Transgenic milk containing recombinant humanlactoferrin modulates the intestinal flora inpiglets1

Wenping Hu, Jie Zhao, Jianwu Wang, Tian Yu, Jing Wang, and Ning Li

Abstract: Lactoferrin (LF) is a beneficial multifunctional protein in milk. The objective of this study was to determinewhether bovine transgenic milk containing recombinant human lactoferrin (rhLF) can modulate intestinal flora in the neona-tal pig as an animal model for the human infant. We fed 7-day-old piglets (i) ordinary whole milk (OM), (ii) a 1:1 mixtureof OM and rhLF milk (MM), or (iii) rhLF milk (LFM). LFM provided better average daily mass gain than OM(P = 0.007). PCR–denaturing gradient gel electrophoresis and 16S rDNA sequencing analysis revealed that the LFM pigletsexhibited more diversity of the intestinal flora than the OM group. Except for the colon in the LFM group, an increasingtrend in microbial diversity occurred from the duodenum to the colon. Fecal flora was not different across different ages ordifferent treatment groups, but a cluster analysis showed that the fecal flora of OM- and MM-fed piglets had a higher degreeof similarity than that of LFM-fed piglets. Based on culture-based bacterial counts of intestinal content samples, concentra-tions of Salmonella spp. in the colon and of Escherichia coli throughout the intestine were reduced with LFM (P < 0.01).Concentrations of Bifidobacterium spp. in the ileum and of Lactobacillus spp. throughout the intestine were also increasedwith LFM (P ≤ 0.01). We suggest that rhLF can modulate the intestinal flora in piglets.

Key words: recombinant human lactoferrin (rhLF), piglets, intestinal flora.

Résumé : La lactoferrine (Lf) est une protéine du lait multifonctionnelle bénéfique. L’objectif de cette étude était de déter-miner si un lait transgénique de vache contenant de la lactoferrine humaine (rhLF) pouvait moduler la flore intestinale duporcelet nouveau-né, utilisé comme modèle animal du bébé humain. Nous avons nourri des porcelets de 7 jours avec : (i)du lait entier normal (OM); (ii) un mélange 1 : 1 de lait entier normal et de lait contenant de la rhLF (MM); ou (iii) du laitcontenant de la rhLF (LFM). Le LFM était plus avantageux sur le plan du gain de poids quotidien comparativement au OM(P = 0,007). Une analyse par PCR et par électrophorèse sur gel en gradient dénaturant, ainsi que le séquençage de l’ADNr16S ont révélé que les porcelets nourris au LFM montraient une flore intestinale plus diversifiée comparativement au groupeOM. À l’exception du côlon du groupe LFM, la diversité microbienne tendait à s’accroitre du duodénum au côlon. La florefécale n’était pas différente selon l’âge ou entre les différents groupes de traitement, mais une analyse de grappes géniquesa montré que la flore fécale des porcelets nourris au OM et au MM montrait un degré de similarité plus élevé que celle desporcelets nourris au LFM. Selon la numération bactérienne basée sur la culture du contenu des échantillons intestinaux, lesconcentrations de Salmonella sp. du côlon et d’Escherichia coli tout au long de l’intestin étaient réduites par le LFM(P < 0,01). Les concentrations de Bifidobacterium sp. de l’iléon et de Lactobacillus sp. au long de l’intestin étaient aussiaugmentées par le LFM (P ≤ 0,01). Nous suggérons que la rhLF peut moduler la flore intestinale des porcelets.

Mots‐clés : lactoferrine recombinante humaine (rhLF), porcelets, flore intestinale.

[Traduit par la Rédaction]

Introduction

Lactoferrin (LF) is a natural iron-binding glycoprotein ofthe transferrin family. LF is ∼80 kDa, contains 1 to 4 gly-cans, and is widely present in human milk and the milk ofmost other mammals, including the swine, bovine, goat, rab-bit, and mouse (Steijns and van Hooijdonk 2000), but not inthe milk of the dog or rat (Levay and Viljoen 1995). LF isalso found in saliva, tears, neutrophils, bile, and pancreatic

juice (Levay and Viljoen 1995; Steijns and van Hooijdonk2000). LF is a beneficial multifunctional protein that regu-lates iron homeostasis and also has bacteriostatic, bacterici-dal, antioxidant, anticancer, immune-modulating, andantiinflammatory activities (Iyer and Lönnerdal 1993; Wardet al. 2005).LF is a prominent component of the secondary granules of

neutrophils (PMNs), which release LF after migrating to in-

Received 16 September 2011. Revision received 10 November 2011. Accepted 10 November 2011. Published atwww.nrcresearchpress.com/bcb on 8 March 2012.

W. Hu, J. Zhao, J. Wang, T. Yu, J. Wang, and N. Li. State Key Laboratory of AgroBiotechnology, China Agricultural University, No. 2Yuan Ming Yuan West Road, Haidian District, Beijing 100193, P. R. China.

Corresponding author: Ning Li (e-mail: ninglcau@cau.edu.cn).1This article is part of a Special Issue entitled Lactoferrin and has undergone the Journal’s usual peer review process.

485

Biochem. Cell Biol. 90: 485–496 (2012) doi:10.1139/O2012-003 Published by NRC Research Press

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fected tissues or in blood as part of the inflammation process(Legrand et al. 2005). In milk and fluids of the digestivetract, LF is expressed and secreted abundantly and partici-pates in the first line of host defense (Legrand et al. 2005).In the intestine, LF directly interacts with luminal microbesand has multiple effects on enteropathogens (Appelmelk etal. 1994; Brandenburg et al. 2001; Ochoa et al. 2004; Ochoaand Cleary 2009). Of particular note, LF can inhibit thegrowth of pathogens such as Escherichia coli, Salmonella ty-phimurium, and Shigella flexneri (Tigyi et al. 1992; Gomezet al. 2002; Bessler et al. 2006; Ochoa and Cleary 2009) andpromotes the growth of beneficial bacteria such as Bifidobac-terium (Ochoa and Cleary 2009). The effect of LF should beascribed to an inhibitory effect on pathogenic flora ratherthan to a direct stimulus to the development of Bifidobacteria(Coppa et al. 2006).Most research regarding the interaction between LF and

enteric pathogens has been performed in vitro or in mice.Thus, it remains unknown whether the same interactions oc-cur in the human intestinal tract. Some (Roberts et al. 1992;Zavaleta et al. 2007) but not all (Balmer et al. 1989) pub-lished studies have shown beneficial effects of LF on intesti-nal flora in human infants. These inconsistent results may beexplained by factors such as LF quality, origin, study design(group size, infant age, and length of study), and the detec-tion methods (Tomita et al. 2009). More accurate assessmentsof the effect of LF on human intestinal microflora and in ap-propriate animal models, along with modern molecular bio-logical methods, are needed to draw definite conclusions.Human lactoferrin (hLF) is one of the most abundant whey

proteins in milk. Its concentration is >7.0 g/L in colostrumand approximately 1–2 g/L in mature milk (Steijns and vanHooijdonk 2000). The concentration of bovine lactoferrin(bLF) is >1.5 g/L in cow’s colostrum and is reduced to about20–200 mg/L in milk at mid-lactation (Steijns and van Hooij-donk 2000). We have produced transgenic cows that secreterecombinant human lactoferrin (rhLF) at high levels (≤3.4 g/L)in milk (Yang et al. 2008). To better understand the role ofhLF in modulating the intestinal flora of infants, we chose,for this study, the neonatal pig that is a well-established an-imal model for studying infant nutrition (Wilson et al.2010) and intestinal development (Bergen and Wu 2009).

Materials and methods

Milk powder processingWe collected mature milk from the rhLF transgenic cow

named Wanwa at mid-lactation for 3 months continuouslyand converted the rhLF-containing milk to solid powder withvacuum freeze-drying technology, then homogeneouslymixed the milk powder. After sterilization with a 2 kGy doseof irradiation, the milk was stored at 4 °C. We used ordinarywhole-milk powder, which was supplied by Inner MongoliaYili Industrial Group Co., Ltd., as the control. The concentra-tion of rhLF in the transgenic milk powder was 20 mg/g,thus, bLF was 2 mg/g in the transgenic milk powder and thecontrol ordinary whole-milk powder.

Experimental animalsEighteen male Landrace 7-day-old piglets from 3 litters

with nearly identical genetics and similar body masses were

divided into the following 3 feeding groups (Table 1; n = 6each): (i) ordinary-milk (OM); (ii) a 1:1 mix of ordinary milkand rhLF milk (MM); and (iii) rhLF milk (LFM). All pigletswere raised in the same room with 50%–70% humidity.Room temperature was 33 °C at 7 to 14 days of age andthen decreased 1 °C every week. Each piglet had an inde-pendent sty, nipple-type waterer, and milk container. All pig-lets were housed under conditions similar to a specificpathogen-free environment. In the current study, animal ex-periments have been approved by the Animal Care and UseCommittee of China Agricultural University.

DietAll piglets were nursed by sows from day 0 to day 6 after

birth and then transferred into the piglet room for weaningunder 24 h care. From postnatal day 7 to 28, the animals inthe 3 groups were given reconstituted milk that was a mix-ture of milk powder and water and placed in the containers.From 29 to 37 days of age, animals were directly fed drymilk powder instead of reconstituted milk. We fed the pigletsevery 3 to 4 h (6 or 7 times a day), providing 24 h of unin-terrupted feeding, which mimics the natural milk-drinkingprocess of piglets. Reconstituted milk and dry milk powderwere fed for a total of 30 days, and each piglet in the 3groups was fed the same amount of milk throughout the ex-periment, ∼130 g milk powder per day.

Table 1. Experimental groups.

Groupname

PigletNo.

SowNo.

Birthmass(kg)

Masst at7 days (kg)

OM 1-1 54 1.50 2.621-2 54 1.40 2.441-3 57 1.46 2.321-4 56 1.12 2.121-4 56 1.10 1.961-6 57 1.14 1.90

Averagemass

1.287 2.227

MM 2-7 57 1.54 2.062-8 54 1.52 2.542-9 56 1.28 2.222-10 57 1.40 2.102-11 54 1.44 2.582-12 56 1.02 1.88

Averagemass

1.367 2.23

LFM 3-13 54 1.48 2.563-14 54 1.56 2.483-15 54 1.42 2.283-16 57 1.10 2.083-17 56 1.14 2.063-18 56 1.09 1.92

Averagemass

1.298 2.23

Note: OM, ordinary-milk feeding group; MM, feedinggroup given a 1:1 mix of ordinary milk and recombinanthuman lactoferrin (rhLF) milk; and LFM, feeding group gi-ven rhLF milk. The masses for each group did not differsignificantly at birth (P = 0.754) or at 7 days of age (P =1.000).

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Mass and sample collectionPiglets were weighed every 3 days, and SPSS 11.5 (SPSS

Inc.) was used for statistical analysis. Fresh feces (5 mL)were immediately collected after defecation from piglets inthe morning once a week. The fecal samples were pooledwithin the same group in 50 mL sterile centrifuge tubes andstored at –70 °C for subsequent DNA extraction and PCR –denaturing gradient gel electrophoresis (PCR–DGGE) analy-sis.

NecropsyAt the end of the feeding period (38 days of age), animals

were euthanized, and the contents of the middle sections ofthe duodenum, jejunum, ileum, and colon were obtained.The samples used for PCR–DGGE analysis were stored at–70 °C. The samples used for culture-based bacterial countanalysis were stored for less than 12 h at 4 °C and thenused for the experiment.

PCR–DGGE analysisMicrobial DNAs were extracted from intestinal contents

and fecal samples using a QIAamp DNA stool mini kit (Qia-gen,Germany) and E.Z.N.A.™ Soil DNA kit (Omega Bio-Tek), respectively, in accordance with the manufacturer’s in-structions. Extracted DNA was quantified by a Tecan infinite200 microplate reader (Tecan, Switzerland) at 260 and280 nm (Xu et al. 2011). The variable V3 region of bacterial16S rDNA was amplified with PCR using the primer pair338F 5′-ACTCCTACGGGAGGCAGCAG-3′ and 518R 5′-ATTACCGCGGCTGCTGG-3′. A GC clamp (GCCCGCCGC-GCGCGGCGGGCGGGGCGGGGGCACGGGGGG) was addedto the 5′ terminus. PCR amplification was carried out in 50mL reaction mixtures containing 1 mL template DNA(2.5 ng/mL), 0.25 mL Taq polymerase (5 U/mL; Takara, Da-lian, China), 1 mL of each primer (20 mmol/L), 4 µL dNTPmix (2.5 mmol/L; Takara), and 5 mL of 10× PCR buffer(Takara). The PCR cycle was 94 °C for 10 min, followedby 30 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °Cfor 1 min 30 s, and then a final extension at 72 °C for7 min. PCR products were examined with electrophoresis(2% (w/v) agarose) and then used for DGGE analysis withthe Dcode Universal Mutation Detection System (Bio-Rad,Hercules, Calif.). The PCR products were loaded onto 8%(w/v) polyacrylamide gels in 1× Tris–EDTA buffer. Thegels were run at 150 V for 420 min and then silver stained(Xu et al. 2011).

Cloning and sequencingProminent bands from the PCR amplification were excised

from the DGGE gel, purified with a Watson Gel RecoveryPurification kit (Watson Biotechnologies, China), cloned intothe pMD18-T vector (Takara), and transformed into E. coliDH5a competent cells. Ten clones from each band were ran-domly selected, and plasmids were isolated with the E.Z.N.A.™DNA Isolation System (Omega Bio-Tek). An ABI 3730XLDNA sequencer (Applied Biosystems) was used for se-quencing using the universal primer pair for the PMD18-Tvector, M13F (–40) 5′-GTTTTCCCAGTCACGAC-3′ andM13R (–26) 5′-CAGGAAACAGCTATGAC-3′ (Xu et al.2011).

Diversity indexA diversity index was used to describe the diversity of a

microbial community. Here, we used the Shannon’s diversityindex to evaluate the diversity of the microflora (Shannon1997). Quantity One analysis software (Bio-Rad) was usedfor quantitative analysis of DGGE bands for each sampleand to calculate the preliminary data of the diversity index,which was then calculated with the Shannon formula.

Cluster analysisCluster analysis was used to study the similarity of micro-

flora among samples. The method we used is derived fromDice’s algorithm and uses the unweighted pair group methodwith arithmetic averages to construct a similarity matrix(Yang et al. 2001; Fromin et al. 2002; Boon et al. 2002;Smalla et al. 2007; Kim et al. 2008; Xu et al. 2011). Thesoftware programs BioEdit 7.0, Phylip 4.0, and MEGA 4.0were used for cluster analysis.

Phylogenetic analysis and phyla distribution16S rDNA gene sequences were submitted to the National

Center for Biotechnology Information (NCBI) to obtainclosely related sequences by BLAST, and all sequenceswith >97% similarity were chosen for construction of phylo-genetic trees (Xing et al. 2010; Xu et al. 2011). Neighbor-joining phylogenetic trees were constructed using MEGA 4.0(Tamura et al. 2007; Xu et al. 2011) and assessed using abootstrap analysis with 1000 replications (Xu et al. 2011).The percent of each genus or phylum was derived from phy-logenetic analysis (Xu et al. 2011). Phyla distribution wasused to show the composition of microflora as comparedwith the percent of each genus or phylum in different sam-ples.

Nucleotide sequence acc. Nos.The 16S rDNA gene sequences used for phylogenetic anal-

ysis have been submitted to the GenBank nucleotide se-quence database. The accession numbers are JN624931–JN625028.

Culture-based bacterial count analysisWe selected 6 types of bacteria for culture-based analysis,

each of which grew in an appropriate medium (Table 2). Onegram of each intestinal sample was rapidly mixed with9.0 mL sterile diluent in an aseptic homogenate cup, homo-genized at 8000 r/min (9100g) for 1–2 min, and a 10–1 ho-mogeneous sample diluent was prepared. Then, each diluentwas used to make a series of liquid samples with differentdilution ratios, such as 10–2, 10–3, 10–4, etc. All procedureswere completed within 15 min. Using the appropriate me-dium (Table 2), we prepared 2 or 3 consecutive appropriatedilutions of each sample and plated 0.1 mL of homogeneoussample diluent per plate, making 2 replicates and 1 blankcontrol. Culture time, temperature, and condition are pre-sented in Table 2. The morphology of the flora and individ-ual cells, Gram staining, and oxygen consumption were usedto identify the type of bacteria. The logarithm of the numberof bacteria per gram of intestinal contents (logCFU/g) wasused to show the results. SPSS 11.5 (SPSS Inc.) was usedfor statistical analysis.

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Results

MassWe weighed each piglet every 3 days, determined the mass

change over the study period (Fig. 1A), and calculated theaverage daily gain (Fig. 1B) for each of the 3 groups. Themasses of the LFM and OM group differed beginning at29 days of age (P = 0.045). The LFM piglets grew fasterthan the OM group. The average daily gain of the LFMgroup was greater (P = 0.007) than that of the OM group(Fig. 1B).

PCR–DGGE analysis and 16S rDNA sequences ofintestinal content samplesAfter necropsy, the contents of the duodenum, jejunum,

ileum, and colon from each piglet were individually pooledwith the replicates for each different group, resulting in a to-tal of 12 pooled samples (samples are labeled as in Fig. 2A).The 16S rDNA PCR products of the 12 intestinal content

samples were electrophoresed on the same DGGE gel platefor comparison. Based on the PCR–DGGE analysis, differentintestinal compartments and groups showed different DGGEband patterns (Fig. S1).2 We performed further analysis using16S rDNA sequencing and the following statistical methods.Cluster analysis of the 16S rDNA sequences showed that

the similarity was ∼54%–94% among the different samples(Fig. 2A). The similarity between DF1 and DF2 was closeto 94%, and comparison of DF3 with DF1/DF2 revealed asimilarity of 77%. The similarity among the 3 jejunum fecalsamples (JF1, JF2, and JF3) was ∼84%–85%. The similarityamong IF1, IF2, and IF3 was 54%–70%, and the similarityamong CF1, CF2, and CF3 was 61%–65% (Fig. 2A). Onlythe similarity between DF1 and DF2 was >90%.Phyla distribution was derived from phylogenetic analysis

(Fig. S3A).2 When the 16S rDNA clone libraries were exam-ined at the phylum level, the prominent groups of phyla wereFirmicutes and Gammaproteobacteria, which appeared in allthe intestinal compartments of the 3 feeding groups and hadthe highest percents (Fig. 3A). Some sequences could not beclassified and were designated as unknown.When we examined the clone libraries at the genus level

(Fig. 3B), the genus distribution result seemed more specific.We classified all 16S rDNA clones into the following18 gen-era: Pseudomonas, Lactobacillus, Robinsoniella, Escherichia,Clostridium, Ruminococcus, Enterococcus, Streptomyces,Acinetobacter, Streptococcus, Erwinia, Aeromonas, Lacto-coccus, Ewingella, Dorea, Rothia, Salmonella, and Mega-sphaera. Some sequences could not be classified and weredesignated as unknown. The prominent groups at the genuslevel were Lactobacillus and Pseudomonas in all the intesti-nal content samples.Regarding the genus complexity of the different intestinal

compartments, the duodenum was the simplest, and we ob-served a trend toward increasing complexity from the duode-num to the colon (Fig. 3B). Regarding the genus complexityof the different feeding groups, in the duodenum and ileum,the LFM group showed the most complex genera, especiallyin the ileum, where the percent of unknown genera observedin the LFM group was higher (34.48%) than in the OM(14.29%) and MM (16.67%) groups, revealing more com-plexity. Unknown microbes in the other intestinal contentsamples comprised only 0%–25%.Different diversity indices among the different intestinal

compartments of the 3 feeding groups indicated an increasingtrend from the duodenum to the colon, except for the colonof the LFM group. However, the diversity index of the LFMgroup in the duodenum, jejunum, and ileum was higher thanthat in the OM or MM groups (Fig. 4A).To summarize, PCR–DGGE analysis and 16S rDNA se-

quencing of intestinal content samples revealed low similarityamong the intestinal content samples. Specifically, the LFMpiglets exhibited more complexity and diversity than the con-trol group did in the duodenum and ileum and a trend towardan increase in complexity and diversity from the duodenumto the colon.

PCR–DGGE analysis and 16S rDNA sequencing of fecalsamplesFresh fecal samples of piglets were collected at 13, 20, 28,

and 35 days of age, and samples were pooled within thesame group and collection time. PCR products of the 12pooled samples (samples are labeled as in Fig. 2B) were

Table 2. Cultured bacteria and their different media.

Bacteria MediumCulturecondition

Culturetime (h)

Culture tem-perature(°C) Supplier

Total aerobic bacteria Brain heart infusion agarmedium

Aerobicculture

48±2 36±1 Qingdao Hope Bio, China

Total anaerobic bacteria Wilkins–Chalgren anaerobeagar medium

Anaerobicculture

48±2 36±1 Qingdao Hope Bio, China

Salmonella spp. Salmonella spp.chromogenic medium

Aerobicculture

22–24 36±1 Qingdao Hope Bio, China

Escherichia coli E. coli chromogenicmedium

Aerobicculture

18–24 36±1 Qingdao Hope Bio, China

Bifidobacterium spp. Bifidobacterium spp.selective medium

Anaerobicculture

48±2 36±1 Qingdao Hope Bio, China

Lactobacillus spp. Lactobacillus spp. selectivemedium

Anaerobicculture

48±2 36±1 Qingdao Hope Bio, China

2Supplementary data are available with the article through the journal Web site (www.nrcresearchpress.com/doi/suppl/10.1139/o2012-003).

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electrophoresed on the same DGGE gel plate for comparison.From the DGGE profile, there appeared to be few major dif-ferences among the different groups of fecal samples or sam-

ples collected at different times (Fig. S2).2 The diversityindex values were not different among the 3 groups of piglets(Fig. 4B).

Fig. 1. Mass change trend and average daily gain of piglets (n = 6 each). One-way ANOVA, Least-significant difference, and Duncan's mul-tiple range test were used for statistical analysis. (A) Mass change trend of piglets. The masses of the LFM group and OM group differedbeginning at 29 days of age (P = 0.045). Error bars represent standard error. (B) Average daily mass gain of the piglets. The average dailygain of the LFM group was greater (P = 0.007) than that of the OM group. Error bars represent standard error. a and b, P < 0.05 amongdifferent groups. OM, ordinary-milk feeding group; MM, feeding group given a 1:1 mix of ordinary milk and recombinant human lactoferrin(rhLF) milk; and LFM, feeding group given rhLF milk.

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Fecal flora phyla distribution was derived from fecal floraphylogenetic analysis (Fig. S3B).2 Regarding the phyla distri-bution at the phylum level (Fig. 5A), the dominant groupswere Firmicutes and Gammaproteobacteria, which appearedin all fecal samples and showed the highest percents(Fig. 5A). Similar results were obtained for the phyla distri-bution in the intestinal content samples (Fig. 3A).Regarding the phyla distribution at the genus level

(Fig. 5B), we classified all 16S rDNA clones into the follow-ing 10 genera: Lactobacillus, Robinsoniella, Escherichia,Clostridium, Ruminococcus, Klebsiella, Gemmiger, Faecali-bacterium, Erysipelotrichaceae, and Phascolarctobacterium.The prominent groups at the genus level were Lactobacillus,Robinsoniella, and Escherichia in all fecal samples.Regarding genus complexity, there were no major differen-

ces among the different periods or groups (Fig. 5B). The fe-cal flora was not richer than the intestinal content in theclassified microbes, whereas unknown microbes were presentat higher percents in all the fecal samples (41.67%–61.11%).Based on cluster analysis (Fig. 2B), the similarity among

all 12 pooled fecal samples ranged from 60% to 92%. F1-1and F1-2 were clustered together, as were F2-1 and F2-2 andF3-1 and F3-2. Compared with the LFM group, the fecalflora of the OM and MM groups had a higher degree of sim-ilarity at 13, 20, and 28 days of age. At 35 days of age, thesimilarity between F4-1 and F4-2 was also higher than theirsimilarity with F4-3. Thus, the OM and MM groups exhib-ited a higher degree of similarity in fecal flora than did theLFM group.To summarize, PCR–DGGE analysis and 16S rDNA se-

quencing revealed no apparent differences in the complexityand diversity of fecal samples, but the OM and MM groupshad greater similarity in fecal flora than the LFM group.

Culture-based bacterial count analysis of intestinalcontent samplesIntestinal content samples were analyzed with culture-

based bacterial count analysis. Six types of bacteria were se-lected for culture as follows: total aerobes, total anaerobes,Salmonella spp., Escherichia coli, Bifidobacterium spp., andLactobacillus spp.For total aerobes (Table 3), we observed no differences in

the duodenum or jejunum, but we observed a difference inthe ileum and colon among the 3 feeding groups. The totalnumber of aerobes in the ileum of the OM group was thehighest (P < 0.001). For total anaerobes (Table 3), we ob-served no differences in the duodenum, jejunum, or colon.

Only in the ileum the total anaerobe number in the MMgroup was lower than those in the OM and LFM groups (P =0.003), but the OM and LFM groups were not different.For Salmonella spp., we observed no differences in the du-odenum, jejunum, or ileum, but the number of Salmonellaspp. in the OM group in the colon was higher than that inthe LFM group (P = 0.005). For E. coli, the OM grouphad a higher number than the LFM group in the duodenum(P < 0.001), jejunum (P < 0.001), ileum (P < 0.001), andcolon (P = 0.006). For Bifidobacterium spp. (Table 3), wealso observed no differences in the duodenum, jejunum, orcolon. The number of Bifidobacterium spp. in the ileum ofthe OM group was the lowest and different from the num-ber in the MM and LFM groups (P = 0.011). For Lactoba-cillus spp., the number in the LFM group was higher thanthat in the OM duodenum (P = 0.001), jejunum (P =0.002), ileum (P < 0.001), and colon (P = 0.002). TheLFM group had greatly increased levels of Lactobacillusspp. in the intestine, which was also the dominant genus inthe intestine of piglets weaned at a very young age.

DiscussionWhen human infants are born, the gastrointestinal tract is

sterile and is then exposed to an external environment that isrich in various bacteria. These microbes continuously popu-late the infant gastrointestinal tract. After growth and repro-duction, the microbes gradually form a complex microbialcommunity (Coppa et al. 2006). The type of feeding is themain factor that contributes to the development of the intesti-nal ecosystem of newborns. Bifidobacteria and Lactobacillusrepresent up to 90% of the intestinal flora of breast-fed in-fants, whereas in formula-fed infants, these 2 types of bacte-ria represent only 40%–60% of the flora (Coppa et al. 2006).This bifidogenic effect has been reported in several studies(Roberts et al. 1992; Rubaltelli et al. 1998; Harmsen et al.2000). In breast-fed infants, the minor components in fecalsamples were mainly streptococci, but fecal samples fromformula-fed infants often contained staphylococci, E. coli,and clostridia (Harmsen et al. 2000), which include harmfulmicroorganisms, such as Staphylococcus aureus, enterotoxi-genic E. coli (ETEC), enteropathogenic E. coli (EPEC), en-terohemorrhagic E. coli (EHEC), shiga toxin-producing E.coli (STEC), Clostridium tetani, Clostridium perfringens,and Clostridium botulinum. Bifidobacteria and Lactobacillushave many beneficial effects on infants, including modulationof mucosal physiology and barrier function, inhibition of thegrowth of pathogenic bacteria, and participation in systemic

Fig. 2. Cluster analysis of 16S rDNA clone libraries. Numbers along the bottom indicate the similarity coefficient. (A) Cluster analysis of 16SrDNA clone libraries derived from intestinal content samples. DF1, duodenum content of the ordinary-milk feeding (OM) group; DF2, duo-denum content of the 1:1 mix of ordinary milk and recombinant human lactoferrin (rhLF) milk feeding (MM) group; DF3, duodenum contentof the rhLF feeding (LFM) group; JF1, jejunum content of the OM group; JF2, jejunum content of the MM group; JF3, jejunum content ofthe LFM group; IF1, ileum content of the OM group; IF2, ileum content of the MM group; IF3, ileum content of the LFM group; CF1, coloncontent of the OM group; CF2, colon content of the MM group; and CF3, colon content of the LFM group. (B) Cluster analysis of 16SrDNA clone libraries derived from fresh fecal samples. F1-1, fresh fecal sample mixture of OM at 13 days of age; F1-2, fresh fecal samplemixture of MM at 13 days of age; F1-3, fresh fecal sample mixture of LFM at 13 days of age; F2-1, fresh fecal sample mixture of OM at 20days of age; F2-2, fresh fecal sample mixture of MM at 20 days of age; F2-3, fresh fecal sample mixture of LFM at 20 days of age; F3-1,fresh fecal sample mixture of OM at 28 days of age; F3-2, fresh fecal sample mixture of MM at 28 days of age; F3-3, fresh fecal samplemixture of LFM at 28 days of age; F4-1, fresh fecal sample mixture of OM at 35 days of age; F4-2, fresh fecal sample mixture of MM at 35days of age; and F4-3, fresh fecal sample mixture of LFM at 35 days of age.

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immunologic and inflammatory responses (Coppa et al.2006).Breast milk contains a complex mixture of unique compo-

nents and nutrients, and the bifidogenic effect is related to a

complex of interacting factors, such as lactose, oligosacchar-ides, and a low concentration of proteins, especially LF(Coppa et al. 2006). When LF is added to formula as a sup-plement, infants can also establish a bifidus flora that is

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somewhat similar to the flora established in breast-fed infants(Roberts et al. 1992). This effect is, however, still controver-sial (Balmer et al. 1989).We chose piglets that were weaned early as an animal

model to evaluate the effect of oral rhLF-containing trans-

genic milk in modulating intestinal flora. A critical stage inpig development is weaning, which alters the gastrointestinaltract architecture and function, including the immune re-sponse and adaptations to enteric microbiota. Weaning stresswill result in impaired absorption of nutrients, diarrhea, and

Fig. 3. (A) Phyla and (B) genus distribution of 16S rDNA clone libraries derived from intestinal content samples. Samples are labeled as inFig. 2A.

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decreased growth performance (Liu et al. 2008). In our study,7-day-weaned piglets in the rhLF milk feeding group showedbetter average daily mass gain than control piglets, suggest-ing that rhLF may have a beneficial effect on the growth ofpiglets.To understand the mechanisms responsible for the effect of

rhLF on the intestinal microflora ecosystem, we used themolecular-biology-based approach (PCR–DGGE) followedby 16S rDNA sequence analysis. This method permits quali-tative analysis of the abundance of bacterial species and howtheir presence interacts with diet. Simpson et al. (1999)studied changes in pig intestinal flora from birth to weaningusing PCR–DGGE and found that pig intestinal flora variedwith age, individuals, and the compartments of the intestine.Zhu et al. (2003) used PCR–DGGE to analyze the fecal floravariations of 12 weaning piglets and found that on the wean-ing day, the pattern of the DGGE bands was simple. After

weaning, the DGGE bands gradually increased over time, be-came complex and diverse, and individual differences in-creased. When examining the effect of fermentablecarbohydrates on piglet fecal bacterial communities withDGGE analysis of 16S rDNA, Konstantinov et al. (2003)found that compared with a regular diet, supplementing fer-mentable carbohydrates to piglets produces more diverse andmore stable intestinal flora.In intestinal samples, we observed a low degree of similar-

ity among intestinal content samples. Cluster analysis identi-fied the similarity of most samples as below 90%. From thediversity index and phyla distribution statistical analysis, thehigher diversity and complexity in the LFM group than inthe control may mean that the LFM piglets developed intesti-nal flora faster than the OM and MM groups did after earlyweaning and that the different doses of rhLF may be the rea-son for the differential microflora ecosystems.

Fig. 4. Diversity index analysis. (A) Diversity index analysis of the intestinal content samples. OM, ordinary-milk feeding group; MM, feed-ing group given a 1:1 mix of ordinary milk and recombinant human lactoferrin (rhLF) milk; LFM, feeding group given rhLF milk; DF, duo-denum; JF, jejunum; IF, ileum; and CF, colon. (B) Diversity index analysis of fecal samples.

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In the DGGE profile of the fecal samples, there were nodifferences in the complexity or diversity of flora. But withcluster analysis, the OM and MM groups had greater similar-ity in their fecal flora than they did with the LFM group.Thus, rhLF may have a dosage compensation effect on thecomposition of the developing intestinal flora. When therhLF dose reaches the level in the LFM group, this proteinmay have a greater influence on the composition of the flora.Large amounts of Lactobacillus and anaerobic bacteria

usually live in the digestive tracts of healthy animals, andthe number of Lactobacillus controls the intestinal microbialbalance (Morotomi et al. 1975; Conway et al. 1990). Lacto-

bacillus and some other anaerobic bacteria are also the mainmicrobes in the gastrointestinal tract of nursing pigs. Escher-ichia coli are part of the normal flora in the pig gastrointesti-nal tract, but under normal circumstances, aerobic bacteriasuch as E. coli account for only ∼1% of the total bacteria.Why do pathogenic bacteria such as E. coli not easily prolif-erate in nursing pigs? An important reason is the supply ofbreast milk and the effective mucosal barrier. When close toweaning, Lactobacillus, Enterobacteriaceae, and Bifidobacte-rium become the advantageous bacteria. Early weaned pigsoften experience ablactation hyperirritability, which greatlyinfluences microbial numbers in the intestine. After weaning,

Fig. 5. (A) Phylo and (B) genus distribution of 16S rDNA clone libraries derived from fecal samples. Samples are labeled as in Fig. 2B.

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the Lactobacillus population declines over a short period oftime, and the ratio of total bacteria to Enterobacteriaceae (es-pecially E. coli) rises (Su and Zhu 2006). Changes in pigletintestinal microbial composition and number are the underly-ing causes of disease when weaning.From the results of the quantitative culture-based bacterial

count analysis, the LFM piglets showed reduced levels of E.coli and Salmonella and increased numbers of Lactobacillusand Bifidobacterium, which creates an advantageous micro-biota in the gut. rhLF-containing transgenic milk appears tobeneficially modulate the intestinal flora both in the numberand concentrations of bacteria.In summary, the results of this study indicate that feeding

of the rhLF-containing transgenic cow’s milk resulted inhigher average daily mass gain than the ordinary whole-milk. Additionally, the PCR–DGGE and 16S rDNA sequenceanalysis revealed that the intestinal microbiota in the LFMpiglets had higher diversity and complexity. The differentdoses of rhLF in milk may be the major factor contributingto the differential microbial ecosystems in the gut. Our find-

ings have important implications for the use of bovine-derived rhLF in the infant formula industry.

AcknowledgementsWe thank Professors Defa Li and Xiangsu Piao and the

Ministry of Agriculture Feed Industry Centre, China Agricul-tural University, Beijing, for their help with the pig feedingand necropsy. This research was financially supported by theNational “863” High-Tech Research and Development Pro-gram (grant No. 2007AA100501) and the National Trans-genic Breeding Program (2008ZX08007-001), China.

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Item OM MM LFM SE P-valueTotal aerobesDuodenum 10.69 10.48 10.73 0.10 0.221Jejunum 10.09 10.23 10.17 0.14 0.765Ileum 9.03a 8.17c 8.64b 0.11 <0.001Colon 9.47a,b 9.80a 9.12b 0.21 0.102Total anaerobesDuodenum 10.50 10.56 10.63 0.09 0.560Jejunum 8.85 8.66 8.80 0.15 0.649Ileum 11.68a 10.93b 11.34a 0.13 0.003Colon 10.31 10.25 10.23 0.16 0.770SalmonellaDuodenum 6.75 6.69 6.76 0.13 0.915Jejunum 6.91 7.02 6.97 0.10 0.715Ileum 6.85 6.96 6.80 0.12 0.611Colon 6.96a 6.75a,b 6.51b 0.08 0.005Escherichia coliDuodenum 6.60a 5.38b 5.29b 0.17 <0.001Jejunum 6.90a 6.21b 5.35c 0.12 <0.001Ileum 9.34a 7.28b 6.91c 0.11 <0.001Colon 9.86a 9.10b 8.67b 0.22 0.006BifidobacteriumDuodenum 7.88 7.94 7.80 0.09 0.599Jejunum 7.86 7.87 7.91 0.09 0.901Ileum 7.66b 8.03a 7.97a 0.08 0.011Colon 8.62 8.66 8.74 0.08 0.582LactobacillusDuodenum 9.05b 9.67a 10.12a 0.16 0.001Jejunum 8.48b 8.71b 9.43a 0.16 0.002Ileum 7.54c 8.21b 9.50a 0.12 <0.001Colon 9.40b 9.50b 10.11a 0.12 0.002

Note: One-way ANOVA, least-significant difference, and Duncan's mul-tiple range test were used for statistical analysis. Mean values within a rowwith different superscripts significantly differ (P < 0.05). OM, ordinary-milk feeding group; MM, feeding group given a 1:1 mix of ordinary milkand recombinant human lactoferrin (rhLF) milk; and LFM, feeding groupgiven rhLF milk.

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