primary aim of this Ph.D.-thesis was therefore to investigate, whether ma-nipulation of specific dietary and probiotic factors could shape the gut microbiotain a direction that favours healthier piglets. The aim was fulfilled by character-ising the colonisation and succession of the gut microbiota of piglets from birthuntil two weeks after weaning, using classical microbial culture and 16S rRNAgene sequencing. The investigated factors were specifically: Bovine colostrum andBacillus spp. spores combined with early antibiotic therapy. The effect of alpha-(1,2)-fucosyltransferase (FUT1 ) genotype variants was investigated as well.
The animal experiments showed, that: 1) The early milk-based diet had asignificant influence on the microbial community in the gut. When comparedto piglets fed a regular milk replacer, bovine colostrum reduced the incidence ofdiarrhoea and enhanced the growth of specific lactic acid bacteria genera. Pigletssuckling their dams performed expectedly better than the experimental groups. 2)Administrating gentamicin and Bacillus spp. spores influenced the microbiota ofthe distal part of the gut and was especially evident by a change in species richnessand diversity. Yet, administration of Bacillus spores had a negative effect on thefrequency of diarrhoea. 3) FUT1 genotype did not influence the colonisation asseen by analysing the faecal microbiota, though susceptible piglets did house ahigher number of haemolytic bacteria and enterobacteria in their guts. 4) Pigletage, and weaning, had a significant influence on the composition of the microbialcommunity of the gut.
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In conclusion, the investigated external factors, and especially diet, influencedthe colonisation of the piglet gut microbiota. No effect on the overall microbialcommunity was seen when FUT1 genotype was manipulated. Bovine colostrumshowed the most promising results in shaping the gut microbiota in a directionfavouring healthier piglets. We did not find a beneficial effect of Bacillus sporeswhen administered before, under and after gentamicin administration.
Future studies focusing on the gut microbial community in pigs should besupported by extensive functional analyses, and measures of clinical and subclinicalcondition. Such studies may provide a deeper insight into the actual significanceof microbial community changes in the gut.
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ResuméDanmark producerer årligt omkring 30 millioner svin. Denne høje produktionmuliggøres af intensive produktionssystemer, hvor der hos de helt unge grise idagene umiddelbart efter fødslen, og omkring fravænning, hyppigt forekommermavetarmlidelser og kliniske symptomer med diarré. Sådanne kliniske symptomerer ofte årsag til antibiotikabehandling, og en stor del af forbruget anvendes iforbindelse med fravænning. Det høje forbrug af antibiotika i svineproduktio-nen har skabt stor bekymring i forbindelse med opståen af antibiotikaresistentebakterier, og ydermere kompromitterer hyppigt forekommende mavetarmlidelserdyrevelfærden. Behovet for at forbedre tarmsundheden hos smågrise er derforstort.
Tarmen huser et samfund af bakterier, som udgør størstedelen af det der kendessom mikrobiotaen. Tarmens mikrobiota er et komplekst og diverst samfund. I dagfindes der utallige beviser for, at mikrobiotaen er et yderst vigtigt organ, der haren stor indflydelse på værtens sundhed. Adskillelige interne og eksterne faktorerpåvirker tarmens mikrobiota, især på etablerings- og udviklingsstadiet. Sådannefaktorer kan potentielt udnyttes til at fremme tarmsundheden hos diende- og nyligtfravænnede grise. Det primære formål med dette Ph.D.-studie var derfor at un-dersøge, om man ved at manipulere specifikke diætetiske og probiotiske faktorerkan påvirke mavetarmkanalens mikrobiota i en retning som favoriserer sunderedyr. Formålet blev opfyldt ved at karakterisere koloniseringen og udviklingenaf mavetarmkanalens mikrobiota, hos grise i perioden mellem fødsel og indtil touger efter fravænning, ved hjælp af klassisk mikrobiologisk dyrkning og 16S rRNAgensekventering. De specifikke undersøgte faktorer var: Bovin råmælk og Bacillusspp. sporer kombineret med tidlig antibiotikatildeling. Effekten af to forskelligealpha-(1,2)-fucosyltransferase (FUT1 ) genotyper blev ligeledes undersøgt.
Dyreforsøgene viste, at: 1) Den mælkebaserede diæt havde en betydelig ind-flydelse på det mikrobielle samfund i mave og tarm. I forhold til grise fodret medalmindelig mælkeerstatning, reducererede bovin råmælk forekomsten af diarré, oghavde en fordrende effekt på fremvæksten af visse slægter af mælkesyrebakterier.De somælksfodrede grise præsterede som forventeligt bedst. 2) Tildeling af gen-tamicin og Bacillus sporer påvirkede mikrobiotaen i den distale del af tarmkanalen,som især viste sig ved et ændret antal af arter og en ændret mikrobiel diversitet.Imidlertid havde tildelingen af Bacillus sporer en negativ effekt på forekomstenaf diarré. 3) FUT1 genotype havde ingen effekt på den mikrobielle kolonisering,pÃěvist ved analyse af den af den fæcale mikrobiota. Der blev dog fundet en be-
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tydelig højere forekomst af hæmolytiske bakterier og enterobakterier i tarmen hosmodtagelige grise. 4) Grisenes alder, og fravænning, havde en betydelig indflydelsepå sammensætningen af tarmkanalens mikrobiota.
På baggrund af de fremstillede resultater kunne det konkluderes, at de under-søgte eksterne faktorer, især diet, påvirkede tarmkanalens mikrobielle koloniseringhos smågrisene. Manipulering af FUT1 genotypen viste sig ikke at have nogeneffekt på det overordnede samfund. Bovin råmælk udviste størst potentiale til atpåvirke mavetarmkanalens mikrobiota i en gunstig retning, som ydermere var led-saget af sundere dyr. Derimod kunne der ikke påvises en gavnlig effekt af Bacillussporer i forbindelse med, og efter, indgivelse af gentamicin.
Fremtidige studier med fokus på grisens mikrobiota i mavetarmkanalen børunderstøttes af vidtgående funktionelle analyser og mål for grisene kliniske og sub-kliniske tilstand. Sådanne studier kan give en dybere indsigt i den reelle betydningaf ændringer i mavetarmkanalens mikrobiota.
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Abbreviations
AGP : Antibiotic Growth PromotersBC : Bovine Colostrumcfu : colony-forming unitsETEC : Enterotoxigenic Escherichia coliFAO : Food and Agriculture Organisation of the United NationsFUT1 : alpha-(1,2)-fucosyltransferaseIg : ImmunoglobulinLPS : LipopolysaccharideMR : Milk ReplacerNEC : Necrotising enterocolitisNGS : Next-generation sequencingOTU : Operational Taxonomical UnitPCR : Polymerase Chain ReactionrRNA : ribosomal RNASCFA : Short-Chain Fatty AcidWHO : World Health Organisation
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List of original manuscriptsThe presented thesis is based on the following three original manuscripts:
1. Ann-Sofie Riis Poulsen, Nadieh de Jonge, Sugiharto Sugiharto, Jeppe LundNielsen, Charlotte Lauridsen and Nuria Canibe.
The microbial community of the gut differs between piglets fed sowmilk, milk replacer or bovine colostrum.
Submitted to British Journal of nutrition.
2. Ann-Sofie Riis Poulsen, Sugiharto Sugiharto, Nuria Canibe and CharlotteLauridsen.
Investigating the potential effects of alpha-(1,2)-fucosyltransferasegenotype on the establishment and succession of the gastrointesti-nal microbiota of young piglets.
Prepared for submission to Veterinary Microbiology.
3. Ann-Sofie Riis Poulsen, Nadieh de Jonge, Jeppe Lund Nielsen, Ole Højberg,Charlotte Lauridsen and Nuria Canibe.
Influence of Bacillus spp. spores and gentamicin on the gut mi-crobiota of suckling and newly weaned piglets.
In preparation.
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Other scientific presentations
1. Ann-Sofie Riis Poulsen, Nadieh de Jonge, Sugiharto Sugiharto, JeppeLund Nielsen, Charlotte Lauridsen, Nuria Canibe. Gut microbiota profileof piglets fed sow milk, bovine colostrum or milk replacer. Abstract/posterpresentation. Gut Microbiology - 20 years and counting. 10th INRA-RowettSymposium. France, 2016. Page 143.
2. Ann-Sofie Riis Poulsen, Nuria Canibe, Charlotte Lauridsen, SugihartoSugiharto, Lone Bruhn Madsen, Emøke Bendixen, Jamal Momeni. Alpha-(1,2)-fucosyltransferase genotype influences the gastrointestinal microbiota ofpiglets. Abstract/poster presentation. Digestive Physiology in Pigs Sympo-sium. Kliczkow, 2015. Page 272.
3. Ann-Sofie Riis Poulsen, Sugiharto Sugiharto, Charlotte Lauridsen, NuriaCanibe. Comparison of bovine colostrum, milk replacer and sow milk on gas-trointestinal microbiota and mucosal E.coli F18 attachment of piglets. Oralpresentation. International workshop on nutrition and intestinal microbiotahost interaction in the pig. 2013. Page 30.
xii Contents
Contents
General introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Scope of thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.1 Defining gut health . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.2 The intestinal barrier . . . . . . . . . . . . . . . . . . . . . . . . . . 41.3 Functions of the gut microbiota . . . . . . . . . . . . . . . . . . . . 6
1.3.1 Host nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . 71.3.2 Defence against pathogens . . . . . . . . . . . . . . . . . . . 8
1.4 Composition of the pig gut microbiota . . . . . . . . . . . . . . . . 101.4.1 Colonisation and succession . . . . . . . . . . . . . . . . . . 10
1.5 Factors influencing the gut microbiota . . . . . . . . . . . . . . . . 121.5.1 Diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121.5.2 Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151.5.3 Probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191.5.4 Host genetics . . . . . . . . . . . . . . . . . . . . . . . . . . 251.5.5 Investigated factors in the current thesis . . . . . . . . . . . 25
1.6 Methods for studying the gut microbiota . . . . . . . . . . . . . . . 301.6.1 Culture-based methods . . . . . . . . . . . . . . . . . . . . . 301.6.2 Next-generation sequencing . . . . . . . . . . . . . . . . . . 30
2 Objectives and hypotheses . . . . . . . . . . . . . . . . . . . . . . . 332.1 Project aim and objectives . . . . . . . . . . . . . . . . . . . . . . . 332.2 Experimental objectives and hypotheses . . . . . . . . . . . . . . . 33
3 Methodological approach . . . . . . . . . . . . . . . . . . . . . . . 353.1 Gastrointestinal samples . . . . . . . . . . . . . . . . . . . . . . . . 353.2 Characterising the gut bacterial community . . . . . . . . . . . . . 36
3.2.1 Sequencing of the 16S rRNA gene . . . . . . . . . . . . . . . 36
Contents xiii
3.2.2 Microbial plate culture . . . . . . . . . . . . . . . . . . . . . 37
4 Manuscript 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
5 Manuscript 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
6 Manuscript 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .107
7 General discussion . . . . . . . . . . . . . . . . . . . . . . . . . . .1537.1 Manipulating the gut microbiota . . . . . . . . . . . . . . . . . . . 153
7.1.1 Milk-based dietary intervention . . . . . . . . . . . . . . . . 1547.1.2 ETEC F18 susceptibility . . . . . . . . . . . . . . . . . . . . 1557.1.3 Bacillus spores and gentamicin . . . . . . . . . . . . . . . . 156
7.2 Age-related succession of the gut microbiota . . . . . . . . . . . . . 1577.3 Sampling the gut microbial communities . . . . . . . . . . . . . . . 1587.4 High-throughput sequencing versus microbial culture . . . . . . . . 1597.5 Linking gut microbial community composition to gut health and
animal robustness . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .161
9 Perspectives and future work . . . . . . . . . . . . . . . . . . . . .163
Appendix A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .181
General introduction 1
General introduction
Intestinal disorders are highly prevalent in pigs reared in modern production sys-tems, and diarrhoea is a common clinical condition, especially in newborn andnewly weaned piglets (Kongsted et al., 2013; Lalles et al., 2007). The high preva-lence of diarrhoea in piglets poses a major challenge as it results in decreasedanimal welfare and high use of antibiotics, with the consequent risk of bacterialantibiotic resistance development (WHO, 2014).
Antibiotic resistance causes a major threat to human health. The ongoingincrease in multi-drug resistant bacterial infections (Blair et al., 2015) has empha-sised an increased need for prudent use of antibiotics, and has put major focuson the antibiotic consumption in production animals. In Denmark, pigs consume76 % of the total antibiotic mass consumed by production animals, horses, petsand fish, while only contributing with 43 % to the live biomass. Large amountsof antibiotics (approximately 42 %) are consumed by piglets in the post-weaningperiod (DANMAP, 2014).
From being perceived as a collection of disease promoting organisms, the gutmicrobiota has changed into a structure appreciated as an additional ’organ’ of thebody, serving numerous functions in relation to host biology and health (Sommerand Baeckhed, 2013). The early gut colonisation of the young pig is essential asa local protective mechanism, but also in maturing the adaptive immune system(Guarner and Malagelada, 2003). Intestinal disorders disrupting the gut microbialcommunity in early life may be jeopardising both the short- and long-term healthof the animal.
If the use of antibiotics is to be brought down, robustness of piglets againstinfectious diseases, especially those encountered immediately after weaning, hasto be improved. This may be accomplished by using specific dietary or probioticmeans to improve piglet gut health by beneficially influencing the colonisation ofthe gastrointestinal microbiota.
2 Scope of thesis
Scope of thesis
Gut health and piglet robustness are highly dependent on the gut microbiota,as well as the intestinal immune system and the epithelial barrier function. Theincluded studies and reviewed literature in this thesis focus on the gut microbiota,but a general introduction is also given to the concept of gut health, the barrierfunction of the intestinal wall, public health consequences of the high consumptionof antibiotics in the pig production, and the influencing factors investigated in thecurrent Ph.D.-study.
The present thesis investigates the impact of four factors on the gut microbialcomposition of piglets, with the end-point aim of obtaining healthy pigs with areduced use of antibiotics. The original studies included focus on the microbialcomposition and some fermentation metabolites. Functional studies (e.g. tran-scriptomics or proteomics) of the gut microbiota are not included. Wheneverpossible, background literature originating from pig studies is included. However,as current scientific literature on the gut microbiota comprises relevant studies inother mammalian species, these will be included when needed.
Chapter 1. Background 3
Chapter 1
Background
1.1 Defining gut health
’Gut health’ is a term often encountered in the scientific literature on human andanimal health. Although understood and appreciated by most, a precise definitionof a healthy gut is lacking (Bischoff, 2011). Due to the rather unspecific definitionson gut health, Bischoff (2011) proposed five major criteria for a healthy gastroin-testinal tract in humans, being 1) effective digestion and absorption of food, 2)absence of gastrointestinal illness, 3) normal and stable intestinal microbiota, 4)effective immune status, and 5) a status of well-being.
In veterinary medicine, as opposed to human medicine, only objective parame-ters are usable in the assessment of gut health status. The experimental veterinaryliterature proposes a variety of parameters as markers of gut health. In pigs, com-monly used markers are intestinal histomorphology, such as intestinal villous heightand crypt depth (e.g. Le Bon et al., 2010) and gene expression analyses on e.g.tight junction proteins, inflammatory mediators, oxidative stressors, and prolifer-ation and apoptosis markers (e.g. Alizadeh et al., 2015). From a microbiologicalpoint of view, a healthy gut is generally regarded as one with a balanced micro-biota, free from dysbiotic hallmarks as expansion of pathogens, loss of beneficialbacteria, and/or a reduced microbial diversity (Petersen and Round, 2014). Stud-ies focusing on the weaning process of piglets have contributed to our knowledgeabout which bacteria are supportive of a healthy animal, and which are poten-tial disease-causing organisms. The healthy pig gut has a high number of lacticacid bacteria, especially lactobacilli, and a low number of potential pathogenicbacteria. Such potential pathogenic bacteria are especially those belonging to theEnterobacteriaceae family, as for example some E. coli types and Salmonella. The
4 Chapter 1. Background
lactobacilli to enterobacteria ratio has traditionally been used as a measure of guthealth in pigs (Castillo et al., 2007).
An absolute state of optimal gut health is practically impossible to define, asgut health is a dynamic and relative concept. In addition, gut health status isthe result of a fine balance between gut microbiology, immunology and physiol-ogy (Lalles et al., 2007). The evaluation of selected gut health markers is oftendependent on the experimental set-up and subject in question. A definition of ahealthy gut has to be accompanied by a measure of the overall health conditionof the animal. An overall healthy pig is suggestively one that has an undisturbedgeneral condition, is in no need for antibiotic or analgesic therapy, and is able tocope with the given housing/environmental conditions.
The present thesis seeks to find gut health-promoting interventions. Success-fulness of these interventions is evaluated primarily from their effect on the gutmicrobiota and the occurrence of clinical symptoms originating from the gut. Asnot all animal experiments included a large number of animals, the studies werenot meant as diarrhoea studies per se. Hence, the microbiological aspect of guthealth is the focus of the current thesis.
1.2 The intestinal barrier
The intestinal mucosa is constantly bombarded with foreign antigens from ingestedmaterial and the gut microbiota, and defensive mechanisms are thus very well de-veloped, constituting physical, cellular, and chemical barriers. Optimal function ofthese barriers is pivotal to animal health. Figure 1.1 shows a schematic illustrationof the microscopic layers of the small intestine and the barrier structures presentedin the next paragraphs.
The most important barrier against antigens and microbes is the intestinalepithelial lining, consisting of an epithelial monolayer. Individual enterocytes arejoined by three groups of junctional proteins; 1) adherens junctions, 2) desmo-somes, and 3) tight junctions. While the first two groups of proteins in particularare responsible for mechanically linking neighbouring enterocytes, tight junctionsare responsible for limiting the intercellular space, being a crucial regulator ofparacellular permeability and only allowing certain electrolytes and substrates toby-pass the epithelial lining and enter the submucosal layers. Tight junction com-plexes consist of occludin, claudins, junctional adhesion molecules, and tricellulinproteins, which are spanning the cellular membranes near the apical membrane
Chapter 1. Background 5
(Groschwitz and Hogan, 2009). An increased intestinal permeability, also knownas a ’leaky gut’, may be caused by a down-regulation in the expression of tightjunction proteins, and has been associated with increased levels of inflammatorycytokines (Mankertz et al., 2000).
ç
Lumen
Peritoneal cavity
Epithelial monolayer
Superficial connective tissue layer (lamina propria)
Thin muscle layer= nerve plexus= blood vessel= lymph vessel= glands= dendritic cell= T-lymphocyte= B-lymphocyte= mucus
Tight junction
Microvilli
Crypt of LieberkühnOrigin of new enterocytes
Deep connective tissue layer
Peritoneum
Circular and longitudinal muscle layers
Figure 1.1: Schematic illustration of the microscopic structure of the small intestine
The intestinal luminal content is separated from the epithelial lining by aninner and an outer mucus layer. The outer layer (closest to the lumen) is looselyorganised and populated by bacteria, while the inner layer is firmly attached, andunder normal circumstances free from bacteria. Mucus is made up by glycosy-lated mucins (especially muc2 mucins) secreted by goblet cells (Johansson et al.,2011), acting as adhesins that enable binding of commensal and pathogenic bac-teria. For bacteria to be able to colonise and proliferate in the gut, the generalbelief is that the bacteria have to be able to adhere to the mucus layer, which re-quires the presence of surface-bound mucin-binding proteins (Juge, 2012). Besidesconstituting a physical barrier and acting as bacterial binding sites, mucins are
6 Chapter 1. Background
also important energy substrates to commensal bacteria (Johansson et al., 2011).Much research on this topic stems from human and mice studies. However, studiesin pigs confirm that MUC2 genes are expressed also in the pig intestine (Liu et al.,2014b). The importance of muc2 mucins as a protective barrier in the gut hasbeen demonstrated in mice, where muc2-deficient mice were more susceptible tointestinal infections, and experienced more mucosal damage upon infection, thandid wild-type mice (Bergstrom et al., 2010). Other mice studies show that theabsence of a gut microbiota results in a thinner intestinal mucus layer (Sharmaet al., 1995), indicating that the gut microbiota stimulates the intestinal mucusproduction.
The gut associated lymphoid tissue (GALT) consists of Peyer’s patches, lym-phoid follicles, intraepithelial lymphocytes and mesenteric lymph nodes, and pro-vide a cellular barrier against antigens and microorganisms. Peyer’s patches arewell-organised lymphoid structures located in the superficial connective tissue layerof the gut (lamina propria). These structures are dominated by T-lymphocytesand dendritic cells. The overlying epithelium is represented by microfold cells,which are specialised epithelial cells responsible for taking up luminal antigensand microbes, and delivering them to the underlying antigen-presenting dendriticcells (Min and Rhee, 2015). B-lymphocytes of the lamina propria are responsiblefor producing immunoglobulin A (IgA), the most abundant immunoglobulin of thegut. IgA is taken up by enterocytes and secreted to the intestinal lumen, where itcan interact with antigens and microbes by ’immune exclusion’, hence performingits chemical barrier function (Ramanan and Cadwell, 2016).
1.3 Functions of the gut microbiota
The gut microbiota is the total collection of bacteria, archeae, protozoa, vira, andyeasts in the stomach and intestines (Mackie et al., 1999; Mikkelsen et al., 2004).In the following text, the gut microbiota refers to the bacterial community only.
The commensal gut microbiota interacts with the host through numerous path-ways. The major mechanisms whereby the microbiota serves its beneficial effectsare by 1) contributing to host nutrition by synthesising vitamins and providing en-ergy through short-chain fatty acid (SCFA) production (Cummings et al., 2004);2) defending the host against enteric pathogens (Cummings et al., 2004; Sarkerand Gyr, 1992); and 3) stimulating local immune cell proliferation, thus playinga key role in maturing the adaptive immune system (Guarner and Malagelada,
Chapter 1. Background 7
2003)
1.3.1 Host nutrition
Feed components escaping digestion and absorption in the small intestine are fer-mented by the intestinal microbiota (Jha and Berrocoso, 2016). Carbohydratesincluding oligosaccharides, non-starch polysaccharides, and starch are the mostimportant substrates for bacterial fermentation, yet proteins can act as substratesas well (Williams et al., 2005). Figure 1.2 provides an overview of the producedmetabolites from carbohydrates and proteins, and figure 1.3 gives an example ofthe approximate concentrations of selected compounds.
Lactic acid Ethanol Formic acid Succinic acid
Proteins Amino acids
Carbohydrates Starch, non-starch polysaccharides
oligosaccharides
Acetic acid Propionic acid Butyric acid
Butyric acid CH4 Acetic acid
Primary metabolites
H2S
Acetic acid
Propionic acid CO2 H2 Butyric acid
BCFA Ammonia Amines Phenols Indols
CH4
Acetaldehyde
Propionic acid
Secondary metabolites
Figure 1.2: Overview of metabolites originating from carbohydrate and protein sources. Modifiedfrom Rist et al. (2013) and Venema and Do Carmo (2015). BCFA=branched chain fatty acids
Carbohydrates are fermented to organic compounds as lactic acid, succinicacid, and straight SCFAs (Macfarlane and Macfarlane, 2003). The major SCFAsin the gut are acetic, propionic and butyric acid, and these are generally regardedas being beneficial to the host (Williams et al., 2005). Acetic, propionic, and bu-tyric acid are converted to glucose by gluconeogenesis in liver cells, while skeletaland cardiac muscle cells are able to use acetic acid for energy production (Jha andBerrocoso, 2016). Some SCFAs are used as bacterial substrates and metabolisedto secondary metabolites (Venema and Do Carmo, 2015). SCFAs furthermore pro-
8 Chapter 1. Background
vide a substantial amount of energy to colonocytes, that especially prefer butyricacid. SCFAs have a trophic effect on intestinal cells (Burrin and Stoll, 2002),and butyrate has been shown to have anti-inflammatory potentials (Andriamihajaet al., 2010). Therefore, dietary interventions resulting in high concentrations ofSCFAs are generally considered beneficial to gut health (Williams et al., 2005).
Intestinal bacteria metabolise undigested and endogenous proteins by aminoacid deamination and decarboxylation (Jha and Berrocoso, 2016). End-products,besides those mentioned above for carbohydrates, include specific metabolites likebranched (short) chain fatty acids, as iso-butyric and iso-valeric acid, and po-tentially toxic compounds as ammonia, phenols, indols, and biogenic amines asputrescine and cadaverine (Williams et al., 2005). While iso-butyric acid has beensuggested to be a potential energy source for colonocytes (Blachier et al., 2007),some of the latter nitrogenous compounds (e.g. ammonia) are believed to havenegative effects on the host by impairing the mucosal barrier and interfering withcolonocyte metabolism (Blachier et al., 2007; Pieper et al., 2014). Thus, diets re-sulting in extensive protein fermentation, and high concentrations of nitrogenouscompounds, are considered as having a negative effect on gut health and shouldpreferably be avoided.
Both carbohydrate and protein metabolites are found in different concentra-tions along the length of the gut, and the highest fermentation activity is generallyseen in the caecum and colon (see figure 1.3).
1.3.2 Defence against pathogens
Local defence mechanisms of the gut microbiota include competing with pathogensfor mucosal binding sites and nutrients, production of antimicrobials, and elimi-nation of potential harmful/toxic substances (Cummings et al., 2004; Sarker andGyr, 1992). Production of organic acids by the gut microbiota (as described above)decreases gut pH, which inhibits growth of pathogens as Escherichia coli andSalmonella spp. (Canibe and Jensen, 2012). Lactic acid bacteria, especially Lac-tobacilli, are found in high numbers in the gut and are important in maintaining ahealthy gut (Daly et al., 2014). They are capable of reducing pH by lactic acid pro-duction, compete with potential pathogens, and produce bacteriocins (Hou et al.,2015), thereby reducing the number of pathogens and hindering their attachmentto the intestinal mucosa.
Human and mice studies have shown that the development of the gut mu-cosal immune system starts prenatally. Unlike the prenatal development, proper
Chapter 1. Background 9
Organic acids and ammoniam
mol
/l di
gest
a
0
15
30
45
60
Stomach Ileum Caecum Prox. colon Dist. colon
Lactic acid Acetic acid Propionic acidButyric acid Ammonia
Protein metabolites
umol
/l di
gest
a
0
400
800
1200
1600
Stomach Ileum Caecum Prox. colon Dist. colon
Amines Putrescine Cadaverine Phenol
Figure 1.3: Example of carbohydrate and protein metabolite concentrations along the length ofthe gut. Samples originate from six weeks old piglets three weeks post-weaning. Original datais from Pieper et al. (2014)
(i.e. resulting in a state of homeostasis) postnatal development and maturation oflymfoid structures in the gut is dependent on the presence of microbes (Sommerand Baeckhed, 2013). Germ-free mice have been reported to have underdevelopedmesenteric lymph nodes, intestinal lymphoid follicles and Peyer’s patches (reviewedby Round and Mazmanian (2009)), and on the cellular level, lines of natural killercells, and T- and B- lymphocytes are all modulated by the gut microbiota (re-viewed by Sommer and Baeckhed (2013)). These observations are supported bystudies conducted on pigs. Mulder et al. (2009) reported that manipulation ofthe gut microbiota, by rearing piglets in different environments, changed the ex-pression of genes involved in immunological processes. A similar observation wasdone when the gut microbiota of four-day-old piglets was manipulated by antibi-otic administration (Schokker et al., 2014). Thus, the gut microbiota is crucialin educating the intestinal immune system, thereby playing a major part in theanimal’s long-term ability to effectively launch an immunological response againstpathogens and pathological processes.
10 Chapter 1. Background
1.4 Composition of the pig gut microbiota
The microbial composition is highly influenced by intrinsic factors as pH, oxygentension, transit time, and hepatic and pancreatic secretions, which vary betweendifferent compartments (Buddington and Sangild, 2011), and the gut microbialcommunity thus consists of different niches along the length and width of the gut(Looft et al., 2014a). When moving from the proximal duodenum to the colon,microbial richness and diversity increase (Jensen, 1998; Looft et al., 2014a). Thenumber of culturable bacteria range from 103 per gram content in the stomach to1011 in the colon (Pluske et al., 2002)
Firmicutes and Bacteroidetes are the most abundant phyla in the gut (Kimand Isaacson, 2015; Looft et al., 2014a). Clostridium and Lactobacillus are amongthe most abundant genera in the Firmicutes phylum (Slifierz et al., 2015). As de-scribed in earlier sections, Lactobacillus and other lactic acid bacteria are generallyregarded as being beneficial to the host. Clostridium perfringens is a commonlyencountered Clostridium species, whereof some types are potential pathogens.Neonatal piglets are highly susceptible to C. perfringens type A and C, whichcause necrotic enteritis and ultimately result in high mortality rates (Songer andUzal, 2005). Prevotella and Bacteroides are among the highest abundant generaof the Bacteroidetes phylum, and are beneficial bacteria involved in the breakdownof polysaccharides and milk glycans (Frese et al., 2015).
1.4.1 Colonisation and succession
The process of acquiring a fully colonised gastrointestinal tract is a gradual andcomplex successional process beginning during (or immediately after) birth, whenbacteria originating from the mother (vaginal secretions, faeces and skin) and sur-rounding environment are ingested by the young (Mackie et al., 1999). Somebacteria are able to establish themselves and become permanent residents withoutthe need for further reintroduction (indigenous), while others are only temporaryresidents (non-indigenous) (Pluske et al., 2002). Species diversity and richnessincrease with age (Slifierz et al., 2015) and when fully established, the gut micro-biota constitutes a complex micro-environment existing in symbiosis with the host,and generally accepted as having a crucial influence on host physiology and health(Sommer and Baeckhed, 2013). The establishment of a gut microbiota is pivotalto the adaptation of the gut to the extra-uterine life (Buddington and Sangild,2011). Appendix A gives an overview of the dominating taxa along the piglet
Chapter 1. Background 11
gut during suckling and the first weeks after weaning. Data from sequencing andculture is included, and comprise mainly samples from the lower part of the gut.The below description, however, is impeded by the lack of sufficient comparablepapers originating from pig studies. Also there is a gap in the available literatureon the colonisation of the small intestine.
Immediately after birth, the piglet gut microbiota is dominated by bacteria be-longing to the Firmicutes and Proteobacteria phyla (Petri and Hill, 2010; Slifierzet al., 2015). Sequencing pooled samples from the stomach, small intestine, andcolon, Petri and Hill (2010) found the dominating genera to belong to Clostridi-aceae and Enterobacteriaceae. Swords et al. (1993) cultured specific microbialgroups and found Clostridium spp. and E. coli to be the most numerous gen-era in distal colonic digesta up until 4 days of age. From 1 day of age, bacteriabelonging to Streptococcaceae dominated the pooled community investigated byPetri and Hill (2010), until Lactobacillaceae and Lactobacillus appeared from 1week of age (Petri and Hill, 2010; Swords et al., 1993). Studies on the successionalpattern in faeces generally support the pattern seen in digesta. Sequencing datashow that the faecal microbiota is dominated by Clostridium, Escherichia, andFusobacterium immediately after birth. Nevertheless, as mentioned earlier, themicrobial communities are not identical along the gut, and these differences areapparent shortly after birth. Using microbial culturing, Gancarcikova et al. (2008)investigated specific groups in digesta from the jejunum, ileum and caecum, andfound differences as early as 2 days after birth, which were especially apparent be-tween the small intestinal compartments and the caecum, with more Lactobacilliin jejunum and ileum, and more Enterobacteriaceae in the caecum. From 1 weekof age, the relative abundances of Clostridium and Escherichia in faeces decrease,while Lactobacillus increases in relative abundance and stabilises around 4 weeksof age (Slifierz et al., 2015). The pig gut microbiota appears to have become arelatively stable community 3 to 4 weeks after weaning. See Appendix A for bac-terial fluctuations in different gut compartments between 1 and 3 weeks of age.Weaning at 21 days of age has been associated with an increase in bacteria belong-ing to Prevotellaceae and Ruminococcaceae (Frese et al., 2015). As these taxa areprimarily identified by sequencing, these findings are not supported by culture.
During early life, the gut microbiota is thus a highly dynamic and unstablecommunity. According to Thompson et al. (2008), the gut microbiota is most sus-ceptible to perturbations between 2-3 weeks of age. Stress caused by weaning isa major cause of bringing the piglet gut microbiota out of balance (Konstantinov
12 Chapter 1. Background
et al., 2006; Thompson et al., 2008), and such imbalances are believed to im-pair host-defensive mechanisms and increase the susceptibility to enteric disease(Castillo et al., 2007; Heo et al., 2013). Gut microbial perturbations during earlylife are potentially problematic as the early colonisers are active players in shapingthe ’adult’ microbiota (Guarner and Malagelada, 2003). Also, several studies in-volving germ-free and specific-pathogen-free mice suggest, that there is a ’windowof opportunity’ during early life, where the gut microbes present at the time havelasting effects on immune cell functions (Olszak et al., 2012) and stress responses(Sudo et al., 2004). It can thus be speculated that some microbial profiles in earlylife may be more beneficial than others.
1.5 Factors influencing the gut microbiota
Numerous studies in pigs, rodents, and humans have illustrated that the gut mi-crobiota is a dynamic ecosystem, which is influenced by a variety of internal (host)and external factors. These influencers include: Genetics, diet, stress, environ-ment, antibiotics, probiotics, and age. Different specific factors can potentially beutilised for beneficially manipulating the gut microbiota in favour of an improvedanimal health.
The impact of different dietary components, breed, antibiotics, and probioticson the pig gut microbiota and, when available, intestinal immunology, is describedin the following subsections.
1.5.1 Diet
Non-digested dietary components act as bacterial substrates (Jha and Berrocoso,2016) and vary between different diets. Table 1.1 summarises information on timeof intervention, type of sample analysed, microbial analysis, and diets from thepapers cited in the next paragraphs.
The neonatal piglet’s intestinal microbiota is dependent on the origin of themilk provided. Yeruva et al. (2016) found that the ileal microbiotas of 21-day-oldpiglets were different between those fed sow milk and those fed milk formula (cowmilk and soy based). The piglets fed sow milk had the highest relative abundanceof Lactobacillus and Clostridiaceae, while those fed milk formula had the highestabundance of Enterobacteriaceae and Lactococcus. The authors furthermore foundthat the lymphoid follicle sizes in the ileum and jejunum of piglets fed milk formula
Chapter 1. Background 13
were smaller than those from piglets fed sow milk, and thus indicated an effect ofa changed microbiota on the intestinal mucosal immune system.
The abrupt transition from milk to solid feed, occurring at weaning, is as-sociated with a change from a microbiota utilising primarily milk sugars to oneutilising plant fibers. Frese et al. (2015) reported an increase in Prevotellaceae,Ruminococcaceae and Lactobacillaceae, and a decrease in Enterobacteriaceae andBacteroidaceae after weaning. These changes were accompanied by a relative in-crease in bacterial genes encoding plant-degrading enzymes in the post-weaningperiod. These changes may not be a reflection of the isolated effect of diet, butmay also be caused by age and weaning.
Studies in grower and finisher pigs have shown that different protein and car-bohydrate types influence the intestinal microbial community. As these groups ofpigs are not within the scope of this thesis, they will not be addressed further. Seetable 1.1 for more details.
14 Chapter 1. Background
Tabl
e1.
1:K
eyin
form
atio
non
tim
eof
inte
rven
tion
,die
tary
com
pone
nts
inve
stig
ated
,ana
lyse
dsa
mpl
e(s)
,and
type
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isus
edin
the
cite
dst
udie
sin
vest
igat
ing
the
influ
ence
ofdi
eton
the
pig
gut
mic
robi
ota
Per
iod
offe
edin
terv
ention
Die
tary
com
pone
nts
Sam
ple(
s)A
naly
sis
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ding
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efer
ence
One
mon
thfr
om14
.6kg
Soyb
ean
mea
lC
otte
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eal
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hm
eal
Smal
lint
esti
nal
dige
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V4-
V5
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Age
nese
q.Illu
min
aFis
hm
eal)
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mm
unity
rich
ness
dom
inat
edby
Pro
teob
acte
ria.
Lactobacillus
and
Clostridium
wer
em
ore
abun
dant
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glet
sfe
dso
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cott
onse
edm
eal.
Cao
etal
.(20
16)
Wea
ning
Nur
sing
Wea
ning
diet
Faec
esV
416
SrR
NA
gene
seq.
Illu
min
aSe
ete
xt.
Fres
eet
al.(
2015
)
3to
5m
onth
sof
age
Hig
h-fa
t/lo
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ber
Low
-fat
/hig
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erFa
eces
qPC
R16
SrR
NA
gene
regi
onLow
-fat
/hig
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et)
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unda
nce
ofLactobacillus
spp.
,Clostridium
leptum
,Faecalibacterium
prausnitzii
and
Bifidobacterium
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t/lo
w-fi
ber
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)"
abun
danc
eof
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and#
[ace
tate
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ate]
and
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nrit
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icro
bial
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nce
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ced
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onof
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une
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ntly
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atFa
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Chapter 1. Background 15
1.5.2 Antibiotics
Antibiotics have since their discovery been an invaluable tool in combating bac-terial infections. Furthermore, many countries still allow the use of antibiotics asgrowth promoters (AGPs). Table 1.2 summarises information on period of antibi-otic intervention, type of antibiotic(s), sample types, and analysis from the paperscited in the next paragraphs.
Several studies have shown that antibiotics in sub-therapeutic doses, i.e. whenadded as AGPs, influence the composition of the gut microbiota. Holman andChenier (2014) showed that the effect on the gut microbiota was temporal anddiffered between tylosin and chlortetracycline. Their results indicated that theobserved microbial differences were due to changes in the relative abundancesof the species present, and not due to whether the species were present or not.On the contrary, Looft et al. (2014b) reported both short- and long-term effectsin the faecal microbiota after feeding carbadox. In the first week of antibioticadministration, the relative abundances of Prevotella, Roseburia, Faecalibacteriumand Asteroleplasma were increased, while Lactobacillus was increased in the controlgroup. Even though the most clear separation between carbadox-treated and non-treated piglets was seen in the early period of carbadox administration, an effecton the microbial community was still observed 3 and 6 weeks after the end ofcarbadox administration. AGPs targeting especially gram postive bacteria havebeen shown to reduce the number of lactic acid bacteria (Jensen, 1998).
Changes to the gut microbiota have also been observed when pigs are adminis-tered parenteral therapeutic antibiotics at an early age. Benis et al. (2015) showedthat therapeutic administration of tulathromycin on day 4 after birth had a tem-poral effect on the jejunal microbiota, but did not observe any long-term effects.Long-term effects were, however, seen in the expression of genes related to im-munological functions and cell proliferations in the jejunal tissue. Using a similarexperimental set-up, Schokker et al. (2014) and Schokker et al. (2015) showed thatthe jejunal microbial diversity was increased in tulathromycin-treated (at 4 days ofage) piglets 8 days after birth and decreased on day 176. The increased diversityon day 8 was associated with an increased relative abundance in Bifidobacterium,Faecalibacterium, Eubacterium, and Solobacterium, and a decreased relative abun-dance of Bacillus and Staphylococcus. The decreased diversity on day 176 wasassociated with an increased relative abundance of Actinomyces, Allofustis andEggerthella, and a decreased abundance of Bifidobacterium, Fusobacterium, Neis-seria, and Oxalobacter. However, no effect on the microbial diversity was seen on
16 Chapter 1. Background
day 55, yet effects on intestinal gene expressions related to metabolic and immuno-logical functions were seen at this time.
Hence, though the microbiota in some circumstances apparently is able to re-establish itself, disturbances in the microbial community may in some cases belong-lasting and have lasting functional effects. Disturbances in the gut micro-biota in early life, and the long-term effect on the expression of genes involved inimmunological processes, are all suggesting that perturbations to the gut micro-biota in early life change the way the intestinal immunological functions develop.
Chapter 1. Background 17
Tabl
e1.
2:K
eyin
form
atio
non
tim
eof
anti
biot
icad
min
istr
atio
n,an
tibi
otic
(s)
used
,ana
lyse
dsa
mpl
e(s)
,and
type
ofsa
mpl
ean
alys
isus
edin
the
cite
dst
udie
sin
vest
igat
ing
the
influ
ence
ofan
tibi
otic
son
the
gut
mic
robi
ota.
BW
=bo
dyw
eigh
t;SC
=su
bcut
aneo
us
Peri
odof
antibi
otic
adm
inis
trat
ion
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ibio
tic(
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mpl
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Ana
lysi
sR
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ence
3to
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ofag
eT
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in44
/22/
11m
g/kg
feed
Chl
orte
trac
yclin
e5.
5m
g/kg
feed
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essa
mpl
edat
3,6,
9,12
and
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Age
nese
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umin
a
Hol
man
and
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nier
(201
4)
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dox
50g/
ton
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essa
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d21
(wee
ks6-
9)an
dda
y2,
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and
42(w
eeks
9-15
)
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rRN
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nese
q.45
4G
S-FL
Xti
tani
um
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2014
b)
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ledo
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age
Tula
thro
myc
in2.
5m
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BW
SCJe
juna
ldig
esta
sam
pled
at8,
55an
d17
6da
ysof
age
PIT
Chi
pG
ene
expr
essi
onSc
hokk
eret
al.(
2014
)Sc
hokk
eret
al.(
2015
)B
enis
etal
.(20
15)
18 Chapter 1. Background
Antibiotic use and public health consequences
Before 2006, in-feed AGPs were used as preventative measures in coping with post-weaning enteric diseases (DANMAP, 2014). However, as the association betweenin-feed antibacterials and antibiotic resistance became clear, this practice had tobe abandoned and use of in-feed AGPs in the European Union was banned (Heoet al., 2013). In the years following this ban, the amount of prescribed antibioticsfor use in pigs rose, and the pool of antibacterial resistant genes remained high(DANMAP, 2014). As practically no new antibiotics (or antibacterials) have beendiscovered since the late 1980’s (Silver, 2011), it is crucial that the antibiotics oftoday remain effective for many years to come.
Development of antibiotic resistance is a natural evolutionary process and thusinevitable. However, the tremendous doses of antibiotics used in animal medicineare significant risk factors in selecting single and multi-drug resistant bacteria(WHO, 2014). The most commonly used antibiotics for pigs are tetracyclines withnearly 29 tonnes active compound, followed by penicillins and macrolides. Morethan 50 % of the tetracyclines and macrolides are used for pigs in the post-weaningperiod. Even though the amount of antibiotics used in pigs decreased from 2013 to2014 (DANMAP, 2014), the amounts of antibiotics consumed by pigs in Denmarkare still too high. Hence continuous efforts are made to bring the amounts useddown.
Antibiotic resistance arising in bacteria from pigs poses major problems in re-lation to human and animal health. Humans are in risk of being infected withresistant bacteria originating from pigs. Methicillin-Resistant Staphylococcus Au-reus (clonal complex 398; MRSA CC398) is an example of this. Pigs are consideredthe primary reservoir of MRSA CC398 and the number of infected humans in Den-mark rose in the period from 2004 to 2011. The vast majority of infections wererelated to the skin and soft tissues (Larsen et al., 2015), though fatal cases withsepticaemia have been reported as well (Nielsen et al., 2016). Another exampleis Salmonella typhimurium, which is an important zoonotic food-borne pathogen(DANMAP, 2014). DANMAP (2014) reported 64 % of isolates from pigs to beresistant against ampicillin, sulfonamide, and tetracycline, and hence categorisedas multi-resistant. Isolates from humans showed the same pattern of resistance.Furthermore, there is a horisontal transfer of antibiotic resistance genes among bac-teria. Transposable elements as plasmids can be transferred from one bacteriumto another, thus enabling transfer of resistance genes (Penesyan et al., 2015). Thisis especially worrying as otherwise harmless bacteria can transfer resistance genes
Chapter 1. Background 19
to pathogenic bacteria (Miller et al., 2016). Thus, whether it is the pathogenic orcommensal bacteria that carry the genes encoding resistance against specific an-tibiotics, they pose a major threat to the modern treatment of bacterial infectionsas we know it.
1.5.3 Probiotics
Oral probiotics have for several years been defined as ’live microbial feed supple-ments which beneficially affect the host animal by improving its intestinal micro-bial balance’ (Fuller, 1989). A never definition, proposed by WHO and FAO in2002, says that probiotics are ’live microorganisms which, when administered inadequate amounts, confer a health benefit on the host’ (FAO/WHO, 2002), andthus covers all probiotics.
Probiotics are believed to exert their beneficial effects by competing withpathogens for mucosal binding sites and nutrients in the intestine, and by pro-ducing antimicrobial compounds and organic acids thereby inhibiting pathogengrowth (Vondruskova et al., 2010). A considerable number of studies have beencarried out in pigs to test the effect of different probiotics on parameters as intesti-nal microbiota composition, immunological components, occurrence of diarrhoeaand growth performance, but results have been ambiguous, probably as a result ofdifferences in the studied organism, strain, dose, experimental setup and duration,environment, husbandry, and genotypes (Kenny et al., 2011; Vondruskova et al.,2010). Thus, general recommendations on the use of probiotics are so far lacking.
The most studied probiotic bacteria in pigs are Streptococcus thermophilus,Bifidobacterium animalis subsp. lactis and B. thermophilus, Enterococcus fae-cium, Lactobacillus bulgaricus, L. fermentum, L. murinus, L. acidophilus, L. pen-tosus, L. casai, L. paracasai, L. plantarum, L. salivarius and L. reuteri, Pedio-coccus acidilactici and P. pentasaceus, Bacillus subtilis, B. cereus var. Toyoi andB. licheniformis, Escherichia coli Nissle, and Propionibacterium freudenreichii.Saccharomyces cerevisiae and Aspergillus oryzae are examples of probiotic fungi.Three of the most commonly used probiotics belong to the Lactobacillus, Entero-coccus and Bacillus genera. Some of the studies focusing on the gut microbiota aredescribed in the next paragraphs. Table 1.3 summarises information on period ofprobiotic administration, type of probiotic(s), dose(s), sample type(s) and effectsfrom the cited and additional relevant papers.
B. cereus var. Toyoi has been associated with a decreased number of enterotox-igenic E. coli (ETEC) and morbidity in weaned piglets (Papatsiros et al., 2011). In
20 Chapter 1. Background
a study by Scharek et al. (2007a), B. cereus var. Toyoi was fed to sows during ges-tation and to piglets from 15 days of age, and showed a decrease in the number ofpathogen-associated E. coli serogroups. In a similar study, feeding sows, and laterpiglets, B. cereus var. Toyoi resulted in a decrease in the number of pigs showingsigns of diarrhoea (Taras et al., 2005). Feeding E. faecium to piglets 1-14 days ofage decreased the number of E. coli in faeces, decreased pH in the duodenum, andincreased the concentration of lactic and propionic acid in the colon (Strompfovaet al., 2006). Scharek et al. (2005) found that feeding E. faecium to sows duringgestation and to piglets from 14 days of age resulted in a reduced frequency ofb-hemolytic E. coli and E. coli O:141. Feeding weaned piglets L. acidophilus hasalso been shown to reduce the coliform numbers, but also the diversity and richnessof the gut microbiota (Wang et al., 2012). Also B. pumilus has been reported todecrease the ileal number of E. coli after daily administration for 3 weeks in newlyweaned piglets (Prieto et al., 2014). The immunological effects are seen from table1.3.
Probiotic administration in periods of antibiotic therapy, as a way of diminish-ing unwanted side effects (e.g. antibiotic-associated diarrhoea) has been investi-gated in humans and mice, and has been shown to have beneficial effects (Cresciet al., 2013; Pirker et al., 2013). Administration of L. casei concomitantly withantibiotics resulted in a lower reduction in gut microbial diversity compared withsubjects treated with antibiotics alone. Moreover, the incidence of developingantibiotic-associated diarrhoea decreased from 18.6 % to 5 % when L. casei andantibiotics were given together (Pirker et al., 2013). A mouse study by Cresciet al. (2013) found that the negative effects of tributyrin on a butyrate transporterand receptor, ion exchangers, and water channels were reduced when mice weresimultaneously fed L. GG. A study by Plummer et al. (2005) reported that, whenpatients were administered strains of L. acidophilus and Bifidobacterium spp. con-comitantly with antibiotics, there was a significant decrease in the development ofantibiotic resistance among enterococci.
Hence, numerous studies have reported several probiotic bacteria to have health-promoting effects in pigs. However, the modes of actions are generally still un-known and, according to our knowledge, the consensus on which probiotics to usenon-existing.
Chapter 1. Background 21
Tabl
e1.
3:St
udie
son
the
use
ofpr
obio
tics
and
thei
reff
ect
onth
egu
tm
icro
biot
aan
din
test
inal
imm
une
para
met
ers
ofpi
glet
s.D
=di
gest
a;F=
faec
es;
M=
muc
osa;
T=
tiss
ue.
jeju
=je
junu
m;i
l=ile
um;c
ae=
caec
um;c
ol=
colo
n.[..]
=co
ncen
trat
ion.
ExP
EC
=ex
trai
ntes
tina
lpat
hoge
nic
E.coli
Age
and
peri
odof
prob
ioti
cad
min
istr
a-ti
on
Pro
biot
ic(s
)D
ose
Sam
ple(
s)E
ffect
sR
efer
ence
Sow
s:28
days
pre-
part
umto
wea
ning
.P
igle
ts:
14-3
9da
ysof
age.
Bacillus
cereus
var.
Toy
oiSo
ws:
3.5x
108
cfu/
gfe
ed.
Pig
lets
:1.
5x10
9cf
u/g
feed
.T
(jej
u)"
Per
mea
bilit
yof
inte
stin
alti
ssue
.#
CD
45+
toen
tero
cyte
rati
o.A
ltm
eyer
etal
.(20
14)
Sow
s:28
days
pre-
part
umto
wea
ning
.P
igle
ts:
12-3
4da
ysof
age.
Enterococcus
faecium
NC
IMB
1041
5So
ws:
4.2-
4.3x
106
cfu/
gfe
ed.
Pig
lets
:5.
1x10
6cf
u/g
(pre
-st
arte
r)an
d3.
6x10
6cf
u/g
(sta
rter
).
D+
M(c
ol)
F#
Num
ber
ofis
olat
esw
ith
ExP
EC
-typ
ical
viru
lenc
ege
nes
inco
lon
asce
nden
sB
edno
rzet
al.(
2013
)
Sow
s:28
days
pre-
part
umto
wea
ning
.P
igle
ts:
2-28
days
ofag
e.
Pediococcus
acidilactici
(PA
)an
d/or
Saccharo-
myces
cerevisiae
subs
p.boulardii
(SC
)
Sow
s:2.
5x10
9-
6x10
9cf
upe
rda
y.P
igle
ts:
1x10
9cf
upe
rda
y(s
uckl
ing)
and
2x10
9cf
u/kg
feed
(wea
ned)
.
F D(il+
col)
PA#
ileal
mic
robi
ota
dive
rsity.
PApr
omot
edes
tabl
ishm
ent
ofFir
mic
utes
,an
dSC
Porphyrom
onadaceae
and
Ru-
minococcaceae
inth
eco
lon.
Bro
usse
auet
al.(
2015
)
Sow
s:7
days
pre-
part
umun
tilb
irth
Bifidobacterium
lactis
BI-07,
Lactobacillus
acido-
philus
WN
0074
and
NC
FM
1.0x
1010
cfu/
day
ofea
chpr
obio
tic
D+
FP
robi
otic
sid
enti
fied
indi
gest
aan
dfa
eces
from
the
offsp
ring
.N
umbe
rof
prob
ioti
csde
crea
sed
wit
hti
me.
Bud
ding
ton
etal
.(20
10)
Pre
-ter
mpi
glet
s(9
0%
gest
atio
n).
5da
ys.
Lactobacillusparacasei,
Bifidobacterium
ani-
malis,
Streptococcus
therm
o-
philus
3.0x
109
cfu/
pig.
Tes
ted
both
live
and
dead
pro-
biot
ics.
D+
T"
NE
Cin
cide
nce.
#M
icro
bial
dive
rsity
(liv
e).
#SC
FAco
ncen
trat
ion
inco
lon
(liv
e).
"E
xpre
ssio
nsof
imm
une
med
iato
rs.
Cili
ebor
get
al.(
2011
)
Continues
on
the
next
page
22 Chapter 1. BackgroundTa
ble
1.3:
Stud
ies
onth
eus
eof
prob
ioti
csan
dth
eir
effec
ton
the
gut
mic
robi
ota
and
inte
stin
alim
mun
epa
ram
eter
sof
pigl
ets.
D=
dige
sta;
F=fa
eces
;M
=m
ucos
a;T
=ti
ssue
.je
ju=
jeju
num
;il=
ileum
;cae
=ca
ecum
;col
=co
lon.
[..]
=co
ncen
trat
ion.
ExP
EC
=ex
trai
ntes
tina
lpat
hoge
nic
E.coli
Age
and
peri
odof
prob
ioti
cad
min
istr
a-ti
on
Pro
biot
ic(s
)D
ose
Sam
ple(
s)E
ffect
sR
efer
ence
Sow
s:28
days
pre-
part
umto
21da
yspo
st-p
artu
m.
Pig
lets
:1-
31da
ysof
age.
Pediococcus
acidilactici
MA
18/5
M,
Saccharom
yces
cere-
visiae
subs
p.boulardii.
Alo
neor
com
bine
d.
Sow
s:2.
5-6.
9x10
9cf
u.P
igle
ts:
1.0-
2.0x
109
cfu.
ET
EC
F4
chal
leng
eda
y25
T(i
l)
Com
bine
d:"
Exp
ress
ion
ofIL
-6.
Sing
lepr
obio
tic:
#E
TE
CF4
atta
chm
ent.
Dau
delin
etal
.(20
11)
35-4
0da
ysol
d.4
wee
ks.
Bacillus
subtilis
MA
139
2.2x
105
cfu/
gfe
ed(l
ow)
2.2x
106
cfu/
gfe
ed(m
ediu
m)
2.2x
107
cfu/
gfe
ed(h
igh)
F"
Num
ber
ofla
ctob
acill
ion
day
28ir
resp
ecti
veof
prob
ioti
cdo
seG
uoet
al.(
2006
)
Wea
ned
pigl
ets
17-1
8da
ysof
age.
17da
ys.
Escherichia
coli
UM
-2an
d-7
3.1x
109
cfu/
ml;
50m
l/pi
g/da
yD
(il+
col)
"M
icro
bial
dive
rsity
inile
uman
dco
lon.
#E.coli
K88
num
bers
inile
uman
dco
lon.
Kra
use
etal
.(20
10)
Wea
ned
pigl
ets
26-2
7da
ysof
age.
4w
eeks
.
Enterococcus
faecium
,Lactobacillus
salivar-
ius,
Lactobacillus
reuteri,
Bifidobacterium
ther-
mophilus
1.0x
109
cfu/
kgfe
edD
(il+
col)
Ileu
m:
"Tot
alae
robe
san
den
tero
cocc
i.C
olon
:"
Gra
m-n
egat
ive
anae
robe
s,la
ctob
acill
iand
ente
roco
cci.
"[a
ceti
cac
id].
"Lac
toba
cilli
:ent
erob
acte
ria
ratio.
Mai
ret
al.(
2010
)
Wea
ned
pigl
ets
28da
ysof
age.
5w
eeks
.
Bacillus
cereus
var.
Toy
oisp
ores
1.0x
109
spor
es/g
feed
F#
Dia
rrho
easc
ore.
#N
umbe
rof
ET
EC
.Pap
atsi
ros
etal
.(20
11)
Continues
on
the
next
page
Chapter 1. Background 23
Tabl
e1.
3:St
udie
son
the
use
ofpr
obio
tics
and
thei
reff
ect
onth
egu
tm
icro
biot
aan
din
test
inal
imm
une
para
met
ers
ofpi
glet
s.D
=di
gest
a;F=
faec
es;
M=
muc
osa;
T=
tiss
ue.
jeju
=je
junu
m;i
l=ile
um;c
ae=
caec
um;c
ol=
colo
n.[..]
=co
ncen
trat
ion.
ExP
EC
=ex
trai
ntes
tina
lpat
hoge
nic
E.coli
Age
and
peri
odof
prob
ioti
cad
min
istr
a-ti
on
Pro
biot
ic(s
)D
ose
Sam
ple(
s)E
ffect
sR
efer
ence
1-21
days
afte
rw
eani
ngBacillus
Pum
ilus
5.0x
1010
spor
espe
rpi
g(o
rala
dmin
istr
atio
n)an
d>
1010
spor
espe
rpi
g(t
opdr
ess)
F D(il+
cae)
#N
umbe
rsof
E.coli
inile
um.
"Ilea
l[pr
opio
nic
acid
].P
riet
oet
al.(
2014
)
26da
ysol
d.4
wee
ks.
Enterococcus
faecium
,Lactobacillus
salivar-
ius,
Lactobacillus
reuteri,
Bifidobacterium
ther-
mophilum
1x10
9cf
u/kg
feed
D(i
l+ca
e+co
l)"
Mic
robi
alri
chne
ssin
ileum
."
Rel
ativ
eab
unda
nce
ofEnterococcaceae
inile
um.
Satt
ler
etal
.(20
15)
Sow
s:25
days
afte
rin
sem
inat
ion
unti
lw
eani
ng.
Pig
lets
:15
-56
days
ofag
e.
Bacillus
cereus
var.
Toy
oiSo
ws:
2.6-
4.0x
105
cfu/
gfe
ed.
Pig
lets
:1.
3-1.
4x10
6cf
u/g
feed
.T
(jej
u)Pey
er’s
Pat
ches
"C
D3/
CD
8nu
mbe
ron
day
28in
the
ep-
ithe
liall
ayer
."
Num
ber
ofC
D8+
lym
phoc
ytes
day
35.
"N
umbe
rof
CD
25+
andgdT
cells
inla
m-
ina
prop
ria.
#N
umbe
rof
part
hoge
n-as
soci
ated
E.
coli
sero
type
s.
Scha
rek
etal
.(20
07a)
Sow
s:25
days
afte
rin
sem
inat
ion
unti
lw
eani
ng.
Pig
lets
:1-
56da
ysof
age.
Bacillus
cereus
var.
Toy
oiEnterococcus
faecium
B.cereus
Sow
s:2.
6-4.
0x10
5cf
u/g
feed
.P
igle
ts:
1.3-
1.4x
106
cfu/
gfe
ed.
E.faecium
Sow
s:1.
6-1.
2x10
6cf
u/g
feed
.P
igle
ts:
1.7-
2.0x
105
cfu/
gfe
ed.
F Seru
m"
Faec
alIg
Ain
sow
san
dpi
glet
s(d
ay28
)(B
.cereus).
#Se
rum
IgG
inpi
glet
sda
y42
-56
(bot
hpr
o-bi
otic
s).
Scha
rek
etal
.(20
07b)
Continues
on
the
next
page
24 Chapter 1. BackgroundTa
ble
1.3:
Stud
ies
onth
eus
eof
prob
ioti
csan
dth
eir
effec
ton
the
gut
mic
robi
ota
and
inte
stin
alim
mun
epa
ram
eter
sof
pigl
ets.
D=
dige
sta;
F=fa
eces
;M
=m
ucos
a;T
=ti
ssue
.je
ju=
jeju
num
;il=
ileum
;cae
=ca
ecum
;col
=co
lon.
[..]
=co
ncen
trat
ion.
ExP
EC
=ex
trai
ntes
tina
lpat
hoge
nic
E.coli
Age
and
peri
odof
prob
ioti
cad
min
istr
a-ti
on
Pro
biot
ic(s
)D
ose
Sam
ple(
s)E
ffect
sR
efer
ence
1-14
days
ofag
eEnterococcus
faecium
EK
132.
0x10
9cf
u/pi
glet
D+
FFa
eces
day
7:"
Num
bers
ofen
tero
cocc
i.#
Num
bers
ofE.coli.
Dig
esta
:"
Num
bers
ofen
tero
cocc
i."
[Lac
tic
and
prop
ioni
cac
id]i
nco
lon.
Stro
mpf
ova
etal
.(20
06)
Wea
ned
pigl
ets
18-2
1da
ysol
d.5
wee
ks.
Lactobacillus
acido-
philus
orPediococcus
acidilactici.
Del
iver
edin
ahi
ghm
oist
ure
ferm
ente
dm
aize
feed
.
Bef
ore
ferm
enta
tion
:1.
7x10
7cf
u/m
lD
(il+
co)
#M
icro
bial
rich
ness
and
dive
rsity.
#N
umbe
rof
colif
orm
sw
hen
fed
L.
aci-
dophilus
com
pare
dto
P.acidilactici.
Wan
get
al.(
2012
)
Wea
ned
pigl
ets
21-3
6da
ysof
age
Bacillus
licheniform
is
DSM
5749
,Bacillus
subtilis
DSM
5750
3.9x
108
cfu/
day
(low
dose
)7.
8x10
8cf
u/da
y(h
igh
dose
)T
(jej
u)A
mel
iora
ted
path
ophy
siol
ogic
alch
ange
sca
used
byE.coli
F4
infe
ctio
n.In
duce
dT
-reg
type
1ce
lls.
Zhou
etal
.(20
15)
Chapter 1. Background 25
1.5.4 Host genetics
Different pig breeds have different genetic make-up and hence differ in certainphenotypic traits. Such traits may be related to intestinal physiology. Yorkshire,Landrace and Duroc are widely used pig breeds in Denmark. Pajarillo et al.(2015) characterised the faecal microbiota of these three pig breeds at 15 weeksof age, and found breed to have a significant effect on the overall gut microbiota.This breed effect was still apparent after 12 weeks of housing the three breedstogether, though the dissimilarity between the microbial communities decreased.A similar effect of breed has been reported in the faecal microbiota of neonatalYorkshire and Meishan piglets (Bian et al., 2016). Interestingly, the breed effectwas significant only on day 14 and 49 of age and hence, not detectable immediatelyafter birth. At 14 days of age, Meishan piglets had the highest relative abundanceof Fusobacteriales and lowest abundance of Erysipelotrichaceae, while they on day49 had the highest abundance of Lactobacilllus. Despite the influential effect ofgenotype on the composition of the faecal microbiota, it appears that this effectis minor when compared to the effect of age, weaning, and environment as thesetend to even out the effects of genotype (Bian et al., 2016; Pajarillo et al., 2015).
1.5.5 Investigated factors in the current thesis
Bovine colostrum
Bovine colostrum (BC) is the first milk secreted by the dairy cow after partu-rition. A precise definition of colostrum is lacking as authors generally disagreeabout when the mammary secretion switch from colostrum to non-colostrum milk(McGrath et al., 2016).
The essential function of colostrum is to provide the neonate with nutrients, inaddition to growth-promoting and protective compounds. The latter is especiallyimportant in mammals with epitheliochorial and synepitheliochoral placentas, e.g.pigs and cows, which do not allow transfer of immunoglobulins in utero. Hence,ingestion of colostrum immediately after birth is pivotal for the survival of theseneonates (Pakkanen and Aalto, 1997).
BC is generally regarded as unsuited for human consumption and produced inhigher amounts than required by the calf. Hence, it is a by-product from the dairyindustry and thus available in excess amounts (McGrath et al., 2016). Colostrumhas a chemical composition that varies significantly from that of ’mature’ (non-colostrum) milk, and especially the high content of antimicrobial components and
26 Chapter 1. Background
growth factors has led to an increased interest in bovine colostrum as a nutraceu-tical during recent years (Pandey et al., 2011). In addition to these bio-activecomponents, BC is also constituted of notable amounts of numerous oligosaccha-rides (Gopal and Gill, 2000), which have been suggested to have growth-promotingeffects on probiotic bacteria (Champagne et al., 2014).
Major antimicrobial components in BC are immunoglobulins (Igs) (Pakkanenand Aalto, 1997) and non-specific components as lactoferrin and lactoperoxidase(van Hooijdonk et al., 2000). The major Igs are IgG, IgA, and IgM, with IgG(1 and 2) being the primary Ig in colostrum (Hurley and Theil, 2011). Ingestedimmunoglobulins provide the neonate with local passive immunity by binding tobacteria and toxins, hereby limiting their ability to bind to the enterocyte surface(Hurley and Theil, 2011). The antimicrobial mode of action of lactoferrin is largelyunknown, but is generally believed to be related to the iron-binding capacity of themolecule (van Hooijdonk et al., 2000). Lactoperoxidase is an enzymatic antioxidantinhibiting bacterial metabolism (Pandey et al., 2011).
Growth factors are important to the growth and development of the newbornyoung. Numerous growth factors are found in BC, with the most abundant onesbeing Insulin-like Growth Factor 1 and 2. Then follows growth factors as Epider-mal Growth Factor and Transforming Growth Factor-b. These growth factors areimportant in stimulating cellular growth and cell differentiation (Boudry et al.,2008).
The beneficial effects of BC have been investigated in both pre-term, new-born, and newly weaned piglets (see table 1.4). Several studies suggest that BCexerts its function by inhibiting/limiting intestinal inflammatory processes andpathogen-induced cellular destruction. However, the conglomerate of studies hasprovided varying results. Discrepancies between studies are likely due to differ-ences in doses administered, health status of the target group, period and lengthof administration, and end-points. Also, the chemical composition of BC is influ-enced by processing (Elfstrand et al., 2002), cow breed, feeding, parity, length ofdry period, and time of collection (Gopal and Gill, 2000), hence different productsmay result in varying biological effects.
Chapter 1. Background 27
Table 1.4: Reported effects of bovine colostrum. BC = Bovine Colostrum; NEC = NecrotisingEnterocolitis; PP = Peyer’s Patches. IFN = Interferon. IL = Interleukin
Experimental set-up Effect(s) Reference
Newly weaned piglets
BC orally administered.Fed for three weeks.
No effect on feed intake or growth." serum IgA.Stimulated ileal PP.PP: # IFN-g and " IL-2, IL-4 and IL-10expression.Lymph nodes: " IL-2, IL-10 and IL-12expressionIntestinal tissue: " IL-12 expression.
Boudry et al. (2007)
BC supplemented diet.Fed for four weeks.
Improved weight gain (highest effectduring week 1)." Systemic IgA during week 1.
Boudry et al. (2008)
BC supplemented diet.Fed for 7 or 14 days.
" Duodenal villi perimeter. Huguet et al. (2007)
BC supplemented diet.Fed for 12 days post-weaning.
" Feed intake and daily weight gain.Improved faecal consistency.Effects lasted beyond the study period.
Huguet et al. (2012)
BC supplemented diet.Challenged with E. coli K88.
No beneficial effect on intestinalmorphology reported.
King et al. (2007)
BC supplemented diet.Fed for 7 days.
No significant effect on weight gain. King et al. (2008)
Fed solely BC for eight days.Relative to milk formula.
Fewer E. coli colonising jejunal and ilealtissue.Jejunum: # expression of TLR.Ileum: # expression of TLR and IL-2
Sugiharto et al. (2015)
Neonatal piglets
BC orally administered.Fed from 1-10 days of age.
" Lifespan of low birth-weight piglets.No effect on body weight or weight gain.
Viehmann et al. (2015)
Pre-term born piglets piglets
BC orally administered.Relative to milk formula.
# NEC incidence and severity." Lactose absorption.# Intestinal permeability.
Shen et al. (2015)
BC orally administered.Relative to milk formula.
# NEC severity." Small intestinal enzyme activities e.g.lactase.
Stoy et al. (2014)
In vitro
IPEC-J2 cells.Stimulated with heat-treatedE. coli and Salmonella entericaserovar Typhimurium.
# Expression of genes related to immune,defence and inflammatory responses." Expression of genes related to proliferation,wounding and migration processes.# Induction of NF-kB transcription factor.
Blais et al. (2015)
Bacterial cultures Stimulated probiotic bacteria.Inhibited growth of E. coli.
Champagne et al. (2014)
28 Chapter 1. Background
Bacillus spp. spores
Generally, there exists no consensus on which probiotic is considered the best, orwhen and how it should be administered. Therefore, due to the list of studiesreporting beneficial effects of Bacillus spp., and its availability, a total of nineBacillus strains belonging to B. subtilis, B. licheniformis and B. amyloliquefaecenswere included in the animal experiment presented in this thesis.
Bacillus spp. is a spore-forming aerobic/facultative anaerobic bacterial genus,which forms endospores when the surrounding environmental conditions are un-favourable to the continued survival of the vegetative cell (Cutting, 2011). Theproduced spores are in a state of dormancy, and their physical structure makesthem highly resistant to otherwise intolerant pH values, temperatures, irradiation,and desiccation (Nicholson et al., 2000). Figure 1.4 depicts the schematic structureof an endospore. These characteristics make Bacillus spp. able to survive pelletingprocesses, and hence ideal as an in-feed probiotic. Bacillus species are primarilyfound in soil and are not normally regarded as inhabitants of the pig gut micro-biota (Tam et al., 2006). Studies have shown that B. subtilis and B. licheniformisare able to germinate and re-sporulate in the pig gut, but that their ability to growis limited. Also, when spores are no longer administered, they disappear from themicrobial community, indicating that they are not able to colonise the gut (Leseret al., 2008). Studies suggest that both spores and vegetative cells have probioticpotentials (Tam et al., 2006).
Core
Inner membrane
Cortex
Coat
Thick peptidoglycan layer
Lamellar proteinaceous structure
Plasma membrane of the vegetative cell
Dehydrated cytoplasm, stabilised DNA and RNA. Ions in complexes.
Figure 1.4: Illustration of the basic structure of a Bacillus endospore. Adapted from Venemaand Do Carmo (2015)
Chapter 1. Background 29
Gentamicin
In Denmark, neonatal piglets with diarrhoea are commonly administered gentam-icin, an antibiotic sold as a watery formulation intended for oral administration.Gentamicin is a bactericidal concentration-dependant antibiotic acting primarilyagainst gram-negative aerobes by irreversible binding to the 30S ribosomal sub-unit, thereby inhibiting bacterial protein synthesis (Plumb, 2011). The effectsof gentamicin on the gut microbiota of piglets are, according to our knowledge,unknown.
Alpha-(1,2)-fucosyltransferase
Escherichia coli is a heterogeneous bacterial group. It is a gram-negative rod nor-mally inhabiting the gut, but is also found in several pathotypes causing intestinaland extra-intestinal disease (Croxen and Finlay, 2010). ETEC is an importantintestinal pathotype in piglets, where it causes intestinal disease (enteric colibacil-losis) in both newborn and newly-weaned piglets (Fairbrother et al., 2005; Nagyand Fekete, 1999). To be able to cause disease, ETEC is dependant on two majorvirulence factors; surface adhesins and enterotoxins. Adhesins are mainly consti-tuted of fimbriae. Fimbriae are antigenic proteins protruding from the bacterial cellwall and come in numerous variants. In pigs, especially the F4 and F18 fimbriaehave been studied intensively. Bacterial colonisation is dependent on the presenceof fimbriae-specific receptors on the small intestinal microvilli (Nagy and Fekete,2005). F4 receptors are expressed in the intestinal tissue from birth, while F18receptors are not fully expressed in piglets younger than three weeks of age (Fair-brother et al., 2005). ETEC produces heat-stable and heat-labile enterotoxins,and both are produced by ETECs able to infect piglets (Nagy and Fekete, 2005).The enterotoxins increase the cellular electrolyte and fluid secretion (Croxen andFinlay, 2010) leading to hyper-secretory diarrhoea. The expression of both F4 andF18 receptors are genetically inherited in an autosomal dominant manner, andpigs resistant toward F4 and F18 have been bred.
The alpha-(1,2)-fucosyltransferase (FUT1 ) gene product is involved in the for-mation of blood-group antigen sugar structures on cell membranes, and is ex-pressed in the small intestine of pigs (Bao et al., 2012; Frydendahl et al., 2003).Due to its chromosomal location in the ETEC F18 receptor region, the FUT1 genehas been proposed as a candidate gene for manipulating the adhesion of ETECF18 to the specific intestinal receptor (Meijerink et al., 1997). Frydendahl et al.(2003) showed that a guanine to adenine mutation at nucleotide position 307 (chro-
30 Chapter 1. Background
mosome 6), resulting in an alanine to threonine substitution at position 103, inboth loci of the FUT1 gene (FUT1-307A/A), significantly decreased the suscepti-bility of weanling piglets to ETEC F18 infection when compared to piglets with theFUT1-307A/G and FUT1-307G/G genotypes. The FUT1-307A/A genotype has beenassociated with a decreased FUT1 enzyme activity (Meijerink et al., 2000). Theeffect of FUT1 polymorphisms on colonisation of commensal bacteria is currentlyunknown.
1.6 Methods for studying the gut microbiota
1.6.1 Culture-based methods
Microbial culture is generally regarded as the traditional method for studyingmicroorganisms. Using various growth media and fulfilling specific requirementsto substrates, pH, incubation temperatures and oxygen tension, bacteria can begrown, allowing their quantification and further biochemical, physiological andtaxonomical characterisation (Hoiby, 2008). The use of culture-based methods forstudying the pig gut microbiota dates back to the 1950’s, where focus was only on afew bacterial groups (Fewins et al., 1967). As more bacteria were isolated from thegut, researchers started realising the limitations of using selective growth mediaand growth conditions in characterising a microbial community as diverse as theone found in the pig gut (Allison et al., 1979; Salanitro et al., 1977). A study byPryde et al. (1999) showed that, compared to a molecular identification technique(using the 16S ribosomal DNA gene), bacterial culture was insufficient in describingthe microbial diversity of the gut. It was later suggested that approximately50 % of all bacterial species found in the human gut were yet to be cultured(Sears, 2005). Despite a change of focus towards newer molecular techniques,microbial culture should still be considered an invaluable tool in quantificationand functional characterisation of specific bacterial groups, as stressed by recentpublications on culturomics, a technique combining diverse culture conditions andmass spectrometry (Lagier et al., 2015).
1.6.2 Next-generation sequencing
During recent years, several molecular techniques taking advantage of the 16SrRNA gene have been employed in the investigation of the phylogenic diversity ofthe pig gut microbiota, these being; cloning and sequencing (Leser et al., 2002;
Chapter 1. Background 31
Pryde et al., 1999); Terminal Restriction Fragment Length Polymorphism (Ho-jberg et al., 2005; Liu et al., 2012); and Denaturing Gradient Gel Electrophoresis(Levesque et al., 2012; Liu et al., 2014a). However, due to the rapidly developingsequencing technologies and the appreciation of their potential in characterisingcomplex communities, focus has changed to the high-throughput next-generationsequencing (NGS) technologies. Currently, Illumina (HiSeq and MiSeq) (Kim andIsaacson, 2015; Slifierz et al., 2015) and 454-pyrosequencing (Hermann-Bank et al.,2015; Mach et al., 2015; Mann et al., 2014) are the dominating NGS technologiesin current literature investigating the pig gut microbiota.
The 16S ribosomal RNA (rRNA) gene has become the preferred gene for phy-logenetic classification of bacteria. It is found in all prokaryotes and made up bynine hypervariable (V1-V9) regions interspersed by highly conserved regions. Asclosely related bacterial species have more similar sequences in their hypervariableregions, than species more distantly related, these regions are used in the phyloge-netic classification (Kim et al., 2015). The conserved regions act as annealing sitesfor universal primers, allowing amplification of one or several of the hypervariableregions across species (Nocker et al., 2007). Before proceeding to sequencing, anamplicon library of a specific region of the 16S rRNA gene is produced by PCR(Kim and Isaacson, 2015). As current technologies do not allow sequencing of thefull 16S rRNA gene, only up to three variable regions are used as representativesof the full length. There exists no general agreement on which regions are the bestwhen studying the microbial community in the pig gut, but the most commonlyused regions are V1 to V6 (reviewed by Kim and Isaacson (2015)). Preparation ofsamples for sequencing, and the following data analyses, often vary considerablybetween studies, and hence make it difficult to directly compare results betweenstudies. These issues are further addressed in the general discussion.
Sequencing platforms differ not only in their sequencing technologies, but alsoin the number and length of reads produced in a single run. Generally, we wishto sequence as deep as possible. Sequencing depth is associated with the numberof reads produced, and should preferably be as high as possible. In addition, tobe able to assign taxonomy with high precision, sequencing reads should be aslong as possible. Hence, key features of a sequencing platform are the number andlength of reads it is able to produce at low error rates. This was demonstratedby Claesson et al. (2010), who reported the insufficiency of short reads with higherror rates in characterising a human faecal microbial community. Hence, severalparameters, including potential biases, have to be taken into consideration when
32 Chapter 1. Background
utilising these new sequencing platforms. Table 1.5 compares key features betweenNGS and traditional culture. Challenges of bioinformatic processing of sequencingdata are further described in the methodological approach.
Table 1.5: Comparing high-throughput next-generation sequencing and traditional bacterial cul-ture. NGS = next-generation sequencing
NGS Culture
PCR amplification step Yes NoCoverage of complex communities High LowQuantitative No YesPrice Expensive (decreasing) Lower expenseData output Large amounts Small amountsSpecial requirements for data analysis Yes NoWorkload Small Large
Chapter 2. Objectives and hypotheses 33
Chapter 2
Objectives and hypotheses
2.1 Project aim and objectives
The overall aim of the presented Ph.D. project is to investigate if it is possible, byspecific dietary and probiotic (in relation to antibiotic therapy) means, to shapethe piglet gut microbiota in a direction that favours healthier animals. In addition,the gut microbial effects of a genetic variation in ETEC F18 susceptibility is inves-tigated to see if this has an unintentional effect on the colonising gut microbiota.
The project objective is to taxonomically and quantitatively characterise thecolonisation and succession of the gut microbiota of piglets from birth until twoweeks after weaning within four scenarios, each representing a dietary, probiotic,antibiotic, or genetic factor. This objective will be accomplished by using classi-cal microbial culture (quantitative approach) accompanied by culture-independentNGS 16S rRNA gene sequencing (in-depth qualitative approach), and detection ofmajor microbial fermentation products.
2.2 Experimental objectives and hypotheses
Animal experiment 1Objective: To characterise the colonisation of the gut microbial community in 23-30 days old piglets fed BC, a porcine milk replacer (MR) or sow milk.
Hypothesis: When compared to a commercial porcine MR, BC is a more suitablesubstitute to sow milk in maintaining a health-promoting gut microbiota. Further-more, the gut microbial community of piglets fed BC will have a closer resemblanceto that of piglets fed sow milk than will MR-fed piglets.
34 Chapter 2. Objectives and hypotheses
Animal experiment 2Objective: To characterise how FUT1 genotype influences the establishment andsuccession of the gut microbial community in 5 to 34 days old piglets.
Hypothesis: Piglets with the FUT1-M307A/A genotype will have a gut microbiotadifferent from piglets with the FUT1-M307A/G genotype.
Animal experiment 3Objective: To characterise the gut microbial effects of administrating gentamicinand Bacillus subtilis, B. licheniformis, and B. amyloliquefaecens spores in pigletsbetween 0 and 42 days of age.
Hypothesis: Administering gentamicin to newborn piglets will change the microbialcommunity of the gut. Concurrent administration of Bacillus spp. spores willalleviate the expected negative effect(s) of gentamicin.
Chapter 3. Methodological approach 35
Chapter 3
Methodological approach
To obtain a thorough knowledge on the gut microbial diversity, the following ap-proaches were used for sample collection and community analyses.
3.1 Gastrointestinal samples
In the three animal studies, several sample types were collected from the lengthof the gut. In order to study the succession of the gut microbiota, sequentialfaecal samples were collected. Fresh faecal samples were collected directly fromthe rectum by gentle digital exploration. The advantage of this approach is that thesame animal can be sampled multiple times. As the microbial community differsbetween different gut segments (Looft et al., 2014a), luminal contents (digesta)were collected from several segments of the gut as well. Piglets were sacrificedfollowing a standard protocol using a captive bolt gun followed by bleeding, orby blunt trauma to the head (piglets < 5 kg). Immediately after sacrification,the stomach and intestines were located and excised. The stomach and caecumwere located and excised from the rest of the intestinal organs. The small andlarge intestine was removed from the intestinal mesenterium, measured in fulllength, and divided into two or three (dependent on piglet age) equally long parts.Stomach and intestinal digesta was immediately collected.
36 Chapter 3. Methodological approach
3.2 Characterising the gut bacterial community
3.2.1 Sequencing of the 16S rRNA gene
In-depth characterisation of the microbial community was achieved by ampliconsequencing of the 16S rRNA gene using high-throughput sequencing. A flowchartfor sample preparation and data analysis is seen in figure 3.1. Each of these steps,and different applied methods in each of these steps, potentially have an effect onthe final community diversity.
DNA extraction
Amplicon production by PCR
Clean-up of amplicon library
DNA quantification
Quality control
Quality control
Multiplex samples in equimolar
concentrations
Sequencing
Quality control
Step 1
Step 2
Step 3
Step 4
Step 5
Step 6
Quality filteringTrimming
Merge reads
OTU clustering
Chimera detection
Data visualisation
Assigning taxonomy
Figure 3.1: Flowchart on sample preparation for next-generation sequencing and data processing.OTU = Operational Taxonomical Unit
Sequencing of samples obtained from animal experiment 1 and 3 was doneusing the Illumina MiSeq (Illumina, USA). This platform provided 2x300 base-pairs (paired-end) reads and allowed us to use the V1-V3 regions. These regionshave been proposed to be the preferred representatives of the full-length 16S rRNAgene when analysing highly diverse communities as the gut microbiota (Kim et al.,2011). In animal experiment 2, the samples were sequenced on the Ion Torrent
Chapter 3. Methodological approach 37
PGM platform (Thermo Fisher Scientific, USA). However, as this platform onlyprovided 200 base-pairs reads, only the V3 region was sequenced. Using different16S rRNA regions potentially results in different microbial communities (Burbachet al., 2016), and the use of different approaches limits our ability to compare theresults of experiment 2 with those of experiment 1 and 3.
Aside from sequencing platform and the variable region(s) sequenced, DNAextraction procedure (Burbach et al., 2016; Clooney et al., 2016), PCR primers(Osborne et al., 2005), and DNA purity and concentration (Clooney et al., 2016)also influence the observed diversity. Bioinformatic processing of reads obtainedby high-throughput sequencing takes advantage of advanced computational al-gorithms. However, numerous pipelines using different algorithms (e.g. OTU-clustering algorithms) exist, which have a significant influence on the microbialdiversity (Schloss, 2010). Hence, the methodological approach has a significantimpact on the final community observed, and should be considered when analysingand interpreting sequencing data.
3.2.2 Microbial plate culture
Classical microbial enumeration by culture was included as a measure of absolutemicrobial numbers of selected microbial groups. The absolute numbers were givenas colony forming units (cfu) (log 10 transformed), and in order to obtain countablecolony numbers, a series of 10-fold dilutions were made from the digesta or faecalsample, followed by plating on different selective and non-selective media. Wechose to include six (seven in experiment 3) major microbial classes, which werecultured on the following media;
• Blood agar: Enumeration of haemolytic bacteria. Such colonies are gener-ally unwanted as they typically represent pathogenic bacteria, e.g. ETEC(Fairbrother et al., 2005). Further typing was not performed.
• Casein Soya Bean Digest agar: Enumeration of Bacillus spp. spores. Vege-tative cells were killed and spores activated by heat-treatment.
• Colon roll-tubes: Enumeration of total anaerobic bacteria in media contain-ing colon extract.
• MacConkey agar: Enumeration of bacteria belonging to the Enterobacteri-aceae family. Lactose fermentors grow as pink/red colonies (e.g. E. coli),
38 Chapter 3. Methodological approach
while non-lactose fermenters grow as white colonies (e.g. Salmonella andShigella).
• Malt Chloramphenicol (MCA) agar: Enumeration of yeasts and moulds.
• Man Rogosa and Sharpe (MRS) agar: Enumeration of lactic acid bacteria.Colonies were not further identified.
• Tryptose Sulfite Cycloserine (TSC) agar: Enumeration of C. perfringens.
Chapter 4. Manuscript 1 39
Chapter 4
Manuscript 1
The microbial community of the gut differs between piglets fed sowmilk, milk replacer or bovine colostrum.
Ann-Sofie Riis Poulsen, Nadieh de Jonge, Sugiharto Sugiharto, Jeppe Lund Nielsen,Charlotte Lauridsen, Nuria Canibe.
Submitted to British Journal of nutrition.
40 Chapter 4. Manuscript 1
Experimental set-up:
36 piglets from four sowsFollowed from 23 to 30 days of age
SM-fedPiglets fed sow milk
MR-fedPiglets fed milk replacer
BC-fedPiglets fed bovine
colostrum
Digesta Stomach, ileum, caecum and mid colon (day 30)
1. 16S rRNA gene sequencing• V1-V3 region• Illumina MiSeq
2. Classical culture3. Organic acid analysis
12 piglets 12 piglets12 piglets
Digesta Stomach, ileum, caecum and mid colon (day 30)
Digesta Stomach, ileum, caecum and mid colon (day 30)
Faeces Day 23, 25, 27 og 30
Faeces Day 23, 25, 27 og 30
Faeces Day 23, 25, 27 og 30
1
The microbial community of the gut differs between piglets fed sow milk, milk replacer or
bovine colostrum
Ann-Sofie R. Poulsen1*, Nadieh de Jonge2, Sugiharto Sugiharto3, Jeppe L. Nielsen2, Charlotte
Lauridsen1, Nuria Canibe1
1 Department of Animal Science, Faculty of Science and Technology, Aarhus University, Tjele,
Denmark
2 Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University,
Aalborg, Denmark
3 Faculty of Animal and Agricultural Sciences, Diponegoro University, Semarang, Central Java,
Indonesia
* Corresponding author.
Aarhus University,
Faculty of Science and Technology, Department of Animal Science,
Blichers Allé 20, P.O. Box 50,
DK-8830 Tjele, Denmark
Tel.: +45 87 15 80 58
Fax.: +45 87 15 42 49
E-mail address: ann-sofie.riispoulsen@anis.au.dk
Short title: Bovine colostrum and gut microbiota.
Key words: Bovine colostrum. Gut microbiota. Undersized piglets. 16S rRNA gene sequencing.
Chapter 4. Manuscript 1 41
2
Abstract
The aim of the current study was to characterise the gut microbiota of piglets fed bovine colostrum
(BC), milk replacer (MR) or sow milk (SM). Twenty-three days old piglets (n=36) were randomly
allocated to the three diets. Faecal samples were collected at 23, 25, 27 and 30 days of age. Piglets
were euthanized at 30 days of age and luminal content from the stomach, ileum, caecum and mid-
colon was collected. Bacterial DNA was subjected to amplicon sequencing of the 16S rRNA gene.
Bacterial enumerations by culture and short-chain fatty acid analysis were conducted as well. BC-
piglets had a high abundance of Lactococcus in the stomach (p<0.0001) and ileal (p<0.0001)
digesta, while SM-piglets had a high abundance of Lactobacillus in the stomach digesta (p<0.0001).
MR-piglets had a high abundance of Enterobacteriaceae in the ileal digesta (p<0.0001) and a
higher number of haemolytic bacteria in the ileal (p=0.0002) and mid-colon (p=0.001) digesta than
SM-piglets. BC-piglets showed the highest colonic concentration of iso-butyric and iso-valeric acid
(p=0.02). Both sequencing and culture indicated higher shedding of potential pathogenic
Escherichia coli in MR-piglets, while the gut microbiota of BC-piglets was characterised by a
change in lactic acid bacteria genera when compared to SM-piglets. We conclude that especially the
ileal microbiota of BC-piglets had a closer resemblance to that of SM-piglets in regard to the
abundance of potential enteric pathogens than did MR-piglets. The higher content of colonic
branched-chain fatty acids in BC-piglets, however, calls for further investigation.
42 Chapter 4. Manuscript 1
3
Introduction
The increased litter sizes of the modern pig industry have negatively influenced piglet viability.
Large litters come with great variation in within-litter birth weights and are accompanied by an
increased number of low birth-weight piglets. Undersized piglets have difficulties competing with
heavier littermates, and experience reduced growth rates and increased morbidity and mortality (1).
To ensure adequate feed intake of undersized piglets, and hence improve weight gain and survival
chances, milk replacers have been implemented by many pig farmers (2). However, the
gastrointestinal tract of undersized piglets may be immature, influencing optimal nutrient digestion
and absorption. Also, as sow colostrum quality (and quantity) influences short- and long-term
survival (reviewed by Theil et al. (3)), there is an increasing interest in the nutritional and functional
quality of milk replacers and milk supplements for newborn piglets.
Diet is known to influence gut maturation (4). Besides maturing functional entities of the
intestinal tissues, gut maturation also includes the establishment of a gastrointestinal microbiota (5).
The gastrointestinal microbiota is appreciated as an important inhabitant of the body, being highly
involved in host defensive mechanisms. The local defensive mechanisms of the gut microbiota
include competing with pathogens for mucosal binding sites and nutrients, production of
antimicrobial-like agents and elimination of noxious substances (6; 7). Furthermore, microbial
colonisation of the gut stimulates local immune cell proliferation, hence playing a key role in the
maturation of the adaptive immune system. As the gut microbiota influences animal health,
choosing a diet in favor of a beneficial microbiota may be crucial. This could especially be
important at the time of microbiota establishment after birth, as the very first bacteria to colonise
the gut are able to influence the proceeding colonisation, and hence the composition of the 'adult'
gut microbiota (8).
Colostrum is the milk secreted during the first 24-48 hours following parturition (3; 9).
Bovine colostrum (BC) is a by-product from the dairy industry, available in excess amounts, and a
rich source of biologically active compounds (10; 11). The major bio-active compounds are growth
factors such as Insulin-like Growth Factor, Epidermal Growth Factor and Tissue Growth Factor β,
and antimicrobials as immunoglobulins (12). In preterm born piglets, BC has been reported to
increase the activity of specific brush-border enzymes, improve intestinal health and decrease
severity of necrotising enterocolitis when compared to feeding milk formula (13). In term born
piglets, provision of BC rather than milk replacer reduced the intestinal colonisation of
enterotoxigenic Escherichia coli and modulated the intestinal immune system, while no difference
between BC and natural rearing with the sow was observed (14). Furthermore, newly weaned piglets
have shown improved growth performance (15) and intestinal mucosal restoration (16) when
supplemented with BC.
Chapter 4. Manuscript 1 43
4
The effect of BC on the gut microbiota remains to be explored. The aim of the current study
was therefore to taxonomically and quantitatively characterise the early colonisation of the
gastrointestinal microbial community in piglets fed sow milk, milk replacer (originating from
bovine milk) or BC. We hypothesised that the gastrointestinal microbial community of piglets fed
BC would have closer resemblance to that of piglets fed sow milk than would piglets fed milk
replacer.
Materials and methods
Study design
The present study was conducted according to the ethical license obtained from the Danish Animal
Experiments Inspectorate, Ministry of Food, Agriculture and Fisheries, Danish Veterinary and Food
administration. National guidelines on experimental animal housing, care and sacrificing procedure
were followed. The study was performed at the experimental facility at the Department of Animal
Science (Foulum, Aarhus University).
A total of 36 piglets (crossbred Danish Landrace x Yorkshire; mixed females and males)
from four different sows were included in the study. The sows originated from the herd at Foulum,
Aarhus University. Piglets were housed with their dams until 23 days of age. The 36 piglets (nine
piglets from each sow) were randomly assigned to one of the following treatment groups (three
piglets from each sow per treatment): (a) kept with the sow for the whole experimental period (SM-
fed); (b) separated from the sow and fed a commercial porcine milk replacer powder (Grifor,
Hatting KS) (MR-fed); (c) separated from the sow and fed powdered BC (European Colostrum
Industry S.A.) (BC-fed). Chemical compositions are shown in Table 1.
Piglets separated from the sow were transported to an experimental stable and housed in
pens (1.45 m x 1.70 m) in groups of three until 30 days of age (end of experiment). Piglets were
randomly allocated to the dietary treatments. All pens were padded with rubber mats and piglets
had access to rooting material. Stable temperature was 24˚C. Piglets were continuously exposed to
faecal material from the sow by daily collecting faecal matter from the dam pen and spreading it
into the piglet pens. Each pen was equipped with an automated wet feeder (Mambo Automix 25;
Wit-Mambo Inc.) from which the piglets received ad libitum feeding. Piglets had access to fresh
water. The powdered BC and MR were dissolved in approximately 45˚C warm water in the
automated feeder (approximate dry matter percentage: BC 20% and MR 15%). To get the piglets
accustomed to the feeding machine, they were fed one portion of sow milk in the trough of the
machine upon arrival to the pen. SM-fed piglets suckled their dams until 30 days of age (end of
experiment). In an attempt to minimize the impact of other factors than the planned dietary
intervention, SM-fed piglets were transported exactly as the BC-fed and MR-fed piglets before
44 Chapter 4. Manuscript 1
5
returning to the sow. This ensured that all piglets were subjected to similar amount of stress due to
transportation.
Sample and data collection
Piglets were individually weighed at 23 and 30 days of age. BC and MR consumption was recorded
daily as powder provided minus leftovers from the automated wet feeder (determined by freeze
drying). The clinical condition of the piglets was evaluated daily, including occurrence of diarrhoea.
Faecal samples were collected from the rectum on day 23 (before transportation), 25, 27 and
30. All piglets were euthanised at 30 days of age; the abdomen was incised and the gastrointestinal
tract removed. Total luminal contents (digesta) from the stomach, small intestine (proximal and
distal), cecum and colon (proximal, mid and distal) were collected immediately after killing.
Digesta from the respective segments and faeces was mixed and subsamples were stored at -20˚C
for organic acid analysis (stomach, distal small intestine, caecum and mid colon) and snap-frozen in
liquid nitrogen and stored at -80˚C for 16S rRNA gene amplicon sequencing (stomach, distal small
intestine and mid colon). Bacterial enumeration by culture was performed on a fresh subsample of
faeces and digesta (stomach, distal small intestine, caecum and mid colon).
Dry matter and organic acid analysis
Dry matter content of digesta was determined by freeze-drying using a ScanVac Coolsafe 55
(Labogene ApS, Lynge, Denmark). Concentrations of the short-chain fatty acids (SCFAs) acetic,
propionic, butyric, isobutyric, valeric and isovaleric acid, and lactic acid in faeces and digesta were
quantified as previously described by Canibe et al. (17).
Microbiological enumerations
Approximately 1 g faecal material was suspended in 10 ml pre-reduced salt medium(18). The content
was homogenized in a Smasher paddle blender (bioMérieux Industry, USA) for 2 min.
Approximately 5 g digesta were suspended in a flask containing 50 ml pre-reduced salt medium.
The flask content was transferred to a CO2 flushed bag and homogenized for 2 minutes. 1 ml
homogenate was transferred to a Hungate tube containing 9 ml pre-reduced salt medium and 10-
fold dilutions were prepared using the technique previously described by Miller and Wolin (19). The
samples were plated on selective (and indicative) and non-selective agar plates.
Enterobacteriaceae were enumerated on MacConkey agar (Merck 1.05465) after aerobic
incubation for one day. Yeasts were enumerated on malt, chlortetracycline and chloramphenicol
agar (Merck 1.03753 (yeast extract), 1.05397 (malt extract), 1.07224 (bacto-pepton), 1.08337
(glucose), 1.01614 (agar-agar) and Oxoid Sr0177E) after aerobic incubation for two days.
Chapter 4. Manuscript 1 45
6
Haemolytic bacteria were enumerated on blood agar (Oxoid Pb5039A) after aerobic incubation for
one day. Clostridium perfringens were enumerated using the pour-plate technique on tryptose sulfit
cycloserine agar (Merck 1.11972, 1.00888) after anaerobic incubation for one day. Lactic acid
bacteria were enumerated on de Man, Rogosa and Sharp agar (Merck 1.10660) after anaerobic
incubation for two days. Total anaerobic bacteria were enumerated in roll tubes containing pig
colon fluid-glucose-cellobiose agar (18) and incubated for seven days. Plates and roll-tubes were
incubated at 37˚C.
DNA extraction
Samples for DNA extraction included 47 faecal samples (one sample was missing from the SM-fed
group on day 25) from 12 piglets and 72 digesta (stomach, distal small intestine, mid colon)
samples from 24 piglets. DNA was extracted with the E.Z.N.A. Stool DNA Kit (Omega Bio-Tek,
inc., VWR international) following a standard protocol with the following exception; bead beating
was performed on a FastPrep FP120 (Bio 101 Savant/MP Biomedicals, USA) for 2x30 s. DNA
extract purity was evaluated with Nanodrop ND1000 (Thermo Scientific, USA) and quantified
fluorometrically with Qubit 3.0 HS dsDNA assay (Life Technologies, Thermo Fisher Scientific,
USA). DNA concentrations were normalized to 5 ng/µl.
16S rRNA gene amplicon sequencing
Amplicon libraries were generated by targeted amplification of the V1-V3 hypervariable regions of
the bacterial 16S rRNA gene. The PCR reaction (25 µl) contained 10 ng template DNA, Platinum®
High Fidelity buffer (x1), dNTP (400 uM of each), MgSO4 (1.5 mM) and Platinum® Taq DNA
polymerase High Fidelity (1U) and barcoded library adapters (400 nM). V1-V3 primers: 27F
AGAGTTTGATCCTGGCTCAG and 534R ATTACCGCGGCTGCTGG. Thermocycler settings:
Initial denaturation at 95˚C for 2 min, 30 cycles of 95˚C for 20 s, 56˚C for 30 s, 72˚C for 60 s and
final elongation at 72˚C for 5 min. PCR reactions were run in duplicate for each sample and pooled
afterwards. Purification of the amplicon libraries was performed using the Agencourt AMPure XP
bead protocol (Beckman Coulter, USA) and eluted in 23 µL nuclease-free water. Individual
libraries were quantified with Quant-iT HS dsDNA assay (Life Technologies, USA) and quality
checked on a Tapestation 2200 (Agilent, USA). Libraries were pooled in equimolar concentrations,
and diluted to 4 nM. The library pool was sequenced using an Illumina MiSeq (Illumina, USA) and
MiSeq reagent kit v3 (2x300 PE).
46 Chapter 4. Manuscript 1
7
Bioinformatic processing and analysis
The obtained raw sequencing reads were quality filtered and trimmed using trimmomatic (v0.32) (20), only keeping reads with a minimum length of 275 bp. The trimmed reads were merged using
FLASH v. 1.2.7 (21) and read pairs between 425 and 525 bp in length were formatted for use with
the UPARSE workflow (22). Reads were dereplicated and clustered into Operational Taxonomical
Units (OTUs) using USEARCH7 at 97% sequence similarity. Taxonomy was assigned using the
RDP-classifier as implemented in QIIME (23) with a minimum confidence of 0.8 and Greengenes
(version 08-2013) as a reference database. Results were analysed in R studio (version 0.99.489 for
Mac) using the Ampvis package (24).
Statistical analyses
Principal Component Analysis was performed on square root transformed OTU abundances. A
constrained (by diet) redundancy analysis with plotting of bacterial culture and organic acid
parameters was performed to check for potential correlations between sequencing data, bacterial
enumerations and organic acid data. Significance of diet was tested on the first two principal
components (PCs) using the envfit parametric test and on the Bray-Curtis dissimilarity matrix using
the Adonis test (25). The parametric Wald-test (26) was used to test for significant OTU abundance
differences between the SM group and the MR and BC groups. OTUs with an adjusted p<0.001
were considered significantly different between the respective diets.
The impact of diet and age on bacterial and organic acid parameters, microbial richness, and
diversity index were investigated by fitting the data to a linear mixed model using the lmer function
from the lme4 package (27) using R studio (Version 0.99.489 for Mac). Diet and age/intestinal
segment were included as fixed effects, while pig and sow were included as random effects (by
including random intercept terms) to account for multiple observations made on the same litter and
on the same pig. When analysing the body weight variable, the piglets’ body weights at the
beginning of the experiment (day 23) were included as a co-variate. The fixed effects were tested
using an F-test with Kenward-Roger approximation, where the reduced model was tested against
the full model. This was done using the KRmodcomp function in the pbkrtest package (28). When a
fixed effect was found to be significant, a post-hoc test was performed using the multcomp package
and Bonferroni adjustment to correct for multiple comparisons (29). Effects were considered
significant when p<0.05 and as trends when 0.05≤p<0.10.
Chapter 4. Manuscript 1 47
8
Results
During the course of the experiment, one BC-fed piglet was euthanised due to vomitus and general
weakness and one MR-fed piglet died. At 30 days of age, SM-fed piglets weighed more than BC-
fed (p=0.016) and MR-fed piglets (p=0.011) (Table 2). Diarrhoea incidence rate and the consumed
amounts of milk replacer and BC are also listed in Table 2.
Microbiome composition – 16S rRNA gene amplicon sequencing
Sequencing of 119 samples yielded a total of 2,090,874 sequences. A sequencing depth of 5000
sequences was considered appropriate from rarefaction curves, excluding four samples from
analysis (data not shown). Recovered sequences clustered into 2485 OTUs, which were classified
into 34 bacterial phyla, 154 families and 271 genera. Eight phyla had an overall relative abundance
above 1%.
Faecal microbiota
The relative abundance of the eight most abundant phyla (relative abundance > 1%) and 20 most
abundant genera are presented in Fig. 1A and B. Irrespective of diet and age, Firmicutes and
Bacteroidetes dominated the communities. Prevotella and Oscillospira were the most abundant and
stable genera both regarding diet and age. Of the 20 most abundant genera, ten belonged to the
phylum Bacteroidetes and nine to the phylum Firmicutes. The microbial community richness did
not differ between diet or age (Fig. 1C and S1A). The diversity, expressed as the Shannon index,
was higher in MR-fed compared to BC-fed piglets (p=0.015; Fig. 1D). There was a significant
effect of diet on the overall faecal microbial community composition of the three dietary groups on
days 25 (padonis=0.01) and 30 (padonis=0.008) (Fig. 2). However, no OTUs were found to differ
significantly in their read abundances between diets.
Digesta microbiota
Fig. 3A and B presents the eight most abundant phyla and 20 most abundant genera of the microbial
communities of the stomach, distal small intestine and mid-colon. Irrespective of diet, the microbial
communities of the stomach and mid-colon were dominated by Firmicutes, followed by
Bacteroidetes. The distal small intestinal community was dominated by Firmicutes in SM-fed
piglets, Firmicutes and Proteobacteria in MR-fed piglets, and Firmicutes followed by Proteobacteria
and Actinobacteria in BC-fed piglets. Overall, Lactobacillus and Prevotella were the most
dominating genera in the stomach and mid colon. In addition, Mitsuokella was the third most
dominating genus in the stomach of BC-fed piglets. The microbial community of the distal small
intestine was dominated by Lactobacillus (most pronounced in SM-fed piglets) and
48 Chapter 4. Manuscript 1
9
Enterobacteriaceae in MR-fed piglets. As for the faecal samples, community richness did not differ
between diets; nor did the Shannon index (Fig. 3C and D, and S1B). Diet had a significant effect on
the overall microbial community of the stomach (padonis=0.001; Fig. 4A), distal small intestine
(padonis=0.001; Fig. 5A) and mid colon (padonis=0.001; Fig. 6A).
In the stomach, when comparing BC- to SM-fed piglets, six out of 551 OTUs were found to
have significantly different read abundances (Fig. 4B), all with a higher read abundance in BC-fed
piglets. OTU_72, belonging to Lactococcus, was the most significantly changed OTU, having a
higher read abundance in BC-fed piglets (p=5.3E-6). Nine out of 566 OTUs were found to have
significantly different read abundances between MR- and SM-fed piglets (Fig. 4C). OTU_66,
belonging to Lactobacillus, was the most significantly changed OTU, having a higher read
abundance in SM-fed piglets (p=2.7E-11).
In the distal small intestine, the three most influential taxa were responsible for the observed
grouping seen from the Principal Component Analysis (Fig 5A). Clustering of MR-fed piglets was
explained by the higher relative abundance of Enterobacteriaceae, clustering of SM-fed piglets was
explained by Lactobacillus, and clustering of BC-fed piglets was explained by Lactococcus.
Comparing BC- and MR-fed piglets with SM-fed piglets resulted in 11 out of 433 (BC-fed) and 435
(MR-fed) OTUs having significantly different read abundances (Fig. 5B and C). Again, OTU_72
belonging to Lactococcus was found to be the most significantly changed OTU, having a higher
read abundance in BC-fed piglets (p=2.2E-15). OTU_6, belonging to Enterobacteriaceae, was the
most significantly changed OTU when comparing MR- to SM-fed piglet (p=4.0E-10), having a
higher read abundance in MR-fed piglets.
Mid colon samples showed distinct grouping according to diet on PC3 (when plotted as a
function of PC1) with MR-fed piglets clustering by themselves (Fig. 6A). 14 out of 802 OTUs were
found to have significantly different read abundances when comparing BC- and SM-fed piglets
(Fig. 6B). The most significantly changed OTU was OTU_1 (p=4.7E-12) belonging to Lactobacillus,
and was found to have a higher read abundance in SM-fed piglets. Comparing MR- and SM-fed
piglets resulted in 13 out of 840 OTUs being significantly different (Fig. 6C). OTU_47, belonging
to Blautia, was the most significantly changed OTU (p=1.1E-13) and was found to have a higher
read abundance in MR-fed piglets.
Dry matter and pH
There was no difference in pH of digesta between diets. Dry matter content of digesta varied
between diets, being dependent on gut segment (Table S1). There was no difference in dry matter
content of digesta from the proximal small intestine and caecum between diets. Dry matter content
of digesta from the stomach (p<0.0001), proximal colon (p≤0.002), and mid colon (p≤0.006) was
Chapter 4. Manuscript 1 49
10
highest in SM-piglets, while being higher in digesta from the distal small intestine (p=0.049) in BC-
piglets compared to MR-piglets. The lowest dry matter content of digesta from the distal colon was
found in MR-piglets (p≤0.0003).
Microbiological enumerations and concentration of organic acids
Faeces
The number of C. perfringens was lower on day 30 compared to day 23 (p=0.0006) and day 25
(p=0.0008) for all diets, but there was no difference in any of the investigated microbial groups
between diets (Table S2). Results of haemolytic bacteria have not been included due to the majority
of counts being below detection level.
Faecal concentrations of acetic (p≤0.004), propionic (p≤0.013), butyric (p≤0.0034) and the
sum of acetic, propionic and butyric acid (p≤0.001) were higher in BC-fed compared to SM- and
MR-fed piglets (Table 3). The concentration of the sum of iso-butyric and iso-valeric acid was
highest in BC-fed piglets (p≤0.009) on day 25, 27 and 30. The concentrations of propionic
(p≤0.037), butyric (p≤0.045), and the sum of acetic, propionic and butyric (p≤0.045) acid were
higher on day 25, 27 and 30 compared to day 23. The acetic acid concentration was higher on day
25 (p=0.014) and day 30 (p=0.001) compared to day 23. In BC-piglets, the concentration of the sum
of iso-butyric and iso-valeric acid was lowest on day 23 (p≤0.003).
Digesta
Haemolytic bacterial counts in digesta from the distal small intestine (p=0.0002), caecum
(p=0.003), and mid colon (p=0.001) were higher in MR-fed compared to SM-fed piglets (Table 4).
The number of C. perfringens was higher in all segments of BC-fed (p=0.041) compared to MR-fed
piglets but similar to those in the SM-fed group.
The concentration of the sum of iso-butyric and iso-valeric acid in digesta from the colon
(p≤0.02) was higher in BC-fed compared to MR-fed piglets, while the caecal concentration was
higher in SM-fed (p=0.036) compared to MR-fed piglets (Table 5).
Correlation between 16S rRNA gene sequences, organic acids and bacterial enumerations
The constrained redundancy analysis performed on all samples (16S rRNA amplicon data) with
fitted microbial enumerations and SCFA data (lactic acid omitted as detectable amounts were only
found in digesta) showed a clear separation between the different diets with positive correlations to
microbial enumerations and SCFA concentrations (Fig. 7). Samples from the SM-fed piglets
correlated with the number of lactic acid bacteria (r2=0.11), while samples from the MR-fed piglets
correlated with the number of haemolytic bacteria (r2=0.30) and yeast (r2=0.25) and to a lesser
50 Chapter 4. Manuscript 1
11
degree with Enterobacteriaceae (r2=0.07). Samples from the BC-fed piglets correlated with the
concentration of iso-butyric (r2=0.38), iso-valeric (r2=0.32), the sum of iso-butyric and iso-valeric
acid (r2=0.36) and to a lesser degree with the number of C. perfringens (r2=0.10) and concentration
of acetic (r2=0.18), propionic (r2=0.15), butyric (r2=0.16), valeric (r2=0.21) and the sum of acetic,
propionic and butyric acid (r2=0.18).
Discussion
Several studies have investigated the effects of supplementary BC feeding on a variety of host
protective functions in pigs (e.g. (16), (13) and (30)). However, according to our knowledge no studies
have focused on the effect of BC on the gut microbiota when fed as sole nutrition. This should be
considered an important step in the process of optimising supplementary feeding of undersized
piglets, and in developing dietary strategies for optimal nutrition of preterm infants prone to
necrotising enterocolitis (31).
The piglets in the SM- and BC-fed dietary groups were weaned at 23 days of age. As
weaning is a highly stressful experience resulting in a decreased feed intake and nutrient digestion
capacity (32), the piglets in the current study were used as models for undersized piglets in regards to
having an unstable (immature) intestinal microbiota, suboptimal nutrient digestion and impaired
immune status.
The microbial community is known to change according to gut segment (33), and multiple
samples from the length of the gastrointestinal tract were therefore sampled. In addition, to be able
to follow the development of the microbiota from the same pigs over the course of the experiment,
faecal samples were included as well. Using 16S rRNA gene sequencing, we showed that there
were clear differences in the microbial communities from piglets fed different milk-based diets. As
expected, the microbial communities were gut region dependent, why differences between diets
varied in the different gut segments. The present results showed a clear influence of diet on the
microbial communities of the stomach, small intestine and colon. Diet did not influence the faecal
microbial community to the same level as the digesta communities, suggesting that the faecal
microbiota might need longer time to adjust in order to become diet specific.
Compared to the SM-fed piglets, the microbial communities of the stomach and small
intestine of BC-fed piglets indicated a shift in lactic acid bacteria (LAB) genera. OTUs belonging to
Lactococcus, Leuconostoc, Streptococcus and Carnobacterium were more abundant in BC-fed
piglets, whereas the LAB of SM-fed piglets was mainly dominated by Lactobacillus. Lactococcus
sp. is traditionally not found to be part of the commensal gut microbiota, but has been reported to
have bacteriocin producing properties (34). Lactococcus and Leuconostoc have most frequently been
associated with fermented dairy products, Carnobacterium have been found in dairy products, meat
Chapter 4. Manuscript 1 51
12
and fish (35) and Streptococcus sp. has previously been isolated from the gastrointestinal tract of pigs (36). BC-fed piglets furthermore had a higher abundance of Mitsuokella in the stomach digesta,
which previously has been isolated from the stomach and colon digesta of pigs (36; 37). A previous
study reported an inhibiting effect of Mitsuokella jalaludinii on Salmonella typhimurium. Levine et
al. (38) found that the combined effect of acetic, lactic and succinic acid produced by M. jalaludinii
during fermentation, and the accompanied decrease in pH, was able to inhibit the growth of and
cellular invasion by S. typhimurium. The authors therefore proposed M. jalaludinii to have potential
as a probiotic species. In our study, we were not able to determine the Misuokella species in
question, but assuming that it was M. jalaludinii, it could be speculated on the mentioned beneficial
effects. Hence, the finding of potential probiotic bacteria in BC-fed piglets may have enhanced
pathogen resistance and intestinal mucosal integrity, as demonstrated by the immunological results
obtained by Sugiharto et al. (14) from the piglets included in the present study.
The shift in LAB genera seen in BC-fed piglets was not observed in MR-fed piglets. An in
vitro study showed a stimulating effect of BC on LAB growth rates, suggestively due to the
oligosaccharides found in colostrum (39). As BC has a higher content of oligosaccharides than
mature milk (40), such oligosaccharides might be the reason why BC-fed and not MR-fed piglets
were found to have a higher read abundance of LAB as Lactococcus, Mitsuokella and Leuconostoc
in their stomach and small intestinal digesta than SM-fed piglets.
16S rRNA gene sequencing on digesta from the distal small intestine revealed that MR-fed
piglets had a higher abundance of Enterobacteriaceae when compared to SM-fed piglets.
Enterotoxigenic Escherichia coli (ETEC) belongs to the Enterobacteriaceae family and is an
intestinal pathogen frequently observed after weaning causing post-weaning diarrhoea. ETEC
produces haemolysin, a virulence factor enabling haemolysis of red blood cells (41). By bacterial
culture, we observed a higher number of haemolytic bacteria in digesta from MR-fed compared to
SM- and BC-fed piglets. The higher abundance of Enterobacteriaceae observed using 16S rRNA
gene sequencing could therefore represent a higher abundance of potential intestinal pathogens as
ETEC. This is further supported by the higher diarrhoea incidence observed in MR- compared to
BC- and SM-fed piglets. The improved faecal consistency in BC-fed piglets is in accordance with
results by Huguet et al. (15) who found an improved sanitary status and faecal consistency in
weanling pigs supplemented with BC. The fact that BC-fed piglets in the present study experienced
less diarrhoea than MR-fed piglets, could be due to the effect of immunoglobulins and growth
promoting factors present in colostrum. The high content of IgG provides passive immune
protection to the newborn calf (42) and growth factors, as insulin-like growth factor, stimulate
enterocyte proliferation (9) potentially resulting in a less disturbed intestinal barrier. BC has
furthermore been reported to inhibit E. coli growth in vitro (39). De Vos et al. (30) investigated the
52 Chapter 4. Manuscript 1
13
effect of feeding three-day-old piglets a milk replacer supplemented with a BC whey fraction on the
intestinal permeability, and found an increased occludin gene expression and decreased mannitol
absorption, thus indicating an effect on the enterocyte-to-enterocyte adherence and hence gut barrier
function. Thus, BC seems to have various bioactive factors of importance to intestinal immunity
and integrity.
Despite the high content of growth factors reported in BC, we found no difference in growth
performance between BC-fed and MR-fed piglets. The present study, however, was not designed as
a performance study per se. The study by De Vos et al. (30) did not obtain any effect on growth
performance when feeding BC-supplemented milk replacer to 3-10 days old piglets. Other studies
have reported an increased growth performance when piglets were fed weaning diets supplemented
with BC compared to un-supplemented diets (15; 43).
The chemical compositions of the diets were very different and with the most noticeably
difference being the protein content. The high protein content of BC was attributed to the high
concentration of immunoglobulins (12). As branched SCFAs are indicators of protein fermentation (44; 45), the higher concentrations of iso-valeric and iso-butyric acid in faeces and digesta from BC-
fed piglets most likely reflect that these piglets were fed a high-protein diet. Protein fermentation,
however, also produces unwanted and potentially toxic compounds such as ammonia (46), amines,
phenols and indols (45). Other reports have shown an increased shedding of E. coli from pigs fed
high-protein diets (47) and a decreased ileal cytokine response upon LPS-stimulation in pigs fed a
high-protein milk formula in early life (48). In the present study, we did not see any association
between the higher protein content in the BC diet and an increased shedding of pathogenic bacteria
as E. coli. Besides a higher concentration of branched SCFAs, feeding BC also resulted in higher
faecal concentrations of the straight SCFAs acetic, propionic and butyric acid, which are considered
to be beneficial to the host (45).
Conclusion
In conclusion, sequencing and bacterial culture indicated a higher shedding of potential pathogenic
ETEC in MR-fed piglets, while the microbiota of BC-fed piglets was characterised by a change in
LAB genera when compared to SM-fed piglets. Especially the ileal (distal small intestinal)
microbiota of BC-piglets had a closer resemblance to that of SM-piglets with regard to the
abundance of potential enteric pathogens than had the MR-fed piglets. Dietary treatment had less
influence on the faecal microbial community than on the digesta community, and this should be
taken into account when studying the impact of nutrition on the microbial community in other
species (e.g. humans) as well. However, our study does not enable us to account for the long-term
effects of feeding BC. Furthermore, read abundances obtained from 16S rRNA gene amplicon
Chapter 4. Manuscript 1 53
14
sequencing should be supported by absolute quantifications. Additional studies on the potential
bioactivity of BC are needed to investigate whether the positive results on the intestinal microbiota
observed in this study are reproducible in groups of undersized weak piglets. Also, the effect of
higher branched SCFA concentrations in BC-fed piglets needs further investigation, as these
components indicate an increased protein fermentation in the gut.
Acknowledgements
The authors would like to thank Karin Durup, Mette Lykkegaard, Mette Kvist and Karin Johansen
for their skillful technical assistance during the course of the experiment.
Financial support
The Danish Council for Strategic Research funded the experiment through the NEOMUNE
consortium together with the Graduate School of Science and Technology, Aarhus University,
Denmark. Funders had no role in the design, analysis or writing of this article.
Conflict of interest
The bovine colostrum powder was partly sponsored by The European Colostrum Industry S.A. The
European Colostrum Industry S.A. had no role in the design, analysis or writing of this article.
Authorship
Authors contributed as follows: A.-S.R.P. conducted the animal experiment, performed sample and
data analyses and drafted the manuscript. S.S. conducted the animal experiment. N.C. designed the
experiment, contributed to the conduction of the animal experiment and assisted with data analysis.
N.d.J and J.L.N performed data analysis. C.L. designed the experiment. All authors have revised
and accepted the final manuscript.
(49)
54 Chapter 4. Manuscript 1
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Tables:
Table 1
Chemical compositions of sow milk (SM), milk replacer (MR) and bovine colostrum (BC).
Item Dietary group SM MR BC
Dry matter (%) 17.9* 95.0 96.1
Protein (%) 5.1* 22.1 68.2
Fat (%) 6.5* 13.2 2.0
Ash (%) 1.0† 6.8 6.0
Immunoglobulins (% of DM)‡
IgG IgA IgM
0.11 2.18 0.56
0.05 0.01 ND
38.4 3.59 2.52
Chemical analyses (drymatter, protein, fat and ash) were performed by Eurofins Steins Laboratory A/S,
Odense, Denmark.
DM = dry matter.
ND = not detected.
* Adopted from Lauridsen and Danielsen (50).
† Adopted from Aguinaga et al. (51).
‡ Immunoglobulin concentrations are adopted from Sugiharto et al. (14).
Chapter 4. Manuscript 1 57
18
Table 2
Body weight at 23 (initial body weight) and 30 days of age, milk replacer and bovine colostrum
powder intake, and diarrhoea incidence rate*
Item Dietary group† p-value SM MR BC
Body weight day 23 (kg) 8.9 (8.5-9.4) 8.1 (7.7-8.5) 8.2 (7.8-8.6) Body weight day 30 (kg) 10.2b (9.8-10.6) 9.4a (8.9-9.8) 9.4a (9.0-9.9) 0.005 Powder intake (g) ND 3980 (2168-5791) 2653 (842-4465) 0.25 Diarrhoea incidence rate‡ 0.038 0.172 0.054
ND = not determined. a,b Values with different superscripts within a row are significantly different (p<0.05).
* Values are presented as least square means and 95% confidence intervals (in parentheses).
† SM = Sow Milk; MR = Powdered porcine milk replacer; BC = Spray-dried bovine colostrum powder.
Number of piglets: SM=12; MR=11; BC=11.
‡ Defined as the ratio between the number of new cases of diarrhoea in the study period and the total number
of days the piglets have been in risk (i.e. the number of days from the animal enters the study until the
animal (a) shows clinical signs of diarrhoea, (b) dies or (c) the study ends) (49).
58 Chapter 4. Manuscript 1
19
Tab
le 3
Sh
ort-c
hain
fatty
aci
d (S
CFA
) con
cent
ratio
ns (m
mol
/kg
sam
ple)
in fa
eces
from
pig
lets
at 2
3, 2
5, 2
7 an
d 30
day
s of
age
*
D
ieta
ry g
roup
†
p-va
lue
Item
SM
M
R
BC
#
D‡
A§
DxA
Ace
tic a
cid
y
y
z
0.
0005
0.
0008
0.
06
Day
23
30.5
(2
2.8-
38.3
) 28
.0
(20.
2-35
.7)
46.3
(3
8.5-
54.0
) a
D
ay 2
5 42
.0
(34.
1-49
.8)
39.4
(3
1.5-
47.3
) 57
.7
(49.
9-65
.5)
b
Day
27
Day
30
37.6
(2
9.8-
45.4
) 35
.1
(27.
2-42
.9)
53.4
(4
5.3-
61.5
) ab
45.3
(3
7.5-
53.1
) 42
.8
(34.
8-50
.7)
61.1
(5
3.1-
69.0
) b
Pr
opio
nic
acid
y
y
z
0.00
2 <0
.000
1 0.
41
Day
23
7.4
(5.6
-9.7
) 6.
6 (5
.0-8
.7)
11.9
(9
.0-1
5.6)
a
D
ay 2
5 10
.9
(8.2
-14.
4)
9.8
(7.4
-12.
9)
17.5
(1
3.2-
23.0
) b
D
ay 2
7 13
.4
(10.
2-17
.7)
12.0
(9
.1-1
5.9)
21
.5
(16.
1-28
.7)
b
Day
30
15.4
(1
1.7-
20.3
) 13
.8
(10.
4-18
.3)
24.7
(1
8.6-
32.7
) b
B
utyr
ic a
cid
y
y
z
0.
0008
0.
002
0.11
D
ay 2
3 1.
6 (0
.8-2
.9)
0.9
(0.4
-1.7
) 3.
9 (2
.2-6
.8)
a
Day
25
3.9
(2.2
-6.8
) 2.
3 (1
.2-4
.1)
9.0
(5.2
-15.
4)
b
Day
27
3.5
(2.0
-6.1
) 2.
1 (1
.1-3
.7)
8.2
(4.6
-14.
3)
b
Day
30
4.5
(2.5
-7.7
) 2.
7 (1
.4-4
.8)
10.3
(5
.9-1
7.7)
b
A
+P+B�||
y
y
z
<0
.000
1 0.
0002
0.
09
Day
23
41.8
(2
8.4-
55.2
) 34
.8
(21.
4-48
.2)
71.3
(5
8.5-
85.4
) a
D
ay 2
5 61
.8
(48.
1-75
.4)
54.8
(4
1.1-
68.5
) 91
.9
(78.
4-10
5.5)
b
D
ay 2
7 59
.6
(46.
0-73
.1)
52.6
(3
9.0-
66.2
) 89
.7
(75.
6-10
3.8)
b
D
ay 3
0 70
.8
(57.
2-84
.4)
63.8
(4
9.9-
77.7
) 10
1.0
(87.
2-11
4.8)
b
IB
+IV
�
<.
0001
0.
002
0.00
04
Day
23
3.5
(2.2
-5.5
) 2.
0 (1
.2-5
.5)
3.7A
(2
.4-5
.7)
Day
25
3.3b
(2.1
-5.3
) 3.
5b (2
.2-5
.3)
10.8
aB
(7.0
-16.
4)
Day
27
3.8b
(2.4
-5.9
) 2.
0b (1
.2-5
.9)
11.9
aB
(7.3
-19.
4)
Day
30
5.0b
(3.2
-7.7
) 3.
0b (1
.8-7
.7)
12.9
aB
(8.3
-20.
0)
Chapter 4. Manuscript 1 59
20
a,b V
alue
s w
ith d
iffer
ent s
uper
scrip
ts w
ithin
a ro
w a
re s
igni
fican
tly d
iffer
ent.
y, z
: Col
umns
with
diff
eren
t let
ters
with
in a
SC
FA g
roup
are
sig
nific
antly
diff
eren
t. A
,B: V
alue
s w
ith d
iffer
ent s
uper
scrip
ts w
ithin
a c
olum
n ar
e si
gnifi
cant
ly d
iffer
ent.
* V
alue
s ar
e pr
esen
ted
as le
ast s
quar
e m
eans
and
95%
con
fiden
ce in
terv
al (i
n pa
rent
hese
s).
† SM
= s
ow m
ilk, M
R =
milk
repl
acer
, BC
= b
ovin
e co
lost
rum
. Num
ber o
f pig
lets
: BC
=12,
exc
ept d
ay 5
(n=9
) and
day
8 (n
=11)
; MR
=12,
exc
ept d
ay 3
(n=1
1) a
nd
day
8 (n
=10)
; SM
=12,
exc
ept d
ay 3
(n=1
0).
#: R
ows
with
diff
eren
t let
ters
with
in a
SC
FA g
roup
are
sig
nific
antly
diff
eren
t.
‡ D
= D
iet.
§ A
= A
ge.
|| A
+P+B
= a
cetic
+ p
ropi
onic
+ b
utyr
ic a
cid.
¶ IB+I
V =
iso-
buty
ric +
iso-
vale
ric a
cid.
60 Chapter 4. Manuscript 1
21
Tab
le 4
C
ount
s (lo
g cf
u/g
sam
ple)
of s
elec
ted
mic
robi
al g
roup
s in
dig
esta
from
the
gast
roin
test
inal
trac
t of 3
0 da
ys o
ld p
igle
ts (e
nd o
f exp
erim
ent)*
Die
tary
gro
up†
p-va
lue
Item
SM
M
R
BC
D
‡ S§
D
xS
Ente
roba
cter
iace
ae
0.
42
<.00
01
0.08
Stom
ach
<4.9
(1)
(3.9
-5.8
) <5
.5 (3
) (4
.5-6
.4)
<5.1
(2)
(4.1
-6.0
)
Dis
tal s
mal
l int
estin
e 7.
7 (6
.8-8
.7)
8.3
(7.4
-9.3
) 7.
9 (7
.0-8
.9)
C
aecu
m
8.1
(7.1
-9.0
) 8.
7 (7
.7-9
.6)
<8.3
(1)
(7.3
-9.2
)
Mid
col
on
8.2
(7.2
-9.1
) 8.
8 (7
.8-9
.7)
8.4
(7.4
-9.3
)
Hae
mol
ytic
bac
teri
a
0.
003
<.00
01
<0.0
001
Stom
ach
<4.0
(3)
(3.2
-4.9
) <4
.3 (2
) (3
.5-5
.2)
<4.5
(4)
(3.6
-5.3
)
Dis
tal s
mal
l int
estin
e <5
.1a (3
) (4
.2-5
.9)
8.3b
(7.4
-9.1
) <6
.8b (2
) (5
.9-7
.6)
C
aecu
m
<6.2
a (4)
(5.3
-7.0
) 8.
6b (7
.7-9
.4)
<7.4
ab (2
) (6
.6-8
.3)
M
id c
olon
<6
.0a (3
) (5
.1-6
.8)
8.6b
(7.8
-9.5
) <7
.4ab
(2)
(6.5
-8.2
)
Clo
stri
dium
per
frin
gens
ab
a
b
0.03
<.
0001
0.
15
Stom
ach
4.3
(3.7
-5.0
) 3.
3 (2
.7-3
.9)
4.6
(4.0
-5.2
)
Dis
tal s
mal
l int
estin
e 5.
2 (4
.5-5
.8)
<4.1
(4)
(3.5
-4.8
) 5.
4 (4
.8-6
.0)
C
aecu
m
5.5
(4.9
-6.1
) <4
.5 (2
) (3
.8-5
.1)
<5.7
(1)
(5.1
-6.4
)
Mid
col
on
5.4
(4.7
-6.0
) <4
.3 (2
) (3
.7-5
.0)
5.6
(5.0
-6.2
)
Lact
ic a
cid
bact
eria
0.
07
<.00
01
0.16
Stom
ach
8.5
(8.0
-9.0
) 7.
8 (7
.3-8
.3)
8.1
(7.6
-8.6
)
Dis
tal s
mal
l int
estin
e 8.
3 (7
.8-8
.8)
7.6
(7.1
-8.1
) 7.
9 (7
.4-8
.4)
C
aecu
m
Mid
col
on
8.6
(8.1
-9.1
) 7.
9 (7
.4-8
.4)
8.2
(7.7
-8.7
)
9.0
(8.5
-9.5
) 8.
3 (7
.8-8
.8)
8.6
(8.1
-9.1
)
Yea
st
0.12
0.
001
0.30
St
omac
h <4
.1 (2
) (3
.2-5
.1)
5.5
(4.5
-6.4
) <4
.4 (1
) (3
.4-5
.3)
D
ista
l sm
all i
ntes
tine
4.1
(3.2
-5.1
) 5.
5 (4
.5-6
.5)
4.4
(3.4
-5.3
)
Cae
cum
<4
.3 (1
) (3
.3-5
.2)
5.6
(4.7
-6.6
) 4.
5 (3
.6-5
.5)
M
id c
olon
4.
6 (3
.7-5
.6)
6.0
(5.1
-7.0
) 4.
9 (3
.9-5
.9)
To
tal a
naer
obic
bac
teria
0.
47
<.00
01
0.34
St
omac
h 8.
5 (8
.2-8
.9)
8.3
(7.9
-8.7
) 8.
5 (8
.1-8
.9)
D
ista
l sm
all i
ntes
tine
8.9
(8.5
-9.3
) 8.
7 (8
.3-9
.0)
8.9
(8.5
-9.3
)
Cae
cum
9.
4 (9
.0-9
.8)
9.2
(8.8
-9.6
) 9.
4 (9
.0-9
.8)
M
id c
olon
9.
8 (9
.4-1
0.2)
9.
5 (9
.1-9
.0)
9.8
(9.4
-10.
2)
Chapter 4. Manuscript 1 61
22
a,b:
Col
umns
with
diff
eren
t let
ters
with
in a
mic
robi
al g
roup
are
sig
nific
antly
diff
eren
t. a,
b Val
ues
with
diff
eren
t sup
ersc
ripts
with
in a
row
are
sig
nific
antly
diff
eren
t.
<: In
dica
tes
that
at l
east
one
of t
he o
bser
vatio
ns u
sed
to c
alcu
late
the
leas
t squ
are
mea
n w
as b
elow
det
ectio
n le
vel.
Num
bers
in b
rack
ets
indi
cate
how
man
y
sam
ples
wer
e be
low
det
ectio
n le
vels
.
* Sa
mpl
es fr
om th
e st
omac
h, d
ista
l sm
all i
ntes
tine,
cae
cum
and
mid
col
on w
ere
anal
ysed
. Val
ues
are
pres
ente
d as
leas
t squ
are
mea
ns a
nd 9
5% c
onfid
ence
inte
rval
(in
pare
nthe
ses)
.
† SM
= s
ow m
ilk, M
R =
milk
repl
acer
, BC
= b
ovin
e co
lost
rum
. Num
ber o
f pig
lets
: BC
=4; M
R=4
; SM
=4.
‡ D
= D
iet.
§ S =
Inte
stin
al s
egm
ent.
62 Chapter 4. Manuscript 1
23
Tab
le 5
O
rgan
ic a
cid
conc
entra
tions
(m
mol
/g s
ampl
e) i
n di
gest
a fr
om f
our
segm
ents
of
the
gast
roin
test
inal
tra
ct o
f 30
day
s ol
d pi
glet
s (e
nd o
f
expe
rimen
t)*
D
ieta
ry g
roup
† p-
valu
e Ite
m
SM
MR
B
C
D‡
S§
DxS
La
ctic
aci
d ||
0.
11
0.64
0.
09
Stom
ach
9.0
(4.1
-17.
5)
7.2
(3.2
-14.
5)
2.8
(0.7
-6.6
)
Dis
tal s
mal
l int
estin
e 8.
7 (4
.0-1
7-1)
7.
1 (3
.1-1
4.2)
2.
8 (0
.7-6
.5)
A
cetic
aci
d
!0.
42
<.00
01
0.46
Stom
ach
3.4
(1.6
-6.3
) 2.
7 (1
.2-5
.2)
4.1
(2.0
-7.5
)
Dis
tal s
mal
l int
estin
e 7.
1 (3
.8-1
2.5)
5.
8 (3
.0-1
0.4)
8.
3 (4
.6-1
4.6)
Cae
cum
52
.5
(31.
0-88
.5)
44.1
(2
5.7-
75.2
) 61
.0
(36.
1-10
2.7)
Mid
col
on
44.0
(2
6.0-
73.9
) 36
.9
(21.
7-62
.3)
51.2
(3
0.3-
85.8
)
Prop
ioni
c ac
id
0.10
<.
0001
0.
30
Stom
ach
2.5
(1.2
-4.8
) 1.
5 (0
.6-3
.0)
3.3
(1.7
-6.2
)
Dis
tal s
mal
l int
estin
e 0.
8 (0
.2-1
.8)
0.4
(0.0
-1.0
) 1.
2 (0
.4-2
.4)
C
aecu
m
14.6
(8
.0-2
6.3)
9.
4 (5
.0-1
7.4)
18
.7
(10,
3-33
.6)
M
id c
olon
17
.4
(9.7
-31.
1)
11.3
(6
.1-2
0.3)
22
.3
(12.
4-39
.7)
B
utyr
ic a
cid �
0.
56
0.64
0.
13
Cae
cum
6.
9 (2
.6-1
1.3)
5.
6 (1
.1-1
0.1)
8.
2 (3
.8-1
2.5)
Mid
col
on
6.8
(2.5
-11.
2)
5.5
(1.1
-9.9
) 8.
1 (3
.7-1
2.4)
A+P
+B**
0.
26
<.00
01
0.04
St
omac
h 6.
8 (3
.7-1
2.4)
4.
9 (2
.6-8
.9)
7.7
(4.2
-14.
2)
D
ista
l sm
all i
ntes
tine
8.4
(4.6
-15.
4)
6.0
(3.3
-11.
1)
9.6
(5.2
-17.
6)
C
aecu
m
76.9
(4
1.7-
141.
6)
55.0
(2
9.4-
102.
8)
87.6
(4
7.6-
161.
3)
M
id c
olon
71
.9
(39.
2-13
1.7)
51
.4
(28.
0-94
.5)
81.9
(4
4.7-
150.
0)
IB
+IV
����†
†
0.
02
0.65
0.
03
Cae
cum
5.
8a (3
.3-1
0.2)
1.
7b (0
.8-3
.2)
4.7ab
(2
.6-8
.3)
M
id c
olon
4.
1ab
(2.3
-7.3
) 2.
2a (1
.2-4
.0)
10.0
b (5
.7-1
7.5)
Chapter 4. Manuscript 1 63
24
a,b V
alue
s w
ith d
iffer
ent s
uper
scrip
ts w
ithin
a ro
w a
re s
igni
fican
tly d
iffer
ent.
* V
alue
s ar
e pr
esen
ted
as le
ast s
quar
e m
eans
and
95%
con
fiden
ce in
terv
al (i
n pa
rent
hese
s). S
ampl
es fr
om th
e st
omac
h, d
ista
l sm
all i
ntes
tine,
cae
cum
and
mid
colo
n w
ere
anal
ysed
.
† SM
= s
ow m
ilk, M
R =
milk
repl
acer
, BC
= b
ovin
e co
lost
rum
. Num
ber o
f pig
lets
: BC
=4; M
R=4
; SM
=4, e
xcep
t seg
men
t 8 (n
=2).
‡ D
= D
iet.
§ S
= In
test
inal
seg
men
t.
|| Sa
mpl
es fr
om th
e ca
ecum
and
mid
col
on h
ad v
alue
s be
low
det
ectio
n le
vel.
¶ Sa
mpl
es fr
om th
e st
omac
h an
d di
stal
sm
all i
ntes
tine
had
valu
es b
elow
det
ectio
n le
vel.
** A
+P+B
= a
cetic
+ p
ropi
onic
+ b
utyr
ic a
cid.
†† IB
+IV
= is
o-bu
tyric
+ is
o-va
leric
aci
d.
64 Chapter 4. Manuscript 1
25
Figures:
Fig. 1. Heatmaps, estimated species richness and Shannon diversity index of faecal samples
collected at 23, 25, 27, and 30 days of age from piglets fed sow milk (SM; n=15), milk replacer
(MR; n=15) or bovine colostrum (BC; n=16). Heatmaps show the relative abundances (%) of (A)
the eight most abundant phyla and (B) the 20 most abundant genera in faecal samples. Colours
represent the relative abundances. Boxplots show the (C) estimated species richness and (D)
Shannon diversity index.
Fig. 2. Principal Component Analysis of square root transformed OTU abundances in faeces (n=46)
displaying PC1 and PC2. Points are coloured for diet and grouped according to age.
Fig. 3. Heatmaps, estimated species richness and Shannon diversity index of stomach, distal small
intestinal and mid colon digesta samples from piglets fed sow milk (SM; n=23), milk replacer (MR;
n=23) or bovine colostrum (BC; n=23). Heatmaps show the relative abundances (%) of (A) the
eight most abundant phyla and (B) 20 most abundant genera. Colours represent relative abundances.
Boxplots show the (C) estimated species richness and (D) Shannon diversity index.
Fig. 4. (A) Principal Component Analysis of square root transformed OTU abundances displaying
PC1 and PC2; stomach content (n=21). Points are coloured for diet. Boxplots show the OTUs
significantly different between (B) sow milk fed and bovine colostrum fed piglets and (C) sow milk
fed and milk replacer fed piglets.
Fig. 5. (A) Principal Component Analysis of square root transformed OTU abundances displaying
PC1 and PC2; distal small intestinal content (n=24). Points are coloured for diet. Boxplots show the
OTUs significantly different between (B) sow milk fed and bovine colostrum fed piglets and (C)
sow milk fed and milk replacer fed piglets.
Fig. 6. (A) Principal Component Analysis of square root transformed OTU abundances displaying
PC1 and PC3; mid colon content (n=24). Points are coloured for diet. Boxplots show the OTUs
significantly different between (B) sow milk fed and bovine colostrum fed piglets and (C) sow milk
fed and milk replacer fed piglets.
Chapter 4. Manuscript 1 65
26
Fig. 7. Constrained redundancy analysis of square root transformed OTU abundances fitted with
microbial culture and short-chain fatty data (n=115), displaying RDA1 and RDA2. The arrows
point toward the highest values and the length of the arrow indicates the parameters significance.
Ana = total anaerobic bacteria; Clos = Clostridium perfringens; Entero = Enterobacteriaceae; Hem
= haemolytic bacteria; Lab = lactic acid bacteria. Ace = Acetic acid; Apb = Acetic + Propionic +
Butyric acid; But = Butyric acid; Pro = Propionic acid; Iso_But = Iso-butyric acid; Iso_Val = Iso-
valeric acid; Ibiv = Iso-butyric + iso-valeric acid; Val = Valeric acid.
Fig. S1. Boxplots showing the observed species richness in (A) faecal samples (n=46) collected at
23, 25, 27 and 30 days of age and (B) stomach (n=21), distal small intestinal (n=24) and mid colon
(n=24) digesta from piglets fed sow milk (SM), milk replacer (MR) or bovine colostrum (BC).
66 Chapter 4. Manuscript 1
2325
2730
0.1
1.5
1.7
0.4
0.9
1.1
66.8
26.1
0.5
32.4 20.8
0.8
0.2
0.5
61.4
0.3
1.1
40.4
1.3
1.250 0.5
4.3
0.4
41.9
0.5 055.1
0.3
0.4
0.6
0.9
26.8
65.1 1 0.1
0.5
1.3
3.1
0.3
38.4
54.1
3.6 1 1.4
0.5
0.1
45.4
0.445 5.6
1.6
0.8 00.9
46 1.6
1.7
47.3
1.4
0.4
0.1
0.5
11.6
40.9
1.6
0.7
43.4
0.3
0.1
0.4
1.3
0.4
0.6
0.441 0.1
55.5
0.6
15.1
45.6
1.1
0.1
44.7
0.3
0.5
0.3
0.1
2.2
53.3
39.1
2.1
1.6
0.7
Syne
rgis
tete
s
Actin
obac
teria
Tene
ricut
es
Spiro
chae
tes
Prot
eoba
cter
ia
Fuso
bact
eria
Bact
eroi
dete
s
Firm
icut
es
23 colostrum
23 milk replacer
23 sow milk
25 colostrum
25 milk replacer
25 sow milk
27 colostrum
27 milk replacer
27 sow milk
30 colostrum
30 milk replacer
30 sow milk
0.1
1.0
10.0
% R
ead
Abu
ndan
ce
Day
23
Day
25
Day
27
Day
30
BC
M
R
S
M
BC
M
R
S
M
BC
M
R
S
M
BC
M
R
S
M
2325
2730
1.2 55.4
0.4
0.2
9.5
1.4
1.2
0.4
3.9
1.7
4.3
2.3 31.1
1.8
15.4
0.1
0.3
0.6
0.9
0.1
0.5
5.7
1.5
9.7
1.2
1.6
6.1
1.33 1.7 3 0.2
0.67 20.5
10.7
1.3
1.3 0 1.8
3.3
0.3
0.8
1.2
2.1
4.6
1.4
0.684.9
1.19 4.3
8.8
14.3
0.2
1.4
2.8
1.1
0.4
1.9
0.8
0.4
3.8
1.4
0.1
2.3
17.7
0.3
1.7
15.6
2.6
12.2
0.3
1.8 40.4
0.5
2.2
1.4
3.2
4.1
0.1
3.8
0.8
0.7
7.4
1.5
2.2
1.6
2.6
0.9
2.3
0.5
2.2
5.3
0.1
0.4
2.2
2.6
0.901.7
3.1
6.5
2.5 2 0.4
13.9
1.1
4.9
3.6
0.5
1.513.6
6.8
0.6
4.3
7.5
0.81 0.6
1.3
1.2
1.5
0.4
1.1
4.5
0.4
2.7
6.2
2.6
15.2
5.6
0.2
10.7
1.2
0.4
4.7
8.5 10 2.3
0.2
1.5
1.3
0.9
0.5
0.2
0.1
17.8
1.6
1.2
2.7
0.3
3.8
0.7
5.2
0.6
0.8
0.9
1.1
5.6 12.2
0.8
24.6
0.501.6
1.7
6.3
0.5
0.8
11.6
2.4
0.29 0.1
0.5
6.5
1.7
9.8
5.5
0.6
0.7
0.7
18.7
0.7
0.3
8.7
0.3
2.1
1.9
4.5
0.4
0.3
0.9
3.6 026.1
4.2
0.7
1.64 00.1
2.2
1.3
2.9
0.1
1.3
0.5
17.9
0.1
02 0.1 03.3
2.5
1.864.8
8.4
1.2
1.6
7.6
2.3
1.7
0.4 01.2
18.5
1.6
Firm
icute
s; f_
_Rum
inoc
occa
ceae
_OTU
_20
Bact
eroi
dete
s; f_
_BS1
1_OT
U_12
4Fi
rmicu
tes;
Meg
asph
aera
Bact
eroi
dete
s; P
arab
acte
roid
esFi
rmicu
tes;
f__R
umin
ococ
cace
ae_O
TU_1
605
Firm
icute
s; f_
_Rum
inoc
occa
ceae
_OTU
_29
Firm
icute
s; f_
_Rum
inoc
occa
ceae
_OTU
_21
Bact
eroi
dete
s; f_
_p−2
534−
18B5
_OTU
_52
Bact
eroi
dete
s; f_
_S24−7
_OTU
_100
Bact
eroi
dete
s; o
__Ba
cter
oida
les_
OTU_
4Ba
cter
oide
tes;
f__S
24−7
_OTU
_27
Bact
eroi
dete
s; B
acte
roid
esFi
rmicu
tes;
Rum
inoc
occu
sFu
soba
cter
ia; F
usob
acte
rium
Bact
eroi
dete
s; [P
revo
tella
]Fi
rmicu
tes;
Lac
toba
cillu
sBa
cter
oide
tes;
f__[
Para
prev
otel
lace
ae]_
OTU_
19Fi
rmicu
tes;
p−7
5−a5
Firm
icute
s; O
scillo
spira
Bact
eroi
dete
s; P
revo
tella
23 colostrum
23 milk replacer
23 sow milk
25 colostrum
25 milk replacer
25 sow milk
27 colostrum
27 milk replacer
27 sow milk
30 colostrum
30 milk replacer
30 sow milk
0.1
1.0
10.0
% R
ead
Abu
ndan
ce
Day
23
Day
25
Day
27
Day
30
Bact
eroi
dete
s; fa
mily
[Par
apre
vote
llace
ae] O
TU 1
9
Bact
eroi
dete
s; fa
mily
S24
-7 O
TU 2
7
Bact
eroi
dete
s; o
rder
Bac
tero
idal
es O
TU 4
Bact
eroi
dete
s; fa
mily
S24
-7 O
TU 1
00
Bact
eroi
dete
s; fa
mily
p-2
534-
16B5
OTU
52
Firm
icut
es; f
amily
Rum
inoc
occa
ceae
OTU
21
Firm
icut
es; f
amily
Rum
inoc
occa
ceae
OTU
29
Firm
icut
es; f
amily
Rum
inoc
occa
ceae
OTU
160
5
Bact
eroi
dete
s; fa
mily
BS1
1 O
TU 1
24
Firm
icut
es; f
amily
Rum
inoc
occa
ceae
OTU
20
BC
MR
S
M
BC
MR
S
M
BC
MR
S
M
BC
MR
S
M
2325
2730
250
375
500
625
750
875
1000
colos
trum
milk re
place
rso
w milk
colos
trum
milk re
place
rso
w milk
colos
trum
milk re
place
rso
w milk
colos
trum
milk re
place
rso
w milk
Diet
Estimated number of OTUs Estimated number of OTUs
Day
23
Day
25
Day
27
Day
30
BC
MR
S
M
BC
MR
S
M D
iet
BC
MR
S
M
BC
MR
S
M
2325
2730
2.00
2.25
2.50
2.75
3.00
3.25
3.50
3.75
4.00
4.25
4.50
4.75
5.00
5.25
colos
trum
milk
repla
cer
sow
milk
colos
trum
milk
repla
cer
sow
milk
colos
trum
milk
repla
cer
sow
milk
colos
trum
milk
repla
cer
sow
milk
Die
t
Diversity index Diversity index
Day
23
Day
25
Day
27
Day
30
BC
MR
S
M
BC
MR
S
M
BC
MR
S
M
BC
MR
S
M
Die
t
(A)
(B)
(C)
(D)
Figu
re 1
Chapter 4. Manuscript 1 67
23 25 27 30
−5.0
−2.5
0.0
2.5
5.0
−5.0 −2.5 0.0 2.5 −5.0 −2.5 0.0 2.5 −5.0 −2.5 0.0 2.5 −5.0 −2.5 0.0 2.5PC1 [14.8%]
PC2
[13.
3%] Diet
colostrum
milk replacer
sow milk
PC
2 - v
aria
tion
expl
aine
d 13
.3%
PC1 - variation explained 14.8%
Day 23 Day 25 Day 27 Day 30
23 25 27 30
−5.0
−2.5
0.0
2.5
5.0
−5.0 −2.5 0.0 2.5 −5.0 −2.5 0.0 2.5 −5.0 −2.5 0.0 2.5 −5.0 −2.5 0.0 2.5PC1 [14.8%]
PC2
[13.
3%] Diet
colostrum
milk replacer
sow milk
23 25 27 30
−5.0
−2.5
0.0
2.5
5.0
−5.0 −2.5 0.0 2.5 −5.0 −2.5 0.0 2.5 −5.0 −2.5 0.0 2.5 −5.0 −2.5 0.0 2.5PC1 [14.8%]
PC2
[13.
3%] Diet
colostrum
milk replacer
sow milk
PC
2 - v
aria
tion
expl
aine
d 13
.3%
PC1 - variation explained 14.8%
Day 23 Day 25 Day 27 Day 30
23 25 27 30
−5.0
−2.5
0.0
2.5
5.0
−5.0 −2.5 0.0 2.5 −5.0 −2.5 0.0 2.5 −5.0 −2.5 0.0 2.5 −5.0 −2.5 0.0 2.5PC1 [14.8%]
PC2
[13.
3%] Diet
colostrum
milk replacer
sow milk
Figure 2
68 Chapter 4. Manuscript 1
Dis
tal S
mal
l Int
estin
eM
id C
olon
Stom
ach
10.6
1.8 0069.9
16.7
0.6 0
046 0.2 0 051.5
1.1
0.9
03.2 000.1
92.9
1.2
2.1
2 1.2
1.4
1.2
1.1
1.2
55.8
35.6
0.7 13.9
57.6
7.5
0.4
27.6
0.3
0.4
0.6
1.6
0.9
61.9 132.1
0.8
005.4 01.1
20.1
68.3
3.9
0 0.1
5.7
2.7
0.6 082.6
7.3
0.2 077.7
3.4
1.5
16.7 0 0
Spiro
chae
tes
Tene
ricut
es
Plan
ctom
ycet
es
Fuso
bact
eria
Actin
obac
teria
Prot
eoba
cter
ia
Bact
eroi
dete
s
Firm
icut
es
colostrum Distal Small Intestine
milk replacer Distal Small Intestine
sow milk Distal Small Intestine
colostrum Mid Colon
milk replacer Mid Colon
sow milk Mid Colon
colostrum Stomach
milk replacer Stomach
sow milk Stomach
0.1
1.0
10.0
% R
ead
Abu
ndan
ce
Dis
tal S
mal
l Int
estin
eM
id C
olon
Stom
ach
10.6
1.8 0069.9
16.7
0.6 0
046 0.2 0 051.5
1.1
0.9
03.2 000.1
92.9
1.2
2.1
2 1.2
1.4
1.2
1.1
1.2
55.8
35.6
0.7 13.9
57.6
7.5
0.4
27.6
0.3
0.4
0.6
1.6
0.9
61.9 132.1
0.8
005.4 01.1
20.1
68.3
3.9
0 0.1
5.7
2.7
0.6 082.6
7.3
0.2 077.7
3.4
1.5
16.7 0 0
Spiro
chae
tes
Tene
ricut
es
Plan
ctom
ycet
es
Fuso
bact
eria
Actin
obac
teria
Prot
eoba
cter
ia
Bact
eroi
dete
s
Firm
icut
es
colostrum Distal Small Intestine
milk replacer Distal Small Intestine
sow milk Distal Small Intestine
colostrum Mid Colon
milk replacer Mid Colon
sow milk Mid Colon
colostrum Stomach
milk replacer Stomach
sow milk Stomach
0.1
1.0
10.0
% R
ead
Abu
ndan
ce
Dis
tal S
mal
l Int
estin
eM
id C
olon
Stom
ach
10.6
1.8 0069.9
16.7
0.6 0
046 0.2 0 051.5
1.1
0.9
03.2 000.1
92.9
1.2
2.1
2 1.2
1.4
1.2
1.1
1.2
55.8
35.6
0.7 13.9
57.6
7.5
0.4
27.6
0.3
0.4
0.6
1.6
0.9
61.9 132.1
0.8
005.4 01.1
20.1
68.3
3.9
0 0.1
5.7
2.7
0.6 082.6
7.3
0.2 077.7
3.4
1.5
16.7 0 0
Spiro
chae
tes
Tene
ricut
es
Plan
ctom
ycet
es
Fuso
bact
eria
Actin
obac
teria
Prot
eoba
cter
ia
Bact
eroi
dete
s
Firm
icut
es
colostrum Distal Small Intestine
milk replacer Distal Small Intestine
sow milk Distal Small Intestine
colostrum Mid Colon
milk replacer Mid Colon
sow milk Mid Colon
colostrum Stomach
milk replacer Stomach
sow milk Stomach
0.1
1.0
10.0
% R
ead
Abu
ndan
ce
Dis
tal S
mal
l Int
estin
eM
id C
olon
Stom
ach
10.6
1.8 0069.9
16.7
0.6 0
046 0.2 0 051.5
1.1
0.9
03.2 000.1
92.9
1.2
2.1
2 1.2
1.4
1.2
1.1
1.2
55.8
35.6
0.7 13.9
57.6
7.5
0.4
27.6
0.3
0.4
0.6
1.6
0.9
61.9 132.1
0.8
005.4 01.1
20.1
68.3
3.9
0 0.1
5.7
2.7
0.6 082.6
7.3
0.2 077.7
3.4
1.5
16.7 0 0
Spiro
chae
tes
Tene
ricut
es
Plan
ctom
ycet
es
Fuso
bact
eria
Actin
obac
teria
Prot
eoba
cter
ia
Bact
eroi
dete
s
Firm
icut
es
colostrum Distal Small Intestine
milk replacer Distal Small Intestine
sow milk Distal Small Intestine
colostrum Mid Colon
milk replacer Mid Colon
sow milk Mid Colon
colostrum Stomach
milk replacer Stomach
sow milk Stomach
0.1
1.0
10.0
% R
ead
Abu
ndan
ce
Sto
mac
hD
ista
l sm
all i
ntes
tine
Mid
col
on
BC
MR
SM
B
C
M
R
S
M
BC
MR
SM
Dist
al S
mal
l Int
estin
eM
id C
olon
Stom
ach
66.2
5.4
4.8
1.23 08.3 0 07.4
8.6
9.2
0.1
3.700.3
0.1
13.4 0
09.6
34.8
0.2
0.1
0.7
1.9
4.8 00.2
5.1
1.3
0.1 325.5
0.8
1.9 0 00
0.3
0.4
1.2
0.5011.6
0.8 02.8
4.401.6
0.164 1.9 00 0 0.20
0.1
0.1 0 40 13.4
1.5 01.4
8.8
0.42 0 0.1
2.4 00.7
3.8
1.3
11.9
4.8
3.7
0.4
1.4
8.1
0.2 0 2.1 00.4 00.6
0.1
5.2
1.3
0.7
0.9 00.1
4.3
0.1
0.1
2.8
5.8 400.1
1.9
1.7 0014.1
2.1
1.5
16.6
0.2 00.1 54.4
6.2
0.2
1.6
2.3
0.1
0.4
0.2
0.2
16.4
14.8 011 0.1016.5
0.7
0.9
0.2
1.6
0.8
11.3
0.1
2.9
5.4
0.8
0.1
0.21 00.5
1.3
3.2
0.2
3.5
1.6
0.8
37.5
0.1
1.4
0.5
0.701.4
0.4 0 0 0015.8
9.3 053.4
0.3
0.7
2.80 00.3
3.2
0.1
Firm
icute
s; B
acillu
sAc
tinob
acte
ria; f
__Co
rioba
cter
iace
ae_O
TU_1
0Ba
cter
oide
tes;
Bac
tero
ides
Firm
icute
s; R
umin
ococ
cus
Firm
icute
s; S
arcin
aFi
rmicu
tes;
Stre
ptoc
occu
sBa
cter
oide
tes;
[Pre
vote
lla]
Bact
eroi
dete
s; o
__Ba
cter
oida
les_
OTU
_4Fi
rmicu
tes;
02d
06Pr
oteo
bact
eria
; Acin
etob
acte
rFi
rmicu
tes;
Osc
illosp
iraFi
rmicu
tes;
Lac
toco
ccus
Firm
icute
s; p−7
5−a5
Firm
icute
s; S
MB5
3Fi
rmicu
tes;
Vei
llone
llaFi
rmicu
tes;
Meg
asph
aera
Firm
icute
s; M
itsuo
kella
Prot
eoba
cter
ia; f
__En
tero
bact
eria
ceae
_OTU
_6Ba
cter
oide
tes;
Pre
vote
llaFi
rmicu
tes;
Lac
toba
cillu
s
colostrum Distal Small Intestine
milk replacer Distal Small Intestine
sow milk Distal Small Intestine
colostrum Mid Colon
milk replacer Mid Colon
sow milk Mid Colon
colostrum Stomach
milk replacer Stomach
sow milk Stomach
0.1
1.0
10.0
% R
ead
Abu
ndan
ce
Dist
al S
mal
l Int
estin
eM
id C
olon
Stom
ach
66.2
5.4
4.8
1.23 08.3 0 07.4
8.6
9.2
0.1
3.700.3
0.1
13.4 0
09.6
34.8
0.2
0.1
0.7
1.9
4.8 00.2
5.1
1.3
0.1 325.5
0.8
1.9 0 00
0.3
0.4
1.2
0.5011.6
0.8 02.8
4.401.6
0.164 1.9 00 0 0.20
0.1
0.1 0 40 13.4
1.5 01.4
8.8
0.42 0 0.1
2.4 00.7
3.8
1.3
11.9
4.8
3.7
0.4
1.4
8.1
0.2 0 2.1 00.4 00.6
0.1
5.2
1.3
0.7
0.9 00.1
4.3
0.1
0.1
2.8
5.8 400.1
1.9
1.7 0014.1
2.1
1.5
16.6
0.2 00.1 54.4
6.2
0.2
1.6
2.3
0.1
0.4
0.2
0.2
16.4
14.8 011 0.1016.5
0.7
0.9
0.2
1.6
0.8
11.3
0.1
2.9
5.4
0.8
0.1
0.21 00.5
1.3
3.2
0.2
3.5
1.6
0.8
37.5
0.1
1.4
0.5
0.701.4
0.4 0 0 0015.8
9.3 053.4
0.3
0.7
2.80 00.3
3.2
0.1
Firm
icute
s; B
acillu
sAc
tinob
acte
ria; f
__Co
rioba
cter
iace
ae_O
TU_1
0Ba
cter
oide
tes;
Bac
tero
ides
Firm
icute
s; R
umin
ococ
cus
Firm
icute
s; S
arcin
aFi
rmicu
tes;
Stre
ptoc
occu
sBa
cter
oide
tes;
[Pre
vote
lla]
Bact
eroi
dete
s; o
__Ba
cter
oida
les_
OTU
_4Fi
rmicu
tes;
02d
06Pr
oteo
bact
eria
; Acin
etob
acte
rFi
rmicu
tes;
Osc
illosp
iraFi
rmicu
tes;
Lac
toco
ccus
Firm
icute
s; p−7
5−a5
Firm
icute
s; S
MB5
3Fi
rmicu
tes;
Vei
llone
llaFi
rmicu
tes;
Meg
asph
aera
Firm
icute
s; M
itsuo
kella
Prot
eoba
cter
ia; f
__En
tero
bact
eria
ceae
_OTU
_6Ba
cter
oide
tes;
Pre
vote
llaFi
rmicu
tes;
Lac
toba
cillu
s
colostrum Distal Small Intestine
milk replacer Distal Small Intestine
sow milk Distal Small Intestine
colostrum Mid Colon
milk replacer Mid Colon
sow milk Mid Colon
colostrum Stomach
milk replacer Stomach
sow milk Stomach
0.1
1.0
10.0
% R
ead
Abu
ndan
ce
Dist
al S
mal
l Int
estin
eM
id C
olon
Stom
ach
66.2
5.4
4.8
1.23 08.3 0 07.4
8.6
9.2
0.1
3.700.3
0.1
13.4 0
09.6
34.8
0.2
0.1
0.7
1.9
4.8 00.2
5.1
1.3
0.1 325.5
0.8
1.9 0 00
0.3
0.4
1.2
0.5011.6
0.8 02.8
4.401.6
0.164 1.9 00 0 0.20
0.1
0.1 0 40 13.4
1.5 01.4
8.8
0.42 0 0.1
2.4 00.7
3.8
1.3
11.9
4.8
3.7
0.4
1.4
8.1
0.2 0 2.1 00.4 00.6
0.1
5.2
1.3
0.7
0.9 00.1
4.3
0.1
0.1
2.8
5.8 400.1
1.9
1.7 0014.1
2.1
1.5
16.6
0.2 00.1 54.4
6.2
0.2
1.6
2.3
0.1
0.4
0.2
0.2
16.4
14.8 011 0.1016.5
0.7
0.9
0.2
1.6
0.8
11.3
0.1
2.9
5.4
0.8
0.1
0.21 00.5
1.3
3.2
0.2
3.5
1.6
0.8
37.5
0.1
1.4
0.5
0.701.4
0.4 0 0 0015.8
9.3 053.4
0.3
0.7
2.80 00.3
3.2
0.1
Firm
icute
s; B
acillu
sAc
tinob
acte
ria; f
__Co
rioba
cter
iace
ae_O
TU_1
0Ba
cter
oide
tes;
Bac
tero
ides
Firm
icute
s; R
umin
ococ
cus
Firm
icute
s; S
arcin
aFi
rmicu
tes;
Stre
ptoc
occu
sBa
cter
oide
tes;
[Pre
vote
lla]
Bact
eroi
dete
s; o
__Ba
cter
oida
les_
OTU
_4Fi
rmicu
tes;
02d
06Pr
oteo
bact
eria
; Acin
etob
acte
rFi
rmicu
tes;
Osc
illosp
iraFi
rmicu
tes;
Lac
toco
ccus
Firm
icute
s; p−7
5−a5
Firm
icute
s; S
MB5
3Fi
rmicu
tes;
Vei
llone
llaFi
rmicu
tes;
Meg
asph
aera
Firm
icute
s; M
itsuo
kella
Prot
eoba
cter
ia; f
__En
tero
bact
eria
ceae
_OTU
_6Ba
cter
oide
tes;
Pre
vote
llaFi
rmicu
tes;
Lac
toba
cillu
s
colostrum Distal Small Intestine
milk replacer Distal Small Intestine
sow milk Distal Small Intestine
colostrum Mid Colon
milk replacer Mid Colon
sow milk Mid Colon
colostrum Stomach
milk replacer Stomach
sow milk Stomach
0.1
1.0
10.0
% R
ead
Abu
ndan
ce
Dist
al S
mal
l Int
estin
eM
id C
olon
Stom
ach
66.2
5.4
4.8
1.23 08.3 0 07.4
8.6
9.2
0.1
3.700.3
0.1
13.4 0
09.6
34.8
0.2
0.1
0.7
1.9
4.8 00.2
5.1
1.3
0.1 325.5
0.8
1.9 0 00
0.3
0.4
1.2
0.5011.6
0.8 02.8
4.401.6
0.164 1.9 00 0 0.20
0.1
0.1 0 40 13.4
1.5 01.4
8.8
0.42 0 0.1
2.4 00.7
3.8
1.3
11.9
4.8
3.7
0.4
1.4
8.1
0.2 0 2.1 00.4 00.6
0.1
5.2
1.3
0.7
0.9 00.1
4.3
0.1
0.1
2.8
5.8 400.1
1.9
1.7 0014.1
2.1
1.5
16.6
0.2 00.1 54.4
6.2
0.2
1.6
2.3
0.1
0.4
0.2
0.2
16.4
14.8 011 0.1016.5
0.7
0.9
0.2
1.6
0.8
11.3
0.1
2.9
5.4
0.8
0.1
0.21 00.5
1.3
3.2
0.2
3.5
1.6
0.8
37.5
0.1
1.4
0.5
0.701.4
0.4 0 0 0015.8
9.3 053.4
0.3
0.7
2.80 00.3
3.2
0.1
Firm
icute
s; B
acillu
sAc
tinob
acte
ria; f
__Co
rioba
cter
iace
ae_O
TU_1
0Ba
cter
oide
tes;
Bac
tero
ides
Firm
icute
s; R
umin
ococ
cus
Firm
icute
s; S
arcin
aFi
rmicu
tes;
Stre
ptoc
occu
sBa
cter
oide
tes;
[Pre
vote
lla]
Bact
eroi
dete
s; o
__Ba
cter
oida
les_
OTU
_4Fi
rmicu
tes;
02d
06Pr
oteo
bact
eria
; Acin
etob
acte
rFi
rmicu
tes;
Osc
illosp
iraFi
rmicu
tes;
Lac
toco
ccus
Firm
icute
s; p−7
5−a5
Firm
icute
s; S
MB5
3Fi
rmicu
tes;
Vei
llone
llaFi
rmicu
tes;
Meg
asph
aera
Firm
icute
s; M
itsuo
kella
Prot
eoba
cter
ia; f
__En
tero
bact
eria
ceae
_OTU
_6Ba
cter
oide
tes;
Pre
vote
llaFi
rmicu
tes;
Lac
toba
cillu
s
colostrum Distal Small Intestine
milk replacer Distal Small Intestine
sow milk Distal Small Intestine
colostrum Mid Colon
milk replacer Mid Colon
sow milk Mid Colon
colostrum Stomach
milk replacer Stomach
sow milk Stomach
0.1
1.0
10.0
% R
ead
Abu
ndan
ce
BC
MR
SM
B
C
M
R
S
M
BC
MR
SM
Act
inob
acte
ria; f
amily
Cor
ioba
cter
iace
ae O
TU 1
0
Bac
tero
idet
es; o
rder
Bac
tero
idal
es O
TU 4
Pro
teob
acte
ria; f
amily
Ent
erob
acte
riace
ae O
TU 6
Sto
mac
hD
ista
l sm
all i
ntes
tine
Mid
col
on
Stom
ach
Dist
al S
mal
l Int
estin
eM
id C
olon
125
250
375
500
625
750
875
1000
1125
colos
trum
milk re
place
rso
w milk
colos
trum
milk re
place
rso
w milk
colos
trum
milk re
place
rso
w milk
Diet
Estimated number of OTUs Estimated number of OTUs
BC
MR
S
M
BC
MR
S
M
Die
t
BC
MR
S
M
Stom
ach
Dis
tal s
mal
l int
estin
e M
id c
olon
Stom
ach
Dis
tal S
mal
l Int
estin
eM
id C
olon
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
3.50
3.75
4.00
4.25
4.50
4.75
5.00
colos
trum
milk
repla
cer
sow
milk
colos
trum
milk
repla
cer
sow
milk
colos
trum
milk
repla
cer
sow
milk
Die
t
Diversity index Diversity index
BC
M
R
SM
B
C
MR
S
M
Die
t
BC
M
R
SM
Sto
mac
hD
ista
l sm
all i
ntes
tine
Mid
col
on
(A)
(B)
(C)
(D)
Figu
re 3
Chapter 4. Manuscript 1 69
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Megasphaera; OTU_1106
Firmicutes; f__[Mogibacteriaceae]_OTU_103; OTU_103
Firmicutes; Lactobacillus; OTU_58
Firmicutes; Lactobacillus; OTU_1926
Firmicutes; Lactobacillus; OTU_107
Firmicutes; Lactococcus; OTU_31
Bacteroidetes; Prevotella; OTU_152
Firmicutes; Lactobacillus; OTU_66
0.01 0.10 1.00 10.00Read Abundance (%)
Dietmilk replacer
sow milk
Stomach
Read abundance (%)
Firmicutes; family [Mogibacteriaceae] OTU_103
Firmicutes; Oscillospira; OTU_3
Actinobacteria; Collinsella; OTU_79
Firmicutes; Lactococcus; OTU_31
Firmicutes; Mitsuokella; OTU_1282
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Lactococcus; OTU_72
0.01 0.10 1.00 10.00Read Abundance (%)
Dietcolostrum
sow milk
Stomach
Read abundance (%)
−3
0
3
6
−5.0 −2.5 0.0 2.5 5.0PC1 [26.4%]
PC2
[17.
2%] Diet
colostrum
milk replacer
sow milk
StomachStomach
PC
2 - v
aria
tion
expl
aine
d 17
.2%
PC1 - variation explained 26.4%
(A)
(B)
(C)
Figure 4
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Megasphaera; OTU_1106
Firmicutes; f__[Mogibacteriaceae]_OTU_103; OTU_103
Firmicutes; Lactobacillus; OTU_58
Firmicutes; Lactobacillus; OTU_1926
Firmicutes; Lactobacillus; OTU_107
Firmicutes; Lactococcus; OTU_31
Bacteroidetes; Prevotella; OTU_152
Firmicutes; Lactobacillus; OTU_66
0.01 0.10 1.00 10.00Read Abundance (%)
Dietmilk replacer
sow milk
Stomach
Read abundance (%)
Firmicutes; family [Mogibacteriaceae] OTU_103
Firmicutes; Oscillospira; OTU_3
Actinobacteria; Collinsella; OTU_79
Firmicutes; Lactococcus; OTU_31
Firmicutes; Mitsuokella; OTU_1282
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Lactococcus; OTU_72
0.01 0.10 1.00 10.00Read Abundance (%)
Dietcolostrum
sow milk
Stomach
Read abundance (%)
−3
0
3
6
−5.0 −2.5 0.0 2.5 5.0PC1 [26.4%]
PC2
[17.
2%] Diet
colostrum
milk replacer
sow milk
StomachStomach
PC
2 - v
aria
tion
expl
aine
d 17
.2%
PC1 - variation explained 26.4%
(A)
(B)
(C)
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Megasphaera; OTU_1106
Firmicutes; f__[Mogibacteriaceae]_OTU_103; OTU_103
Firmicutes; Lactobacillus; OTU_58
Firmicutes; Lactobacillus; OTU_1926
Firmicutes; Lactobacillus; OTU_107
Firmicutes; Lactococcus; OTU_31
Bacteroidetes; Prevotella; OTU_152
Firmicutes; Lactobacillus; OTU_66
0.01 0.10 1.00 10.00Read Abundance (%)
Dietmilk replacer
sow milk
Stomach
Read abundance (%)
Firmicutes; family [Mogibacteriaceae] OTU_103
Firmicutes; Oscillospira; OTU_3
Actinobacteria; Collinsella; OTU_79
Firmicutes; Lactococcus; OTU_31
Firmicutes; Mitsuokella; OTU_1282
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Lactococcus; OTU_72
0.01 0.10 1.00 10.00Read Abundance (%)
Dietcolostrum
sow milk
Stomach
Read abundance (%)
−3
0
3
6
−5.0 −2.5 0.0 2.5 5.0PC1 [26.4%]
PC2
[17.
2%] Diet
colostrum
milk replacer
sow milk
StomachStomach
PC
2 - v
aria
tion
expl
aine
d 17
.2%
PC1 - variation explained 26.4%
(A)
(B)
(C)
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Megasphaera; OTU_1106
Firmicutes; f__[Mogibacteriaceae]_OTU_103; OTU_103
Firmicutes; Lactobacillus; OTU_58
Firmicutes; Lactobacillus; OTU_1926
Firmicutes; Lactobacillus; OTU_107
Firmicutes; Lactococcus; OTU_31
Bacteroidetes; Prevotella; OTU_152
Firmicutes; Lactobacillus; OTU_66
0.01 0.10 1.00 10.00Read Abundance (%)
Dietmilk replacer
sow milk
Stomach
Read abundance (%)
Firmicutes; family [Mogibacteriaceae] OTU_103
Firmicutes; Oscillospira; OTU_3
Actinobacteria; Collinsella; OTU_79
Firmicutes; Lactococcus; OTU_31
Firmicutes; Mitsuokella; OTU_1282
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Lactococcus; OTU_72
0.01 0.10 1.00 10.00Read Abundance (%)
Dietcolostrum
sow milk
Stomach
Read abundance (%)
−3
0
3
6
−5.0 −2.5 0.0 2.5 5.0PC1 [26.4%]
PC2
[17.
2%] Diet
colostrum
milk replacer
sow milk
StomachStomach
PC
2 - v
aria
tion
expl
aine
d 17
.2%
PC1 - variation explained 26.4%
(A)
(B)
(C)
70 Chapter 4. Manuscript 1
Firmicutes; Dorea; OTU_207
Firmicutes; Lactobacillus; OTU_107
Firmicutes; Weissella; OTU_880
Firmicutes; Lactobacillus; OTU_66
Firmicutes; Geobacillus; OTU_220
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Streptococcus; OTU_163
Firmicutes; Lactococcus; OTU_31
Firmicutes; Lactobacillus; OTU_58
Firmicutes; Anoxybacillus; OTU_83
Proteobacteria; f__Enterobacteriaceae_OTU_6; OTU_6
0.01 0.10 1.00 10.00Read Abundance (%)
Diet
milk replacer
sow milk
Distal small intestine
Read abundance (%)
Proteobacteria; family Enterobacteriaceae OTU_6
Firmicutes; Bulleidia; OTU_505
Firmicutes; Leuconostoc; OTU_749
Firmicutes; Lactobacillus; OTU_1
Firmicutes; Anoxybacillus; OTU_83
Firmicutes; Carnobacterium; OTU_670
Firmicutes; Streptococcus; OTU_703
Firmicutes; Lactococcus; OTU_387
Firmicutes; Lactococcus; OTU_31
Firmicutes; Lactococcus; OTU_208
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Lactococcus; OTU_72
0.01 0.10 1.00 10.00Read Abundance (%)
Diet
colostrum
sow milk
Distal small intestine
Read abundance (%)
−5.0
−2.5
0.0
2.5
−5.0 −2.5 0.0 2.5 5.0PC1 [26%]
PC2
[15.
8%] Diet
colostrum
milk replacer
sow milk
Distal small intestine
PC
2 - v
aria
tion
expl
aine
d 15
.8%
PC1 - variation explained 26.0%
Distal small intestine(A)
(B)
(C)
Enterobacteriaceae Lactobacillus
Lactococcus
Figure 5
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Megasphaera; OTU_1106
Firmicutes; f__[Mogibacteriaceae]_OTU_103; OTU_103
Firmicutes; Lactobacillus; OTU_58
Firmicutes; Lactobacillus; OTU_1926
Firmicutes; Lactobacillus; OTU_107
Firmicutes; Lactococcus; OTU_31
Bacteroidetes; Prevotella; OTU_152
Firmicutes; Lactobacillus; OTU_66
0.01 0.10 1.00 10.00Read Abundance (%)
Dietmilk replacer
sow milk
Stomach
Read abundance (%)
Firmicutes; family [Mogibacteriaceae] OTU_103
Firmicutes; Oscillospira; OTU_3
Actinobacteria; Collinsella; OTU_79
Firmicutes; Lactococcus; OTU_31
Firmicutes; Mitsuokella; OTU_1282
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Lactococcus; OTU_72
0.01 0.10 1.00 10.00Read Abundance (%)
Dietcolostrum
sow milk
Stomach
Read abundance (%)
−3
0
3
6
−5.0 −2.5 0.0 2.5 5.0PC1 [26.4%]
PC2
[17.
2%] Diet
colostrum
milk replacer
sow milk
StomachStomach
PC
2 - v
aria
tion
expl
aine
d 17
.2%
PC1 - variation explained 26.4%
(A)
(B)
(C)Firmicutes; Leuconostoc; OTU_209
Firmicutes; Megasphaera; OTU_1106
Firmicutes; f__[Mogibacteriaceae]_OTU_103; OTU_103
Firmicutes; Lactobacillus; OTU_58
Firmicutes; Lactobacillus; OTU_1926
Firmicutes; Lactobacillus; OTU_107
Firmicutes; Lactococcus; OTU_31
Bacteroidetes; Prevotella; OTU_152
Firmicutes; Lactobacillus; OTU_66
0.01 0.10 1.00 10.00Read Abundance (%)
Dietmilk replacer
sow milk
Stomach
Read abundance (%)
Firmicutes; family [Mogibacteriaceae] OTU_103
Firmicutes; Oscillospira; OTU_3
Actinobacteria; Collinsella; OTU_79
Firmicutes; Lactococcus; OTU_31
Firmicutes; Mitsuokella; OTU_1282
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Lactococcus; OTU_72
0.01 0.10 1.00 10.00Read Abundance (%)
Dietcolostrum
sow milk
Stomach
Read abundance (%)
−3
0
3
6
−5.0 −2.5 0.0 2.5 5.0PC1 [26.4%]
PC2
[17.
2%] Diet
colostrum
milk replacer
sow milk
StomachStomach
PC
2 - v
aria
tion
expl
aine
d 17
.2%
PC1 - variation explained 26.4%
(A)
(B)
(C)
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Megasphaera; OTU_1106
Firmicutes; f__[Mogibacteriaceae]_OTU_103; OTU_103
Firmicutes; Lactobacillus; OTU_58
Firmicutes; Lactobacillus; OTU_1926
Firmicutes; Lactobacillus; OTU_107
Firmicutes; Lactococcus; OTU_31
Bacteroidetes; Prevotella; OTU_152
Firmicutes; Lactobacillus; OTU_66
0.01 0.10 1.00 10.00Read Abundance (%)
Dietmilk replacer
sow milk
Stomach
Read abundance (%)
Firmicutes; family [Mogibacteriaceae] OTU_103
Firmicutes; Oscillospira; OTU_3
Actinobacteria; Collinsella; OTU_79
Firmicutes; Lactococcus; OTU_31
Firmicutes; Mitsuokella; OTU_1282
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Lactococcus; OTU_72
0.01 0.10 1.00 10.00Read Abundance (%)
Dietcolostrum
sow milk
Stomach
Read abundance (%)
−3
0
3
6
−5.0 −2.5 0.0 2.5 5.0PC1 [26.4%]
PC2
[17.
2%] Diet
colostrum
milk replacer
sow milk
StomachStomach
PC
2 - v
aria
tion
expl
aine
d 17
.2%
PC1 - variation explained 26.4%
(A)
(B)
(C)
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Megasphaera; OTU_1106
Firmicutes; f__[Mogibacteriaceae]_OTU_103; OTU_103
Firmicutes; Lactobacillus; OTU_58
Firmicutes; Lactobacillus; OTU_1926
Firmicutes; Lactobacillus; OTU_107
Firmicutes; Lactococcus; OTU_31
Bacteroidetes; Prevotella; OTU_152
Firmicutes; Lactobacillus; OTU_66
0.01 0.10 1.00 10.00Read Abundance (%)
Dietmilk replacer
sow milk
Stomach
Read abundance (%)
Firmicutes; family [Mogibacteriaceae] OTU_103
Firmicutes; Oscillospira; OTU_3
Actinobacteria; Collinsella; OTU_79
Firmicutes; Lactococcus; OTU_31
Firmicutes; Mitsuokella; OTU_1282
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Lactococcus; OTU_72
0.01 0.10 1.00 10.00Read Abundance (%)
Dietcolostrum
sow milk
Stomach
Read abundance (%)
−3
0
3
6
−5.0 −2.5 0.0 2.5 5.0PC1 [26.4%]
PC2
[17.
2%] Diet
colostrum
milk replacer
sow milk
StomachStomach
PC
2 - v
aria
tion
expl
aine
d 17
.2%
PC1 - variation explained 26.4%
(A)
(B)
(C)
Chapter 4. Manuscript 1 71
Firmicutes; o__Clostridiales_OTU_1273; OTU_1273
Firmicutes; Lactobacillus; OTU_58
Deferribacteres; Mucispirillum; OTU_462
Firmicutes; f__Lachnospiraceae_OTU_603; OTU_603
Firmicutes; Oscillospira; OTU_324
Firmicutes; Ruminococcus; OTU_11
Actinobacteria; Collinsella; OTU_79
Actinobacteria; f__Coriobacteriaceae_OTU_41; OTU_41
Firmicutes; [Ruminococcus]; OTU_128
Firmicutes; Dorea; OTU_207
Firmicutes; Dorea; OTU_313
Firmicutes; Blautia; OTU_305
Firmicutes; Blautia; OTU_47
0.01 0.10 1.00 10.00Read Abundance (%)
Dietmilk replacer
sow milk
Mid colon
Read abundance (%)
Actinobacteria; family Coriobacteriaceae OTU_41
Firmicutes; family Lachnospiraceae OTU_603
Firmicutes; order Clostridiales OTU_1273
Spirochaetes; Sphaerochaeta; OTU_295
Firmicutes; c__Firmicutes_OTU_85; OTU_85
Firmicutes; Lactococcus; OTU_72
Firmicutes; f__Ruminococcaceae_OTU_119; OTU_119
Firmicutes; f__Ruminococcaceae_OTU_171; OTU_171
Firmicutes; f__Ruminococcaceae_OTU_142; OTU_142
Firmicutes; Oscillospira; OTU_361
Firmicutes; f__Christensenellaceae_OTU_17; OTU_17
Firmicutes; Lactobacillus; OTU_546
Fusobacteria; Fusobacterium; OTU_7
Proteobacteria; Campylobacter; OTU_2348
Firmicutes; Lactobacillus; OTU_94
Firmicutes; f__Ruminococcaceae_OTU_21; OTU_21
Firmicutes; Lactobacillus; OTU_1
0.01 0.10 1.00 10.00Read Abundance (%)
Dietcolostrum
sow milk
Mid colon
Read abundance (%)
Firmicutes; family Ruminococcaceae OTU_21
Firmicutes; family Christensenellaceae OTU_17
Firmicutes; family Ruminococcaceae OTU_142
Firmicutes; family Ruminococcaceae OTU_171
Firmicutes; family Ruminococcaceae OTU_119
Firmicutes; OTU_85
−5.0
−2.5
0.0
2.5
5.0
−6 −3 0 3PC1 [13.3%]
PC3
[10%
] Diet
colostrum
milk replacer
sow milk
Mid colon
PC
3 - v
aria
tion
expl
aine
d 10
.0%
PC1 - variation explained 13.3%
Mid colon
(A)
(B)
(C)
Figure 6
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Megasphaera; OTU_1106
Firmicutes; f__[Mogibacteriaceae]_OTU_103; OTU_103
Firmicutes; Lactobacillus; OTU_58
Firmicutes; Lactobacillus; OTU_1926
Firmicutes; Lactobacillus; OTU_107
Firmicutes; Lactococcus; OTU_31
Bacteroidetes; Prevotella; OTU_152
Firmicutes; Lactobacillus; OTU_66
0.01 0.10 1.00 10.00Read Abundance (%)
Dietmilk replacer
sow milk
Stomach
Read abundance (%)
Firmicutes; family [Mogibacteriaceae] OTU_103
Firmicutes; Oscillospira; OTU_3
Actinobacteria; Collinsella; OTU_79
Firmicutes; Lactococcus; OTU_31
Firmicutes; Mitsuokella; OTU_1282
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Lactococcus; OTU_72
0.01 0.10 1.00 10.00Read Abundance (%)
Dietcolostrum
sow milk
Stomach
Read abundance (%)
−3
0
3
6
−5.0 −2.5 0.0 2.5 5.0PC1 [26.4%]
PC2
[17.
2%] Diet
colostrum
milk replacer
sow milk
StomachStomach
PC
2 - v
aria
tion
expl
aine
d 17
.2%
PC1 - variation explained 26.4%
(A)
(B)
(C)
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Megasphaera; OTU_1106
Firmicutes; f__[Mogibacteriaceae]_OTU_103; OTU_103
Firmicutes; Lactobacillus; OTU_58
Firmicutes; Lactobacillus; OTU_1926
Firmicutes; Lactobacillus; OTU_107
Firmicutes; Lactococcus; OTU_31
Bacteroidetes; Prevotella; OTU_152
Firmicutes; Lactobacillus; OTU_66
0.01 0.10 1.00 10.00Read Abundance (%)
Dietmilk replacer
sow milk
Stomach
Read abundance (%)
Firmicutes; family [Mogibacteriaceae] OTU_103
Firmicutes; Oscillospira; OTU_3
Actinobacteria; Collinsella; OTU_79
Firmicutes; Lactococcus; OTU_31
Firmicutes; Mitsuokella; OTU_1282
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Lactococcus; OTU_72
0.01 0.10 1.00 10.00Read Abundance (%)
Dietcolostrum
sow milk
Stomach
Read abundance (%)
−3
0
3
6
−5.0 −2.5 0.0 2.5 5.0PC1 [26.4%]
PC2
[17.
2%] Diet
colostrum
milk replacer
sow milk
StomachStomach
PC
2 - v
aria
tion
expl
aine
d 17
.2%
PC1 - variation explained 26.4%
(A)
(B)
(C)
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Megasphaera; OTU_1106
Firmicutes; f__[Mogibacteriaceae]_OTU_103; OTU_103
Firmicutes; Lactobacillus; OTU_58
Firmicutes; Lactobacillus; OTU_1926
Firmicutes; Lactobacillus; OTU_107
Firmicutes; Lactococcus; OTU_31
Bacteroidetes; Prevotella; OTU_152
Firmicutes; Lactobacillus; OTU_66
0.01 0.10 1.00 10.00Read Abundance (%)
Dietmilk replacer
sow milk
Stomach
Read abundance (%)
Firmicutes; family [Mogibacteriaceae] OTU_103
Firmicutes; Oscillospira; OTU_3
Actinobacteria; Collinsella; OTU_79
Firmicutes; Lactococcus; OTU_31
Firmicutes; Mitsuokella; OTU_1282
Firmicutes; Leuconostoc; OTU_209
Firmicutes; Lactococcus; OTU_72
0.01 0.10 1.00 10.00Read Abundance (%)
Dietcolostrum
sow milk
Stomach
Read abundance (%)
−3
0
3
6
−5.0 −2.5 0.0 2.5 5.0PC1 [26.4%]
PC2
[17.
2%] Diet
colostrum
milk replacer
sow milk
StomachStomach
PC
2 - v
aria
tion
expl
aine
d 17
.2%
PC1 - variation explained 26.4%
(A)
(B)
(C)
72 Chapter 4. Manuscript 1
−10
−50
5
−6−4−2024
RDA
1
RDA2
colo
stru
m
milk
repl
acer
sow
milk
Dis
tal S
mal
l Int
estin
e
Faec
es
Mid
Col
on
Stom
ach
Ace
Pro
Iso_
But
But
Iso_
Val
Val
Ibiv
Apb
Ente
ro
Hem
Clo
sLa
b
Yeas
t
Ana
RDA2
RD
A1
Sto
mac
h D
ista
l sm
all i
ntes
tine
Mid
col
on
Faec
es
Col
ostru
m
Milk
repl
acer
S
ow m
ilk
−10
−50
5
−6−4−2024
RDA
1
RDA2
colo
stru
m
milk
repl
acer
sow
milk
Dis
tal S
mal
l Int
estin
e
Faec
es
Mid
Col
on
Stom
ach
Ace
Pro
Iso_
But
But
Iso_
Val
Val
Ibiv
Apb
Ente
ro
Hem
Clo
sLa
b
Yeas
t
Ana
−10
−50
5
−6−4−2024
RDA
1
RDA2
colo
stru
m
milk
repl
acer
sow
milk
Dis
tal S
mal
l Int
estin
e
Faec
es
Mid
Col
on
Stom
ach
Ace
Pro
Iso_
But
But
Iso_
Val
Val
Ibiv
Apb
Ente
ro
Hem
Clo
sLa
b
Yeas
t
Ana
−10
−50
5
−6−4−2024
RDA
1
RDA2
colo
stru
m
milk
repl
acer
sow
milk
Dis
tal S
mal
l Int
estin
e
Faec
es
Mid
Col
on
Stom
ach
Ace
Pro
Iso_
But
But
Iso_
Val
Val
Ibiv
Apb
Ente
ro
Hem
Clo
sLa
b
Yeas
t
Ana
−10
−50
5
−6−4−2024
RDA
1
RDA2
colo
stru
m
milk
repl
acer
sow
milk
Dis
tal S
mal
l Int
estin
e
Faec
es
Mid
Col
on
Stom
ach
Ace
Pro
Iso_
But
But
Iso_
Val
Val
Ibiv
Apb
Ente
ro
Hem
Clo
sLa
b
Yeas
t
Ana
−10
−50
5
−6−4−2024
RDA
1
RDA2
colo
stru
m
milk
repl
acer
sow
milk
Dis
tal S
mal
l Int
estin
e
Faec
es
Mid
Col
on
Stom
ach
Ace
Pro
Iso_
But
But
Iso_
Val
Val
Ibiv
Apb
Ente
ro
Hem
Clo
sLa
b
Yeas
t
Ana
−10
−50
5
−6−4−2024
RDA
1
RDA2
colo
stru
m
milk
repl
acer
sow
milk
Dis
tal S
mal
l Int
estin
e
Faec
es
Mid
Col
on
Stom
ach
Ace
Pro
Iso_
But
But
Iso_
Val
Val
Ibiv
Apb
Ente
ro
Hem
Clo
sLa
b
Yeas
t
Ana
−10
−50
5
−6−4−2024
RDA
1
RDA2
colo
stru
m
milk
repl
acer
sow
milk
Dis
tal S
mal
l Int
estin
e
Faec
es
Mid
Col
on
Stom
ach
Ace
Pro
Iso_
But
But
Iso_
Val
Val
Ibiv
Apb
Ente
ro
Hem
Clo
sLa
b
Yeas
t
Ana
Figu
re 7
Chapter 4. Manuscript 1 73
23 25 27 30
200
300
400
500
600
700
colostrum
milk replacersow milk
colostrum
milk replacersow milk
colostrum
milk replacersow milk
colostrum
milk replacersow milk
Diet
Obs
erve
d nu
mbe
r of O
TUs
Obs
erve
d nu
mbe
r of O
TUs
Day 23 Day 25 Day 27 Day 30
BC MR SM BC MR SM BC MR SM BC MR SM
Diet
Stomach Distal Small Intestine Mid Colon
125
250
375
500
625
750
colostrum
milk replacersow milk
colostrum
milk replacersow milk
colostrum
milk replacersow milk
Diet
Obs
erve
d nu
mbe
r of O
TUs
BC MR SM
Obs
erve
d nu
mbe
r of O
TUs
BC MR SM
Diet
BC MR SM
Stomach Distal small intestine Mid colon
(A)
(B)
Figure S1
74 Chapter 4. Manuscript 1
Chapter 5. Manuscript 2 75
Chapter 5
Manuscript 2
Investigating the potential effects of alpha-(1,2)-fucosyltransferase geno-type on the establishment and succession of the gastrointestinal micro-biota of young piglets.
Ann-Sofie Riis Poulsen, Sugiharto Sugiharto, Nuria Canibe and Charlotte Laurid-sen.
Prepared for submission to Veterinary Microbiology.
76 Chapter 5. Manuscript 2
Experimental set-up:
17 piglets from two sowsFollowed from 5 to 34 days of age
FUT1-307AA piglets= E. coli F18 susceptible
Faeces Day 5, 7, 14, 21,
28 and 34
Digesta Stomach, ileum, caecum and mid colon (day 34)
FUT1-307AG piglets= E. coli F18 resistant
Digesta Stomach, ileum, caecum and mid colon (day 34)
Faeces Day 5, 7, 14, 21,
28 and 34
10 piglets 7 piglets
1. 16S rRNA gene sequencing
• V3 region• Ion Torrent
2. Classical culture3. Organic acid analysis
1. Classical culture2. Organic acid analysis
! 1
Investigating the potential effects of alpha-(1,2)-fucosyltransferase genotype on the
establishment and succession of the gastrointestinal microbiota of young piglets
Ann-Sofie Riis Poulsena, Sugiharto Sugihartob, Nuria Canibea, Charlotte Lauridsena*
a Aarhus University, Faculty of Science and Technology, Department of Animal Science, Blichers
Allé 20, 8830,Tjele, Denmark
b Diponegoro University, Faculty of Animal and Agricultural Sciences, Semarang, Central Java,
Indonesia
* Corresponding author
Aarhus University,
Faculty of Science and Technology, Department of Animal Science,
Blichers Allé 20, P.O. Box 50
DK-8830 Tjele, Denmark
Tel.: +45 87 15 80 19
Fax.: +45 87 15 42 49
E-mail address: charlotte.lauridsen@anis.au.dk
Chapter 5. Manuscript 2 77
! 2
Abstract
Piglets with the alpha-(1,2)-fucosyltransferase (FUT1) genotype M307A/A have decreased
susceptibility to enterotoxigenic Escherichia coli F18 infection. The effect of FUT1 polymorphisms
on the establishing gut microbiota, however, is unknown. This study aimed at characterising the
gastrointestinal microbiota of FUT1-M307A/A and FUT1-M307A/G piglets and the influence of age
using high-throughput sequencing and classical culture techniques.
Ten sensitive (M307G/A) and seven resistant (M307A/A) piglets were included. Piglets suckled
their mother-sow from birth until weaning at 28 days of age. Faecal samples collected on day 5, 7,
14, 21, 28 and 34 of age were analysed using 16S rRNA gene sequencing. All piglets were
euthanized at 34 days of age. Bacterial enumerations by culture and short-chain fatty acid analysis
were performed on faeces and digesta. Multivariate statistics showed similar faecal communities
between genotypes. A numerical higher relative abundance of Prevotella was observed in sensitive
piglets at 7, 14, 21 and 28 days of age, though. Bacterial enumeration by plating showed a higher
number of haemolytic bacteria in faeces (p=0.003) and digesta (p=0.02) in 34 days old sensitive
piglets compared to resistant piglets. The colonic acetic acid concentration was higher in sensitive
piglets (p=0.01). Age was found to have an effect on the faecal communities (padonis=0.001). In
conclusion, faecal microbiota of sensitive piglets was significantly different from that of resistant
piglets in the number of potentially pathogenic E. coli. However, 16S rRNA gene sequencing also
indicated an effect of FUT1 genotype on Prevotella. Our data do not allow us to definitively rule
out an effect of FUT1 genotype on other bacterial groups in the gastrointestinal tract as 16S rRNA
gene sequencing was only performed on faecal samples.
Keywords: Piglets. Gastrointestinal microbiota. 16S rRNA amplicon sequencing. Alpha-(1,2)-
fucosyltransferase (FUT1). Escherichia coli F18. Post-weaning diarrhoea.
78 Chapter 5. Manuscript 2
! 3
Introduction
Diarrhoea is a common clinical condition in pigs post-weaning (Lalles et al., 2007). The vast
majority of pigs having diarrhoea are treated with antibiotics, and in Denmark clinical diarrhoea is
the main cause of antibiotic treatment of pigs (DANMAP, 2014). The growing concern over
antibiotic resistance has increased the demand for alternative therapeutic or preventative options
and underscores the need for improving the general health condition of pigs.
The gut microbiota is a complex micro-environment existing in symbiosis with the host and
recognised as a major player in host health (Sommer and Baeckhed, 2013). Microbial gut
colonisation of the newborn is essential in stimulating local immune cell proliferation and is critical
to the maturation of the adaptive immune system (Chung et al., 2012; Guarner and Malagelada,
2003). Microbial gut colonisation is a gradual and complex process starting at birth (Mackie et al.,
1999). During early life the microbiota is characterised by instability, resulting in a highly dynamic
community with frequent changes in dominating taxa (Petri et al., 2010; Slifierz et al., 2015). The
developing gut microbiota is easily disturbed and in pigs, microbial imbalances due to e.g. weaning
stress (Konstantinov et al., 2006; Thompson et al., 2008) are believed to affect host-defense
mechanisms and increase susceptibility to enteric disease and hence, diarrhoea (Castillo et al., 2007;
Heo et al., 2013). In relation to weaning, enterotoxigenic Escherichia coli (ETEC) F18 is an
important pathogen causing infectious diarrhoea during the first week post-weaning (Fairbrother et
al., 2005). Breeding ETEC F18-resistant piglets has been studied as a potential preventative strategy
in coping with post-weaning diarrhoea (Frydendahl et al., 2003). Host genetics, however, has been
suggested as one of the main factors influencing gut microbiota composition (Scott et al., 2013).
Different breeds of pigs have been reported to harbor different gut microbial communities (Pajarillo
et al., 2014b) and in chickens, different genetic variants of disease-determining genes have been
found to influence the gut colonisation process of the young animal (Schokker et al., 2015).
The FUT1 gene, encoding the alpha-(1,2)-fucosyltransferase enzyme, has been proposed as
a candidate gene for manipulating adhesion of ETEC F18 to its intestinal receptor (Meijerink et al.,
1997). It has been confirmed that a guanine-to-adenine mutation at nucleotide 307 (M307) in both
loci of the FUT1 gene does result in resistance to ETEC F18 adherence to small intestinal
enterocytes (Meijerink et al., 2000). Furthermore, the M307A/A genotype has been associated with
decreased susceptibility of weanling piglets to ETEC F18 infection compared to piglets with
genotypes M307A/G and M307G/G (Frydendahl et al., 2003).
Chapter 5. Manuscript 2 79
! 4
As ETEC F18-resistant and sensitive piglets are genetically different and the resistance
mechanism is believed to involve intestinal receptors, FUT1 polymorphisms might influence the
establishment of the gut microbiota of young piglets beyond the establishment of ETEC F18. As the
gut microbiota is highly important to host health, this knowledge is of great relevance. Hence, the
primary aim of the present study was to investigate how FUT1 genotype influences the
establishment of the gastrointestinal microbiota in piglets aged 5 to 34 days combining high-
throughput next generation sequencing and classical culture techniques. A secondary aim was to
investigate how the microbiota changes with age. We hypothesise that piglets with the FUT1-
M307A/A genotype will have a gut microbiota different from piglets with the FUT1-M307A/G
genotype and that the gut microbial community will change as the piglet ages.
Materials and methods
Animals, study design and FUT1 genotyping
The present study was conducted according to the license obtained from the Danish Animal
Experiments Inspectorate, Ministry of Food, Agriculture and Fisheries, Danish Veterinary and Food
administration. The study was performed at the experimental facility at the Department of Animal
Science (Foulum, Aarhus University). Sows and piglets were housed according to Danish
guidelines.
A total of 17 vaginally born piglets were included in the study. They were the result of
breeding two M307G/A sows with the same M307A/A boar (Danish Landrace x Yorkshire breed x
Duroc). Only FUT1-M307G/A and FUT1- M307A/A piglets were included in the study because FUT1-
M307G/A and FUT1-M307G/G piglets were considered equally susceptible to ETEC F18 (Frydendahl
et al., 2003). An ear tissue sample was collected shortly after birth and used for FUT1 genotyping.
In short, the genotyping procedure included a PCR amplification of the FUT1 gene product
harboring the mutation of interest. Subsequently, the PCR fragments were digested with the HinP1I
restriction enzyme and run on a 1.5% agarose gel.
All piglets were kept with their mother-sow until weaning at 28 days of age. Piglets suckled
their dams and had free access to water and creep feed (no added zinc oxide) from three weeks of
age. After weaning, piglets were housed together in pens according to litter and fed a standard post-
weaning diet (no added zinc oxide). The temperature in the weaning unit was 23°C and piglets had
free access to rooting material.
80 Chapter 5. Manuscript 2
! 5
Sample collection
Faecal samples were collected directly from the rectum at 5, 7, 14, 21, 28 and 34 days of age.
Subsamples were stored at -20˚C for organic acid analysis and snap-frozen in liquid nitrogen and
stored at -80˚C for 16S rRNA amplicon sequencing. Bacterial enumerations by plating were
performed on a subsample of fresh faeces.
All piglets were euthanised at 34 days of age. The abdomen was incised and the
gastrointestinal tract removed. Luminal content (digesta) from stomach, distal small intestine,
caecum and middle part of the colon was sampled immediately and subsamples were stored at -
20˚C for organic acid analysis. Bacterial enumerations by plating were performed on fresh
subsamples.
DNA extraction
DNA was extracted using the MOBIO Power Soil DNA Isolation Kit (MOBIO Laboratories Inc.,
Carlsbad, CA) including an initial enzymatic treatment and otherwise following a standard protocol.
Extracted DNA concentrations were quantified fluorometrically with Qubit 3.0 HS dsDNA assay
(Life Technologies, Thermo Fisher Scientific, USA) and normalized to 5 ng/µL.
16S rRNA amplicon sequencing
Partial 16S rRNA sequences were amplified (at GenoSkan A/S, Tjele, Denmark) from extracted
DNA using ProBio_Uni/Probio_Rev primers (Milani et al., 2013), amplifying the V3 region of the
16S rRNA gene sequence. To establish the amplicon library, a PCR was conducted using the
following thermocycler settings: Initial denaturation at 95°C for 5 min, 35 cycles at 94°C for 30 s,
58°C for 30 s, and 72°C for 90 s, and final elongation at 72°C for 10 min. Individual amplicon
libraries were purified using the Agencourt AMPure XP DNA purification beads (Beckman Coulter
Genomics GmbH, Germany) and the Ion OneTouchTM 200 template Kit v2 DL (Life
Technologies) was employed for emulsion PCR according to the manufacturers recommendations.
Amplicon libraries were sequenced using the Ion Torrent PGM system applying the Ion Sequencing
200 kit (Life Technologies) according to the manufacturer’s recommendations. All PGM quality-
approved, trimmed and filtered data were exported as fastq files. Sequences were filtered using the
FastQ Quality software and sequences with length 150-220 bp and an average quality score above
19 were used for further analysis.
Chapter 5. Manuscript 2 81
! 6
Amplicon bioinformatics processing
Bioinformatic processing was done using a pipeline from The Brazilian Microbiome Project for Ion
Torrent 16S rRNA sequence analysis (Pylro et al., 2014), UPARSE (USEARCH 8.1.1756) (Edgar,
2013) and QIIME (Caporaso et al., 2010). Reads were quality filtered using USEARCH –
fastq_filter with a maximum expected error of 1.0 and global trimming at bp 166 was performed in
the same step. Reads were dereplicated (i.e. identification of unique sequences) and formatted to the
UPARSE pipeline. Singletons were discarded and dereplicated reads were clustered into
Operational Taxonomical Units (OTUs) using USEARCH at 97% sequence similarity. An
additional chimera detection step was included using USEARCH with rpd_gold version 9 (Cole et
al., 2014) as reference database. Taxonomy was assigned using the UCLUST-classifier as
implemented in QIIME (Caporaso et al., 2010) with Greengenes version 13.8 (DeSantis et al.,
2006) as a reference database and default settings. Results were analysed in R studio (version
0.99.489 for Mac) using the Ampvis package (Albertsen et al., 2015).
Dry matter and organic acid analysis
Dry matter content of digesta was determined by freeze-drying (ScanVac Coolsafe 55, Labogene
ApS, Lynge, Denmark). The concentration of short-chain fatty acids and lactic acid in faeces and
digesta were measured as previously described by Canibe et al. (2007).
Microbiological enumerations by plating
Faecal samples (approximately 1 g) were transferred to plastic bags and 5 or 10 ml pre-reduced salt
medium was added. The content was homogenised in a Smasher paddle blender (bioMérieux
Industry, USA) for 2 minutes. Digesta samples (approximately 5 g) were transferred to flasks
containing 50 ml pre-reduced salt medium (Holdeman et al., 1977). The flask content was
transferred to a CO2 flushed bag and homogenized in a stomacher blender for 2 minutes. 1 ml
homogenate was transferred to a Hungate tube containing 9 ml pre-reduced salt medium and 10-
fold dilutions were prepared using the technique previously described by Miller and Wolin (1974).
Each sample was plated on selective (and indicative) and non-selective agar plates.
Enterobacteriaceae were enumerated on MacConkey agar (Merck 1.05465) after aerobic
incubation for 1 day. Yeasts were enumerated on Malt, Chlortetracycline and -Amphenicol agar
(Merck 1.03753 (yeast extract), 1.05397 (malt extract), 1.07224 (bacto-pepton), 1.08337 (glucose),
1.01614 (agar-agar) and Oxoid Sr0177E) after aerobic incubation for 2 days. Haemolytic bacteria
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were enumerated on blood agar (Oxoid Pb5039A) after aerobic incubation for 1 day. Clostridium
perfringens was enumerated using the pour-plate technique on Tryptose Sulfite Cycloserine agar
(Merck 1.11972 and 1.00888) after anaerobic incubation for 1 day. Lactic acid bacteria were
enumerated on de Man, Rogosa and Sharp agar (Merck 1.10660) after anaerobic incubation for 2
days. Total anaerobic bacteria were enumerated in roll tubes containing pig colon fluid-glucose-
cellobiose agar (Holdeman et al., 1977) and incubated for 7 days. All plates and roll-tubes were
incubated at 37˚C.
Porcine Intestinal Organ Culture (PIOC)
The PIOC procedure was included to investigate the adherence of E. coli O138:F18 to the mucosa
of resistant and sensitive piglets following the procedure previously described by Sugiharto et al.
(2012). In short, a distal small intestinal segment (15 cm) was sampled and incubated with a
solution containing E. coli O138:F18 for 1 hour. The tissue was then homogenized, diluted and E.
coli enumerated on MacConkey (Merck 1.05465) agar plates after aerobic incubation at 37˚C
overnight.
Statistical analyses
The effect of genotype and age on bacterial and organic acid parameters, pH, microbial community
richness and diversity and animal body weight were analysed by fitting the data to a linear mixed
model using the lmer function from the lme4 package (Bates, 2014) using R studio (Version
0.99.489 for Mac). Genotype and age/intestinal segment were included as fixed effects, while pig
and sow were included as random effects (by including random intercept terms) to account for
multiple observations within the same litter or within the same pig. When analysing the weight
variable, birth weight was included as co-variate. The fixed effects were tested using a F-test with
Kenward-Roger approximation, where the reduced model was tested against the full model. This
was done using the KRmodcomp function in the pbkrtest package (Halekoh, 2014). When age was
found to have a significant effect, a post-hoc test was performed using the multcomp package and
Bonferroni adjustment to correct for multiple comparisons (Hothorn, 2008). Effects were
considered significant when p<0.05 and as trends when 0.05≤p<0.10. Principal Component
Analysis (PCA) was performed on square root transformed OTU abundances. Significance of
genotype and age was tested on the first two principal components (PCs) using the envfit parametric
test and on the Bray-Curtis dissimilarity matrix using adonis (Oksanen, 2015). The parametric
Chapter 5. Manuscript 2 83
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Wald-test (Love et al., 2014) was used to test for significant differences in OTUs abundances
between genotypes and between the ages 5 and 28, and 28 and 34 days. OTUs with an adjusted
p<0.001 were considered significantly different between genotypes and/or ages.
Results
Genetic testing revealed that ten piglets were of the sensitive genotype (M307G/A) and seven piglets
were of the resistant genotype (M307A/A). Body weight data is presented in Table 1. Sensitive
piglets weighed significantly more than resistant piglets at day 28 (p=0.003) and 34 (p=0.01) of age.
Composition of faecal microbiota– 16S rRNA sequencing
Sequencing of 74 samples (sensitive n=39; resistant n=35) yielded a total of 6,060,572 sequences
after removal of poor quality reads. Due to the small size of the piglets, 28 samples are missing due
to either unsuccessful sampling or sampling an amount too small to allow successful DNA
extraction. Furthermore, nine of the sequenced samples had less than 5000 sequences and were
excluded from further analyses. Recovered sequences clustered into 2993 OTUs, which were
classified into 15 bacterial phyla, 74 families and 112 genera. Nine phylum level groups had an
overall relative abundance ≥1% (found in at least one of the two genotypes at each age), of which
two groups were taxonomically unassigned (OTU 57 and OTU 188) (Fig. 1a).
Firmicutes, followed by Bacteroidetes, dominated the communities irrespective of FUT1
genotype and age. The remaining phyla were present at frequencies lower than 6% in both
genotypes at all ages. The five most abundant families were Ruminococcaceae, Clostridiaceae,
Lactobacillaceae, Prevotellaceae and Lachnospiraceae (Fig. 1b). A decrease in relative abundance
was observed from day 7 for Lactobacillaceae and an increase on day 34 for Lachnospiraceae in
both genotypes. At the genus level, numerical differences (at least a doubling in the relative
abundance and at least one genotype having a relative abundance above 5%) between genotypes
were seen for Lactobacillus on day 7, Prevotella on day 7, 14, 21 and 28, Clostridium on day 5 and
14, Bacteroides on day 14, and Butyricimonas on day 7 (Fig S2). Based on the first two PCs of the
OTU abundances, the FUT1 genotype had no significant effect on the overall faecal microbial
community (Fig. 2, padonis>0.27), and accordingly no OTUs differed significantly between
genotypes. Species richness (Fig. S1 a and b; observed p=0.73; estimated p=0.75) and Shannon
diversity index (Fig. S1 c; p=0.11) were not significantly different between genotypes.
84 Chapter 5. Manuscript 2
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Based on the first and third PC of the OTU abundances, a significant effect of age on the
overall microbial community was observed (Fig. 3; padonis=0.001). A gradual change in the overall
bacterial community composition was seen from day 5 to 34 of age. Pairwise comparisons between
all sampling days revealed that the microbial community richness was higher on day 21 (observed
p=0.004; estimated p=0.003) and 28 (observed p=0.03; estimated p=0.01) compared to day 5 (Fig.
S1 a and b). Age did not have an effect on the Shannon diversity index. Comparing day 5 and day
28 (the first and last sampling day during suckling), 201 out of 1769 OTUs had significantly
different read abundances (Fig. 4a: the 25 most significantly different OTUs). The most
significantly different OTU (the OTU with the lowest p-value) was OTU_9 (p=3.3E-26) belonging
to the family Ruminococcaceae, having a higher read abundance on day 5. Of the 25 most
significantly different OTUs, 12 belonged to the order Clostridiales with 10 having a lower read
abundance on day 28 compared to day 5. When comparing day 28 and day 34 (weaning day and
one week later), 105 out of the 1790 OTUs differed significantly (Fig. 4b; the 25 most significantly
different OTUs). OTU_28, belonging to the order Clostridiales, was the most significantly different
OTU (p=6.1E-26), having a higher read abundance on day 28. Of the 25 most significantly changed
OTUs, 11 belonged to the order Clostridiales with nine having a lower read abundance on day 34.
Six OTUs belonged to the family Ruminococcaceae and had a lower read abundance on day 34.
Litter was found to have a significant effect on the overall microbial community on day 21
(padonis=0.01).
Microbiological enumerations by plating and organic acid concentrations
Due to the small size of the piglets, adequate quantities of faeces for bacterial enumeration by
plating and organic acid analysis could not be sampled from all animals at all ages.
Faeces. Higher faecal numbers of haemolytic bacteria at 34 days of age (p=0.003), and a tendency
to higher numbers of lactic acid bacteria (p=0.06), were observed in sensitive piglets (Table 2). The
number of haemolytic bacteria was constant between day 5 and 28, whereas a significant increase
was detected on day 34 (p≤0.003). An age-dependent decrease of C. perfringens was seen with
numbers being lowest on day 28 (p<0.0001) and 34 (p<0.0001). The same was observed for lactic
acid bacteria, with lower numbers on day 28 (p≤0.03) and day 34 (p≤0.005) compared to day 5 and
7. There was no significant effect of genotype or age on the number of Enterobacteriaceae or total
anaerobic bacteria.
Chapter 5. Manuscript 2 85
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Furthermore, no difference in the concentration of faecal organic acids between genotypes
was determined, except for a tendency to a lower concentration of butyric acid (p=0.09) in resistant
piglets (Table 3). The concentration of acetic acid was higher at 34 days of age (one week post-
weaning) compared to day 5, 7, 14 and 21 of age (suckling period) (p<0.05). The concentration of
propionic acid was constant between day 5 and 21, but at 34 days of age, a significant increase in
propionic acid was observed (p=0.0002). A tendency to a lower concentration of butyric acid
(p=0.09) with age was measured, whereas no effect of genotype or age was detected for valeric, iso-
butyric and iso-valeric acid in faeces (data not shown).
The age-dependent decrease in lactic acid bacteria and C. perfringens seen by culture were
in agreement with the results from the 16S rRNA gene sequencing. Both methods detected a
marked decrease in Clostridium (C. perfringens) from day 14. Regarding lactic acid bacteria, the
culture results were in accordance with the relative abundances of Lactobacillus as both methods
detected a decrease from day 7. In accordance with the 16S rRNA sequencing results, culture of
Enterobacteriaceae showed a higher number in resistant piglets at 5 and 7 days of age, while the
number was higher in sensitive piglets at 28 and 34 days of age. See Fig. 1b, Fig S2 and Table 2.
Digesta. Genotype had no significant effect on dry matter content or pH (Table S1). Sensitive
piglets had a higher number of Enterobacteriaceae (p≤0.02) in digesta from the distal small
intestine, caecum and mid colon; of haemolytic bacteria (p=0.02) in the stomach, small intestine,
caecum and colon; and of total anaerobic bacteria (p=0.004) in the distal small intestine compared
to resistant piglets (Table 4).
The concentration of acetic acid was higher in the colon of sensitive piglets (p=0.01, Table
5). Digesta concentrations of propionic, butyric, lactic, valeric, and the sum of iso-butyric and iso-
valeric acid were similar between genotypes.
Quantification of E. coli attachment to distal small intestinal tissue. The E. coli numbers in the
small intestinal tissue were similar between genotypes (p=0.41). However, sensitive piglets had
numerically higher E. coli numbers before (sensitive 6.3 log cfu/g; resistant 6.0 log cfu/g) and after
E. coli O138:F18 inoculation (sensitive 8.1 log cfu/g; resistant 7.8 log cfu/g).
86 Chapter 5. Manuscript 2
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Discussion
Newly weaned piglets are highly susceptible to enteric disease and diarrhoea caused by ETEC F18.
The FUT1 M307A/A genotype is believed to cause changes in the intestinal receptors responsible for
ETEC F18 adhesion (Meijerink et al., 1997); however, little scientific information is available on
the influence of FUT1 genotype on the gut microbiota of piglets beyond ETEC F18.
Applying 16S rRNA gene sequencing, the overall faecal microbial communities were found
to be similar between resistant and sensitive piglets. Bacterial culture showed a significant effect of
FUT1 genotype on the number of haemolytic bacteria after weaning, being higher in both faeces
and digesta from sensitive piglets. These results indicate that sensitive piglets have a higher
shedding of what could potentially be ETEC F18 in the post-weaning period and supports the
findings of Frydendahl et al. (2003), that FUT1 M307G/A piglets are indeed more susceptible to
ETEC F18 infection than are FUT M307A/A piglets. This is furthermore supported by an apparent
up-regulation in the humoral immune response in sensitive piglets. We were, however, not able to
detect significantly higher E. coli F18 numbers in the small intestinal tissue using an ex vivo porcine
intestinal model. As this ex vivo model has been successfully applied in a number of studies
(Sugiharto and Lauridsen, 2014; Sugiharto et al., 2015) and the fact that we in the present study
were able to detect numerical differences between genotypes, the lack of significance might be due
to the low number of replicates.
Ruminococcoceae, Clostridiaceae, Lactobacillaceae, Prevotellaceae and Lachnospiraceae
dominated the microbial community of ETEC F18-resistant and sensitive piglets in the present
study. Even though no OTUs were found to be significantly different between the two genotypes,
interesting trends were observed in especially Prevotella, as this genus was present in numerically
higher relative abundances in sensitive piglets at 7, 14, 21 and 28 days of age. Prevotella is
normally associated with the intake of fermentable fibers (Ivarsson et al., 2014) and its abundance
has been shown to increase after weaning (Pajarillo et al., 2014a) as piglets are normally weaned to
solid feed which, contrary to sow milk, is rich in fibers. These results are in contrast to the
observations in the present study, in which the relative abundance of Prevotella was highest before
weaning especially in sensitive piglets.
Mach et al. (2015) identified Prevotella and Ruminococcaceae as being two enterotype-like
clusters in piglets aged 14-70 days and reported that the body weight of piglets belonging to the
Prevotella cluster was lower during lactation but higher after weaning when compared to pigs
belonging to the Ruminococcaceae cluster. Bao et al. (2011) found ETEC F18-resistant Sutai pigs
Chapter 5. Manuscript 2 87
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to be marginally heavier at weaning, while Huang et al. (2008) found no effect of FUT1 genotype
on growth performance in Landrace and Duroc pigs. These observations do not agree with the
results of the present study, where sensitive piglets weighed significantly more than resistant piglets
on the day of weaning. It is unlikely that the higher body weight of sensitive piglets were due to the
higher fiber digestion capacity of Prevotella as piglets were not introduced to creep-feed until three
weeks of age. Furthermore, the limited intake of feed during the suckling period can probably not
explain the difference between genotypes on piglet weight gain until weaning. On the basis of data
from the present study, we were not able to suggest a mode of action of the effect of FUT1
genotype on growth parameters. It should be noted, however, that Prevotella is represented by
numerous species (Ley, 2016). As our data does not enable us to classify OTUs to species level, an
exact interpretation of the significance of the Prevotella genus is not possible.
Acetic acid, which is the major short-chain fatty acid in the gut, was found in higher
concentrations in colonic content of sensitive piglets. As the short-chain fatty acid profile reflects
bacterial metabolism (Williams et al., 2005), the higher colonic concentration of acetic acid in
sensitive piglets most likely reflect bacterial community differences between genotypes. Although a
higher number of Enterobacteriaceae was measured in sensitive piglets, and E. coli, belonging to
this family, has been shown to produce higher amounts of acetic acid at high growth rates (Han et
al., 1992), this alone is unlikely to explain the difference in acetic acid concentrations. Regardless
of genotype, the acetic acid concentration in the stomach was higher than anticipated. Also, a higher
concentration of butyric acid in the stomach than in the large intestine was unexpected, since the
opposite often is observed, e.g. as shown by Starke et al. (2014). An obvious explanation has not
been found.
In order to assess the age-dependent changes in the gut microbiota, faecal specimens were
used for 16S rRNA amplicon sequencing. Assessing the overall faecal microbiota, we found a
gradual change in the microbial community in the period from 5 to 34 days of age with an
increasing microbial diversity peaking at 21 days of age. When these results are compared with
previous studies, it is obvious that the dominating populations differ between studies. Frese et al.
(2015) investigated the effect of diet on the faecal microbiota in the pre- and post-weaning period
and found the dominating families to be Enterobacteriaceae, Lachnospiraceae, Bacteroidaceae,
Lactobacillaceae, Clostridiaceae, Ruminococcaceae and Prevotellaceae. In contrary to the results
of Frese et al. (2015) we did not find Enterobacteriaceae and Bacteroidaceae to be dominating.
88 Chapter 5. Manuscript 2
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Owing to differences between studies, such as housing conditions, dietary composition, in-feed
antibiotics, pig breed and analytical procedures, microbial community dissimilarities were expected.
As microbial species richness in the gut has been reported to increase with age (Slifierz et
al., 2015), and this study involved young piglets, the species richness, irrespective of age, was
higher than expected. As 16S rRNA sequence read length has been reported to influence the
observed microbial community richness (Engelbrektson et al., 2010), the high species richness is
most likely explained by the relatively short reads. This could also be the reason why numerous
OTUs were not taxonomically assigned, or only assigned to order level.
Gut microbiota composition varies between gastrointestinal region and location within the
gut (lumen vs. mucosa) (Looft et al., 2014). As the FUT1 gene product is believed to exert its effect
on ETEC F18 receptors in the small intestine (Meijerink et al., 1997), faecal samples might not be
the most appropriate sampling choice for in-depth microbial analysis. Supporting this, a recent
study from our group (Poulsen et al, in preparation) revealed a dietary treatment effect on the
microbial community composition of luminal contents in the small intestine, when analysed by 16S
rRNA gene sequencing, which was not detected in faecal samples. It could therefore be speculated
that different results would have been obtained if small intestinal luminal content and/or mucosal
samples rather than faecal samples had been analysed by 16S rRNA amplicon sequencing.
Conclusion
The data obtained from the present study contribute to the existing knowledge on FUT1
polymorphisms in pigs. In conclusion, the faecal microbiota of sensitive piglets significantly
differed from that of resistant piglets in only the number of potentially pathogenic ETEC, although
16S rRNA gene sequencing did indicate an effect of FUT1 genotype on Prevotella. Further studies
on the impact of FUT1 genotype on the microbial community composition in the small intestine are
needed, including absolute bacterial quantifications. The reported effect of FUT1 genotype on body
weight gain should also be investigated further.
Acknowledgements
The authors would like to thank laboratory technicians Trine Poulsen and Karin Durup, technicians
Mette Lykkegaard and Karin Johansen and the staff of SB32 at the Department of Animal Science
for their skillful technical assistance during the course of the experiment and Samantha Joan Noel
for proofreading the manuscript. The authors want to thank Lone Bruhn Madsen for FUT1
Chapter 5. Manuscript 2 89
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genotyping the animals. The authors furthermore want to thank Senior Scientist Emøke Bendixen,
Department of Molecular Biology and Genetics and collaborators at GenoSkan A/S, Niels
Pedersens Allé 2, DK-8830 Tjele. The paper drafting was primarily funded through a Ph.D.-
scholarship obtained from the Graduate School of Science and Technology, Aarhus University,
Denmark, for the main author. Funders had no influence on study design, collection, analysis or
interpretation of data, or drafting of manuscript.
Author contributions
Authors contributed as follows: A.-S.R.P. conducted the animal experiment, performed
bioinformatics and statistical data analyses and drafted the manuscript. S.S. conducted the animal
experiment and performed the PIOC experiment. N.C. designed the experiment, contributed in the
conduction of the animal experiment and assisted with the overall data analysis. C.L. designed the
experiment and contributed in the conduction of the animal experiment. All authors have revised
and accepted the final manuscript.
Conflict of interest statement
Conflicts of interest: None.
90 Chapter 5. Manuscript 2
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Oksanen, J.G.B., F.; Kindt, R.; Legendre, P.; Minchin, P.R.; O'Hara, R.B.; Simpson, G.L.; Solymos, P.; M. Stevens H.H.; Wagner, H., 2015. vegan: Community Ecology Package. R package version 2.3-2. .
Pajarillo, E.A.B., Chae, J.-P., Balolong, M.P., Kim, H.B., Kang, D.-K., 2014a. Assessment of fecal bacterial diversity among healthy piglets during the weaning transition. J. Gen. Appl. Microbiol. 60, 140-146.
Pajarillo, E.A.B., Chae, J.P., Balolong, M.P., Kim, H.B., Seo, K.S., Kang, D.K., 2014b. Pyrosequencing-based Analysis of Fecal Microbial Communities in Three Purebred Pig Lines. J. Microbiol. 52, 646-651.
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Slifierz, M.J., Friendship, R.M., Weese, J.S., 2015. Longitudinal study of the early-life fecal and nasal microbiotas of the domestic pig. BMC Microbiol 15.
Sommer, F., Baeckhed, F., 2013. The gut microbiota - masters of host development and physiology. Nature reviews. Microbiology 11, 227-238.
Starke, I.C., Pieper, R., Neumann, K., Zentek, J., Vahjen, W., 2014. The impact of high dietary zinc oxide on the development of the intestinal microbiota in weaned piglets. FEMS Microbiol. Ecol. 87, 416-427.
Sugiharto, Jensen, B.B., Lauridsen, C., 2012. Development of an ex vivo model for investigating the bacterial association to the gut epithelium of pigs. J. Anim. Sci. 90 Suppl 4, 397-399.
Sugiharto, S., Lauridsen, C., 2014. Influence of age on the susceptibility of piglets to Escherichia coli O138:F18. J. Indonesian Trop. Anim. Agric. 39, 159-166.
Sugiharto, S., Poulsen, A.S.R., Canibe, N., Lauridsen, C., 2015. Effect of bovine colostrum feeding in comparison with milk replacer and natural feeding on the immune responses and colonisation of enterotoxigenic Escherichia coli in the intestinal tissue of piglets. Br. J. Nutr. 113, 923-934.
Thompson, C.L., Wang, B., Holmes, A.J., 2008. The immediate environment during postnatal development has long-term impact on gut community structure in pigs. ISME J. 2, 739-748.
Williams, B.A., Bosch, M.W., Awati, A., Konstantinov, S.R., Smidt, H., Akkermans, A.D.L., Verstegen, M.W.A., Tamminga, S., 2005. In vitro assessment of gastrointestinal tract (GIT) fermentation in pigs: Fermentable substrates and microbial activity. Anim. Res. 54, 191-201.
Chapter 5. Manuscript 2 93
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Tables:
Table 1 Piglet weight (kg) at day 0, 7, 14, 21, 28 and 34 of age1
Days of age Genotype2 p-value
SENS RES G3 A4 G*A 0.002 <.0001 0.002
0 1.3 (0.0-4.4) 1.7 (0.0-4.1) 7 2.9 (0.0-5.4) 2.4 (0.0-4.8)
14 4.0 (1.0-7.1) 3.8 (1.3-6.2) 21 5.9 (2.9-9.0) 5.2 (2.7-7.6) 28 8.1a (5.1-11.3) 6.8b (4.3-9.2) 34 7.8a (4.7-10.9) 6.7b (4.2-9.1)
1 Values are presented as least square means and 95% confidence intervals (in parentheses). 2 SENS = M307GA; RES = M307AA. Number of piglets: SENS=10; RES=7. 3 G = Genotype. 4 A = Age. a,b Values with different superscripts within a row are significantly different (p<0.05).
! !
94 Chapter 5. Manuscript 2
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Table 2 Enumerations (log cfu/g sample) of selected microbial groups in faeces from piglets at 5, 7, 14, 21,
28 and 34 days of age1
Genotype2 p-value
Item SENS RES # G3 A4 G*A Enterobacteriaceae 0.30 0.38 0.08
5 8.5 (7.7-9.2) 8.7 (8.0-9.5) 7 8.3 (7.5-9.0) 8.6 (7.9-9.3) 14 8.3 (7.5-9.1) 8.1 (7.4-8.8) 21 8.2 (7.4-8.9) 8.1 (7.4-8.8) 28 8.4 (7.7-9.1) 7.9 (7.2-8.6) 34 8.9 (8.2-9.6) 8.0 (7.3-8.7)
Haemolytic bacteria 0.02 <.0001 0.03 5 <6.2A (2) (5.4-7.1) <6.3AB (1) (5.2-7.5) 7 <6.2A (9) (5.8-6.6) <6.3A (5) (5.8-6.8) 14 <6.3A (3) (5.7-6.9) <6.3A (7) (5.8-6.7) 21 <6.3A (7) (5.8-6.7) <6.2A (6) (5.8-6.7) 28 <6.3A (7) (5.9-6.7) <6.3A (6) (5.8-6.7) 34 8.7aB (8.3-9.1) <7.5bB (3) (7.1-7.9)
Clostridium perfringens 0.60 <.0001 0.97 5 7.7 3.3-12.0 7.5 3.5-11.5 ab 7 <8.0 (1) 3.2-12.7 7.8 3.5-12.1 b 14 7.5 3.6-11.4 <7.4 (1) 3.2-11.6 ab 21 <6.6 (1) 2.2-11.1 6.5 2.2-10.8 a 28 5.2 0.7-9.6 <5.0 (1) 0.7-9.3 c 34 <3.0 (6) 0-7.4 <2.8 (3) 0-7.2 d
Lactic acid bacteria 0.06 0.0002 0.25 5 9.7 9.3-10.0 9.4 9.0-9.8 a 7 9.6 9.3-10.0 9.4 9.0-9.7 a 14 8.9 8.5-9.3 8.7 8.3-9.0 b 21 9.1 8.8-9.4 8.8 8.5-9.2 a 28 9.0 8.6-9.3 8.7 8.4-9.0 b 34 8.8 8.5-9.2 8.6 8.2-8.9 b
Total anaerobic bacteria 0.86 0.43 0.36 5 9.9 9.7-10.2 9.9 9.7-10.2 7 9.7 9.4-9.9 9.7 9.4-9.9 14 9.7 9.5-10.0 9.7 9.5-10.0 21 9.7 9.5-10.0 9.8 9.5-10.0 28 9.8 9.6-10.0 9.8 9.6-10.0 34 9.8 9.5-10.0 9.8 9.6-10.0
Chapter 5. Manuscript 2 95
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1 Values are presented as least square means and 95% confidence intervals (in parentheses). 2 SENS = M307GA; RES = M307AA. Number of piglets: SENS=7, except day 5 (n=8), day 7 (n=9) and day 14
(n=4); RES=6, except day 5 (n=4), day 7 (n=5) and day 14 (n=7). 3 G = Genotype. 4 A = Age.
#: Rows with different letters, within a microbial group, are significantly different (p<0.05). A,B Values with different superscripts within a column are significantly different (p<0.05). a,b Values with different superscripts within a row are significantly different (p<0.05).
< : Indicates that at least one of the observations used to calculate the least square mean was below detection
level. Numbers in brackets indicate the number of samples below detection level.
96 Chapter 5. Manuscript 2
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Table 3 Short-chain fatty acid concentrations (mmol/kg sample) in faeces from piglets at 5, 7, 14, 21, 28
and 34 days of age1
Genotype2 p-value Item SENS RES # G3 A4 G*A Acetic acid 0.44 <.0001 0.23
5 42.3 (32.0-52.7) 39.5 (29.5-49.6) a 7 38.1 (29.0-47.1) 35.3 (26.0-44.6) a 14 32.7 (22.1-43.4) 29.9 (20.4-39.4) a 21 38.0 (28.7-47.4) 35.2 (26.2-44.2) a 28 46.5 (37.1-55.9) 43.7 (34.7-52.7) ab 34 58.9 (49.9-67.9) 56.1 (47.2-65.0) b
Propionic acid 0.19 <.0001 0.13 5 12.4 (8.1-16.7) 10.4 (6.2-14.6) a 7 10.9 (7.2-14.6) 8.9 (5.1-12.8) a 14 12.3 (7.8-18.8) 10.3 (6.4-14.2) a 21 12.6 (8.8-16.4) 10.6 (6.9-14.3) a 28 12.0 (8.2-15.9) 10.0 (6.4-13.7) a 34 22.4 (18.7-26.0) 20.4 (16.7-24.0) b
Butyric acid 0.09 0.09 0.48 5 6.6 (3.5-10.7) 4.6 (2.2-8.0) 7 5.3 (2.9-8.4) 3.5 (1.6-6.2) 14 3.0 (1.0-6.1) 1.7 (0.5-3.8) 21 2.5 (0.9-4.8) 1.3 (0.3-3.1) 28 4.0 (1.9-6.9) 2.5 (1.0-4.7) 34 5.3 (3.0-8.4) 3.6 (1.7-6.1)
A+P+B5 0.23 <.0002 0.14 5 61.7 (46.5-77.0) 55.4 (40.6-70.1) a 7 56.0 (43.0-69.0) 49.7 (36.1-63.2) a 14 49.4 (33.5-65.2) 43.0 (29.2-56.8) a 21 54.3 (40.8-67.8) 48.0 (35.0-61.0) a 28 63.8 (50.3-77.3) 57.4 (44.4-70.4) ab 34 87.1 (74.3-100.0) 80.7 (67.9-93.5) b
1 Values are presented as least square means and 95% confidence intervals (in parentheses). 2 SENS = M307GA; RES = M307AA. Number of piglets: SENS=10, except, day 5 (n=8), day 7 (n=9) and day
14 (n=7); RES=6, except day 5 (n=4), day 7 (n=5) and day 14 (n=7). 3 G = Genotype. 4 A = Age. 5 A+P+B = acetic + propionic + butyric acid.
#: Rows with different letters, within a short-chain fatty acid group, are significantly different (p<0.05).
Chapter 5. Manuscript 2 97
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Table 4 Counts (log cfu/g sample) of selected microbial groups in digesta from the gastrointestinal tract of
34 days old piglets (one week post-weaning)1
Genotype2 p-value Item SENS RES G3 S4 G*S Enterobacteriaceae 0.002 <.0001 0.004
Stomach 5.6 (4.8-6.5) 5.9 (5.0-6.7) Distal small intestine 8.8a (7.9-9.7) 7.5b (6.6-8.3) Caecum 8.8a (8.0-9.7) 7.8b (7.0-8.7) Mid colon 8.9a (8.0-9.8) 7.9b (7.1-8.8)
Haemolytic bacteria 0.02 <.0001 0.28 Stomach 5.2 (4.5-6.0) <4.4 (1) (3.8-5.1) Distal small intestine 7.9 (6.8-9.2) <6.8 (4) (5.8-7.9) Caecum 8.6 (7.4-10) <7.3 (4) (6.3-8.5) Mid colon 8.6 (7.4-10) <7.3 (3) (6.3-8.5)
Clostridium perfringens 0.93 0.63 0.24 Stomach <3.2 (1) (2.6-3.7) 3.2 (2.6-3.7) Distal small intestine <2.8 (5) (2.3-3.4) <2.8 (4) (2.3-3.4) Caecum <3.0 (4) (2.5-3.6) <3.0 (4) (2.5-3.6) Mid colon <3.0 (4) (2.4-3.5) <3.0 (4) (2.4-3.5)
Lactic acid bacteria 0.29 <.0001 0.40 Stomach 9.0 (8.4-9.6) 8.7 (8.1-9.3) Distal small intestine 8.3 (7.8-8.9) 8.0 (7.3-8.6) Caecum 8.6 (8.0-9.2) 8.3 (7.6-8.8) Mid colon 8.6 (8.0-9.2) 8.3 (7.7-8.9)
Total anaerobic bacteria 0.02 <.0001 0.01 Stomach 9.0 (8.7-9.4) 9.3 (9.0-9.6) Distal small intestine 8.9a (8.6-9.3) 8.3b (8.0-8.6) Caecum 9.3 (9.0-9.7) 9.2 (8.9-9.5) Mid colon 9.4 (9.1-9.7) 9.4 (9.1-9.7)
1 Samples from the stomach, distal small intestine, caecum and mid colon were analysed. Values are
presented as least square means and 95% confidence intervals (in parantheses). 2 SENS = M307GA; RES = M307AA . Number of piglets: SENS n=10; RES n=7. 3 G = Genotype. 4 S = Intestinal segment. a,b Values with different superscripts within a row are significantly different (p<0.05).
< : Indicates that at least one of the observations used to calculate the least square mean was below detection
level. Numbers in brackets indicate the number of samples below detection levels.
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98 Chapter 5. Manuscript 2
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Table 5 Organic acid concentrations in digesta (mmol/kg wet sample) from the gastrointestinal tract of 34
days old piglets (one week post-weaning)1
Genotype2 p-value Item SENS RES G3 S4 G*S Lactic acid5 0.99 <.0001 0.91
Stomach 19.6 (5.3-62.7) 19.5 (6.9-50.2) Distal small intestine 5.6 (0.6-20.4) 5.5 (1.1-16.2)
Acetic acid 0.01 <.0001 0.01
Stomach 21.1 (4.2-37.9) 26.8 (11.6-41.9) Distal small intestine 4.2 (0.0-21.0) 5.4 (0.0-20.6) Caecum 47.6 (30.7-64.4) 39.0 (24.1-53.9) Mid colon 54.5a (37.7-71.4) 39.4b (24.4-54.4)
Propionic acid6 0.02 0.0001 0.01
Stomach 8.1 (2.7-13.6) 11.7 (6.9-16.5) Caecum 16.7 (11.3-22.2) 13.2 (8.1-18.3) Mid colon 16.6 (11.1-22.0) 11.9 (6.9-16.9)
Butyric acid7 0.90 <.0001 0.83 Stomach 8.3 (1.4-48.0) 8.1 (0.5-41.2) Caecum 2.3 (6.1-30.3) 2.2 (3.8-24.0) Mid colon 1.9 (6.8-29.0) 1.8 (4.6-23.6)
1 Samples from the stomach, distal small intestine, caecum and mid colon were analysed. Values are
presented as least square means and 95% confidence interval (in parentheses). 2 SENS = M307GA; RES = M307AA. Number of piglets: SENS n=10, except the distal small intestine (n=9);
RES n=7, except the mid colon (n=6) and caecum (n=5). 3 G = Genotype. 4 S = Intestinal segment. 5 Samples from the caecum and mid colon had values below detection level. 6 Samples from the distal small intestine had values below detection level. No superscripts as the G*S
interaction was non-significant after pairwise comparisons using Bonferroni correction. 7 Samples from the distal small intestine had values below detection level. a,b Values with different superscripts within a row are significantly different (p<0.05).
Chapter 5. Manuscript 2 99
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S1 Table Dry matter content (%) and pH of digesta from the gastrointestinal tract of 34 days old piglets (one
week post-weaning)1
Genotype2 p-value Item SENS RES G3 S4 G*S Dry matter 0.33 <.0001 0.34
Stomach 25.1 (20.8-29.5) 24.0 (20.1-27.9) Distal small intestine 6.0 (1.7-10.3) 4.9 (1.0-8.7) Caecum 7.1 (3.2-10.9) 5.9 (2.2-9.6) Mid colon 7.8 (3.6-11.9) 6.6 (2.9-10.4)
pH 0.86 <.0001 0.55 Stomach 3.0 (2.7-3.2) 3.0 (2.7-3.2) Distal small intestine 7.3 (7.0-7.6) 7.3 (7.0-7.5) Caecum 6.7 (6.4-6.9) 6.7 (6.4-6.9) Mid colon 6.9 (6.7-7.2) 6.9 (6.7-7.2)
1 Samples from the stomach, distal small intestine, caecum and mid colon were analysed. Values are
presented as least square means and 95% confidence intervals (in parentheses). 2 SENS = M307GA; RES = M307AA. Number of piglets: SENS=10; RES=7, except: pH SENS distal small
intestine (n=9); dry matter SENS piglets caecum and mid colon (n=5) and RES caecum (n=6). 3 G = Genotype. 4 S = Intestinal segment.
!
100 Chapter 5. Manuscript 2
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Figures:
Fig. 1. Heatmap showing relative abundances (%) of the (A) nine most abundant phyla (relative
abundance > 1%) and (B) 15 most abundant families in faecal samples from ETEC F18-sensitive
(SENS = M307GA) and resistant (RES = M307AA) piglets at 5, 7, 14, 21, 28 and 34 days of age.
Colours represent relative abundances. Number of samples: SENS = 35; RES=30.
Fig. 2. Principal Component Analysis of square root transformed OTU abundances displaying PC1
and PC2. Points are colored for genotype and grouped according to age. Sensitive = M307GA
(SENS); resistant = M307AA (RES). Number of samples: SENS = 35; RES=30.
Fig. 3. Principal Component Analysis of square root transformed OTU abundances displaying PC1
and PC3. Points colored for age. Number of samples: Sensitive = 35; resistant =30.
Fig. 4. Boxplots showing the 25 most significantly changed OTUs when comparing the microbial
community structure of (A) 5 and 28 days old piglets and (B) 28 and 34 days old piglets. Number of
samples: Sensitive = 35; resistant =30.
Fig. S1. Boxplots showing (A) the observed and (B) estimated species richness and (C) the
Shannon diversity index for sensitive and resistant piglets at 5, 7, 14, 21, 28 and 34 days of age.
Number of samples: Sensitive = 35; resistant =30.
Fig. S2. Heatmap showing the relative abundances (%) of the 25 most abundant genera in faecal
samples from ETEC F18-sensitive (SENS = M307GA) and resistant (RES = M307AA) piglets at 5, 7,
14, 21, 28 and 34 days of age. Colours represent relative abundances. Number of samples: SENS =
35; RES=30.
Chapter 5. Manuscript 2 101
5 7 14 21 28 34
0.4
0
0
1.1
0
0
0.7
92.8
5
0.6
0
0
0
14.1
0
0.8
0.7
83.8
1
35.4
3.3
0
58.5
0
1.2
0
0.3
0.3
0
27.1
70
0.9
0
1.6
0
0
0.1
43.8
51.3
0.4
0.6
0.6
0
1.9
0
37.4
0.3
60.2
0
0.5
0.6
0
0
0.6
72.6
0.8
0
25.2
0.2
0
0.7
0
0.2
2.3
60
0
0
36.6
0.2
0.4
0
0.3
0
0.6
0
0
92.9
0.3
0.1
5.7
0.2
0
0
12.6
0.9
3.5
0.1
82.3
0.3
0.1
1.4
81.1
5
1.1
0.2
9.1
0.7
0.1
0.1
0
5.5
0.3
0
0
84.4
7.2
0.4
1.7
k__Unassigned_OTU_188
Spirochaetes
WPS−2
k__Unassigned_OTU_57
Actinobacteria
Fusobacteria
Proteobacteria
Bacteroidetes
Firmicutes
5 RES 5 SENS 7 RES 7 SENS 14 RES14 SENS 21 RES21 SENS 28 RES28 SENS 34 RES34 SENS
0.1
1.0
10.0
% ReadAbundance
Day 5 Day 7 Day 14 Day 21 Day 28 Day 34
OTU 57
OTU 188
RES SENS RES SENS RES SENS RES SENS RES SENS RES SENS
5 7 14 21 28 34
0.7
31.4
8.6
1
28.5
0.9
0.2
0.5
2.7
2.5
0
0.7
0
17.4
0.4
4.3
23.1
26.1
2.6
0.2
4.9
0.1
0
2.1
0.1
3.4
3
22.8
0.1
0.7
6.9
0.8
8
14.6
4.9
9.6
0.1
2.2
1.2
9.6
1.1
22.6
2.8
4.7
0.1
2.5
0.7
36.5
1.5
5.4
0.1
0.9
0.3
1.1
1.8
19.5
0.5
1.7
19.3
0.2
3.9
2.2
6.8
3.7
7.7
2.1
15.8
1.2
14.8
15.1
1.4
0.6
2.7
1.3
0.8
0.1
4.4
2.3
1.1
1.7
19.9
2
0.7
19.1
1
0.2
8.8
0.4
20.8
0.3
0.2
13.3
0.7
1.1
4.1
5.1
0.6
21.5
0.5
16.8
8.1
2.8
0.2
8
0.2
0.1
0.3
6.9
3.8
1
0.2
0.8
0.8
22.3
2.3
14.1
0.2
29.5
5.7
0.1
0.1
0.1
0.9
0.4
1.2
28.6
3.1
2.2
1.4
0.3
40.3
0.2
1.2
0.1
0.2
1
1.3
0.2
0.3
26
4.3
3.2
1.3
9.3
24.2
0.2
6.1
4.6
1.1
2.9
1.5
6.3
1
27.7
17.1
0.5
0.2
0.2
0.7
0
0
7.2
11.2
1.1
0.3
3.5
0.3
0.2
17.3
3.6
0
5.9
16
0.1
0
30.9
0.6
0
0.5
5.3
Bacteroidetes; S24−7
Bacteroidetes; [Paraprevotellaceae]
Proteobacteria; Enterobacteriaceae
Bacteroidetes; Rikenellaceae
Bacteroidetes; [Odoribacteraceae]
Bacteroidetes; Bacteroidaceae
Firmicutes; Erysipelotrichaceae
Firmicutes; Veillonellaceae
Firmicutes; o__Clostridiales_OTU_17
Bacteroidetes; o__Bacteroidales_OTU_2
Firmicutes; Lachnospiraceae
Bacteroidetes; Prevotellaceae
Firmicutes; Lactobacillaceae
Firmicutes; Clostridiaceae
Firmicutes; Ruminococcaceae
5 RES 5 SENS 7 RES 7 SENS 14 RES14 SENS 21 RES21 SENS 28 RES28 SENS 34 RES34 SENS
0.1
1.0
10.0
% ReadAbundance
Day 5 Day 7 Day 14 Day 21 Day 28 Day 34
RES SENS RES SENS RES SENS RES SENS RES SENS RES SENS
Bacteroidetes; order Bacteroidales OTU 2
Firmicutes; order Clostridiales OTU 17
(a)
(b)
Figure 1
102 Chapter 5. Manuscript 2
−2.5
0.0
2.5
5.0
−5.0 −2.5 0.0 2.5 5.0PC1 [15%]
PC3
[9.2
%]
age
d_05
d_07
d_14
d_21
d_28
d_34
genotype
resistant
sensitive
PC
3 - v
aria
tion
expl
aine
d 9.
2%
PC1 - variation explained 15%
−2.5
0.0
2.5
5.0
−5.0 −2.5 0.0 2.5 5.0PC1 [15%]
PC3
[9.2
%]
age
d_05
d_07
d_14
d_21
d_28
d_34
genotype
resistant
sensitive
5
7 days
age (days)
714
21
28
34
5 7 14 21 28 34
−6
−3
0
3
−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0PC1 [15%]
PC2
[10%
] genotype
RES
SENS
Day 5 Day 7 Day 14 Day 21 Day 28 Day 34
PC1 - variation explained 15%
PC
2 - v
aria
tion
expl
aine
d 10
%
5 7 14 21 28 34
−6
−3
0
3
−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0PC1 [15%]
PC2
[10%
] genotype
RES
SENS
5 7 14 21 28 34
−6
−3
0
3
−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0PC1 [15%]
PC2
[10%
] genotype
RES
SENS
Day 5 Day 7 Day 14 Day 21 Day 28 Day 34
PC1 - variation explained 15%
PC
2 - v
aria
tion
expl
aine
d 10
%
5 7 14 21 28 34
−6
−3
0
3
−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0−5.0 −2.5 0.0 2.5 5.0PC1 [15%]
PC2
[10%
] genotype
RES
SENS
Figure 2
Figure 3
Chapter 5. Manuscript 2 103
Firmicutes; o__Clostridiales_OTU_346; OTU_346
Firmicutes; o__Clostridiales_OTU_19; OTU_19
Actinobacteria; Corynebacteriaceae; OTU_333
Firmicutes; Ruminococcaceae; OTU_214
Firmicutes; Clostridiaceae; OTU_172
Bacteroidetes; [Paraprevotellaceae]; OTU_110
Firmicutes; o__Clostridiales_OTU_271; OTU_271
Firmicutes; o__Clostridiales_OTU_1680; OTU_1680
Firmicutes; Ruminococcaceae; OTU_887
Firmicutes; Christensenellaceae; OTU_562
Bacteroidetes; o__Bacteroidales_OTU_95; OTU_95
Firmicutes; Christensenellaceae; OTU_230
Firmicutes; Clostridiaceae; OTU_525
Firmicutes; o__Clostridiales_OTU_1229; OTU_1229
Firmicutes; o__Clostridiales_OTU_2240; OTU_2240
Firmicutes; [Acidaminobacteraceae]; OTU_2665
Firmicutes; Ruminococcaceae; OTU_626
Firmicutes; o__Clostridiales_OTU_260; OTU_260
Firmicutes; Ruminococcaceae; OTU_1349
Firmicutes; o__Clostridiales_OTU_1507; OTU_1507
Firmicutes; Ruminococcaceae; OTU_1530
Firmicutes; Veillonellaceae; OTU_46
Firmicutes; Lachnospiraceae; OTU_2737
Firmicutes; Ruminococcaceae; OTU_15
Firmicutes; o__Clostridiales_OTU_28; OTU_28
0.01 0.10 1.00 10.00Read Abundance (%)
age
28
34
Firmicutes; o__Clostridiales_OTU_346; OTU_346
Firmicutes; o__Clostridiales_OTU_19; OTU_19
Actinobacteria; Corynebacteriaceae; OTU_333
Firmicutes; Ruminococcaceae; OTU_214
Firmicutes; Clostridiaceae; OTU_172
Bacteroidetes; [Paraprevotellaceae]; OTU_110
Firmicutes; o__Clostridiales_OTU_271; OTU_271
Firmicutes; o__Clostridiales_OTU_1680; OTU_1680
Firmicutes; Ruminococcaceae; OTU_887
Firmicutes; Christensenellaceae; OTU_562
Bacteroidetes; o__Bacteroidales_OTU_95; OTU_95
Firmicutes; Christensenellaceae; OTU_230
Firmicutes; Clostridiaceae; OTU_525
Firmicutes; o__Clostridiales_OTU_1229; OTU_1229
Firmicutes; o__Clostridiales_OTU_2240; OTU_2240
Firmicutes; [Acidaminobacteraceae]; OTU_2665
Firmicutes; Ruminococcaceae; OTU_626
Firmicutes; o__Clostridiales_OTU_260; OTU_260
Firmicutes; Ruminococcaceae; OTU_1349
Firmicutes; o__Clostridiales_OTU_1507; OTU_1507
Firmicutes; Ruminococcaceae; OTU_1530
Firmicutes; Veillonellaceae; OTU_46
Firmicutes; Lachnospiraceae; OTU_2737
Firmicutes; Ruminococcaceae; OTU_15
Firmicutes; o__Clostridiales_OTU_28; OTU_28
0.01 0.10 1.00 10.00Read Abundance (%)
age
28
34
age (days)
Firmicutes; order Clostridiales OTU_28
Read abundance (%)
Firmicutes; order Clostridiales OTU_1507
Firmicutes; order Clostridiales OTU_260
Firmicutes; order Clostridiales OTU_2240
Firmicutes; order Clostridiales OTU_1229
Firmicutes; order Clostridiales OTU_346
Firmicutes; order Clostridiales OTU_19
Firmicutes; order Clostridiales OTU_271
Firmicutes; order Clostridiales OTU_1680
Bacteroidetes; order Bacteroidales OTU_95
Firmicutes; Clostridiaceae; OTU_1808
Firmicutes; o__Clostridiales_OTU_801; OTU_801
Firmicutes; Veillonellaceae; OTU_132
Firmicutes; Peptostreptococcaceae; OTU_793
Firmicutes; Clostridiaceae; OTU_2082
Firmicutes; Streptococcaceae; OTU_106
Actinobacteria; o__Actinomycetales_OTU_276; OTU_276
Actinobacteria; Coriobacteriaceae; OTU_376
Firmicutes; Lachnospiraceae; OTU_656
Firmicutes; Clostridiaceae; OTU_2875
Firmicutes; Lactobacillaceae; OTU_23
Firmicutes; Christensenellaceae; OTU_65
Firmicutes; Ruminococcaceae; OTU_67
Firmicutes; Clostridiaceae; OTU_651
Actinobacteria; Actinomycetaceae; OTU_223
Firmicutes; Ruminococcaceae; OTU_371
Firmicutes; Lactobacillaceae; OTU_79
Firmicutes; o__Clostridiales_OTU_51; OTU_51
Firmicutes; Clostridiaceae; OTU_2881
Firmicutes; o__Clostridiales_OTU_140; OTU_140
Firmicutes; o__Clostridiales_OTU_28; OTU_28
Firmicutes; o__Clostridiales_OTU_1835; OTU_1835
Firmicutes; Clostridiaceae; OTU_11
Firmicutes; Clostridiaceae; OTU_556
Firmicutes; Ruminococcaceae; OTU_9
0.01 0.10 1.00 10.00Read Abundance (%)
age
5
28
Firmicutes; Clostridiaceae; OTU_1808
Firmicutes; o__Clostridiales_OTU_801; OTU_801
Firmicutes; Veillonellaceae; OTU_132
Firmicutes; Peptostreptococcaceae; OTU_793
Firmicutes; Clostridiaceae; OTU_2082
Firmicutes; Streptococcaceae; OTU_106
Actinobacteria; o__Actinomycetales_OTU_276; OTU_276
Actinobacteria; Coriobacteriaceae; OTU_376
Firmicutes; Lachnospiraceae; OTU_656
Firmicutes; Clostridiaceae; OTU_2875
Firmicutes; Lactobacillaceae; OTU_23
Firmicutes; Christensenellaceae; OTU_65
Firmicutes; Ruminococcaceae; OTU_67
Firmicutes; Clostridiaceae; OTU_651
Actinobacteria; Actinomycetaceae; OTU_223
Firmicutes; Ruminococcaceae; OTU_371
Firmicutes; Lactobacillaceae; OTU_79
Firmicutes; o__Clostridiales_OTU_51; OTU_51
Firmicutes; Clostridiaceae; OTU_2881
Firmicutes; o__Clostridiales_OTU_140; OTU_140
Firmicutes; o__Clostridiales_OTU_28; OTU_28
Firmicutes; o__Clostridiales_OTU_1835; OTU_1835
Firmicutes; Clostridiaceae; OTU_11
Firmicutes; Clostridiaceae; OTU_556
Firmicutes; Ruminococcaceae; OTU_9
0.01 0.10 1.00 10.00Read Abundance (%)
age
5
28
age (days)
Read abundance (%)
Firmicutes; order Clostridiales OTU_1835
Firmicutes; order Clostridiales OTU_140
Firmicutes; order Clostridiales OTU_51
Firmicutes; order Clostridiales OTU_28
Firmicutes; order Clostridiales OTU_801
Actinobacteria; order Actinomycetales OTU_276
(a)
(b)
Firmicutes; o__Clostridiales_OTU_346; OTU_346
Firmicutes; o__Clostridiales_OTU_19; OTU_19
Actinobacteria; Corynebacteriaceae; OTU_333
Firmicutes; Ruminococcaceae; OTU_214
Firmicutes; Clostridiaceae; OTU_172
Bacteroidetes; [Paraprevotellaceae]; OTU_110
Firmicutes; o__Clostridiales_OTU_271; OTU_271
Firmicutes; o__Clostridiales_OTU_1680; OTU_1680
Firmicutes; Ruminococcaceae; OTU_887
Firmicutes; Christensenellaceae; OTU_562
Bacteroidetes; o__Bacteroidales_OTU_95; OTU_95
Firmicutes; Christensenellaceae; OTU_230
Firmicutes; Clostridiaceae; OTU_525
Firmicutes; o__Clostridiales_OTU_1229; OTU_1229
Firmicutes; o__Clostridiales_OTU_2240; OTU_2240
Firmicutes; [Acidaminobacteraceae]; OTU_2665
Firmicutes; Ruminococcaceae; OTU_626
Firmicutes; o__Clostridiales_OTU_260; OTU_260
Firmicutes; Ruminococcaceae; OTU_1349
Firmicutes; o__Clostridiales_OTU_1507; OTU_1507
Firmicutes; Ruminococcaceae; OTU_1530
Firmicutes; Veillonellaceae; OTU_46
Firmicutes; Lachnospiraceae; OTU_2737
Firmicutes; Ruminococcaceae; OTU_15
Firmicutes; o__Clostridiales_OTU_28; OTU_28
0.01 0.10 1.00 10.00Read Abundance (%)
age
28
34
Firmicutes; o__Clostridiales_OTU_346; OTU_346
Firmicutes; o__Clostridiales_OTU_19; OTU_19
Actinobacteria; Corynebacteriaceae; OTU_333
Firmicutes; Ruminococcaceae; OTU_214
Firmicutes; Clostridiaceae; OTU_172
Bacteroidetes; [Paraprevotellaceae]; OTU_110
Firmicutes; o__Clostridiales_OTU_271; OTU_271
Firmicutes; o__Clostridiales_OTU_1680; OTU_1680
Firmicutes; Ruminococcaceae; OTU_887
Firmicutes; Christensenellaceae; OTU_562
Bacteroidetes; o__Bacteroidales_OTU_95; OTU_95
Firmicutes; Christensenellaceae; OTU_230
Firmicutes; Clostridiaceae; OTU_525
Firmicutes; o__Clostridiales_OTU_1229; OTU_1229
Firmicutes; o__Clostridiales_OTU_2240; OTU_2240
Firmicutes; [Acidaminobacteraceae]; OTU_2665
Firmicutes; Ruminococcaceae; OTU_626
Firmicutes; o__Clostridiales_OTU_260; OTU_260
Firmicutes; Ruminococcaceae; OTU_1349
Firmicutes; o__Clostridiales_OTU_1507; OTU_1507
Firmicutes; Ruminococcaceae; OTU_1530
Firmicutes; Veillonellaceae; OTU_46
Firmicutes; Lachnospiraceae; OTU_2737
Firmicutes; Ruminococcaceae; OTU_15
Firmicutes; o__Clostridiales_OTU_28; OTU_28
0.01 0.10 1.00 10.00Read Abundance (%)
age
28
34
age (days)
Firmicutes; order Clostridiales OTU_28
Read abundance (%)
Firmicutes; order Clostridiales OTU_1507
Firmicutes; order Clostridiales OTU_260
Firmicutes; order Clostridiales OTU_2240
Firmicutes; order Clostridiales OTU_1229
Firmicutes; order Clostridiales OTU_346
Firmicutes; order Clostridiales OTU_19
Firmicutes; order Clostridiales OTU_271
Firmicutes; order Clostridiales OTU_1680
Bacteroidetes; order Bacteroidales OTU_95
Firmicutes; Clostridiaceae; OTU_1808
Firmicutes; o__Clostridiales_OTU_801; OTU_801
Firmicutes; Veillonellaceae; OTU_132
Firmicutes; Peptostreptococcaceae; OTU_793
Firmicutes; Clostridiaceae; OTU_2082
Firmicutes; Streptococcaceae; OTU_106
Actinobacteria; o__Actinomycetales_OTU_276; OTU_276
Actinobacteria; Coriobacteriaceae; OTU_376
Firmicutes; Lachnospiraceae; OTU_656
Firmicutes; Clostridiaceae; OTU_2875
Firmicutes; Lactobacillaceae; OTU_23
Firmicutes; Christensenellaceae; OTU_65
Firmicutes; Ruminococcaceae; OTU_67
Firmicutes; Clostridiaceae; OTU_651
Actinobacteria; Actinomycetaceae; OTU_223
Firmicutes; Ruminococcaceae; OTU_371
Firmicutes; Lactobacillaceae; OTU_79
Firmicutes; o__Clostridiales_OTU_51; OTU_51
Firmicutes; Clostridiaceae; OTU_2881
Firmicutes; o__Clostridiales_OTU_140; OTU_140
Firmicutes; o__Clostridiales_OTU_28; OTU_28
Firmicutes; o__Clostridiales_OTU_1835; OTU_1835
Firmicutes; Clostridiaceae; OTU_11
Firmicutes; Clostridiaceae; OTU_556
Firmicutes; Ruminococcaceae; OTU_9
0.01 0.10 1.00 10.00Read Abundance (%)
age
5
28
Firmicutes; Clostridiaceae; OTU_1808
Firmicutes; o__Clostridiales_OTU_801; OTU_801
Firmicutes; Veillonellaceae; OTU_132
Firmicutes; Peptostreptococcaceae; OTU_793
Firmicutes; Clostridiaceae; OTU_2082
Firmicutes; Streptococcaceae; OTU_106
Actinobacteria; o__Actinomycetales_OTU_276; OTU_276
Actinobacteria; Coriobacteriaceae; OTU_376
Firmicutes; Lachnospiraceae; OTU_656
Firmicutes; Clostridiaceae; OTU_2875
Firmicutes; Lactobacillaceae; OTU_23
Firmicutes; Christensenellaceae; OTU_65
Firmicutes; Ruminococcaceae; OTU_67
Firmicutes; Clostridiaceae; OTU_651
Actinobacteria; Actinomycetaceae; OTU_223
Firmicutes; Ruminococcaceae; OTU_371
Firmicutes; Lactobacillaceae; OTU_79
Firmicutes; o__Clostridiales_OTU_51; OTU_51
Firmicutes; Clostridiaceae; OTU_2881
Firmicutes; o__Clostridiales_OTU_140; OTU_140
Firmicutes; o__Clostridiales_OTU_28; OTU_28
Firmicutes; o__Clostridiales_OTU_1835; OTU_1835
Firmicutes; Clostridiaceae; OTU_11
Firmicutes; Clostridiaceae; OTU_556
Firmicutes; Ruminococcaceae; OTU_9
0.01 0.10 1.00 10.00Read Abundance (%)
age
5
28
age (days)
Read abundance (%)
Firmicutes; order Clostridiales OTU_1835
Firmicutes; order Clostridiales OTU_140
Firmicutes; order Clostridiales OTU_51
Firmicutes; order Clostridiales OTU_28
Firmicutes; order Clostridiales OTU_801
Actinobacteria; order Actinomycetales OTU_276
(a)
(b)
Firmicutes; o__Clostridiales_OTU_346; OTU_346
Firmicutes; o__Clostridiales_OTU_19; OTU_19
Actinobacteria; Corynebacteriaceae; OTU_333
Firmicutes; Ruminococcaceae; OTU_214
Firmicutes; Clostridiaceae; OTU_172
Bacteroidetes; [Paraprevotellaceae]; OTU_110
Firmicutes; o__Clostridiales_OTU_271; OTU_271
Firmicutes; o__Clostridiales_OTU_1680; OTU_1680
Firmicutes; Ruminococcaceae; OTU_887
Firmicutes; Christensenellaceae; OTU_562
Bacteroidetes; o__Bacteroidales_OTU_95; OTU_95
Firmicutes; Christensenellaceae; OTU_230
Firmicutes; Clostridiaceae; OTU_525
Firmicutes; o__Clostridiales_OTU_1229; OTU_1229
Firmicutes; o__Clostridiales_OTU_2240; OTU_2240
Firmicutes; [Acidaminobacteraceae]; OTU_2665
Firmicutes; Ruminococcaceae; OTU_626
Firmicutes; o__Clostridiales_OTU_260; OTU_260
Firmicutes; Ruminococcaceae; OTU_1349
Firmicutes; o__Clostridiales_OTU_1507; OTU_1507
Firmicutes; Ruminococcaceae; OTU_1530
Firmicutes; Veillonellaceae; OTU_46
Firmicutes; Lachnospiraceae; OTU_2737
Firmicutes; Ruminococcaceae; OTU_15
Firmicutes; o__Clostridiales_OTU_28; OTU_28
0.01 0.10 1.00 10.00Read Abundance (%)
age
28
34
Firmicutes; o__Clostridiales_OTU_346; OTU_346
Firmicutes; o__Clostridiales_OTU_19; OTU_19
Actinobacteria; Corynebacteriaceae; OTU_333
Firmicutes; Ruminococcaceae; OTU_214
Firmicutes; Clostridiaceae; OTU_172
Bacteroidetes; [Paraprevotellaceae]; OTU_110
Firmicutes; o__Clostridiales_OTU_271; OTU_271
Firmicutes; o__Clostridiales_OTU_1680; OTU_1680
Firmicutes; Ruminococcaceae; OTU_887
Firmicutes; Christensenellaceae; OTU_562
Bacteroidetes; o__Bacteroidales_OTU_95; OTU_95
Firmicutes; Christensenellaceae; OTU_230
Firmicutes; Clostridiaceae; OTU_525
Firmicutes; o__Clostridiales_OTU_1229; OTU_1229
Firmicutes; o__Clostridiales_OTU_2240; OTU_2240
Firmicutes; [Acidaminobacteraceae]; OTU_2665
Firmicutes; Ruminococcaceae; OTU_626
Firmicutes; o__Clostridiales_OTU_260; OTU_260
Firmicutes; Ruminococcaceae; OTU_1349
Firmicutes; o__Clostridiales_OTU_1507; OTU_1507
Firmicutes; Ruminococcaceae; OTU_1530
Firmicutes; Veillonellaceae; OTU_46
Firmicutes; Lachnospiraceae; OTU_2737
Firmicutes; Ruminococcaceae; OTU_15
Firmicutes; o__Clostridiales_OTU_28; OTU_28
0.01 0.10 1.00 10.00Read Abundance (%)
age
28
34
age (days)
Firmicutes; order Clostridiales OTU_28
Read abundance (%)
Firmicutes; order Clostridiales OTU_1507
Firmicutes; order Clostridiales OTU_260
Firmicutes; order Clostridiales OTU_2240
Firmicutes; order Clostridiales OTU_1229
Firmicutes; order Clostridiales OTU_346
Firmicutes; order Clostridiales OTU_19
Firmicutes; order Clostridiales OTU_271
Firmicutes; order Clostridiales OTU_1680
Bacteroidetes; order Bacteroidales OTU_95
Firmicutes; Clostridiaceae; OTU_1808
Firmicutes; o__Clostridiales_OTU_801; OTU_801
Firmicutes; Veillonellaceae; OTU_132
Firmicutes; Peptostreptococcaceae; OTU_793
Firmicutes; Clostridiaceae; OTU_2082
Firmicutes; Streptococcaceae; OTU_106
Actinobacteria; o__Actinomycetales_OTU_276; OTU_276
Actinobacteria; Coriobacteriaceae; OTU_376
Firmicutes; Lachnospiraceae; OTU_656
Firmicutes; Clostridiaceae; OTU_2875
Firmicutes; Lactobacillaceae; OTU_23
Firmicutes; Christensenellaceae; OTU_65
Firmicutes; Ruminococcaceae; OTU_67
Firmicutes; Clostridiaceae; OTU_651
Actinobacteria; Actinomycetaceae; OTU_223
Firmicutes; Ruminococcaceae; OTU_371
Firmicutes; Lactobacillaceae; OTU_79
Firmicutes; o__Clostridiales_OTU_51; OTU_51
Firmicutes; Clostridiaceae; OTU_2881
Firmicutes; o__Clostridiales_OTU_140; OTU_140
Firmicutes; o__Clostridiales_OTU_28; OTU_28
Firmicutes; o__Clostridiales_OTU_1835; OTU_1835
Firmicutes; Clostridiaceae; OTU_11
Firmicutes; Clostridiaceae; OTU_556
Firmicutes; Ruminococcaceae; OTU_9
0.01 0.10 1.00 10.00Read Abundance (%)
age
5
28
Firmicutes; Clostridiaceae; OTU_1808
Firmicutes; o__Clostridiales_OTU_801; OTU_801
Firmicutes; Veillonellaceae; OTU_132
Firmicutes; Peptostreptococcaceae; OTU_793
Firmicutes; Clostridiaceae; OTU_2082
Firmicutes; Streptococcaceae; OTU_106
Actinobacteria; o__Actinomycetales_OTU_276; OTU_276
Actinobacteria; Coriobacteriaceae; OTU_376
Firmicutes; Lachnospiraceae; OTU_656
Firmicutes; Clostridiaceae; OTU_2875
Firmicutes; Lactobacillaceae; OTU_23
Firmicutes; Christensenellaceae; OTU_65
Firmicutes; Ruminococcaceae; OTU_67
Firmicutes; Clostridiaceae; OTU_651
Actinobacteria; Actinomycetaceae; OTU_223
Firmicutes; Ruminococcaceae; OTU_371
Firmicutes; Lactobacillaceae; OTU_79
Firmicutes; o__Clostridiales_OTU_51; OTU_51
Firmicutes; Clostridiaceae; OTU_2881
Firmicutes; o__Clostridiales_OTU_140; OTU_140
Firmicutes; o__Clostridiales_OTU_28; OTU_28
Firmicutes; o__Clostridiales_OTU_1835; OTU_1835
Firmicutes; Clostridiaceae; OTU_11
Firmicutes; Clostridiaceae; OTU_556
Firmicutes; Ruminococcaceae; OTU_9
0.01 0.10 1.00 10.00Read Abundance (%)
age
5
28
age (days)
Read abundance (%)
Firmicutes; order Clostridiales OTU_1835
Firmicutes; order Clostridiales OTU_140
Firmicutes; order Clostridiales OTU_51
Firmicutes; order Clostridiales OTU_28
Firmicutes; order Clostridiales OTU_801
Actinobacteria; order Actinomycetales OTU_276
(a)
(b)
Figure 4
104 Chapter 5. Manuscript 2
5 7 14 21 28 34
2.00
2.25
2.50
2.75
3.00
3.25
3.50
3.75
4.00
4.25
4.50
4.75
5.00
5.25
resistant
sensitiveresistant
sensitiveresistant
sensitiveresistant
sensitiveresistant
sensitiveresistant
sensitive
Genotype
Dive
rsity
inde
x
Day 5 Day 7 Day 14 Day 21 Day 28 Day 34
Genotype
Div
ersi
ty in
dex
5 7 14 21 28 34
375
500
625
750
875
1000
1125
1250
1375
1500
1625
1750
resistantsensitive
resistantsensitive
resistantsensitive
resistantsensitive
resistantsensitive
resistantsensitive
Genotype
Estim
ated
num
ber o
f OTU
s
Day 5 Day 7 Day 14 Day 21 Day 28 Day 34
Genotype
Est
imat
ed n
umbe
r of O
TUs
5 7 14 21 28 34
250
375
500
625
750
875
1000
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Genotype
Obs
erve
d nu
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Day 5 Day 7 Day 14 Day 21 Day 28 Day 34
Genotype
Obs
erve
d nu
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(c)
(a)
(b)
Figure S1
Chapter 5. Manuscript 2 105
5 7
1421
2834
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Day
5D
ay 7
Day
14
Day
21
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28
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Figu
re S
2
106 Chapter 5. Manuscript 2
Chapter 6. Manuscript 3 107
Chapter 6
Manuscript 3
Influence of Bacillus spp. spores and gentamicin on the gut microbiotaof suckling and newly weaned piglets
Ann-Sofie Riis Poulsen, Nadieh de Jonge, Jeppe Lund Nielsen, Ole Højberg, Char-lotte Lauridsen, Nuria Canibe.
In preparation.
108 Chapter 6. Manuscript 3
Experimental set-up:
ControlNo pro- or antibiotics
Faeces Day 7, 28 and 42
Digesta Stomach, ileum, caecum
and mid colonDay 3, 28 and 42
AntibioticPiglets administered
gentamicin day 4, 5 and 6
Faeces Day 7, 28 and 42
ProbioticSows and piglets administered
Bacillus spp. spores
Faeces Day 7, 28 and 42
Probiotic + antibioticSows administered Bacillus spp. spores, and piglets administered Bacillus spp.
spores and gentamicin
Faeces Day 7, 28, and 42
6 sows6 sows 6 sows 6 sows
Digesta Stomach, ileum, caecum
and mid colonDay 3, 28 and 42
Digesta Stomach, ileum, caecum
and mid colonDay 3, 28 and 42
Digesta Stomach, ileum, caecum
and mid colonDay 3, 28 and 42
1. 16S rRNA gene sequencing• V1-V3 region• Illumina MiSeq
2. Classical culture3. Organic acid analysis4. Biogenic amine analysis5. Gene expression analysis
• Inflammatory markers• Tight junction proteins
Intestinal tissue Ileum
Intestinal tissue Ileum
Intestinal tissue Ileum
Intestinal tissue Ileum
24 sows12 piglets from each litter included
! 1
Influence of Bacillus spp. spores and gentamicin on the gut microbiota of suckling and newly
weaned piglets
Ann-Sofie Riis Poulsena, Nadieh de Jongeb, Jeppe Lund Nielsenb, Ole Højberga, Charlotte
Lauridsena, Nuria Canibea*
a Aarhus University, Faculty of Science and Technology, Department of Animal Science, Blichers
Allé 20, P.O. Box 50, DK-8830, Tjele, Denmark
b Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University,
Aalborg, Denmark
* Corresponding author.
Aarhus University,
Faculty of Science and Technology, Department of Animal Science,
Blichers Allé 20, P.O. Box 50,
DK-8830 Tjele, Denmark
Tel.: +45 87 15 80 19
Fax.: +45 87 15 42 49
E-mail address: nuria.canibe@anis.au.dk
Chapter 6. Manuscript 3 109
! 2
Abstract
Diarrhea is highly prevalent in neonatal piglets and is often treated with antibiotics like gentamicin.
Administrating antibiotics to newborn piglets may have short- and long-term consequences on gut
microbiota and immune system development. We hypothesise that these consequences may be
alleviated by concurrent probiotic administration. The objective was therefore to investigate the
effect of administrating gentamicin and a mixture of Bacillus licheniformis, B. subtilis and B.
amyloliquefaeceans spores on the gut microbiota of piglets pre- and post-weaning. Twenty-four
sows and their litters were randomly allocated to four treatment groups receiving; a) Bacillus spore
mixture to sows and piglets (PRO); b) gentamicin to piglets day 4, 5 and 6 of age (AB); c) Bacillus
spore mixture to sows and piglets, and gentamicin to piglets (PRO+AB); or d) no administration of
probiotics or antibiotics (CTRL). The study included 12 piglets from each litter. Faecal samples
were collected day 7, 14, 21, 28, 35 and 42. Piglets were sacrificed for intestinal digesta and tissue
day 3, 28 and 42. Selected samples were subjected to amplicon sequencing of the 16S rRNA gene,
culture counts, and organic acid, biogenic amine and tissue gene expression analysis (TNF-α, IL-
10, COX-2, ZO-1, OCLN, CLDN-4 and CLDN-2). Treatment had a significant effect on the
composition of the faecal microbial community on day 28 (padonis=0.003) and 42 (padonis=0.008), and
the colonic community on day 28 (padonis=0.014). Faecal species richness and diversity (p≤0.001),
and colonic richness (p=0.001), were higher in AB compared to PRO piglets on day 28, and were
not significantly different from day 42. Species richness and diversity were numerically lower in
CTRL piglets compared to AB piglets, and numerically higher compared to PRO piglets.
Significant differences in low abundant OTUs were observed between treatment groups. PRO
piglets had the highest faecal concentration of iso-butyric acid on day 7 (p=0.04) and a higher
butyric acid concentration compared to CTRL piglets (p=0.02); otherwise were no significant
effects observed in organic acid and biogenic amine concentrations, gene expression or bacterial
counts. The results show that administrating oral gentamicin to piglets shortly after birth may affect
gut microbial composition, and that it is counteracted by concurrent administration of Bacillus
spores, suggestively due to spores competitively excluding early colonisers. We conclude that both
gentamicin and Bacillus spores influence the gut microbial diversity of piglets, although
administration of the tested antibiotic did not result in severe dysbiosis. The importance of the
microbiological findings in relation to gut and animal health needs further investigation.
110 Chapter 6. Manuscript 3
! 3
Introduction
The newborn young has an immature immune system and is hence highly susceptible to infections.
Despite vaccination programs, newborn piglets are in high risk of developing diarrhea in their first
week of life and the condition often leads to antibiotic treatment (Kongsted, 2013). The etiology is
commonly unknown prior to therapeutic intervention, why the empirically chosen antibiotics often
are of broad-spectrum. However, administering antibiotics to newborn piglets may have short- and
long-term consequences on the developing gastrointestinal microbiota and the local immune
system, which may ultimately result in increased disease susceptibility later in life.
Microbial gut colonisation begins during birth when the newborn comes into contact with
bacteria from the mother and the surrounding environment. This initial colonisation leads to an
array of complex processes responsible for establishing the gut microbiota (Mackie et al., 1999).
The gut microbiota plays a pivotal role in maturing the gut and local immunological functions
(Sommer and Baeckhed, 2013). During early life, however, the gut microbiota is instable and highly
susceptible to disturbances by influencing factors, antibiotics being one of them (Nylund et al.,
2014). Administering antibiotics within the first week of life has been shown to cause short- and
long-term changes on the composition of the gut microbiota and on intestinal gene expressions
related to immunological functions (Schokker et al., 2014; Schokker et al., 2015). Human studies
have furthermore reported an association between antibiotic treatments in early life and immune-
related disorders (Alm et al., 2008). Alleviating the negative effect of antibiotics on the immature
gut microbiota might be crucial in improving both intestinal and general health of piglets.
Probiotics are defined as ‘live microbial feed supplements which beneficially affect the host
animal by improving its intestinal microbial balance’ (Fuller, 1989) and are believed to exert their
anti-pathogenic effects by competing with pathogens for intestinal mucosal binding sites and
nutrients, and inhibiting pathogen growth by producing organic acids and antibiotic-like compounds
(Vondruskova et al., 2010). Bacteria commonly used as porcine probiotics belong to the
Lactobacillus, Enterococcus, and Bacillus genera. Unlike most other probiotics, Bacillus spp. is a
spore-forming bacteria, and hence more resistant to unfavorable environmental conditions than are
probiotics found as vegetative cells only (Leser et al., 2008). Bacillus cereus var. Toyoi has been
reported to reduce the intestinal number of enterotoxigenic Escherichia coli, diarrhea incidence and
morbidity in weaned piglets (Taras et al., 2005; Scharek et al., 2007a; Papatsiros et al., 2011). It has
furthermore been reported to have immunological effects by increasing the number of T-
lymphocytes locally in the intestinal tissue and faecal IgA concentration (Scharek et al., 2007a;
Chapter 6. Manuscript 3 111
! 4
Scharek et al., 2007b). Also B. licheniformis and B. subtilis have been found to decrease morbidity,
mortality and post-weaning diarrhoea (Alexopoulos et al., 2004). Human studies, focusing on the
use of probiotics in relation to antibiotic treatment, have reported Lactobacillus casei to reduce the
incidence of antibiotic-associated diarrhoea and counteract the effect of antibiotics on the diversity
of the gut microbiota (Pirker et al., 2012).
However, detailed information on the effects of early life antibiotic therapy and
concomitantly probiotic administration on the gut microbiota and local immune parameters in
piglets are lacking. The aim of the current study is therefore to investigate the effect of
administrating antibiotics and probiotics on the gut microbiota in piglets. We hypothesise that
administration of gentamicin, a broad-spectrum aminoglycoside, will change the microbial
community of the gut, and that the expected negative effects of antibiotic administration are
alleviated by concomitantly administration of Bacillus spp. spores.
Materials and methods
Study design
The present study was conducted according to the license obtained from the Danish Animal
Experiments Inspectorate, Ministry of Food, Agriculture and Fisheries, Danish Veterinary and Food
administration. Sows and piglets were housed according to the general guidelines on housing of
pigs before and after farrowing, and after weaning.
A total of 288 piglets (crossbred Danish Landrace x Yorkshire x Duroc; mixed females and
males) from 24 sows (parity varying between one to seven) were included in the study. All sows
originated from Christiansminde Multisite K/S. The study was run in three blocks with eight sows
each. Before farrowing, sows were randomly divided into four treatment groups: (a) Sows fed a
probiotic mixture from ten days pre-partum until 28 days post-partum, and piglets fed the same
probiotic mixture during suckling and in the post-weaning period (PRO); (b) Piglets administered
gentamicin orally at 4, 5, and 6 days of age (AB); (c) Sows fed a probiotic mixture from ten days
pre-partum until 28 days post-partum, and piglets fed a probiotic mixture during lactation and in the
post-weaning period and administered oral gentamicin at 4, 5, and 6 days of age (PRO+AB); (d)
Neither sows nor piglets was fed probiotics or antibiotics (CTRL).
No creep-feed was allowed pre-weaning. Post-weaning feed was free from zinc oxide (Table
1) and no sawdust was allowed in the pens. All piglets were weaned at 28 days of age and housed
litter-wise. The study ended two weeks post-weaning. Clinical conditions and occurrence of
112 Chapter 6. Manuscript 3
! 5
diarrhoea were recorded daily. Diarrhoea was recorded per litter and scored as 0, 1 or 2. The score 0
was given when no diarrhoea was observed in a litter, score 1 when mild diarrhoea was observed,
and score 2 when severe diarrhoea was observed.
Probiotics
The probiotic mixture consisted of a total of nine Bacillus subtilis, B. amyloliquefaeceans, and B.
licheniformis spore strains. Sows were fed 1x1010 cfu per kg feed two times daily from ten days pre-
partum until the piglets were weaned. Piglets were orally administered 2x109 cfu per day on day 3,
5, 7, 10, 13, 16, 20, 24, and 28 of age; 4x109 cfu per day at 33 days of age, and 8x109 cfu per day at
38 days of age. Piglets not receiving probiotics were administered an equal volume of sterilised
water.
Antibiotics
Piglets were orally administered 5 mg gentamicin (Gentacin Vet.) as a single bolus on day 4, 5, and
6 of age. Piglets not receiving the antibiotic were administered an equal volume of sterilised water.
Sample and data collection
Piglets were weighed weekly. Rectal faecal samples were collected weekly from three piglets from
each litter. Two piglets from each litter were euthanised at 3 days of age and one piglet from each
litter at 28 and 42 days of age. 3-day-old piglets (<5 kg) were euhanised by a blunt trauma to the
head, and 28 and 42 days old piglets were euthanized using a captive bolt gun followed by bleeding.
The abdomen was incised and the gastrointestinal tract removed. Luminal content (digesta) from the
stomach, small intestine (proximal and distal segments), caecum and colon (proximal, middle and
distal segment) was collected immediately after killing. Collected digesta from the two 3-day-old
piglets was pooled. pH was measured and the digesta was weighed and taken to the laboratory for
further analysis. Bacterial enumeration by culture was performed on digesta samples from the
stomach, distal small intestine, caecum, and mid colon. Faecal and digesta subsamples were stored
at -20°C for organic acid and biogenic amine analyses. Other faecal and digesta subsamples were
snap-frozen in liquid nitrogen and stored at -80°C for 16S rRNA gene amplicon sequencing. An
intestinal tissue sample from 50% of the length of the distal small intestine was carefully rinsed
with PBS, cut into two pieces of 1 cm2 and stored in Dulbecco’s modified Eagle’s medium
(DMEM) on ice until LPS-stimulation.
Chapter 6. Manuscript 3 113
! 6
DNA extraction
Samples for DNA extraction included 214 faecal samples (day 7, 28 and 42; two day 7 samples
from the PRO group were missing) from 72 piglets and 144 digesta samples (distal small intestine
and mid colon from day 3, 28 and 42) from 72 piglets. DNA was extracted with the E.Z.N.A. Stool
DNA Kit (Omega Bio-Tek, inc., VWR international) following a standard protocol with the
following exceptions: In step 2, 450 µl SLX-Mlus buffer was added followed by 2x20 s bead
beating (FastPrep FP120; Bio 101 Savant/MP Biomedicals, USA). In step 3, 50 µl DS Buffer and
Proteinase K solution was added and followed by bead beating for 20 s. Step 4 was followed by
centrifugation at 2,000 g for 30 s. In step 5, 170 µl SP2 buffer was added and followed by bead
beating for 20 s. If no supernatant was present after step 8, another 180 µl SLX-Mlus, 20 µl DS
buffer and 67 µl SP2-buffer were added and the samples vortexed for 30 s., put on ice for 3 min and
centrifuged at maximum speed for 5 min. DNA extract purity was evaluated with Nanodrop
ND1000 (Thermo Scientific, USA) and quantified fluorometrically with Qubit 3.0 HS dsDNA assay
(Life Technologies, Thermo Fisher Scientific, USA). DNA concentrations were normalized to 5
ng/µl.
16S rRNA amplicon sequencing
Amplicon libraries were generated by targeted amplification of the V1-V3 hypervariable regions of
the bacterial 16S rRNA gene. The PCR reaction (25 µl) contained 10 ng template DNA, Platinum®
High Fidelity buffer (x1), dNTP (400 uM of each), MgSO4 (1.5 mM) and Platinum® Taq DNA
polymerase High Fidelity (1U) and barcoded library adapters (400 nM). V1-V3 primers: 27F
AGAGTTTGATCCTGGCTCAG and 534R ATTACCGCGGCTGCTGG. Thermocycler settings:
Initial denaturation at 95˚C for 2 min, 30 cycles of 95˚C for 20 s, 56˚C for 30 s, 72˚C for 60 s and
final elongation at 72˚C for 5 min. PCR reactions were run in duplicate for each sample and pooled
before purification. Purification of the amplicon libraries was performed using the Agencourt
AMPure XP bead protocol (Beckman Coulter, USA) and eluted in 23 µL nuclease-free water.
Individual libraries were quantified with Quant-iT HS dsDNA assay (Life Technologies, USA) and
quality checked on a Tapestation 2200 (Agilent, USA). Libraries were pooled in equimolar
concentrations, and diluted to 4 nM. The library pool was sequenced using an Illumina MiSeq
(Illumina, USA) and MiSeq reagent kit v3 (2x300 PE).
114 Chapter 6. Manuscript 3
! 7
Amplicon bioinformatic processing and analysis
The obtained raw sequencing reads were quality filtered and trimmed using trimmomatic (v0.32)
(Bolger et al., 2014), only keeping reads with a minimum length of 275 bp. The trimmed reads were
merged using FLASH v. 1.2.7 (Magoc and Salzberg, 2011) and read pairs between 425 and 525 bp
in length were formatted for use with the UPARSE workflow (Edgar, 2013). Reads were
dereplicated and clustered into Operational Taxonomical Units (OTUs) using USEARCH7 at 97%
sequence similarity. Taxonomy was assigned using the RDP-classifier as implemented in QIIME
(Caporaso et al., 2010) with a minimum confidence of 0.8 and Greengenes (version 08-2013) as a
reference database. Results were analysed in R studio (version 0.99.489 for Mac) using the Ampvis
package (Albertsen et al., 2015).
Organic acid and biogenic amine analysis
The concentrations of short-chain fatty acids and lactic acid in faeces and digesta samples were
measured as previously described by Canibe et al. (2007). Biogenic amine concentrations
(agmatine, putrescine, cadaverine and tyramine) were measured according to the following
procedure. Samples (including a blank sample) were diluted 10-fold with an internal standard
solution and homogenized in a Smasher paddle blender (bioMérieux Industry, USA) for 2 min.
Proteins were precipitated by mixing 20 µl sample or standard mix with 780 µl 0.1 M hypochloric
acid and 240 µl 2 M perchloric acid, followed by incubation at room temperature for 5 min and
centrifugation at 13,000 rpm for 5 min. Derivates were produced by adding 320 µl 0.5 M sodium
bicarbonate to 200 µl supernatant from the protein precipitation step, followed by the addition of
600 µl 5 mM Fmoc-solution and mixing. The samples were subsequently heated at 40°C for 10
min, followed by mixing with 40 µl concentrated hypochloric acid. Samples were centrifuged at
13,000 rpm for 5 minutes. 600 µl supernatant was transferred to a vial and analysed by high-
performance liquid chromatography (HPLC).
Microbiological analysis of digesta samples
For microbial plating, digesta samples (approximately 5 g) were transferred to bottles containing 50
ml of pre-reduced salt medium (Holdeman et al., 1977). The bottle content was then transferred to a
CO2 flushed bag and homogenized in a Smasher paddle blender for 2 min. One ml of the digesta
homogenate was transferred to a Hungate tube containing 9 ml pre-reduced salt medium and 10-
Chapter 6. Manuscript 3 115
! 8
fold dilutions were prepared using the technique previously described by Miller and Wolin (1974).
The samples were plated on selective (and indicative) and non-selective agar plates.
Enterobacteriaceae were enumerated on MacConkey agar (Merck 1.05465) after aerobic
incubation for 1 day. Yeasts were enumerated on Malt, Chlortetracycline and -Amphenicol agar
(Merck 1.03753 (yeast extract), 1.05397 (malt extract), 1.07224 (bacto-pepton), 1.08337 (glucose),
1.01614 (agar-agar) and Oxoid Sr0177E) after aerobic incubation for 2 days. Hemolytic bacteria
were enumerated on blood agar (Oxoid Pb5039A) after aerobic incubation for 1 day. Clostridium
perfringens were enumerated using the pour-plate technique on Tryptose Sulfit Cycloserine agar
(Merck 1.11972, 1.00888) after anaerobic incubation for 1 day. Lactic acid bacteria were
enumerated on de Man, Rogosa and Sharpe agar (Merck 1.10660) after anaerobic incubation for 2
days. Total anaerobic bacteria were enumerated in roll tubes containing pig colon fluid-glucose-
cellobiose agar (Holdeman et al., 1977) and incubated for 7 days. Bacillus spores were enumerated
on Casein soya bean digest broth agar (Oxoid CM0129) after aerobic incubation for 1 day. Prior to
Bacillus spore enumeration, the sample was incubated in a water bath at 80˚C for 20 min. All plates
and roll-tubes were incubated at 37˚C.
LPS-stimulation of ileal tissue
One 1 cm2 tissue sample was transferred to 1 ml DMEM and 10 µl PBS (control) and the second 1
cm2 tissue sample was transferred to 1 ml DMEM and 10 µl LPS (1 µg/µl; Sigma L4391). The two
test tubes were incubated at 37˚C for 120 min during constant gently shaking. After ended
incubation, the tubes were stored on ice for 10 min and then transferred to 700 µl RNAlater (Sigma-
Aldrich), stored at 5˚C for one day and then stored at -20˚C until gene expression analysis.
Gene expression analysis
The intestinal tissue sample was added to a 2 ml Eppendorf tube together with a 5 mm Stainless
Steel Bead and homogenized on the TissueLyser system (Qiagen). Total mRNA for gene expression
analysis was extracted using the NucleoSpin RNA isolation kit (Macherey-Nagel, Germany)
following the manufacturers protocol. The Nanodrop ND1000 was used for measuring RNA
quantity and assessing RNA quality (Thermo Scientific, USA). All samples were diluted to 100
ng/µl and converted to cDNA using the High-Capacity cDNA Reverse Transcription kit (Applied
Biosystems, USA) according to the manufacturers protocol. Real Time PCR reactions contained 2
µl cDNA and 8 µl mix containing primers, probe (Table 2) and 2X TaqMan mastermix, and run in
116 Chapter 6. Manuscript 3
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triplicate on the Applied Biosystems ViiA 7 Real-Time PCR system (Life Technologies) with the
following thermocycler settings: 50˚C for 2 min, 95˚C for 10 min and 40 cycles of 95˚C for 15 s
and 60˚C for 60 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as
housekeeping gene. The Applied Biosystem ViiA 7 software was used for determining the gene
expression cycle threshold (Ct) values. Relative quantification (comparative threshold method) was
used for evaluating the gene expression level using non-LPS stimulated ileal samples from 28 days
old CTRL pigs as reference samples. For each sample the Ct value of the GAPDH gene was
subtracted from the Ct value of the target gene (ΔCt). The average ΔCt value of the reference
animals was then subtracted from the ΔCt value of all samples (ΔΔCt). The expression of the target
gene was given as fold change calculated by 2-ΔΔCt.
Statistical analysis
Principal Component Analysis was performed on square root transformed OTU abundances.
Significance of treatment was tested on the first two principal components (PCs) using the envfit
parametric test and on the Bray-Curtis dissimilarity matrix using the Adonis test (Oksanen, 2015).
The parametric Wald-test (Love et al., 2014) was used to test for significant OTU abundance
differences between the four treatment groups. OTUs with an adjusted p<0.001 were considered
significantly different between the respective treatments.
The impact of treatment and age on bacterial, organic acid and biogenic amine parameters,
microbial richness, diversity index, gene expression levels and body weight were investigated by
fitting the data to a linear mixed model using the lmer function from the lme4 package (Bates, 2014)
using R studio (Version 0.99.489 for Mac). Diet, age, and intestinal segment (the latter included
when analysing samples obtained from slaughter) were included as fixed effects, while piglet and
sow were included as random effects (by including random intercept terms) to account for multiple
observations from the same litter and the same pig. When analysing the body weight variable, the
piglet body weight at the day of birth was included as a co-variate. The fixed effects were tested
using an F-test with Kenward-Roger approximation, where the reduced model was tested against
the full model. This was done using the KRmodcomp function in the pbkrtest package (Halekoh,
2014). When a fixed effect was found to be significant, a post-hoc test was performed using the
multcomp package and Bonferroni adjustment to correct for multiple comparisons (Hothorn, 2008).
Effects were considered significant when p<0.05.
Chapter 6. Manuscript 3 117
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Results
General observations
Weekly body weights of piglets are shown in Table 3. Body weight was not affected by treatment
(p=0.89) or gender (p=0.72) (data not shown). During the suckling period, PRO and PRO+AB
piglets had a higher frequency of grade 1 and 2 diarrhoea, while after weaning there was a higher
frequency of grade 1 diarrhoea in PRO+AB piglets (Table 4).
Gut microbiota – culture results
Treatment had a significant effect on the number of Bacillus spores, with the highest numbers in
PRO and PRO+AB piglets compared to AB and CTRL piglets (p<0.001). In addition, age and
segment had an interacting effect. The number of Bacillus spp. spores in the caecum was higher on
day 3 and 28 than on day 42 (p≤0.04) and in the mid colon, it was higher on day 28 (p≤0.003) than
on day 3 and 42 (Figure 1A).
Treatment did not have an effect on the number of Enterobacteriaceae, haemolytic bacteria,
C. perfringens, total anaerobic bacteria or lactic acid bacteria in any of the investigated gut
segments (Figure 1B-F). The number of Enterobacteriaceae in the stomach was highest on day 3
compared to day 28 (p=0.01), in the ileum the number was higher day 3 compared to day 28 and 42
(p<0.02), and in the caecum and mid colon, the number was highest on day 3 (p<0.001). In all
segments, the number of haemolytic bacteria was highest on day 3 (p<0.001). The numbers of lactic
acid bacteria in the stomach and ileum were lowest on day 3 (p≤0.04); and in the mid colon, the
number was lowest on day 28 (p<0.001) (Figure 1F). In the stomach, ileum, and caecum, C.
perfringens numbers decreased with age, being lowest on day 42 (p<0.001), and in the mid colon,
the number was highest on day 3 compared to day 28 (p=0.03), and lowest on day 42 compared to
day 3 and 28 (p<0.001). In the stomach, the number of total anaerobic bacteria was lowest on day 3
(p≤0.02); in the caecum, the counts were highest on day 3 compared to day 42 (p<0.001); and in the
mid colon, the number was highest on day 3 compared to day 28 and day 42 (p≤0.01). Treatment
had no effect on pH in any of the investigated segments (data not shown).
Bacterial metabolites
Age and intestinal segment were found to have an interacting effect on the ileal and mid colonic
digesta concentrations of the investigated biogenic amines (cadaverine and putrescine) and organic
acids, while treatment did not have a significant effect (Table 5 and 6). Agmatin and tyramin were
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not included in the analysis as the majority of the observations were zero/below detection level.
Ileal and colonic concentrations of cadaverine and putrescine were consistently lower (numerically)
in AB piglets compared to the other three groups. In the ileal digesta, concentrations of cadaverine
and putrescine were highest on day 42 (p<0.001; p≤0.13), while in the mid colon, the highest
concentration of putrescine was found on day 3 (p<0.001), and the concentration of cadaverine was
higher on day 3 compared to day 28 (p<0.001). The ileal concentration of acetic acid was higher on
day 42 compared to day 3 (p=0.006) and the lactic acid concentration was highest on day 42
(p<0.001). In the colonic digesta, the concentrations of acetic and butyric acid were highest on day
42 (p<0.001), while the concentrations of propionic and valeric acid were highest on day 42
(p<0.001; p≤0.04) and lowest on day 3 (p≤0.01; p≤0.003). The concentration of iso-valeric acid was
higher on day 42 compared to day 3 (p=0.03) in both ileal and colonic digesta. The concentration of
iso-butyric acid was similar between day 3, 28 and 42 regardless of intestinal segment. Colonic
concentrations of propionic acid on day 28 and 42 were unexpectedly higher than the acetic acid
concentrations.
Organic acid concentrations in faecal samples from day 7, 28, and 42 are shown in Table 7.
PRO piglets had the highest faecal concentration of iso-butyric acid on day 7 (p=0.04) and a higher
concentration of butyric acid compared to CTRL piglets also on day 7 (p=0.02). Treatment had no
other significant effect on faecal organic acid concentrations. The concentrations of iso-butyric and
iso-valeric acid were higher on day 7 compared to day 42 in PRO piglets (p=0.008; p=0.006) and
the concentration of propionic acid was highest on day 42 (p<0.001) for PRO+AB, CTRL and AB
piglets (p<0.001), and higher on day 42 compared to day 3 in PRO piglets (p=0.003). The faecal
concentration of butyric acid was higher in PRO piglets compared to CTRL piglets on day 7
(p=0.02) and highest on day 42 in AB (p≤0.009) and PRO+AB (p≤0.02) piglets, and higher on day
42 compared to day 7 (p<0.001) in CTRL piglets. The faecal acetic acid concentration was highest
on day 42 (p<0.001) and lowest on day 7 (p≤0.02). Neither treatment nor age had an effect on the
faecal concentration of valeric acid.
Gut microbiota – 16S rRNA gene amplicons
Sequencing of 356 samples yielded a total of 10,638,599 reads. A sequencing depth of 10,000 reads
was considered appropriate from rarefaction curves (data not shown) and excluded 23 samples from
further analysis. Recovered reads were clustered into 3400 OTUs and classified into 20 phyla, 100
Chapter 6. Manuscript 3 119
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families and 191 genera. Three of the four detected Bacillus species were classified to species level
and belonged to B. safensis, B. coagulans and B. cereus.
Faeces
Figure 2 shows the relative abundances of the ten most abundant phyla and 20 most abundant
genera in faecal samples from day 7, 28 and 42. Firmicutes and Bacteroidetes, followed by
Fusobacteria, were the most abundant phyla on day 7 and 28 irrespective of treatment. Firmicutes,
followed by Bacteroidetes, were the most abundant phyla on day 42, whereas Fusobacteria was
detected at very low levels (Figure 2). At genus level, Bacteroides, Lactobacillus, Prevotella and
Fusobacterium were the most abundant genera on day 7; Prevotella and Fusobacterium most
abundant on day 28; and Lactobacillus and Prevotella most abundant on day 42 irrespective of
treatment. 12 of the most abundant genera belonged to Firmicutes, six to Bacteroidetes, one to
Fusobacteria and one to Proteobacteria. The observed microbial community richness and diversity
index (Shannon index) was lower in PRO piglets compared to AB piglets (p=0.0001; p<0.001) and
PRO+AB piglets (p=0.03; p=0.046) on day 28, while the estimated richness was lower in PRO
piglets compared to AB piglets (p=0.0004) (Figure 3). Age had a significant effect on microbial
richness and diversity, with the observed and estimated species richness and Shannon index lowest
on day 7 (p<0.001), irrespective of treatment. In addition, for treatments CTRL, PRO and PRO+AB
the microbial richness and diversity was highest on day 42 (p<0.02) (figure 3).
There was a significant effect of treatment on the overall community composition of the four
treatment groups on day 28 (padonis=0.003) and 42 (padonis=0.008) (Figure 4). Performing a
redundancy analysis on day 28 and 42 revealed four groupings with slight overlap, indicating that
the four different treatments tended to result in four different communities (Figure 4C and 4E). Age
(padonis=0.001) (Figure S1) and litter (padonis=0.002) had a significant effect on the faecal microbial
composition. Table 8 shows the OTUs that were found in significantly different read abundances
between treatment groups. Generally were the significantly different OTUs present in relatively low
read abundances.
Digesta
Figure 5A and 5B show the relative abundances of the eight most prevalent phyla and 20 most
prevalent genera in ileal digesta from day 3, 28 and 42 of age. Firmicutes was clearly the most
abundant phylum on all three days investigated, irrespective of treatment. On day 3, Fusobacteria
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was measured to be between 2.1 and 12.1 % in all treatments. Lactobacillus was the most abundant
genus irrespective of age and treatment. On day 3, SMB53, Clostridium, Sarcina, Streptococcus and
Fusobacterium followed Lactobacillus, with the relative abundance of Sarcina ranging between 0.7
% (AB group) and 20.8 % (PRO+AB group). On day 28, SMB53, 02d06, Sarcina and Mitsuokella
followed Lactobacillus, with the relative abundance of Mitsoukella ranging between 0.1 % (CTRL
group) and 13.8 % (PRO group). On day 42, 02d06, SMB53, and Streptococcus followed
Lactobacillus.
Figure 5C and 5D show the relative abundances of the eight most prevalent phyla and 20
most prevalent genera in digesta from mid-colon on day 3, 28, and 42 of age. Firmicutes,
Bacteroidetes, and Fusobacteria were the most abundant phyla on day 3 irrespective of treatment,
while Firmicutes and Bacteroidetes were the most abundant phyla on day 28 and 42 irrespective of
treatment. Fusobacteria was also abundant on day 28 in PRO and PRO+AB piglets. At genus level,
Fusobacterium, Bacteroides, Lactobacillus and Clostridium were the most abundant genera on day
3 in all treatment groups. Prevotella, Lactobacillus, and Fusobacterium dominated on day 28, with
Fusobacterium being most abundant in PRO and PRO+AB piglets, and Prevotella most abundant in
PRO+AB piglets. Lactobacillus and Prevotella were the dominating genera on day 42, with the
highest abundance of Lactobacillus in CTRL piglets. The observed richness of colonic digesta on
day 28 was higher in AB piglets compared to PRO (p=0.001) and PRO+AB (p=0.02) piglets, and
the estimated richness was higher in AB compared to PRO (p=0.001) piglets (Figure 6). Age had a
significant effect on the observed and estimated species richness in colon contents. The observed
species richness was lowest on day 3 in all treatments groups (p≤0.02), except PRO+AB, and
highest on day 42 in PRO (p=0.01) and PRO+AB piglets (p=0.001). The estimated species richness
was lowest on day 3 in AB and CTRL piglets (p<0.001), and lowest on day 3 (p≤0.04) and highest
on day 42 (p≤0.05) in PRO and PRO+AB piglets. The Shannon diversity index was lowest on day 3
in AB piglets (p≤0.03), higher on day 28 compared to day 3 (p=0.006) in CTRL piglets, and higher
day 42 compared to day 3 in PRO+AB piglets (p<0.001) (figure 6).
Treatment had an effect on the overall microbial community composition in the mid colon
on day 28 (padonis=0.013) (Figure 7). Otherwise had treatment no significant effect on the microbial
communities (data not shown). Age had an effect on the overall microbial communities in the ileum
and mid-colon (p=0.001) (Figure S2). Samples from the ileum and mid colon clustered closer
together on day 3 compared to day 28 and 42. Litter had no effect on the microbial communities of
the ileum and mid colon (data not shown). Table 8 shows the OTUs that were found in
Chapter 6. Manuscript 3 121
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significantly different read abundance between treatment groups. Generally the significantly
different OTUs were present in relatively low read abundances.
Ileal gene-expression
Comparative expression of TNF-α, COX-2, IL-10, ZO-1, OCLN, CLDN-2, and CLDN-4 genes are
seen in Figure 8. LPS stimulation of ileal tissue increased the expression of TNF-α (p=0.05) and
COX-2 (p=0.04), but had no effect on IL-10, ZO-1, OCLN, CLDN-2 and CLDN-4 gene expression.
Treatment had no effect on the gene expression level of any of the investigated genes. TNF-α
(p<0.001), CLD-4 (p=0.05) and ZO-1 (p=0.01) genes were found to be expressed at higher levels
on day 28 compared to day 42
Discussion
The potential of using probiotics has been intensively investigated in both humans and animals. In
pigs, several probiotics have been tested and some have been found to have beneficial effects on the
gut microbial community and intestinal immunological parameters. However, knowledge on the
combined effects of pro- and antibiotics on the gut microbiota and intestinal health of pigs is
lacking. To our knowledge, this study is the first to investigate the combined effect of Bacillus spp.
spores and gentamicin on the gut microbiota and gut health parameters in piglets.
Analysis of 16S rRNA gene sequences confirmed an effect of treatment on the overall
colonic and faecal microbial communities at the day of weaning (day 28), where AB piglets,
compared to PRO piglets, consistently had the richest and most diverse microbiota. Contrary to the
other treatment groups, the microbial richness and diversity of AB piglets were the same on day 28
as on day 42 (lower on day 28 for the other treatment groups). These observations could indicate an
accelerated microbial gut colonisation in the distal gut segments of AB piglets, perhaps caused by
gentamicin inhibiting bacterial groups that would colonise the gut under normal (non-treated)
circumstances, thereby allowing a more diverse community to colonise faster. In accordance with
these results, Kim et al. (2012) suggested an accelerated maturation of the faecal microbiota in
tylosin-supplemented pigs which the non-treated pigs, however, eventually catched up on. Schokker
et al. (2015) suggested that the effects of intrinsic factors, e.g. early impact of an antibiotic, are most
apparent at times when the gut microbiota has been more or less stabilized, as seen just prior to
weaning and several weeks post-weaning, and our results are generally supportive of this.
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Interestingly, though, the ileal microbiota was the most stable community across treatments without
any significant effects on the overall community, species richness or diversity on day 28 and 42 of
age. Well aware of the varying mode of action among antibiotics, subcutaneous tulathromycin
injection at four days of age have been shown to have short- and long-term effects on the jenunal
microbiota of piglets by decreasing the microbial diversity (Schokker et al., 2014; Schokker et al.,
2015). The short-term effect was seen when the piglets were 8 days of age, the long-term effect was
seen at 176 days of age, while no effect was seen at 55 days of age. So, if the ileal digesta had been
collected from piglets a few days after antibiotic administration in the current study, we would
perhaps have found gentamicin to have a more profound effect on the ileal microbiota. An
explanation might also be, that the spectrum of activity of gentamicin merely was incompatible with
the community found in the ileum. Only faecal samples were collected in the immediate period
following antibiotic administration and it could be speculated that the antibiotic effect was not large
enough to be reflected in these samples.
Enumerating specific bacterial groups by culture did not show any differences between
treatment groups. As gentamicin is especially active against gram-negative infections (Adams,
2001), we did expect a reduction in the number of Enterobacteriaceae cultured from intestinal
digesta. A possible explanation is that the digesta samples were collected 22 and 36 days after the
last gentamicin dose was administered, thus allowing enough time for the piglets to be re-colonised
with gram-negatives from the environment. Daily administration of subtherapeutic doses of
virginiamycin has been shown to have a decreasing effect on anaerobic bacterial and lactoballi
counts and an increasing effect on coliform counts (Jensen, 1988).
The authors found the effect of gentamicin to be unexpectedly low. Oral antibiotics are
generally thought to cause intestinal dysbiosis, as seen in e.g. humans where diarrhoea is a common
complication to antibiotic therapy (Breves et al., 2000; Pirker et al., 2012). However, there exists
many different classes of antibiotics that target different spectra of bacteria, and the development of
antibiotic-associated diarrhea has been shown to be dependent on the type of antibiotic used (Anand
et al., 1994). The relatively low effect of gentamicin may have impeded the detection of potential
effect(s) of Bacillus spores on the gut microbial community in the current study.
Bacillus spores were included to assess whether they were able to counteract the effect of
early gentamicin administration. To allow piglets to be exposed to the spores already from birth,
they were included in the sow feed from ten days before expected farrowing. Plate cultures of
Bacillus spores showed that piglets in the PRO and PRO+AB groups had the highest concentration
Chapter 6. Manuscript 3 123
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of spores along the length of their gastrointestinal tracts on day 3 post-farrowing compared to
piglets in the CTRL and AB groups. Sampling on day 3 was done prior to probiotic administration,
demonstrating that the spores must have been vertically transferred from the sow to the piglets,
which is in accordance with Taras et al. (2005). Our results suggest that probiotics administered to
the dam with the feed during late gestation are transmitted to the offspring.
Piglets given the Bacillus spores (PRO and PRO+AB groups) had higher spore counts than
the other groups. However, the applied method quantified only the number of spores and not
vegetative cells, why we were not able to account for the precise degree of spore germination.
Nevertheless, earlier studies report that B. subtilis and B. licheniformis are able to germinate in the
gut of grower pigs, though only showing limited ability to grow (Leser et al., 2008). Due to the
resilient spore structure (Cutting, 2011), 16S rRNA sequencing were expected to detect germinated
cells only. The detected abundances of Bacillus genera in the ileum, colon and faeces were,
however, extremely low regardless of sample type, age and previous spore administration. If only a
small percentage of the spores germinated, the detection threshold of the sequencing method may
have been too high compared to more traditional methods as suggested by Lagier et al. (2012).
Despite the apparent lack of germination, the oral administration of Bacillus spores did seem
to have an in vivo effect. The consistently lower richness of the communities of the distal colon of
PRO piglets suggests that the normal colonisation pattern during nursing was hindered by the
spores. This was especially evident as the increased colonic and faecal richness and diversity in AB
piglets seemed to be counteracted by spore supplementing. Bacillus spores have previously been
reported to be able to adhere to intestinal cells (Tam et al., 2006), and it could thus be speculated
that the spores were able to competitively halt the colonisation pattern seen after gentamicin
administration. Previous reports on the effect of different probiotics on the microbial diversity are
contradicting. In agreement with our findings, Wang et al. (2012) found Lactobacillus acidophilus
and Pediococcus acidilactici to decrease species richness and diversity of colonic digesta from pigs
in the post-weaning period. The action of decreasing the number of colonising species may have a
health promoting effect when the infection pressure is high. In the current study, piglets were
challenged only during the natural process of weaning, and under these circumstances did spore
administration not improve piglet health. In fact, piglets administered Bacillus spores tended to
have a higher frequency of diarrhoea before and after weaning. As there are no general guidelines
on probiotic use in pigs, the dose and interval may have been sub-optimal for use in piglets.
Furthermore, an increased intestinal permeability has been associated with the use of Bacillus
124 Chapter 6. Manuscript 3
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cereus var. Toyoi (Altmeyer et al., 2014), which would be expected to have a compromising effect
on intestinal health.
Weaning is a stressful event for piglets, where numerous factors contribute to physiological
and microbial changes in the gut (Lalles et al., 2007). Intrinsic and extrinsic factors as litter, nursing
mother and genotype are found to have the biggest influence on the gut microbiota when the piglets
still suckle, while this influence decreases after weaning (Bian et al., 2016). As a consequence, the
reduced effect of gentamicin and spore administration two weeks after weaning was expected, and
the event of introducing solid foods, the stress of being separated from the sow and transported to a
new stable environment, all seemed to have a stronger influence on the gut microbiota.
The establishment of a gut microbiota is a prerequisite for an optimal development of the
intestinal immune system during early life (Sommer and Baeckhed, 2013), and administering
antibiotics to piglets shortly after birth, has been shown to alter the expression of genes related to
immunological functions (Benis et al., 2015; Schokker et al., 2015). The genes investigated in the
current experiment encoded pro- and inflammatory cytokines and cell-to-cell adhesion proteins,
however, neither early gentamicin nor continuous spore administration was able to alter the
expression of these genes. Hence, treatment apparently did not have any effect on the intestinal
barrier or inflammatory status. These results may have been different, it the piglets had been
exposed to an infection challenge.
Age is an important driver of gut microbiota maturation and is an important influencing
factor (Slifierz et al., 2015). In the present study, sequencing data from day 3 and 28 clearly showed
the effect of age. The microbial communities on day 42 was furthermore clearly different from
those on day 3 and 28, though the event of weaning on day 28 causing changes in diet, environment
and stress complicates the interpretation of the gut microbial changes from 28 to 42 days of age.
Microbial richness and diversity of the colonic and faecal communities increased up until day 42,
while it was more or less constant in the ileum. Thus the ileal community is quickly colonised by
the adult-like number and proportion of species, albeit the colonising species change with age.
Conclusion
In conclusion, administering gentamicin on three consecutive days, from 4 days post-partum,
caused an increased diversity and richness of the colonic and faecal microbiota, which was evident
3 weeks later. Administration of Bacillus spores, on the other hand, decreased species richness and
diversity and hence counteracted the effect of gentamicin. We were not able to show that spore
Chapter 6. Manuscript 3 125
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administration beneficially affected the health of newly weaned piglets. Yet, the importance of
these findings in relation to the clinical status of the animals needs further investigation. Such an
investigation should include a long-term study following the pigs into their adult life and include a
challenge trial. The study furthermore underlines the importance of distinguishing between different
classes of antibiotics when discussing dysbiosis in relation to antibiotic treatment.
Acknowledgements
The authors would like to thank Trine Poulsen, Helle Handll, Lene Rosborg Dal, Hanne Purup,
Inger Marie Jepsen and Thomas Rebsdorf for their technical assistance during the experiment, and
subsequent extensive sample processing and analysis. Also thanks to the staff of SB for taking good
care of the sows and piglets from the beginning to the end of the experiment.
126 Chapter 6. Manuscript 3
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Love, M. I., W. Huber, and S. Anders. 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 15.
Mackie, R. I., A. Sghir, and H. R. Gaskins. 1999. Developmental microbial ecology of the neonatal gastrointestinal tract. American Journal of Clinical Nutrition 69: 1035S-1045S.
Magoc, T., and S. L. Salzberg. 2011. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27: 2957-2963.
Miller, T. L., and M. J. Wolin. 1974. Serum bottle modification of hungate technique for cultivating obligate anaerobes. Applied Microbiology 27: 985-987.
Nylund, L., R. Satokari, S. Salminen, and W. M. de Vos. 2014. Intestinal microbiota during early life - impact on health and disease. Proceedings of the Nutrition Society 73: 457-469.
Oksanen, J. G. B., F.; Kindt, R.; Legendre, P.; Minchin, P.R.; O'Hara, R.B.; Simpson, G.L.; Solymos, P.; M. Stevens H.H.; Wagner, H. 2015. vegan: Community Ecology Package. R package version 2.3-2. .
Papatsiros, V. G. et al. 2011. Effect of benzoic acid and combination of benzoic acid with a probiotic containing Bacillus Cereus var. toyoi in weaned pig nutrition. Polish Journal of Veterinary Sciences 14: 117-125.
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Scharek, L., J. Guth, M. Filter, and M. F. G. Schmidt. 2007b. Impact of the probiotic bacteria Enterococcus faecium NCIMB 10415 (SF68) and Bacillus cereus var. toyoi NCIMB 40112 on the development of serum IgG and faecal IgA of sows and their piglets. Archives of Animal Nutrition 61: 223-234.
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Schokker, D. et al. 2014. Early-Life Environmental Variation Affects Intestinal Microbiota and Immune Development in New-Born Piglets. Plos One 9.
Slifierz, M. J., R. M. Friendship, and J. S. Weese. 2015. Longitudinal study of the early-life fecal and nasal microbiotas of the domestic pig. BMC Microbiology 15.
Sommer, F., and F. Baeckhed. 2013. The gut microbiota - masters of host development and physiology. Nature Reviews Microbiology 11: 227-238.
Tam, N. K. M. et al. 2006. The intestinal life cycle of Bacillus subtilis and close relatives. Journal of Bacteriology 188: 2692-2700.
128 Chapter 6. Manuscript 3
! 21
Taras, D., W. Vahjen, M. Macha, and O. Simon. 2005. Response of performance characteristics and fecal consistency to long-lasting dietary supplementation with the probiotic strain Bacillus cereus var. toyoi to sows and piglets. Archives of Animal Nutrition 59: 405-417.
Vondruskova, H., R. Slamova, M. Trckova, Z. Zraly, and I. Pavlik. 2010. Alternatives to antibiotic growth promoters in prevention of diarrhoea in weaned piglets: a review. Veterinarni Medicina 55: 199-224.
Wang, J. Q. et al. 2012. Evaluation of probiotic bacteria for their effects on the growth performance and intestinal microbiota of newly-weaned pigs fed fermented high-moisture maize. Livestock Science 145: 79-86.
Chapter 6. Manuscript 3 129
! 22
Tables:
Table 1
Item %
Dry matter 88.4
Protein 22.2
Fat 7.4
Ash 4.9
Chemical analyses were performed by Eurofins Steins Laboratory A/S, Vejen, Denmark.
Table 2
Information on primers. probes and genes for real-time PCR
Target gene Primer sequences Accession number GAPDH Forward: 5’-GTCGGAGTGAACGGATTTGG-3’
Reverse: 5’-CAATGTCCACTTTGCCAGAGTTAA-3’ Probe: 5’-CGCCTGGTCACCAGGGCTGCT-3’
AF017079
TNF-α Forward: 5’-AACCCTCTGGCCCAAGGA-3’ Reverse: 5’-GGCGACGGGCTTATCTGA-3’ Probe: FAM-TCAGATCATCGTCTCAAAC-MGB
X57321
COX-2 Forward: 5’-GGGACGATGAACGGCTGTT-3’ Reverse: 5’-CACAATCTTAATCGTTTCTCCTATCAGT-3’ Probe: 5’-AGACGAGCAGGCTGA-3’
NM_214321
IL-10 Forward: 5’-GAGGAGGTGAAGAGTGCCTTTA-3’ Reverse: 5’-CTCACCCATGGCTTTGTAGACA-3’ Probe: FAM-CCTCTCTTGGAGCTTGC-MGB
L20001
ZO-1 TaqMan® Gene Expression Assay. catalogue no. 4351370. assay ID ss03373514_m1 (Applied Biosystems. Thermo Fisher Scientific)
OCLN TaqMan® Gene Expression Assay. catalogue no. 4351370. assay ID ss03377507_u1 (Applied Biosystems. Thermo Fisher Scientific)
CLDN-2 TaqMan® Gene Expression Assay. catalogue no. 4351370. assay ID ss03375002_u1 (Applied Biosystems. Thermo Fisher Scientific)
CLDN-4 TaqMan® Gene Expression Assay. catalogue no. 4351370. assay ID ss03375006_u1 (Applied Biosystems. Thermo Fisher Scientific)
GAPDH = glyderaldehyde-3-phosphate dehydrogenase; TNF-α = tumour necrosis factor-α; COX-2 =
cyclooxygenase-2; IL-10 = interleukin-10; ZO-1 = zonula occludens-1; OCLN = occludin; CLDN-2 =
claudin-2; CLDN-4 = claudin-4
130 Chapter 6. Manuscript 3
! 23
Table 3
Piglet body weight (kg) at birth and 7, 14, 21, 28, 35 and 42 days of age1
Treatment group2 p-value Days of age CTRL AB PRO PRO+AB
T3 A4 T*A
0 1.5 (1.4-1.6) 1.5 (1.4-1.6) 1.5 (1.4-1.6) 1.5 (1.4-1.6) 0.89 <0.001 <0.001
7 2.7 (2.6-2.9) 2.8 (2.6-2.9) 2.8 (2.6-3.9) 2.5 (2.4-2.7)
14 5.0 (4.7-5.4) 4.9 (4.6-5.3) 4.9 (4.5-5.2) 4.7 (4.4-5.0)
21 7.5 (7.0-8.0) 7.0 (6.5-7.5) 6.9 (6.4-7.4) 7.0 (6.5-7.5)
28 9.7 (9.0-10.4) 9.2 (8.5-9.8) 8.6 (8.0-9.2) 9.4 (8.8-10.1)
35 9.9 (9.3-10.7) 9.5 (8.9-10.2) 9.1 (8.5-9.8) 9.6 (9.0-10.3)
42 11.3 (10.6-12.2) 10.8 (10.0-11.6) 10.3 (9.6-11.0) 11.1 (10.4-11.9)
1 Values are presented as least square means and 95% confidence intervals (in parentheses). 2 CTRL = control; AB = antibiotic group; PRO = probiotic group; PRO+AB = probiotic+antibiotic group.
Number of observations: CTRL = 414; AB = 395; PRO = 414; PRO+AB = 386. 3 T = treatment group 4 A = age.
! ! ! ! !Table 4
Diarrhoea frequencies during suckling and after weaning1
Treatment group2
Diarrhoea score CTRL AB PRO PRO+AB
Suckling
Grade 1 0.17 0.15 0.28 0.2
Grade 2 0 0.01 0.09 0.07
Weaned
Grade 1 0.49 0.33 0.43 0.62
Grade 2 0.08 0.07 0.08 0.12
1 Values are presented as mean frequencies. 2 CTRL = control; AB = antibiotic group; PRO = probiotic group; PRO+AB = probiotic+antibiotic group.
Chapter 6. Manuscript 3 131
!24
Tab
le 5
Con
cent
ratio
n of
bio
geni
c am
ines
cad
aver
ine
and
putre
scin
e (m
g/kg
sam
ple)
in il
eal a
nd m
id c
olon
ic d
iges
ta fr
om p
igle
ts a
t 3, 2
8, a
nd 4
2
days
of a
ge 1
Trea
tmen
t gro
up2
p-
valu
e
C
TRL
AB
PR
O
PRO
+AB
#
T3 S*
A4
Cad
aver
ine
0.
39
<0.0
01
Day
3
Ile
um
5.4
(0.1
-18.
3)
2.4
(0-1
1.4)
9.
0 (1
.5-2
7.0)
4.
6 (0
-16.
3)
a
M
id c
olon
20
5.7
(83.
0-50
2.5)
13
6.6
(54.
3-33
5.6)
28
7.0
(116
.4-7
00.1
) 18
6.3
(74.
9-45
5.4)
A
D
ay 2
8
Ileum
9.
6 (2
.4-2
5.5)
5.
2 (0
.4-1
6.2)
14
.8
(4.6
-37.
5)
8.4
(1.8
-22.
8)
a
M
id c
olon
24
.2
(9.1
-57.
1)
15.0
(4
.9-3
7.3)
35
.0
(14.
0-80
.9)
21.6
(7
.9-5
1.4)
B
D
ay 4
2
Ileum
11
5.5
(51.
0-25
6.1)
76
.1
(32.
9-17
0.6)
16
1.8
(72.
0-35
7.8)
10
4.4
(45.
9-23
1.9)
b
Mid
col
on
70.3
(3
0.2-
157.
5)
45.8
(1
9.0-
104.
6)
99.0
(4
3.3-
220.
7)
63.4
(2
7.1-
142.
5)
AB
Putre
scin
e
0.28
<0
.001
D
ay 3
Ileum
19
.2
(7.6
-43.
3)
9.6
(2.9
-23.
2)
22.3
(9
.0-4
9.6)
15
.7
(5.9
-36.
0)
a
M
id c
olon
52
6.9
(253
.8-1
089.
9)
302.
3 (1
44.9
-626
.5)
598.
2 (2
88.1
-123
8.5)
44
4.7
(213
.9-9
20.4
) A
D
ay 2
8
!!
!D
ay 2
8!Ile
um
21.1
(9
.4-4
3.4)
10
.7
(4.0
-23.
3)
24.4
(1
1.0-
50.4
) 17
.3
(7.4
-36.
2)
a
M
id c
olon
18
.9
(8.3
-39.
1)
9.5
(3.4
-20.
8)
21.9
(9
.9-4
4.9)
15
.5
(6.5
-32.
5)
B
Day
42
!
!!
Day
42!
Ileum
60
.1
(30.
0-11
7.4)
33
.2
(15.
9-66
) 68
.7
(34.
4-13
4.0)
50
.3
(24.
8-98
.7)
b
M
id c
olon
44
.8
(21.
9-88
.2)
24.4
(1
1.2-
49.1
) 51
.2
(25.
3-10
0.7)
37
.3
(18.
0-74
.0)
B
132 Chapter 6. Manuscript 3
!25
1 Val
ues a
re p
rese
nted
as l
east
squa
re m
eans
and
95%
con
fiden
ce in
terv
als (
in p
aren
thes
es).
2 C
TRL
= co
ntro
l; A
B =
ant
ibio
tic g
roup
; PR
O =
pro
biot
ic g
roup
; PR
O+A
B =
pro
biot
ic+a
ntib
iotic
gro
up.
Num
ber o
f pig
lets
: CTR
L=32
; AB
=32;
PR
O=3
1; P
RO
+AB
=32.
3 T
= tre
atm
ent g
roup
4 S*
A =
inte
ract
ion
betw
een
inte
stin
al se
gmen
t and
age
.
a, b
: Ile
al sa
mpl
es. R
ows w
ith d
iffer
ent l
ette
rs, w
ithin
a b
ioge
nic
amin
e gr
oup,
are
sign
ifica
ntly
diff
eren
t (p<
0.05
).
A, B
: Mid
col
on sa
mpl
es. R
ows w
ith d
iffer
ent l
ette
rs, w
ithin
a b
ioge
nic
amin
e gr
oup,
are
sign
ifica
ntly
diff
eren
t (p<
0.05
).
Maj
ority
of a
gmat
ine
and
tyra
min
e m
easu
rem
ents
wer
e ze
ro/b
elow
det
ectio
n le
vel.
Chapter 6. Manuscript 3 133
!26
Tab
le 6
Con
cent
ratio
n of
org
anic
aci
ds (m
mol
/kg
sam
ple)
in il
eal a
nd c
olon
ic d
iges
ta fr
om p
igle
ts 3
, 28,
and
42
days
of a
ge1
Trea
tmen
t gro
up2
p-
valu
e
C
TRL
AB
PR
O
PRO
+AB
#
T3 S*
A4
Ace
tic a
cid
0.
55
<0.0
01
Day
3
Ile
um
0.9
(0.6
-3.9
) 1.
0 (0
.6-4
.4)
0.8
(0.5
-3.4
) 0.
8 (0
.5-3
.0)
a
M
id c
olon
4.
1 (2
.6-2
1.8)
4.
5 (2
.9-2
4.2)
3.
7 (2
.4-1
9.7)
3.
4 (2
.2-1
7.8)
A
D
ay 2
8
Ileum
1.
5 (1
.0-6
.9)
1.7
(1.1
-7.7
) 1.
4 (0
.9-6
.2)
1.3
(0.8
-5.5
) ab
M
id c
olon
5.
7 (3
.9-2
9.7)
6.
4 (4
.3-3
3.0)
5.
2 (3
.6-2
7.0)
4.
8 (3
.2-2
4.5)
A
D
ay 4
2
Ileum
1.
9 (1
.3-8
.8)
2.1
(1.4
-9.9
) 1.
7 (1
.2-7
.9)
1.5
(1.1
-7.1
) b
Mid
col
on
20.6
(1
4.0-
110.
1)
22.8
(1
5.5-
121.
9)
18.8
(1
2.7-
100.
3)
17.1
(1
1.6-
91.1
) B
Pr
opio
nic
acid
0.56
<0
.001
D
ay 3
Ileum
0.
4 (0
.4-0
.7)
0.4
(0.4
-0.9
) 0.
4 (0
.4-0
.7)
0.4
(0.4
-0.5
)
Mid
col
on
3.3
(2.0
-5.2
) 3.
9 (2
.4-6
.2)
3.5
(2.2
-5.6
) 2.
9 (1
.7-4
.6)
A
Day
28
Ile
um
0.5
(0.4
-1.0
) 0.
4 (0
.4-0
.9)
0.6
(0.4
-1.1
) 0.
4 (0
.4-0
.8)
M
id c
olon
6.
5 (4
.4-9
.6)
7.7
(5.2
-11.
2)
7.0
(4.7
-10.
2)
5.8
(3.9
-8.5
) B
D
ay 4
2
Ileum
0.
4 (0
.4-0
.5)
0.4
(0.4
-0.9
) 0.
4 (0
.4-0
.5)
0.4
(0.4
-0.4
)
Mid
col
on
28.5
(1
9.8-
40.8
) 33
.3
(23.
2-47
.7)
30.2
(2
1-43
.4)
25.3
(1
7.6-
36.4
) C
B
utyr
ic a
cid
0.
93
- M
id c
olon
Day
3
2.7
(0.3
-5.1
) 2.
0 (0
-4.4
) 2.
0 (0
-4.4
) 2.
1 (0
-4.4
) A
D
ay 2
8 4.
3 (2
.2-6
.4)
3.6
(1.5
-5.7
) 3.
6 (1
.4-5
.7)
3.6
(1.5
-5.8
) A
D
ay 4
2 11
.2
(9.1
-13.
3)
10.5
(8
.4-1
2.6)
10
.5
(8.4
-12.
6)
10.6
(8
.4-1
2.7)
B
0.87
-
134 Chapter 6. Manuscript 3
!27
Val
eric
aci
d M
id c
olon
Day
3
0.9
(0.1
-1.7
) 0.
5 (0
-1.3
) 0.
6 (0
-1.4
) 0.
6 (0
-1.3
) A
D
ay 2
8 2.
0 (1
.3-2
.8)
1.7
(1.0
-2.4
) 1.
8 (1
.1-2
.5)
1.7
(1.0
-2.5
) B
D
ay 4
2 2.
8 (2
.1-3
.5)
2.5
(1.7
-3.2
) 2.
5 (1
.8-3
.3)
2.5
(1.8
-3.2
) C
Is
o-bu
tyric
aci
d
0.90
-
Mid
col
on
D
ay 3
0.
8 (0
.4-1
.3)
0.7
(0.3
-1.2
) 0.
9 (0
.4-1
.3)
0.7
(0.3
-1.2
)
Day
28
1.2
(0.8
-1.6
) 1.
2 (0
.8-1
.6)
1.3
(0.9
-1.7
) 1.
1 (0
.7-1
.5)
D
ay 4
2 1.
3 (0
.9-1
.7)
1.3
(0.9
-1.7
) 1.
4 (1
.0-1
.8)
1.2
(0.8
-1.6
)
Iso-
vale
ric a
cid
0.
91
- M
id c
olon
Day
3
0.7
(0.7
-0.7
) 0.
7 (0
.7-0
.8)
0.7
(0.7
-0.7
) 0.
7 (0
.7-0
.7)
A
Day
28
0.7
(0.7
-1.2
) 0.
8 (0
.7-1
.3)
0.7
(0.7
-1.2
) 0.
7 (0
.7-1
.1)
AB
D
ay 4
2 0.
8 (0
.7-1
.2)
0.9
(0.7
-1.4
) 0.
8 (0
.7-1
.3)
0.7
(0.7
-1.1
) B
La
ctic
aci
d
0.41
-
Ileum
Day
3
10.9
(7
.0-1
6.3)
10
.7
(6.9
-16.
0)
7.7
(4.7
-11.
8)
9.1
(5.7
-13.
7)
A
Day
28
10.1
(6
.8-1
4.6)
9.
9 (6
.6-1
4.3)
7.
0 (4
.4-1
0.6)
8.
3 (5
.5-1
2.2)
A
D
ay 4
2 38
.4
(27.
7-52
.7)
37.7
(2
7.2-
51.8
) 28
.6
(20.
4-39
.7)
32.8
(2
3.6-
45.2
) B
1 V
alue
s are
pre
sent
ed a
s lea
st sq
uare
mea
ns a
nd 9
5% c
onfid
ence
inte
rval
s (in
par
enth
eses
).
2 CTR
L =
cont
rol;
AB
= a
ntib
iotic
gro
up; P
RO
= p
robi
otic
gro
up; P
RO
+AB
= p
robi
otic
+ant
ibio
tic g
roup
.
Num
ber o
f pig
lets
: CTR
L=32
; AB
=32;
PR
O=3
1; P
RO
+AB
=32.
3 T
= tre
atm
ent g
roup
. 4 S*
A =
inte
ract
ion
betw
een
inte
stin
al se
gmen
t and
age
.
a, b
: Ile
al sa
mpl
es. R
ows w
ith d
iffer
ent l
ette
rs, w
ithin
an
acid
gro
up, a
re si
gnifi
cant
ly d
iffer
ent (p<
0.05
).
A, B
, C: M
id c
olon
sam
ples
. Row
s with
diff
eren
t let
ters
, with
in a
n ac
id g
roup
, are
sign
ifica
ntly
diff
eren
t (p<
0.05
).
Chapter 6. Manuscript 3 135
!28
Tab
le 7
Con
cent
ratio
n of
shor
t-cha
in fa
tty a
cids
(mm
ol/ k
g sa
mpl
e) in
faec
al m
atte
r fro
m p
igle
ts 7
, 28,
and
42
days
of a
ge1
Tr
eatm
ent g
roup
2
p-va
lues
A
ge
CTR
L A
B
PRO
PR
O+A
B
# T3
A4
T*A
A
cetic
aci
d
0.32
<0
.001
0.
42
7 28
.8
(23.
8-33
.7)
24.6
(1
9.7-
29.5
) 29
.0
(24.
2-33
.9)
29.0
(2
4.2-
33.8
) a
28
34
.9
(30.
2-39
.6)
30.7
(2
6.0-
35.3
) 35
.2
(30.
4-39
.9)
35.1
(3
0.4-
39.7
) b
42
59
.9
(55.
3-64
.6)
55.7
(5
1.1-
60.4
) 60
.2
(55.
5-64
.9)
60.1
(5
5.4-
64.8
) c
Pr
opio
nic
acid
0.76
<0
.001
0.
03
7 8.
6A
(4.7
-12.
4)
7.8A
(4
.1-1
1.5)
11
.6A
(8
.0-1
5.2)
7.
8A
(4.3
-11.
2)
28
14.2
A
(10.
9-17
.5)
10.8
A
(7.5
-14.
1)
14.9
AB
(1
1.4-
18.4
) 12
.9A
(9
.6-1
6.2)
42
24
.8B
(2
1.5-
28.0
) 24
.5B
(2
1.2-
27.8
) 19
.5B
(1
6.2-
22.8
) 25
.4B
(2
2.0-
28.8
)
B
utyr
ic a
cid
0.
84
<0.0
01
0.00
4 7
3.7A
b (0
.6-6
.9)
5.4A
ab
(2.4
-8.5
) 10
.2a
(7.3
-13.
2)
5.2A
ab
(2.4
-8.1
)
28
8.
2AB
(5
.4-1
0.9)
5.
2A
(2.4
-7.9
) 7.
6 (4
.7-1
0.4)
6.
6A
(3.8
-9.3
)
42
12
.1B
(9
.4-1
4.9)
11
.2B
(8.4
-13.
9)
8.0
(5.3
-10.
7)
11.7
B
(8.9
-14.
5)
Val
eric
aci
d
0.52
0.
11
0.09
7
2.2
(1.6
-2.8
) 1.
9 (1
.3-2
.5)
2.4
(1.8
-3.0
) 2.
1 (1
.5-2
.7)
28
2.7
(2.1
-3.3
) 2.
4 (1
.8-2
.9)
2.9
(2.3
-3.4
) 2.
6 (2
.0-3
.1)
42
2.7
(2.2
-3.3
) 2.
4 (1
.9-3
.0)
2.9
(2.3
-3.5
) 2.
6 (2
.0-3
.2)
Iso-
buty
ric a
cid
0.
63
0.91
0.
01
7 1.
6a (0
.9-2
.4)
1.6a
(0.9
-2.4
) 3.
1Ab
(2.4
-3.8
) 1.
7a (1
.0-2
.4)
28
2.3
(1.6
-3.0
) 1.
8 (1
.1-2
.4)
2.3A
B
(1.6
-3.0
) 2.
0 (1
.3-2
.7)
42
2.1
(1.5
-2.8
) 2.
4 (1
.8-3
.1)
1.8B
(1
.1-2
.4)
2.2
(1.6
-2.9
)
Is
o-va
leric
aci
d
0.68
0.
29
0.03
7
1.3
(0.6
-2.0
) 1.
4 (0
.8-2
.1)
2.6A
(1
.9-3
.3)
1.4
(0.8
-2.0
)
28
2.
2 (1
.5-2
.8)
1.7
(1.0
-2.3
) 2.
1AB
(1.5
-2.8
) 1.
8 (1
.2-2
.4)
42
1.6
(1.0
-2.2
) 1.
9 (1
.3-2
.5)
1.3B
(0
.7-1
.9)
1.7
(1.1
-2.4
)
136 Chapter 6. Manuscript 3
!29
1 Val
ues a
re p
rese
nted
as l
east
squa
re m
eans
and
95%
con
fiden
ce in
terv
als (
in p
aren
thes
es).
2 C
TRL
= co
ntro
l; A
B =
ant
ibio
tic g
roup
; PR
O =
pro
biot
ic g
roup
; PR
O+A
B =
pro
biot
ic+a
ntib
iotic
gro
up.
Num
ber o
f pig
lets
: CTR
L=49
; AB
=50;
PR
O=4
9; P
RO
+AB
=51.
3 T
= tre
atm
ent g
roup
4 A
= a
ge.
a, b
, c: R
ows w
ith d
iffer
ent l
ette
rs a
re si
gnifi
cant
ly d
iffer
ent (p<
0.05
). a,
b Val
ues w
ith d
iffer
ent s
uper
scrip
ts w
ithin
a ro
w a
re si
gnifi
cant
ly d
iffer
ent.
A,B
: Val
ues w
ith d
iffer
ent s
uper
scrip
ts w
ithin
a c
olum
n ar
e si
gnifi
cant
ly d
iffer
ent.
Chapter 6. Manuscript 3 137
! 30
Table 8 List of the OTUs that were significantly different between treatment groups1
Age Treatments
compared Total OTUs
Significantly different OTU(s)
OTU classification Log2 fold change
Ileum 3 PRO+AB vs. PRO 182 OTU_143 Bacteroides 7.2 28 PRO+AB vs. CTRL 189 OTU_4 Streptococcus 4.3 42 AB vs. PRO 95 OTU_362 Clostridium 5.9 Mid colon
3 AB vs. PRO 259 OTU_22 Fusobacterium 9.3 PRO+AB vs. AB 266 OTU_22 Fusobacterium 7.9 PRO+AB vs. PRO 230 OTU_872 Order Clostridiales -5.4 OTU_387 Streptococcus -6.4 AB vs. PRO 257 OTU_22 Fusobacterium 9.2 OTU_43 Prevotella -7.7 28 AB vs. PRO 734 OTU_13
9 more.. Bacteroides 7.7
AB vs. CTRL 719 OTU_273 OTU_219 OTU_262
Campylobacter Family Ruminococcaceae Order Tremblayales
9.5 6.4 6.5
PRO vs. CTRL 629 OTU_13 Bacteroides 9.2 PRO+AB vs. AB 694 OTU_386
OTU_13 Anaerovibrio Bacteroides
8.2 5.7
AB vs. PRO OTU_13 Bacteroides 7.7 OTU_2428 Lachnospiraceae 9.8 OTU_179 Rikellaceae 10 OTU_82 Coriobacteriaceae 8.2 OTU_2 Fusobacterium 6.9 OTU_666 Bacteroides 8.1 OTU_297 Bacteroides 9.9 OTU_36 Oscillospira 5.8 OTU_359 Veillonellaceae 8.7 42 AB vs. CTRL 924 OTU_86 Order RF39 7.6 PRO+AB vs. AB 914 OTU_86 Order RF39 6.3 PRO+AB vs. CTRL 901 OTU_33
OTU_9 Lactobacillus Lactobacillus
-5.3 -5.2
Faeces
7 AB vs. CTRL 451 OTU_3118 Family Ruminococcaceae 6.5 PRO+AB vs. AB 407 OTU_340
OTU_46 OTU_84 OTU_299 OTU_1880
Bacteroides Bacteroides Bacteroides Dorea Bacillus
6.7 -4.2 4.7 3.3 5.6
138 Chapter 6. Manuscript 3
! 31
OTU_545 Family Lachnospiraceae 3.3 PRO+AB vs. PRO 406 OTU_24 Fusobacterium -6.2 PRO+AB vs. CTRL 440 OTU_24
OTU_79 Fusobacterium Order Bacteroidales
-4.6 -4.5
AB vs. PRO 423 OTU_3118 Family Ruminococcaceae 4.4 28 AB vs. CTRL 1195 OTU_50
OTU_2333 Megasphaera Oscillospira
3.2 -2.8
PRO vs. CTRL 1071 OTU_129 20 more…
Bacteroides -6.2
PRO+AB vs. AB 1194 OTU_762 OTU_166
Ruminococcus Anaerovibrio
-6.2 4.8
PRO+AB vs. PRO 1099 OTU_135 178 more…
Bacteroides 6.4
PRO+AB vs. CTRL 1175 OTU_268 OTU_891 OTU_402 OTU_2333
Bacteroides Family S24-7 Bacteroides Oscillospira
3.2 4.5 3.5 3.8
AB vs. PRO 1114 OTU_404 Family Mogibacteriaceae -6.0 182 more… 42 AB vs. CTRL 1264 OTU_391 Order Bacteroidales 4.7 PRO vs. CTRL 1252 OTU_691
OTU_5 YRC22 Lactobacillus
2.1 -3.2
PRO+AB vs. AB 1306 OTU_865 OTU_391 OTU_121
Ruminococcus Order Bacteroidales Family Christensenellaceae
-6.0 4.2 -5.0
PRO+AB vs. PRO 1277 OTU_348 Family Veillonellaceae 5.3 AB vs. PRO 1332 OTU_391 Order Bacteroidales 6.1 OTU_31 p-75-a5 -3.3 OTU_256 Faecalibacterium 2.7 OTU_689 Lachnospiraceae -6.0 OTU_62 Coriobacteriaceae -2.8 OTU_720 Anaerovibrio -5.2 OTU_126 Peptococcus -2.5 OTU_273 Campylobacter 2.8
1 CTRL = control; AB = antibiotic group; PRO = probiotic group; PRO+AB = probiotic+antibiotic group.
Chapter 6. Manuscript 3 139
! 32
Figures:
Fig. 1. Enumeration of selected bacterial groups (A-F) in digesta (log cfu/g) from the stomach,
ileum, caecum and mid colon sampled day 3, 28 and 42 from piglets administered gentamicin (AB;
n=71); piglets administered Bacillus spores (PRO; n=68); piglets administered both gentamicin and
Bacillus spores (PRO+AB; n=71) and control piglets not receiving gentamicin or Bacillus spores
(CON; n=72). Values are presented as least-square means and the 95% confidence intervals
presented as vertical bars. Bars embraced by horisontal brackets market by * (0.01≤p<0.05), **
(0.001≤p<0.01) or *** (p<0.001) are significantly different.
Fig. 2. Heatmaps of faecal samples collected at 7, 28 and 42 days of age from piglets administered
gentamicin (AB; n=53), Bacillus spores (PRO; n=50), both gentamicin and Bacillus spores
(PRO+AB; n=53) and control piglets not receiving gentamicin or Bacillus spores (CTRL; n=51).
Heatmaps show the relative abundances (%) of (A) the ten most abundant phyla and (B) the 20
most abundant genera in faecal samples. Colours represent the relative abundances.
Fig. 3. Observed and estimated species richness and Shannon diversity index of faecal samples
collected at 7, 28 and 42 days of age from piglets administered gentamicin (AB; n=53), Bacillus
spores (PRO; n=50), both gentamicin and Bacillus spores (PRO+AB; n=53) and control piglets not
receiving gentamicin or Bacillus spores (CTRL; n=51). Boxplots show the (A) observed and (B)
estimated species richness and (C) Shannon diversity index.
Fig. 4. Principal Component Analysis (PCA) of square root transformed OTU abundances
originating from faecal samples (n=207) displaying PC1 and PC2. (A) Day 7 samples; (B) Day 28
samples; (C) Constrained PCA on day 28 samples; (D) Day 42 samples; (E) Constrained PCA on
day 42 samples. Points are coloured according to treatment. AB: Piglets administered gentamicin;
PRO: Piglets administered Bacillus spores; PRO+AB: Both administered gentamicin and Bacillus
spores; CONTROL: Control piglets not receiving gentamicin or Bacillus spores.
Fig. 5. Heatmaps of ileal and colonic digesta samples collected at 3, 28 and 42 days of age from
piglets administered gentamicin (AB; n=33), Bacillus spores (PRO; n=33), both gentamicin and
Bacillus spores (PRO+AB; n=31) and control piglets not receiving gentamicin or Bacillus spores
(CTRL; n=29). Heatmaps show the relative abundances (%) of (A) the eight most abundant phyla in
140 Chapter 6. Manuscript 3
! 33
ileal digesta, (B) the 20 most abundant genera in ileal digesta, (C) the eight most abundant phyla in
colonic digesta and (D) the 20 most abundant genera in colonic digesta. Colours represent the
relative abundances.
Fig. 6. Observed and estimated species richness and Shannon diversity index of ileal and colonic
digesta samples collected at 3, 28 and 42 days of age from piglets administered gentamicin (AB;
n=33), Bacillus spores (PRO; n=33), both gentamicin and Bacillus spores (PRO+AB; n=31) and
control piglets not receiving gentamicin or Bacillus spores (CONTORL; n=29). Boxplots show the
(A) observed and (B) estimated species richness and (C) Shannon diversity index.
Fig. 7. (A) Principal Component Analysis (PCA) of square root transformed OTU abundances in
colonic digesta (n=65) displaying PC1 and PC2. Points are coloured according to treatment and
grouped according to age. (B) Constrained PCA of square root transformed OTU abundances in
colonic digesta day 28 (n=17). Points are coloured for treatment. AB: Piglets administered
gentamicin; PRO: Piglets administered Bacillus spores; PRO+AB: Both administered gentamicin
and Bacillus spores; CONTORL: Control piglets not receiving gentamicin or Bacillus spores.
Fig. 8. Gene expression levels of (A) TNF-α, (B) IL-10, (C) Cyclo-oxygenase-2, (D) ZO-1, (E)
OCLN, (F) CLDN-4 and (G) CLDN-2 in ileal tissue collected day 28 (n=22) and 42 (n=34) from
piglets administered gentamicin (AB); piglets administered Bacillus spores (PRO); piglets
administered both gentamicin and Bacillus spores (PRO+AB) and control piglets not receiving
gentamicin or Bacillus spores (CONTROL), that has either been left untreated or stimulated with
LPS. Values are presented as least-square means and the 95% confidence intervals presented as
vertical bars. Bars embraced by horisontal brackets market by * (0.01≤p<0.05), ** (0.001≤p<0.01)
or *** (p<0.001) are significantly different.
Fig. S1. Principal Component Analysis of square root transformed OTU abundances in faeces
(n=207) displaying PC1 and PC2. Points are coloured according to age.
Fig. S2. Principal Component Analysis of square root transformed OTU abundances in ileal (n=61)
and colonic (n=65) digesta displaying PC1 and PC2. Points are coloured according to age and
shaped according to segment.
Chapter 6. Manuscript 3 141
1 3 5 7
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
3 28 42 3 28 42 3 28 42 3 28 42Age (days)
Log
cfu/
g di
gest
a Treatment
AB
CON
PRO
PRO+AB
Total anaerobic bacteria
***
*** ** **
Log
cfu/
g di
gest
a
Age (days)
Total anaerobic bacteriaStomach Ileum Caecum Mid colon
1 3 5 7
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
3 28 42 3 28 42 3 28 42 3 28 42Age (days)
Log
cfu/
g di
gest
a Treatment
AB
CON
PRO
PRO+AB
EnterobacteriaceaeEnterobacteriaceaeStomach Ileum Caecum Mid colon
*
**
*** ****** ***
Age (days)Lo
g cf
u/g
dige
sta
1 3 5 7
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
3 28 42 3 28 42 3 28 42 3 28 42Age (days)
Log
cfu/
g di
gest
a Treatment
AB
CON
PRO
PRO+AB
Haemolytic bacteriaHaemolytic bacteriaStomach Ileum Caecum Mid colon
Log
cfu/
g di
gest
a
Age (days)
******
******
****** *** ***
1 3 5 7
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
3 28 42 3 28 42 3 28 42 3 28 42Age (days)
Log
cfu/
g di
gest
a Treatment
AB
CON
PRO
PRO+AB
Lactic acid bacteria
Log
cfu/
g di
gest
a
Age (days)
Stomach Ileum Caecum Mid colon
Lactic acid bacteria
*** * * ****
***
1 3 5 7
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
3 28 42 3 28 42 3 28 42 3 28 42Age (days)
Log
cfu/
g di
gest
a Treatment
AB
CON
PRO
PRO+AB
Clostridium perfringensClostridium perfringensStomach Ileum Caecum Mid colon
Age (days)
Log
cfu/
g di
gest
a
*
******
***
***
***
***
***
***
1 3 5 7
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
3 28 42 3 28 42 3 28 42 3 28 42Age (days)
Log
cfu/
g di
gest
a Treatment
AB
CON
PRO
PRO+AB
BacillusBacillus spp. sporesStomach Ileum Caecum Mid colon
Age (days)
Log
cfu/
g di
gest
a
*********
***
(A) (B)
(D)(C)
(E) (F)
Figure 1
142 Chapter 6. Manuscript 3
7 28 42
0.1
0
0.1
0.1
0.9
0
14.3
33.7
0
50.2
0.4
0
19.9
43.1
0
0
0.6
0
35.8
0
40.4
0.2
0
0.1
0
15.1
0
0
1.4
42.6
0
45.8
0.2
0
0
0
1
0
16.6
35.9 54.1
0.3
30
2
0.7
1.1
2.8
1.4
0.7
5.3
44.7
0.5
1.2
10
0.1
37.7
0.7
2.3
1.4
0.7
17.2
38.1
39
0.6
1.1
0.5
0.5
0.2
0.3
1.8
0.6
7.8
48.3
0.4
36
0.6
0.8
0.4
2
1.2 1.5
0
0.2
64
0.4
29.2
1.5
0.7
0
1.8
1.9
63.5
0.6
1
0
0
0.6
30.6
1.2
0
1.1
4.7
0.1
59.4
32.1
0.5
0
0
0.2
1.2
0
1.3
2.5
0
0.2
61.2
0.4
32.2
0.6
1
Lentisphaerae
Cyanobacteria
Synergistetes
Tenericutes
Actinobacteria
Spirochaetes
Proteobacteria
Fusobacteria
Bacteroidetes
Firmicutes
7 A
B
7 C
ONT
ROL
7 P
RO
7 P
RO+A
B
28 A
B
28 C
ONT
ROL
28 P
RO
28 P
RO+A
B
42 A
B
42 C
ONT
ROL
42 P
RO
42 P
RO+A
B
0.1
1.0
10.0
% ReadAbundance
Day 7 Day 28 Day 42
AB CTRL PRO PRO+AB AB CTRL PRO PRO+AB AB CTRL PRO PRO+AB
7 28 42
0.2
18.8
0
1.5
0
0.6
0.4
9.6
2.11.2
0
0
35
0
0
14.3
0.3
0.9
0
1.3
0.2
17.5
0
0.5
1.21.6
28.1
0
0.1
1.6
19.9
0.4
8
2.2
0
0
0.7
0.1
0.3
0.4
0
0
0.8
27.4
0
22.8
2.7
15.1
0.4
8.9
2.6
1.1
0
1.3
0
0
0.2
0.2
0
0.1
32.4
0.3
2.1
0
8.7
1.1
0.4
1.4
0
20.2
0
0.4
16.6
0.2
0.1
0.4
1.7
0.9
0
0
3.5
1.6
1.7
4.3
2.4
3.6
0.2
0.1
0.3
0.3
1.6
6.2
0.90.5
4.6
0.1
0.1
5.3
1.8
3.5
7.8
1.7
2.6
4.7
0.2
0.1
1.10.3
0.6
3.5
0.6
0.2
0.1
10
1
12.5
2.96
0.3
1.3
2.72.6
0.4
0.70.9
2.7
6
0.6
6
0.1
17.2
0.1
17
4
0.9
5.8
0.1
0.4
0.4
0.7
1.6
2.1
8.5
2.7
0.1
0.6
10.8
3.8
0.2
1.8
1.7
3.4
0.4
3.4
0.2
0.2
7.8
0.2
0.9
1.3
2.2
0.5
10.3
0.7
0.6
1.3
1.1
0.8
3.1
12.2
2.8
0.3
1.2
3.7
2.2
0.6
2.9
2
0.7
0.2
4.1
0.9
0.1
0.7
3.5
13.2
1.6
1.8
15.7
0.61.7
0.8
0.4
2.4
3.3
0.2
0
1.92.8
1.2
1.70.25.7
1.8
1.3
0.7
0.61.7
0.7
0.5
1.7
6.5
0.3
0.114.1
1.9
0.4
2.6
3.3
3.31.6
2.5
0.4
0.2
0.4
3.9
0.2
0.2
1.7
0.6
16.4
1.9
1.1
0.8
0.8
2.8
2.2
13.3
3.2
1.5Firmicutes; Anaerovibrio
Proteobacteria; CampylobacterBacteroidetes; f__p−2534−18B5_OTU_58
Firmicutes; SMB53Firmicutes; Clostridium
Firmicutes; MegasphaeraFirmicutes; Streptococcus
Firmicutes; RoseburiaBacteroidetes; Parabacteroides
Firmicutes; p−75−a5Firmicutes; Coprococcus
Firmicutes; 02d06Bacteroidetes; o__Bacteroidales_OTU_17
Bacteroidetes; [Prevotella]Firmicutes; Ruminococcus
Firmicutes; OscillospiraFusobacteria; Fusobacterium
Bacteroidetes; PrevotellaFirmicutes; Lactobacillus
Bacteroidetes; Bacteroides
7 A
B
7 C
ONT
ROL
7 P
RO
7 P
RO+A
B
28 A
B
28 C
ONT
ROL
28 P
RO
28 P
RO+A
B
42 A
B
42 C
ONT
ROL
42 P
RO
42 P
RO+A
B
0.1
1.0
10.0
% ReadAbundance
Day 7 Day 28 Day 42
AB CTRL PRO PRO+AB AB CTRL PRO PRO+AB AB CTRL PRO PRO+AB
Bacteroidetes; order Bacteroidales OTU 17
Bacteroidetes; family p-2534-18B5 OTU 58
(A)
(B)
Figure 2
Chapter 6. Manuscript 3 143
7 28 42
125
250
375
500
625
750
875
1000
AB
CONTROLPRO
PRO+AB AB
CONTROLPRO
PRO+AB AB
CONTROLPRO
PRO+AB
Treatment
Obs
erve
d nu
mbe
r of O
TUs
Obs
erve
d nu
mbe
r of O
TUs
AB CTRL PRO PRO+AB AB CTRL PRO PRO+AB AB CTRL PRO PRO+AB
Treatment
Day 7 Day 28 Day 42
7 28 42
125
250
375
500
625
750
875
1000
1125
1250
AB
CONTROLPRO
PRO+AB AB
CONTROLPRO
PRO+AB AB
CONTROLPRO
PRO+AB
Treatment
Estim
ated
num
ber o
f OTU
sE
stim
ated
num
ber o
f OTU
s
AB CTRL PRO PRO+AB AB CTRL PRO PRO+AB AB CTRL PRO PRO+AB
Treatment
Day 7 Day 28 Day 42
7 28 42
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
3.50
3.75
4.00
4.25
4.50
4.75
5.00
5.25
AB
CONTROLPRO
PRO+AB AB
CONTROLPRO
PRO+AB AB
CONTROLPRO
PRO+AB
Treatment
Dive
rsity
inde
xD
iver
sity
inde
x
AB CTRL PRO PRO+AB AB CTRL PRO PRO+AB AB CTRL PRO PRO+AB
Treatment
Day 7 Day 28 Day 42
(B)
(A)
(C)
Figure 3
144 Chapter 6. Manuscript 3
−6
−3
0
3
6
−3 0 3 6PC1 [13.9%]
PC2
[10.
8%]
Treatment
AB
CONTROL
PRO
PRO+AB
Faeces day 7
PC
2 - v
aria
tion
expl
aine
d 10
.8%
PC1 - variation explained 13.9%
PCA - faeces day 7
−4
0
4
−5.0 −2.5 0.0 2.5 5.0PC1 [18.2%]
PC2
[11.
7%]
Treatment
AB
CONTROL
PRO
PRO+AB
Faeces day 28
PC
2 - v
aria
tion
expl
aine
d 11
.7%
PC1 - variation explained 18.2%
PCA - faeces day 28
−10
−5
0
5
−4 0 4 8RDA1 [8.2%]
RDA2
[2.1
%]
Treatment
AB
CONTROL
PRO
PRO+AB
Faeces day 28
RD
A2
- var
iatio
n ex
plai
ned
2.1%
RDA1 - variation explained 8.2%
RDA - faeces day 28
−3
0
3
6
−3 0 3PC1 [16.4%]
PC2
[9.1
%]
Treatment
AB
CONTROL
PRO
PRO+AB
Faeces day 42
PC
2 - v
aria
tion
expl
aine
d 9.
1%
PC1 - variation explained 16.4%
PCA - faeces day 42
−5
0
5
−5 0 5 10RDA1 [4%]
RDA2
[2.2
%]
Treatment
AB
CONTROL
PRO
PRO+AB
Faeces day 42
RD
A2
- var
iatio
n ex
plai
ned
2.2%
RDA1 - variation explained 4%
RDA - faeces day 42
(A)
(E)(D)
(C)(B) −6
−3
0
3
6
−3 0 3 6PC1 [13.9%]
PC2
[10.
8%]
Treatment
AB
CONTROL
PRO
PRO+AB
Faeces day 7
PC
2 - v
aria
tion
expl
aine
d 10
.8%
PC1 - variation explained 13.9%
PCA - faeces day 7
−4
0
4
−5.0 −2.5 0.0 2.5 5.0PC1 [18.2%]
PC2
[11.
7%]
Treatment
AB
CONTROL
PRO
PRO+AB
Faeces day 28
PC
2 - v
aria
tion
expl
aine
d 11
.7%
PC1 - variation explained 18.2%
PCA - faeces day 28
−10
−5
0
5
−4 0 4 8RDA1 [8.2%]
RDA2
[2.1
%]
Treatment
AB
CONTROL
PRO
PRO+AB
Faeces day 28
RD
A2
- var
iatio
n ex
plai
ned
2.1%
RDA1 - variation explained 8.2%
RDA - faeces day 28
−3
0
3
6
−3 0 3PC1 [16.4%]
PC2
[9.1
%]
Treatment
AB
CONTROL
PRO
PRO+AB
Faeces day 42
PC
2 - v
aria
tion
expl
aine
d 9.
1%
PC1 - variation explained 16.4%
PCA - faeces day 42
−5
0
5
−5 0 5 10RDA1 [4%]
RDA2
[2.2
%]
Treatment
AB
CONTROL
PRO
PRO+AB
Faeces day 42
RD
A2
- var
iatio
n ex
plai
ned
2.2%
RDA1 - variation explained 4%
RDA - faeces day 42
(A)
(E)(D)
(C)(B)
Figure 4
Chapter 6. Manuscript 3 145
328
42
0.5
2.2
0.2 01.1
95.1 00.8
0.3
0.5 0 0086.5
0.7
12.1
0.5
1.2 089.4 6 3 00
7.8
0.5
1.1
88.8 0 01.7
0.1
0.3
0.4
0.5
0.2 0 098.3
0.1
0.1
0.2 00 098.4
0.3
0.8
096.3
0.1
0.8
1.2
1.6 00
0.3
0.2
3.1
0.7
95.6 0 00
0.2 00.3
0.20 0099.3
0.2
0.2 099.3 0 0.2 0 0
99.1 00 0.1
0.4
0.3 00
01.6
0.2 00.6
0.2
97.3 0
Tene
ricut
es
Cyan
obac
teria
TM7
Actin
obac
teria
Bact
eroi
dete
s
Prot
eoba
cter
ia
Fuso
bact
eria
Firm
icute
s
AB 3
CONTROL 3
PRO 3
PRO+AB 3
AB 28
CONTROL 28
PRO 28
PRO+AB 28
AB 42
CONTROL 42
PRO 42
PRO+AB 42
0.1
1.0
10.0
% R
ead
Abu
ndan
ce
Ileum
Day
3D
ay 2
8D
ay 4
2Ile
um
AB
C
TRL
PR
O
PR
O+A
B
AB
C
TRL
PR
O
PR
O+A
B
AB
C
TRL
PR
O
PR
O+A
B
328
42
0.2
0.70 057.6
0.1
0.7
10.4 00.7
1.9
0.2
0.1
0.7 00.1
2.1
1.2
4.8
12.9
0.1
0.2057.2
6.8 00.1
3.7 12 0.3
0.1
0.2
0.1
0.1
0.3
12.1 10.31 2.3
1.1
0.6
37.7 00.3
8.5
0.2
3.7
0.20 0.7
0.5
27.7
2.2 05.5 0 1.2
0.3
5.9
0.3
7.7
1.1
2.5
0.3
0.3
42.1
0.1
14.2 00.2
0.1
1.3
20.8 0 0 0.2
0.8
4.5
0.1
0.2
1.3
33.6
0.4
0.1
3.2 07.7
3.5
4.7 0 0.1
0.5
3.79 0.2
26.3 00.1
2.2
6.8 1 0.1 0 0.1
0.8
0.1
1.1
53.7
0.2
0.1
1.2
0.1
2.6
28.5
0.1 00.6
0.3
1.5
0.5
3.6 0 0.1
2.6
53.2
0.3
0.5
1.4
0.4
0.7
16.8
0.4
0.9
13.8 01.5 000.1
0.1
3.3
0.2
61.3 00.2
1.4
0.81 02.6
1.1
4.1 00.3
1.1
2.5
8.3
7.9
0.1
064.1 0 00 0.2
0.5
0.1 00.1 00.6
0.1
3.6 0 00021.3 8
34.6
4.1 00.3
50.1 001.9
0.1 00.1 0 0.2
0.2 00.3 06.9 00
000.8 031 2.5
0.3 0 00.8
0.1
0.5
47.9 00.1
4.4
9.5
0.1 00
55 0026.1
0.1 00.5
1.4 00.2
0.3 02.8
0.5
0.2
10.3 0 0.60 0
Prot
eoba
cter
ia; A
ctin
obac
illus
Prot
eoba
cter
ia; S
utte
rella
Actin
obac
teria
; Cor
yneb
acte
rium
Firm
icute
s; G
emel
laBa
cter
oide
tes;
Bac
tero
ides
Firm
icute
s; A
cidam
inoc
occu
sBa
cter
oide
tes;
Pre
vote
llaPr
oteo
bact
eria
; f__
Ente
roba
cter
iace
ae_O
TU_1
36Fi
rmicu
tes;
o__
Clos
tridi
ales
_OTU
_872
Firm
icute
s; V
eillo
nella
Firm
icute
s; M
egas
phae
raFi
rmicu
tes;
Tur
iciba
cter
Firm
icute
s; M
itsuo
kella
Fuso
bact
eria
; Fus
obac
teriu
mFi
rmicu
tes;
Stre
ptoc
occu
sFi
rmicu
tes;
Sar
cina
Firm
icute
s; C
lost
ridiu
mFi
rmicu
tes;
SM
B53
Firm
icute
s; 0
2d06
Firm
icute
s; L
acto
bacil
lus
AB 3
CONTROL 3
PRO 3
PRO+AB 3
AB 28
CONTROL 28
PRO 28
PRO+AB 28
AB 42
CONTROL 42
PRO 42
PRO+AB 42
0.1
1.0
10.0
% R
ead
Abu
ndan
ce
Ileum
Day
28
Day
3D
ay 2
8D
ay 4
2Ile
um
AB
C
TRL
P
RO
PR
O+A
B
AB
C
TRL
P
RO
PR
O+A
B
AB
C
TRL
P
RO
PR
O+A
B
Firm
icut
es; o
rder
Clo
strid
iale
s O
TU 8
72
Pro
teob
acte
ria; f
amily
Ent
erob
acte
riace
ae O
TU 1
36
328
42
0.3
25.5 002.5
41.1 030.6
0.2
30.5 00 032 1.4
35.7
25.9
0.3 042.1
29.3
2.4 0 0
32.2
0.3
1.4
20.5 0045.7 0
0.6
33.9
1.3
1.2
0.6
3.2
56.8
0.4
0.4
38.3
0.112.5
51.1
3.4
2.3
0.1
41.7
25.5
28.2
0.9
1.3
0.4
0.9
3.4
14.2
50.2
0.2
29.8
0.11 0.3
0.7
0.9
28.9
4.6
0.3
0.4
0.5
63.3
0.4
21.4
0.3
72.8
0.5
1.7
1.8
0.8
64.4
0.3
0.6
3.3
28.6
0.4
0.8
0.8
14.6
0.4
0.3
29.7
0.5
61.8
1.2
Cyan
obac
teria
Actin
obac
teria
Tene
ricut
es
Spiro
chae
tes
Prot
eoba
cter
ia
Fuso
bact
eria
Bact
eroi
dete
s
Firm
icute
s
AB 3
CONTROL 3
PRO 3
PRO+AB 3
AB 28
CONTROL 28
PRO 28
PRO+AB 28
AB 42
CONTROL 42
PRO 42
PRO+AB 42
0.1
1.0
10.0
% R
ead
Abu
ndan
ce
Colo
nD
ay 3
Day
28
Day
42
Col
on
AB
C
TRL
PR
O
PR
O+A
B
AB
C
TRL
PR
O
PR
O+A
B
AB
C
TRL
PR
O
PR
O+A
B
328
42
0.2
11.6
1.909.5
25.5 00.5
4.2
0.6
0.3024.5 0 20.1
2.7
0.2 00.8
22.8
0.1 05.6
0.2
5.2
0.1
0.5 016.5
1.1
1.2035.7 01 0.1
0.3 10.1
0.1
0.1
25.2
0.2
0.1 024.8 03.6
0.1 029.2
0.6
0.1 00.2
1.6
1.7 03.1
01.1
1.200.2
6.3
4.6
32.2
0.5
1.7
0.2
0.1
41.4 0 1.10 0.2
0.2
0.4
0.9
2.6
3.8
0.3
1.2
9.9
2.7
4.2
0.5
0.3
0.3
0.1
0.1
4.8
3.5
1.2
1.3
3.8
0.4
0.7
1.7
0.32 1.1
2.6
5.2
7.5
0.110.3
3.4
0.6
1.3
15.2
1.6
4.2
1.4
0.2
1.5
0.6
0.6
13.1
1.5
3.5
2.6
0.10 0.6
1.5
3.2
0.6 03.9
1.4
7.2
0.3
0.9
0.1
25.5
0.1
0.1
2.8
0.7
1.8
0.1
14.2
0.1
32.1 1 0.5
6.4
0.3
7.2
0.2
1.7
0.9
1.1
0.8
1.6
0.1
1.3
4.6
0.4
12.3
0.3
2.9
12.5
3.8
3.4
0.3 2 0.6
1.5
1.1
2.7
0.5
0.1
1.3
3.8
0.3
0.9
0.4
0.6
3.4
27.2
0.6
0.5 8 1.3
1.2
0.4
1.7
0.4
2.9
0.7
1.24 1.2
1.8
0.1
3.7
1.4
3.7
4.7
8.4
0.5
0.8
0.9
0.6
4.1
10.5
0.5
3.7
0.9
0.6
1.4
1.63 1.9
0.1
1.7
0.4
1.1
10.1
0.5
1.4
4.5
4.3
3.5
0.4
2.1
3.5
1.3
1.7
1.2
1.33 110.2
0.4
0.2
Bact
eroi
dete
s; P
arab
acte
roid
esBa
cter
oide
tes;
CF2
31Fi
rmicu
tes;
Dor
eaFi
rmicu
tes;
p−7
5−a5
Firm
icute
s; A
naer
ovib
rioPr
oteo
bact
eria
; Sut
tere
llaFi
rmicu
tes;
Meg
asph
aera
Firm
icute
s; S
MB5
3Pr
oteo
bact
eria
; Cam
pylo
bact
erFi
rmicu
tes;
Rum
inoc
occu
sFi
rmicu
tes;
Cop
roco
ccus
Firm
icute
s; S
trept
ococ
cus
Firm
icute
s; 0
2d06
Firm
icute
s; O
scillo
spira
Bact
eroi
dete
s; [P
revo
tella
]Fi
rmicu
tes;
Clo
strid
ium
Bact
eroi
dete
s; P
revo
tella
Firm
icute
s; L
acto
bacil
lus
Bact
eroi
dete
s; B
acte
roid
esFu
soba
cter
ia; F
usob
acte
rium
AB 3
CONTROL 3
PRO 3
PRO+AB 3
AB 28
CONTROL 28
PRO 28
PRO+AB 28
AB 42
CONTROL 42
PRO 42
PRO+AB 42
0.1
1.0
10.0
% R
ead
Abu
ndan
ce
Colo
nD
ay 3
Day
28
Day
42
Col
on
AB
CTR
L
P
RO
P
RO
+AB
A
B
C
TRL
PR
O
PR
O+A
B
AB
CTR
L
P
RO
P
RO
+AB
(A)
(B)
(C)
(D)
Figu
re 5
146 Chapter 6. Manuscript 3
Ileum Mid colon
125
250
375
500
625
750
875
1000
3 28 42 3 28 42Age (days)
Obs
erve
d nu
mbe
r of O
TUs
Treatment
AB
CONTROL
PRO
PRO+AB
Ileum Mid colon
Obs
erve
d nu
mbe
r of O
TUs
Age (days)
Ileum Mid colon
125
250
375
500
625
750
875
1000
1125
1250
3 28 42 3 28 42Age (days)
Estim
ated
num
ber o
f OTU
s
Treatment
AB
CONTROL
PRO
PRO+AB
Ileum Mid colon
Est
imat
ed n
umbe
r of O
TUs
Age (days)
Ileum Mid colon
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
3.50
3.75
4.00
4.25
4.50
4.75
5.00
5.25
5.50
5.75
6.00
3 28 42 3 28 42Age (days)
Dive
rsity
inde
x
Treatment
AB
CONTROL
PRO
PRO+AB
Ileum Mid colon
Div
ersi
ty in
dex
Age (days)
(A)
(B)
(C)
Ileum Mid colon
125
250
375
500
625
750
875
1000
3 28 42 3 28 42Age (days)
Obs
erve
d nu
mbe
r of O
TUs
Treatment
AB
CONTROL
PRO
PRO+AB
Ileum Mid colonO
bser
ved
num
ber o
f OTU
s
Age (days)
Ileum Mid colon
125
250
375
500
625
750
875
1000
1125
1250
3 28 42 3 28 42Age (days)
Estim
ated
num
ber o
f OTU
s
Treatment
AB
CONTROL
PRO
PRO+AB
Ileum Mid colon
Estim
ated
num
ber o
f OTU
s
Age (days)
Ileum Mid colon
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
3.50
3.75
4.00
4.25
4.50
4.75
5.00
5.25
5.50
5.75
6.00
3 28 42 3 28 42Age (days)
Dive
rsity
inde
x
Treatment
AB
CONTROL
PRO
PRO+AB
Ileum Mid colon
Div
ersi
ty in
dex
Age (days)
(A)
(B)
(C)
Ileum Mid colon
125
250
375
500
625
750
875
1000
3 28 42 3 28 42Age (days)
Obs
erve
d nu
mbe
r of O
TUs
Treatment
AB
CONTROL
PRO
PRO+AB
Ileum Mid colon
Obs
erve
d nu
mbe
r of O
TUs
Age (days)
Ileum Mid colon
125
250
375
500
625
750
875
1000
1125
1250
3 28 42 3 28 42Age (days)
Estim
ated
num
ber o
f OTU
s
Treatment
AB
CONTROL
PRO
PRO+AB
Ileum Mid colon
Estim
ated
num
ber o
f OTU
s
Age (days)
Ileum Mid colon
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
3.50
3.75
4.00
4.25
4.50
4.75
5.00
5.25
5.50
5.75
6.00
3 28 42 3 28 42Age (days)
Dive
rsity
inde
x
Treatment
AB
CONTROL
PRO
PRO+AB
Ileum Mid colon
Div
ersi
ty in
dex
Age (days)
(A)
(B)
(C)
Ileum Mid colon
125
250
375
500
625
750
875
1000
3 28 42 3 28 42Age (days)
Obs
erve
d nu
mbe
r of O
TUs
Treatment
AB
CONTROL
PRO
PRO+AB
Ileum Mid colon
Obs
erve
d nu
mbe
r of O
TUs
Age (days)
Ileum Mid colon
125
250
375
500
625
750
875
1000
1125
1250
3 28 42 3 28 42Age (days)
Estim
ated
num
ber o
f OTU
s
Treatment
AB
CONTROL
PRO
PRO+AB
Ileum Mid colon
Estim
ated
num
ber o
f OTU
s
Age (days)
Ileum Mid colon
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
3.50
3.75
4.00
4.25
4.50
4.75
5.00
5.25
5.50
5.75
6.00
3 28 42 3 28 42Age (days)
Dive
rsity
inde
x
Treatment
AB
CONTROL
PRO
PRO+AB
Ileum Mid colon
Div
ersi
ty in
dex
Age (days)
(A)
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Chapter 6. Manuscript 3 147
3 28 42
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%]
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Day 3 Day 28 Day 42
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4%
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−8 −4 0 4RDA1 [24.1%]
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%]
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RDA - colon day 28
RD
A2
- var
iatio
n ex
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ned
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RDA1 - variation explained 24.1%
PC1 - variation explained 26.4%
(A)
(B)
Figure 7
148 Chapter 6. Manuscript 3
2842
0.0
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1.5
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rol
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rol
LPS
Tiss
ue
Fold change
Treatment
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ROL
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+AB
TNF−
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Fold change*
****
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trol
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ated
2842
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ue
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ROL
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PRO
+AB
COX2
COX-2
Fold change
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*
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trol
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ated
2842
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AB CONT
ROL
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+AB
ZO1
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Fold change
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ZO-1
Con
trol
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-stim
ulat
edC
ontro
lLP
S-s
timul
ated
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(B)
(D)
(C) Fi
gure
8 (1/2)
Chapter 6. Manuscript 3 149
2842
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OCL
NOCLN
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Treatment
AB CONT
ROL
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PRO
+AB
CLDN
4
Fold change
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CLDN-4
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trol
LPS
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ulat
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ontro
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S-s
timul
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2842
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Tiss
ue
Fold change
Treatment
AB CONT
ROL
PRO
PRO
+AB
CLDN
2CLDN-2
Fold change
Tiss
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ontro
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S-s
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-stim
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(E)
(F)
(G) Fi
gure
8 (2/2)
150 Chapter 6. Manuscript 3
−4
−2
0
2
4
−5.0 −2.5 0.0 2.5PC1 [19.6%]
PC2
[15.
8%]
10
20
30
40Age
Sample_Type
Ileum
Mid colon
PC
2 - v
aria
tion
expl
aine
d 15
.8%
PC1 - variation explained 19.6%
−4
−2
0
2
−2 0 2 4PC1 [21.6%]
PC2
[9.6
%]
10
20
30
40
Age
PC
2 - v
aria
tion
expl
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d 9.
6%
PC1 - variation explained 21.6%Figure S1
Figure S2
Chapter 6. Manuscript 3 151
Chapter 7. General discussion 153
Chapter 7
General discussion
Intensive pig production systems, optimised for taking advantage of the pigs fullreproductive potential, demand adult pigs and piglets to cope with unnatural con-ditions as extremely large litter sizes and early weaning. Piglets experience a majorhurdle after weaning, where intestinal disorders and diarrhoea are highly prevalent,leading to decreased animal welfare and frequent use of antibiotics (DANMAP,2014). The gut microbiota, and the process of its development, is appreciated asan essential part of the mammalian gut, serving numerous functions in relation toboth animal and human health. The dynamic nature of the gut microbial com-munity allows it to be manipulated by a range of external and internal factors,many of which can potentially be taken advantage of in the search for measurescapable of improving gut health. Hence, a deeper understanding of how, when,and to what extent the piglet gut microbiota can be manipulated is pivotal, if suchmanipulating actions are to be further developed, and, with time, find general ap-plications in enhancing gut robustness. Thus, the overall aim of the present thesiswas to investigate the influencing effects of external and host-related factors onthe development of the piglet gut microbiota.
The following discussion highlights the major findings from the animal experi-ments presented in chapter 4, 5 and 6, and discusses these in relation to the currentscientific knowledge.
7.1 Manipulating the gut microbiota
The major objective of the current thesis was to characterise if, and how, BC,FUT1 genotype, gentamicin, and Bacillus spp. spores influenced the microbialcolonisation of the piglet gut. This point is discussed in the following sections,
154 Chapter 7. General discussion
including relevant experimental issues.
7.1.1 Milk-based dietary intervention
The aim of manuscript 1 (chapter 4) was to investigate the isolated effects of eachof the included milk-based diets, and required piglets from the same litter to beassigned to three different dietary groups. Piglets were fed no other feed thanmilk, and one week was therefore considered to be enough time for the milk-dietto exert its potential effects on the gut microbiota (e.g. Heinritz et al., 2016). Toallow sufficient milk intake, sow milk-fed piglets were housed together with the sowduring the experimental period. Thus, continuous contact between the sow andsow milk-fed piglets, different housing environments and the stress of weaning wereexpected to cause variation between piglets suckling the sow and piglets in the twoexperimental dietary groups. The sow milk-fed piglets were therefore included asa reference group.
In accordance with previous dietary studies performed on suckling piglets (Caoet al., 2016; Frese et al., 2015; Yeruva et al., 2016), manuscript 1 illustrates thatdifferent diets, in this case different milk types/sources, clearly change the compo-sition of the gut microbiota. This observation is in agreement with human studies,where breast-fed and formula-fed children have been found to have different gutmicrobiotas (e.g. Bokulich et al., 2016). As mother’s own milk is preferred, butnot always available, milk replacers are indispensable feeding supplements, andformula manufacturers are constantly trying to optimise their products (Martinet al., 2016).
The rise of interest for BC stems in particular from its high content of bioactivecomponents as growth hormones and immunomodulators (Pandey et al., 2011). Inour study, the finding of a change in genera belonging to lactic acid bacteria inBC-fed piglets, and a rise in the number of potentially pathogenic Enterobacteri-aceae in MR-fed piglets confirms, that BC provides components to the gut thatare in fact able to promote a gut microbiota harbouring host-beneficial bacteria.This observation agrees with previous studies in piglets, where MR-feeding is ac-companied by a rise in Enterobacteria (Yeruva et al., 2016). The lower incidence ofdiarrhoea in BC-fed piglets, compared to those fed MR, suggests that some of thebioactive components in BC are indeed able to provide protection against intesti-nal pathogens. In accordance with the general perception of mother’s own milkbeing the preferred nutrition for suckling piglets, it was obvious that sow milk wassuperior to the investigated experimental diets. However, when mother’s milk is
Chapter 7. General discussion 155
not available, the loss of important compounds may be counteracted by the rangeof bioactive components found in BC. Such a potential can be taken advantageof when piglets are weaned. Weaning is commonly associated with decreased feedintake, weight loss or growth check, and diarrhoea caused by ETEC (Fairbrotheret al., 2005; Lalles et al., 2007). Previous studies on BC and its use in piglets post-weaning have shown, that BC has a beneficial effect on both weight gain and feedintake (Boudry et al., 2008; Huguet et al., 2012). Due to the experimental set-upof our study, we are not able to make any conclusions on the effect of BC on pigletbody weight under normal weaning conditions. As enteric colibacillose is highlyprevalent after weaning, the apparent ability of BC to reduce the number of Enter-obacteriaceae in digesta suggests that BC has the potential to reduce colonisationof ETEC, and hence reduce the number of piglets with diarrhoea and in need ofantibiotics. In support of this, results published by Sugiharto et al. (2015), thatoriginate from the same animal experiment as the one presented in manuscript1, furthermore confirm that the number of mucosa-associated Enterobacteriaceaeand haemolytic bacteria indeed was reduced in the jejunum and ileum of BC-fedpiglets (Sugiharto et al., 2015). Hence, provision of BC seems to be a promisingstrategy to enhance gut health of (weak) piglets.
7.1.2 ETEC F18 susceptibility
Earlier studies have reported pig breed, and hence genotype, to influence the gutmicrobial composition (Bian et al., 2016; Pajarillo et al., 2015). The rationalebehind manuscript 2 varied from those of manuscript 1 and 3, as the objective wasto investigate whether a genetic mutation causing resistance to ETEC F18 alsocaused an unintentional change in the microbial colonisation of the gut. FUT1expression is age-dependent (Bao et al., 2012), and piglets were studied from birthuntil 34 days of age. This period included weaning at day 28, an event wheresusceptible piglets were highly likely to become infected by ETEC F18.
As the FUT1 enzyme is expressed in the gut (Bao et al., 2012) and acts byfucosylating glycoproteins and -lipids (Meijerink et al., 2000), lack of a functionalenzyme may impose changes to other glycoproteins found in close relation to theintestinal mucosa. From the results of manuscript 2, we were not able to showthat piglets with different FUT1 genotypes (307A/G and 307G/G) also experiencedsignificant differences in their early gut colonisation (i.e. during suckling and thefirst week after weaning). Piglets were partly confirmed to have different suscepti-bilities to ETEC F18, as the number of luminal intestinal haemolytic bacteria was
156 Chapter 7. General discussion
significantly different between piglets with different genotypes. As the haemolyticcolonies unfortunately were not serotyped, we do not know with certainty whetherthe colonies indeed did represent ETEC F18.
The study only included 17 piglets originating from two different sows, andwas an obvious limitation of the study. Bian et al. (2016) show that genotypeis a weaker influencing factor than weaning and age, suggesting that potentialeffects of genotype are harder to detect, which, combined with the low number ofanimals, potentially can be the reason for the observed results. From the currentexperiment, we could not provide evidence that ETEC F18 resistant piglets werein risk of developing a gut microbiota that could potentially compromise animalhealth later in life.
7.1.3 Bacillus spores and gentamicin
Antibiotics are generally believed to have a big influence on the composition of thegut microbiota. As the colonisation process in young animals is pivotal to properintestinal immune education (Sommer and Baeckhed, 2013), perturbations in earlylife may have detrimental long-term effects as suggested by e.g. the associationbetween early antibiotic administration and allergic conditions in children (Almet al., 2008; Goksor et al., 2013). Gentamicin is a commonly used drug for treatingsystemic infections in newborn infants (Chirico et al., 2009) and neonatal diarrhoeain piglets. Hence, gentamicin was believed to be the most relevant antibiotic toinvestigate in newborn piglets. In agreement with previous studies investigatingthe influential effects of antibiotics on the gut microbial community in pigs (Looftet al., 2014b; Schokker et al., 2014), we found gentamicin to affect the colonic andfaecal microbial communities. Surprisingly, this effect was most evident severalweeks after the piglets had been treated with the antibiotic. The effect of gen-tamicin was lower than expected, and gentamicin was not associated with signs ofdysbiosis (e.g. diarrhoea). Actually, our results showed that gentamicin increasedthe microbial diversity (Shannon index), which was in accordance with Schokkeret al. (2014). When compared to gentamicin, administering Bacillus spores hadthe opposite effect on the microbial richness and diversity. Such a counteract-ing effect was previously reported in humans administered Lactobacillus casei forthree days after an antibiotic treatment had been completed (Pirker et al., 2013).Whereas the combined effect of an antibiotic and a probiotic reduced the num-ber of people developing antibiotic-associated diarrhoea in the human study, ourstudy did not provide evidence that the combined effect of gentamicin and Bacil-
Chapter 7. General discussion 157
lus spores resulted in clinically healthier piglets. On the contrary, litters receivingBacillus spores had a higher frequency of diarrhoea than those only administeredgentamicin, especially during suckling. Li et al. (2012) reported an increased diar-rhoea incidence when the probiotic dose of Lactobacillus rhamnosus was increasedfrom 1x1010 to 1x1012 cfu, though both doses were able to reduce diarrhoea inci-dence after ETEC F4 challenge. Hence, the amount of Bacillus spores provided inthe current study may have been too high, but may have shown protective effectsduring an infection trial. The lack of uniformity between probiotic studies in pigs,as mentioned in the introduction, leaves us without solid theoretical knowledgeupon which we can base our choice of dose on. Thus, future studies on the use ofprobiotics for improving piglet gut health are needed.
7.2 Age-related succession of the gut microbiota
Manuscript 2 and 3 (presented in chapter 5 and 6) include longitudinal studies ofthe gut microbiota spanning a minimum of four weeks. The two studies clearlydemonstrate the importance of age in shaping the gut microbial communities ofpiglets. In both studies, a gradual change in the overall microbial community wasobserved during the suckling period. Additionally, there was a clear differencebetween the microbial communities on the day of weaning and 1-2 weeks post-weaning. Besides the impact of age, changes in microbial community profiles afterweaning are also caused by the dietary shift (from sow milk to solid feed) and themere impact of being weaned (stress and change of environment).
The earliest samples were collected three days after birth (manuscript 3), wherea colonisation process in obvious progress was evident. This is in agreement witholder (Jensen, 1998; Swords et al., 1993) and newer literature (Frese et al., 2015;Petri and Hill, 2010) stating that a gut microbiota is established within the first24 hours after birth. From birth until weaning, the microbiota became graduallyricher in species, and species diversity increased, peaking around 21 days of agein manuscript 2. In manuscript 3, the piglets were sampled until 42 days of age,and in this case, species richness and diversity increased from 28 to 42 days of age(however not seen when administered antibiotics). In agreement with our results,Frese et al. (2015) reported an increase in faecal diversity until two weeks afterweaning, and Slifierz et al. (2015) an increase in faecal species richness until oneweek after weaning. Differences between results obtained in manuscript 2 and 3might be caused by the fact that different procedures for 16S rRNA gene analysis
158 Chapter 7. General discussion
were used for sample and bioinformatic processing.On phylum level, the faecal community was consistently dominated by Firmi-
cutes and Bacteroidetes irrespective of age and experimental intervention. Further-more, in accordance with Frese et al. (2015) and Slifierz et al. (2015), Firmicutessteadily increased its dominance as the piglets aged. On genus level the picturewas slightly more inconsistent, and the dominating genera- and age-related differ-ences differed between experiments. These observations highlight the variabilitybetween studies and the difficulty of comparing experiments.
7.3 Sampling the gut microbial communities
The gut microbiota is a composed community consisting of several microhabitats.It is widely known that different compartments along the length and width of thegut vary considerably between each other (Looft et al., 2014a). The results frommanuscript 1, 2, and 3 expectedly agreed with these previous reports. It was alsoevident from these data that in the search of the effects of an influencing factor, thesite investigated may be of major importance. In manuscript 1, the effect of dietwas observed in digesta, whereas manuscript 3 revealed an effect of the antibioticand probiotic in the mid colon and faeces. The mucosa-associated community wasnot investigated in any of the animal studies.
In retrospect, to pursue the objective of manuscript 2, subjecting intestinal di-gesta or, perhaps even better, mucosa from the small intestine to high-throughput16S rRNA gene sequencing would have been useful. As the potentially changedglycoproteins are expected to be found in the mucus layer or on cell surfaces (Hes-selager et al., 2016), it would have been highly relevant to include this niche aswell. Also, sampling of intestinal digesta at an early age shortly after antibiotictherapy would have added more information to the data presented in manuscript3. Nonetheless, sampling of luminal contents from the length of the gut requiresanimals to be sacrificed. Sacrificing too many animals early in the experiment maybe a problem if the number of animals beforehand is low, which was the case inmanuscript 2.
Faecal samples were consistently included to be able to evaluate the effect oftime. This approach has been applied by several papers (e.g. Frese et al., 2015;Slifierz et al., 2015) and obviously allows the same animal to be sampled multipletimes. However, when faecal samples are deemed insufficient, intestinal digestafrom different piglets belonging to the same litter has to be sampled multiple
Chapter 7. General discussion 159
times. Such an approach inevitably introduces variation due to natural variabilitybetween individual piglets.
7.4 High-throughput sequencing versus microbial
culture
Applying high-throughput sequencing of the 16S rRNA gene, as done in all ex-periments of the present thesis, provides an in-depth insight into the diversity ofthe gut microbiota, joined by a thorough knowledge on the taxonomy of presentbacteria. This method has been increasingly applied in the study of complex mi-crobial environments during the last decade. This molecular method, however,potentially introduces biases during sample preparation and sequencing, and doesnot allow absolute quantification. In an effort to circumvent this problem, we in-cluded microbial plate culture as well. From the very beginning we were awarethat traditional culture would be insufficient in regards to the detailed characteri-sation of the complex microbial communities in the gut, and the presented studiesalso confirm these limitations. Manuscript 1 and 2, however, underline the impor-tance of including culture of potentially pathogenic bacteria, here the family ofEnterobacteriaceae and haemolytic bacteria. Furthermore, some pathogens maybe found in numbers too low to be detected by current sequencing platforms, andhence require use of other methods for their detection (Lagier et al., 2015).
The application of two different platforms turned out to complicate the overallinterpretation of the obtained results. Due to the need of additional extensivequality filtering and trimming, the sequences from the Ion Torrent platform wererelatively short (166 base-pairs). The faecal samples appeared abnormally rich,which could be assigned to the short read lengths (Engelbrektson et al., 2010).Also, only ten of the 25 most abundant species were assigned to genus level, andnumerous OTUs were not taxonomically assigned or assigned to order level only.These results demonstrate the importance of platform specifications as e.g. readlength, when assigning taxonomy to 16S rRNA gene fragments.
160 Chapter 7. General discussion
7.5 Linking gut microbial community composition
to gut health and animal robustness
Diarrhoea is frequently used as a clinical measure of gut health and also broaderas an indicator of lack of animal health. In manuscript 1 (chapter 4) the increaseddiarrhoea incidence was associated with an increased detection of Enterobacteri-aceae and haemolytic bacteria, whereas a lower diarrhoea incidence was associatedwith an increased detection of Lactobacillus. This is in accordance with the Enter-obacteriaceae to Lactobacillus ratio often used as a measure of gut health (Castilloet al., 2007). However, whether this measure is consistently associated with anincreased animal health is unknown. Though the number of studied animals werelow, occurrence of diarrhoea was included as a clinical observation of piglet health.In manuscript 3, the included measures of gut health (Enterobacteriaceae, Lacto-bacillus, inflammation and intestinal tight-junction markers) were not significantlydifferent between the experimental groups, and not in accordance with the observeddiarrhoea frequencies. Species richness and diversity, on the other hand, did differbetween treatments. Petersen and Round (2014) suggested that a gut microbiotafree from dysbiosis was characterised by a highly diverse community. However, thegeneral perception of a healthy gut being associated with a microbial communityof high diversity and richness is currently challenged. In humans, a high microbialrichness has been associated with long colonic transit time and increased proteinfermentation (Roager et al., 2016), which generally is not considered beneficial tothe host. Also, as seen in manuscript 1, the piglets with the highest diarrhoea in-cidence were also the ones with the highest microbial richness and diversity. Theseresults stress the complexity of the gut microbiota, and that the definition of ahealthy gut may change according to the investigated circumstances.
Chapter 8. Conclusion 161
Chapter 8
Conclusion
The results of the presented Ph.D.-study contribute to the existing knowledge onhow different factors influence the colonisation and succession of the gut microbiotain piglets. The major findings and conclusions from the three animal experimentsare presented in the following.
• Diet had a major impact on the intestinal luminal microbiota.
• When mother’s own milk was not available, BC was a more suitable substi-tute, compared to a regular milk replacer, in maintaining a health-promotinggut microbiota.
• Especially the ileal community of BC fed piglets had a closer resemblance tothat of sow milk-fed piglets with regard to the abundance of potential entericpathogens.
• Genetic manipulation of the FUT1 gene did not notably affect colonisationof the microbiota, investigated by analysing faecal samples.
• Gentamicin and Bacillus spores changed the colonic and faecal microbiota,which was especially evident just before weaning at 28 days of age.
• Gentamicin increased community species richness and diversity, while Bacil-lus spore administration had the opposite effect.
• No negative effect on the animals general health was observed after gentam-icin administration. On the contrary, piglets administered Bacillus sporestended to have a higher diarrhoea frequency.
162 Chapter 8. Conclusion
• Age had a major effect on the gut microbiota, causing a gradual change ofgut communities as the piglets aged.
In conclusion, the investigated external factors, and especially diet, had themost significant influence on the colonisation of the gut microbiota in piglets. Noeffect was seen when FUT1 genotype was manipulated. BC showed the mostpromising results in shaping the gut microbiota in a direction favouring healthierpiglets.
Chapter 9. Perspectives and future work 163
Chapter 9
Perspectives and future work
The close relationship between the gut microbiota and host health unfolds an arrayof opportunities for potentially influencing piglet health by delicately orchestratingthe complex gut microbiota. The presented results confirm that the gut microbiotaof piglets is manipulative by various factors. Especially the dietary interventionwith BC showed promising results in relation to improving piglet gut health. Thereported results indicate that BC is able to favour a beneficial gut microbiota,which could be useful both during suckling and in relation to weaning. Due to theresemblance in gut structure and the omnivorous dietary habit between humansand pigs, such perspectives might also be of relevance in infants.
Increased litter sizes in the modern pig industry have required many pig farmersto implement the use of milk replacers (De Vos et al., 2014). Substituting regularmilk replacers with BC could potentially enhance the performance of piglets inthe need of sow milk supplementation, increasing survival chances and robustnesslater in life, e.g. after weaning. Also, in relation to weaning, including BC maycontribute to an increased gut health, hence increase animal welfare and reduce theneed for antibiotics. Before such measures can be implemented, additional studiesare needed. Focus should be on finding the proportion of BC needed to obtain thebeneficial effects, how it is best administered, and the long-term effects. The highprotein content might pose problems due to protein fermentation and potentialproduction of harmful by-products. An important aspect is also whether its useis rentable for the pig farmer. If the price is too high, the odds of a successfulimplementation are bad. However, to be able to implement its use on a wide scale,the production has to be able to keep up with the demand. As the lactating cowonly produces colostrum for a short period of time, and the calf needs to be fed it aswell, BC might not be produced in large enough amounts, making it unobtainable
164 Chapter 9. Perspectives and future work
or too expensive.Though we did not find supplementation with Bacillus spores to beneficially
influence gut health, its potential use should not be disregarded. The current ev-idence on probiotics is in some regards indeed contradicting. However, assigningsuch observations to the functionality of the probiotics alone should be done withcare. The variability between studies is substantial and underlines the need foruniformity. Interestingly, the investigated Bacillus spores were shown to counter-act the effect of gentamicin, and hence the concomitant use of these should befurther investigated. Further studies should emphasise investigating the long-termeffects of Bacillus spp. spores and how they influence performance of piglets dur-ing infection challenges. Use of probiotics could find application in the pre- andpost-weaning period. Administrating probiotics to sows might be a way of verti-cally transferring probiotic species to the offspring, and may be a way of increasinghealth of the mother sow.
As genetic manipulation of specific intestinal pathogen receptors is an appliedapproach for coping with ETEC infections after weaning, the lack of evidence ofa changed colonisation of the indigenous gut microbiota favours the continued useof such a procedure. However, additional work is needed to support the presentedfindings. Future studies should include a larger number of animals and analysesof the mucosa-associated bacterial community.
In general, future studies on the gut microbial community should be supportedby extensive functional analyses, and measures of both the piglets clinical and sub-clinical condition. This could provide a deeper insight into the actual significanceof microbial community changes in the gut.
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Appendix A. 181
Appendix A
The table below provides information on the microbial composition in the stom-ach, small intestine and large intestine of piglets immediately after birth, duringsuckling, and during the first two weeks after weaning.
• Frese et al. (2015) used V4 16S rRNA gene sequencing and Illumina MiSeq,and piglets were weaned at 21 days of age.
• Gancarcikova et al. (2008) used culture, and piglets were weaned at 28 daysof age.
• Melin et al. (1997) used culture, and piglets were weaned at 35 days of age.
• Petri and Hill (2010) used sequencing and used an unknown technique; pigletswere not weaned.
• Slifierz et al. (2015) used V4 16S rRNA gene sequencing and Ilumina Miseq,and piglets were weaned at 21 days of age.
• Swords et al. (1993) used culture, and piglets were weaned at 21 days of age.
182 Appendix A.
Age Taxa Sample type Reference
Sequencing (taxa in %)
0.25 d Clostridiaceae (33.8)Enterobacteriaceae (25)Moraxellaceae (8.8)Lactobacillaceae (1.5)Streptococcaceae (1.5)
Peptostreptococcaceae (1.5)
Pooled digesta Petri and Hill (2010)
0.5 d Clostridiaceae (50)Streptococcaceae (19.7)Enterobacteriaceae (13.2)Pasteurellaceae (5.3)
Pooled digesta Petri and Hill (2010)
1 d Streptococcaceae (37.7)Clostridiaceae (27.3)Lactobacillaceae (6.5)Enterobacteriaceae (3.9)Pasteurellaceae (3.9)Peptostreptococcaceae (1.3)Lachnospiraceae (1.3)Micrococcaceae (1.3)
Pooled digesta Petri and Hill (2010)
2 d Streptococcaceae (29.9)Lactobacillaceae (16.9)Clostridiaceae (7.8)Bacteroidaceae (5.2)Veillonellaceae (5.2)Moraxellaceae (3.9)Peptostreptococcaceae (1.3)Lachnospiraceae (1.3)Pasteurellaceae (1.3)Micrococcaceae (1.3)
Pooled digesta Petri and Hill (2010)
3 d Streptococcaceae (22.8)Lactobacillaceae (19.0)Clostridiaceae (12.7)Moraxellaceae (10.1)Peptostreptococcaceae (7.6)Pasteurellaceae (5.1)Veillonellaceae (3.8)Enterobacteriaceae (1.3)Ruminococcaceae (1.3)Lachnospiraceae (1.3)
Pooled digesta Petri and Hill (2010)
Continues on the next page
Appendix A. 183
Age Taxa Sample type Reference
5 d Lactobacillaceae (50.0)Moraxellaceae (6.4)Lachnospiraceae (6.4)Streptococcaceae (3.8)Pasteurellaceae (2.6)Veillonellaceae (2.6)Clostridiaceae (1.3)Enterobacteriaceae (1.3)Peptostreptococcaceae (1.3)Micrococcaceae (1.3)
Pooled digesta Petri and Hill (2010)
10 d Lactobacillaceae (46.7)Ruminococcaceae (10.7)Enterobacteriaceae (5.3)Veillonellaceae (5.3)Clostridiaceae (4.0)Moraxellaceae (4.0)Lachnospiraceae (2.7)Pastrurellaceae (1.3)Micrococcaceae (1.3)
Pooled digesta Petri and Hill (2010)
20 d Lactobacillaceae (44.6)Ruminococcaceae (9.5)Lachnospiraceae (9.5)Streptococcaceae (5.4)Moraxellaceae (2.7)Clostridiaceae (1.4)Pasteurellaceae (1.4)Micrococcaceae (1.4)
Pooled digesta Petri and Hill (2010)
1 d Bacteroidaceae (21.5)Enterobacteriaceae (21.0)Clostridiaceae (15.0)Lachnospiraceae (12.2)Lactobacillaceae (5.0)Streptococcaceae (4.0)Enterococcaceae (2.5)Ruminococcaceae (2.0)Erysipelotrichaceae (1.0)Prevotellaceae (<1.0)
Faeces Frese et al. (2015)
3 d Enterobacteriaceae (27.5)Bacteroidaceae (19.0)Lachnospiraceae (13.5)Lactobacillaceae (8.0)Clostridiaceae (6.0)Ruminococcaceae (4.5)Erysipelotrichaceae (3.0)Enterococcaceae (2.5)Streptococcaceae (2.0)Prevotellaceae (<1.0)
Faeces Frese et al. (2015)
Continues on the next page
184 Appendix A.
Age Taxa Sample type Reference
5 d Enterobacteriaceae (19.0)Bacteroidaceae (13.5)Lachnospiraceae (13.0)Lactobacillaceae (10.5)Clostridiaceae (6.5)Ruminococcaceae (4.5)Enterococcaceae (4.0)Erysipelotrichaceae (3.5)Streptococcaceae (3.0)Prevotellaceae (<1.0)
Faeces Frese et al. (2015)
7 d Enterobacteriaceae (17.5)Lachnospiraceae (14.5)Bacteroidaceae (13.5)Lactobacillaceae (10.5)Erysipelotrichaceae (5.0)Ruminococcaceae (5.0)Enterococcaceae (4.0)Clostridiaceae (3.5)Streptococcaceae (2.0)Prevotellaceae (<1.0)
Faeces Frese et al. (2015)
14 d Enterobacteriaceae (25.0)Lachnospiraceae (14.0)Erysipelotrichaceae (7.5)Enterococcaceae (7.5)Lactobacillaceae (7.0)Bacteroidaceae (6.0)Ruminococcaceae (4.0)Clostridiaceae (2.0)Streptococcaceae (1.0)Prevotellaceae (<1.0)
Faeces Frese et al. (2015)
21 d Bacteroidaceae (16.5)Lachnospiraceae (16.0)Enterobacteriaceae (12.0)Erysipelotrichaceae (10.0)Ruminococcaceae (6.0)Lactobacillaceae (5.0)Enterococcaceae (3.0)Clostridiaceae (2.0)Streptococcaceae (<1.0)Prevotellaceae (<1.0)
Faeces Frese et al. (2015)
Continues on the next page
Appendix A. 185
Age Taxa Sample type Reference
28 d Ruminococcaceae (16.0)Lachnospiraceae (13.5)Prevotellaceae (12.0)Erysipelotrichaceae (11.0)Lactobacillaceae (10.0)Bacteroidaceae (4.0)Streptococcaceae (3.0)Enterobacteriaceae (2.5)Clostridiaceae (1.5)Enterococcaceae (1.0)
Faeces Frese et al. (2015)
35 d Lactobacillaceae (19.0)Prevotellaceae (17.0)Ruminococcaceae (16.0)Lachnospiraceae (6.0)Erysipelotrichaceae (3.0)Streptococcaceae (1.5)Clostridiaceae (1.0)Enterobacteriaceae (<0.1)Bacteroidaceae (<1.0)Enterococcaceae (<1.0)
Faeces Frese et al. (2015)
42 d Lactobacillaceae (20.0)Prevotellaceae (17.5)Lachnospiraceae (12.0)Ruminococcaceae (10.0)Streptococcaceae (8.5)Erysipelotrichaceae (4.0)Clostridiaceae (1.0)Enterobacteriaceae (<0.1)Bacteroidaceae (<1.0)Enterococcaceae (<1.0)
Faeces Frese et al. (2015)
1-3 d Clostridium (17.9)Escherichia (15.0)Fusobacterium (10.5)Clostridium XIVa (4.3)Lactobacillus (4.2)
Faeces Slifierz et al. (2015)
7-21 d Clostridium (8.8)Escherichia (8.6)Lactobacillus (8.2)Clostridium XIVa (7.4)unclassified Firmicutes (5.2)
Faeces Slifierz et al. (2015)
28-35 d Megasphaera (14.0)Lactobacillus (12.3)Clostridium (4.6)Succinivibrio (4.1)unclassified Firmicutes (2.2)
Faeces Slifierz et al. (2015)
Continues on the next page
186 Appendix A.
Age Taxa Sample type Reference
42-49 d Megasphaera (21.2)Lactobacillus (12.3)Roseburia (4.2)Unclassified Firmicutes (3.9)Erysipelotrichaceae (3.9)
Faeces Slifierz et al. (2015)
Culture (taxa in log-transformed cfu numbers)
3 h Total aerobes (3.9)Total anaerobes (2.7)Coliforms (2.6)E. coli (0)Bacteroides spp. (0)Clostridium spp. (0)Lactobacillus spp. (0)
Distal colonic digesta Swords et al. (1993)
6 h Total anaerobes (6.9)Total aerobes (6.7)Clostridium spp. (6.7)Coliforms (6.2)E. coli (5.6)Bacteroides spp. (0)Lactobacillus spp. (0)
Distal colonic digesta Swords et al. (1993)
9 h Total anaerobes (8.4)Coliforms (8.4)Clostridium spp. (8.2)Total aerobes (8.1)E. coli (7.8)Bacteroides spp. (0)Lactobacillus spp. (0)
Distal colonic digesta Swords et al. (1993)
12 h Total aerobes (8.8)Clostridium spp. (8.5)Coliforms (8.4)Total anaerobes (7.8)E. coli (7.3)Bacteroides spp. (0)Lactobacillus spp. (0)
Distal colonic digesta Swords et al. (1993)
1 d Total anaerobes (9.6)Total aerobes (8.3)Clostridium spp. (8.2)Coliforms (7.9)E. coli (6.9)Lactobacillus spp. (5.9)Bacteroides spp. (0)
Distal colonic digesta Swords et al. (1993)
Continues on the next page
Appendix A. 187
Age Taxa Sample type Reference
2 d Total anaerobes (8.1)Coliforms (7.7)Total aerobes (7.4)E. coli (6.6)Clostridium spp. (6.5)Lactobacillus spp. (4.6)Bacteroides spp. (0)
Distal colonic digesta Swords et al. (1993)
3 d Total anaerobes (9.7)Clostridium spp. (9.1)Total aerobes (9.0)Coliforms (8.7)E. coli (8.7)Lactobacillus spp. (5.1)Bacteroides spp. (0)
Distal colonic digesta Swords et al. (1993)
4 d Total anaerobes (9.9)Clostridium spp. (9.6)Total aerobes (9.0)Lactobacillus spp. (7.8)Coliforms (7.5)E. coli (6.8)Bacteroides spp. (6.3)
Distal colonic digesta Swords et al. (1993)
5 d Total anaerobes (9.9)Clostridium spp. (9.8)Total aerobes (8.8)Lactobacillus spp. (7.5)Coliforms (7.3)E. coli (7.0)Bacteroides spp. (<5.0)
Distal colonic digesta Swords et al. (1993)
6 d Total anaerobes (9.5)Total aerobes (8.2)Bacteroides spp. (7.7)Clostridium spp. (7.6)Coliforms (7.3)E. coli (7.2)Lactobacillus spp. (6.9)
Distal colonic digesta Swords et al. (1993)
7 d Total anaerobes (9.8)Clostridium spp. (9.7)Total aerobes (8.9)Coliforms (6.9)Bacteroides spp. (6.6)Lactobacillus spp. (6.0)E. coli (5.8)
Distal colonic digesta Swords et al. (1993)
Continues on the next page
188 Appendix A.
Age Taxa Sample type Reference
8 d Total anaerobes (8.4)Clostridium spp. (8.0)Bacteroides spp. (7.4)Lactobacillus spp. (6.7)Total aerobes (6.6)Coliforms (6.6)E. coli (4.2)
Distal colonic digesta Swords et al. (1993)
9 d Bacteroides spp. (8.9)Clostridium spp. (8.9)Total anaerobes (8.7)Coliforms (7.6)Total aerobes (6.9)Lactobacillus spp. (6.7)E. coli (5.9)
Distal colonic digesta Swords et al. (1993)
10 d Total anaerobes (9.0)Clostridium spp. (7.6)Coliforms (7.1)Bacteroides spp. (<7.0)Total aerobes (6.0)E. coli (6.0)Lactobacillus spp. (5.9)
Distal colonic digesta Swords et al. (1993)
15 d Clostridium spp. (9.3)Total anaerobes (8.8)Bacteroides spp. (8.8)Coliforms (7.8)Lactobacillus spp. (7.6)Total aerobes (6.5)E. coli (4.3)
Distal colonic digesta Swords et al. (1993)
20 d Total anaerobes (9.1)Clostridium spp. (9.1)Bacteroides spp. (8.6)Lactobacillus spp. (7.6)Coliforms (7.0)E. coli (6.5)Total aerobes (6.4)
Distal colonic digesta Swords et al. (1993)
25 d Total anaerobes (10.6)Clostridium spp. (9.5)Bacteroides spp. (8.4)Lactobacillus spp. (7.7)Coliforms (7.6)E. coli (6.9)Total aerobes (5.9)
Distal colonic digesta Swords et al. (1993)
Continues on the next page
Appendix A. 189
Age Taxa Sample type Reference
30 d Clostridium spp. (10.4)Total anaerobes (8.9)Bacteroides spp. (8.7)Lactobacillus spp. (8.1)Coliforms (7.9)Total aerobes (7.5)E. coli (5.1)
Distal colonic digesta Swords et al. (1993)
2 d Total aerobes (7.4)Lactobacilli (6.4)Enterococci (6.4)E. coli (4.6)Enterobacteriaceae (4.0)
Jejunal digesta Gancarcikova et al. (2008)
7 d Total aerobes (6.5)Lactobacilli (6.1)Enterococci (6.1)E. coli (4.9)Enterobacteriaceae (4.4)
Jejunal digesta Gancarcikova et al. (2008)
14 d Total aerobes (7.5)Lactobacilli (7.0)Enterococci (5.0)Enterobacteriaceae (4.0)E. coli (3.8)
Jejunal digesta Gancarcikova et al. (2008)
21 d Total aerobes (7.0)Lactobacilli (6.0)E. coli (5.4)Enterococci (5.0)Enterobacteriaceae (4.8)
Jejunal digesta Gancarcikova et al. (2008)
28 d Total aerobes (8.0)Lactobacilli (6.4)Enterococci (5.5)E. coli (4.9)Enterobacteriaceae (4.8)
Jejunal digesta Gancarcikova et al. (2008)
35 d Total aerobes (6.4)Lactobacilli (6.0)Enterococci (4.0)E. coli (3.8)Enterobacteriaceae
(not detected)
Jejunal digesta Gancarcikova et al. (2008)
42 d Total aerobes (7.8)Lactobacilli (6.8)Enterococci (6.8)E. coli (6.0)Enterobacteriaceae (5.4)
Jejunal digesta Gancarcikova et al. (2008)
Continues on the next page
190 Appendix A.
Age Taxa Sample type Reference
2 d Total aerobes (9.0)Lactobacilli (7.4)Enterobacteriaceae (6.9)E. coli (6.6)Enterococci (6.0)
Ileal digesta Gancarcikova et al. (2008)
7 d Total aerobes (7.8)Enterobacteriaceae (7.2)E. coli (7.1)Lactobacilli (7.0)Enterococci (4.0)
Ileal digesta Gancarcikova et al. (2008)
14 d Lactobacilli (9.2)E. coli (7.7)Total aerobes (7.5)Enterobacteriaceae (7.1)Enterococci (5.0)
Ileal digesta Gancarcikova et al. (2008)
21 d Total aerobes (8.8)Lactobacilli (8.0)E. coli (6.9)Enterobacteriaceae (6.6)Enterococci (6.0)
Ileal digesta Gancarcikova et al. (2008)
28 d Total aerobes (8.9)Lactobacilli (8.3)E. coli (7.0)Enterobacteriaceae (6.8)Enterococci (6.3)
Ileal digesta Gancarcikova et al. (2008)
35 d Total aerobes (9.2)Lactobacilli (8.0)Enterobacteriaceae (5.8)E. coli (5.2)Enterococci (5.0)
Ileal digesta Gancarcikova et al. (2008)
42 d Lactobacilli (8.6)Enterococci (8.0)Total aerobes (7.0)E. coli (6.2)Enterobacteriaceae (5.2)
Ileal digesta Gancarcikova et al. (2008)
2 d Total aerobes (9.4)E. coli (9.0)Enterobacteriaceae (9.0)Lactobacilli (8.9)Enterococci (8.0)
Caecal digesta Gancarcikova et al. (2008)
7 d Total aerobes (9.2)Lactobacilli (8.8)E. coli (8.6)Enterobacteriaceae (8.4)Enterococci (7.5)
Caecal digesta Gancarcikova et al. (2008)
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Appendix A. 191
Age Taxa Sample type Reference
14 d Total aerobes (9.2)Lactobacilli (9.2)E. coli (7.9)Enterobacteriaceae (7.4)Enterococci (6.8)
Caecal digesta Gancarcikova et al. (2008)
21 d Total aerobes (9.1)Lactobacilli (8.9)E. coli (7.2)Enterobacteriaceae (7.0)Enterococci (6.9)
Caecal digesta Gancarcikova et al. (2008)
28 d Total aerobes (9.0)Lactobacilli (8.4)Enterococci (7.2)E. coli (7.0)Enterobacteriaceae (6.1)
Caecal digesta Gancarcikova et al. (2008)
35 d Lactobacilli (9.8)Total aerobes (9.4)Enterococci (7.2)E. coli (6.8)Enterobacteriaceae (6.0)
Caecal digesta Gancarcikova et al. (2008)
42 d Lactobacilli (9.8)Total aerobes (9.1)Enterococci (8.9)E. coli (5.9)Enterobacteriaceae (5.4)
Caecal digesta Gancarcikova et al. (2008)
7 d E. coli (8.8)Enterococci (7.7)C. perfringens (4.6)
Faeces Melin et al. (1997)
14 d Enterococci (8.3)E. coli (8.2)C. perfringens (4.0)
Faeces Melin et al. (1997)
21 d E. coli (8.4)Enterococci (7.4)C. perfringens (0)
Faeces Melin et al. (1997)
28 d E. coli (7.2)Enterococci (7.2)C. perfringens (0)
Faeces Melin et al. (1997)
35 d E. coli (6.1)Enterococci (6.1)C. perfringens (0)
Faeces Melin et al. (1997)
Continues on the next page
192 Appendix A.
Age Taxa Sample type Reference
38 d Enterococci (6.5)E. coli (5.6)C. perfringens (0)
Faeces Melin et al. (1997)
42 d E. coli (5.9)Enterococci (5.5)C. perfringens (0)
Faeces Melin et al. (1997)
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