ACCESSION NO: 0233080  SUBFILE: CRIS 

PROJ NO: ILLU-971-333 AGENCY: NIFA ILLU 

PROJ TYPE: HATCH PROJ STATUS: TERMINATED 
START: 01 OCT 2012 TERM: 30 SEP 2014 FY: 2014

INVESTIGATOR: Donovan, S.; Cann, I.; Mackie, R.

PERFORMING INSTITUTION: 
UNIVERSITY OF ILLINOIS 
2001 S. Lincoln Ave. 
URBANA, ILLINOIS 61801

DEVELOPMENT OF A HUMAN INFANT MICROBIOTA ASSOCIATED [HIMA] PIGLET MODEL TO

PROBE NUTRITIONAL REGULATION OF HOST-MICROBE INTERACTIONS IN THE NEONATE

NON-TECHNICAL SUMMARY: The piglet is the best model for the human infant in terms of GI
development and closely resembles the human for >80% of immune parameters vs. <10% for mice. Thus,
the piglet is the best model for studying host-microbe interactions on gut and immune development.
However, differences in the composition of the gut microbial communities limit the translatability and
relevance of findings obtained in the piglet to the human infant. A HIMA piglet model represents a unique
tool to enable hypothesis-driven research on host-microbe interactions and mechanisms of action of
probiotics and other dietary components within an animal model containing a gut microbiota similar to that
of human infants.

OBJECTIVES: The intestinal tract is functionally immature and immunologically naive at birth.

Accordingly, the neonatal period is a critical phase for intestinal structural, functional and immune
development as well as establishment of the microbiota. As we learn more about interactions between
microbes and the developing gastrointestinal (GI) tract, it has become clear that the microbiome plays a
critical role in the origins of health and disease. Early nutrition and pre- and probiotics alter the microbial
ecology and may affect lifelong health. Dr. Donovan and her collaborators have over 15-years of
experience using the piglet as a preclinical model to assess the impact of human milk and infant formula
components on GI development. However, a major limitation of the piglet model is that the composition of
microbiota differs from that of human infants. A solution to this problem is to develop human microbiome-
associated (HMA) piglets. The development of gnotobiotic and germ-free rodent models that could then
be colonized by the microbiome of the same species or another species has led to significant progress in
defining how the microbiota regulates gut development, immunity, responses to diet and metabolic
diseases including obesity. The feasibility of maintain gnotobiotic pigs and transplanting human
microbiota to piglets has been demonstrated and the resultant microbiota more closely resembled that of
the human donor than that of conventional pigs. Therefore, the goal of this proposal is to establish a
human infant microbiome associated (HIMA) piglet model at the University of Illinois. We anticipate that
the microbiome of the HMA piglet will more closely resemble that of a breast-fed human infant than that of
a sow-reared piglets. A HMA piglet model will enable hypothesis-driven research on host-microbe
interactions and mechanisms of action by probiotics and other dietary components that will be more
directly translatable to the human infant.

APPROACH: Two aims are proposed. In Aim 1, we will establish the human-infant microbiota associated
piglet model. Briefly, colostrum-deprived, cesarean-derived piglets will be colonized with microbiota
derived from sow-reared piglets (control), fecal microbiota pooled from 6-week-old breast-fed human
infants (HIMA) or their mothers (HAMA). The dosage will be 1 ml (~1010-1011 CFU)/piglet daily in the
first 4 days and then once every other day until 10 days of age. Piglets will be fed sterile formula
(Advance Baby Pig LiquiWean (Milk Specialties, Dundee, IL) and will be housed individually to allow for
fecal sample collection. Fresh fecal samples will be collected throughout the study and on d14, feces and
ascending colon contents will be collected and frozen. DNA from the initial inoculate, piglet feces or
ascending colonic contents will be isolated and purified by the QIAamp DNA Stool Mini Kit (Qiagen,
Valencia, CA) in combination with the FastPrep-24 System (MP Biomedicals, Solon, OH). The
composition of the microbial communities will be assessed by pyrosequencing of the V1-V3 region of
bacterial 16S rRNA genes using modified primers Gray28F (5'-TTTGATCNTGGCTCAG-3') and Gray519r
(5'-GTNTTACNGCGGCKGCTG-3'). After the model has been established, Aim 2 will examine the
prebiotic effects of human milk and human milk oligosaccharides (HMO). Piglets will be delivered by
cesarean section, administered serum and gavaged orally with human infant fecal suspension as
described in Aim 1. HIMA piglets will be randomized into three diet groups (n=8/group): FF (formula-fed;
Advance Baby Pig LiquiWean), HM (1:1 formula to human milk) or HMO (formula with 2 g/L LNnT). On d
14, contents from ascending colon will be collected and frozen. For SCFA, ascending colonic contents will
be placed into Eppendorf tubes containing 2N HCl and SCFA will be measured by gas chromatography
using established methods.

PROGRESS: 2012/10 TO 2014/09
Target Audience: Members of the target audience include practitioners interested in improving child
health by modifying the gut microbiota and scientists interested in how the microbiota influences gut
development. Changes/Problems: Nothing Reported What opportunities for training and professional
development has the project provided? The researchers mastered new techniques in conducting this
research. In addition, several undergraduate students were trained and participated in the research.
Researchers presented their findings at a scientific conference. How have the results been disseminated
to communities of interest? Data from this project was disseminated to researchers at the International
Society for Research on Human Milk and Lactation conference in November 2014. What do you plan to
do during the next reporting period to accomplish the goals? This is the final report. At least one
manuscript resulting from this work is in preparation. We have also submitted a grant proposal to fund
further work on the HIMA model.

IMPACT: 2012/10 TO 2014/09
What was accomplished under these goals? We have completed both aims of the project. In Aim 1,
newborn, colostrum-deprived, cesarean-deprived piglets were inoculated with the microbiota of breastfed
infants or the mothers of the infants. A third group was not colonized with any gut microbes. The fecal
inoculum was provided daily from d1-d4, then d6, 8 and 10. Piglets were fed a standard pig milk replacer
diet without any added oligosaccharides. Stool samples were collected and piglets were euthanized on
d18. Piglets administered the adult human fecal inoculum maintained a gut microbiota very similar to the
human microbiota, whereas the gut microbiota of piglets gavaged with the breast-fed human infant
inoculum were not as successful in maintaining a microbiota similar to that of the human infants, which
may be due in part to the lack of human milk oligosaccharides (HMO) or other bioactive components
present in human milk. Thus, in aim 2, piglets were inoculated with the microbiota of breastfed infants and
were fed either pig milk replacer formula (FF), human milk (HM) or pig milk replacer formula with 3 g/L of
either 2´-fucosyllactose (2´-FL) or lactose-N-neotetraose (LNnt), two HMO. Analysis of the fecal
microbiota showed that FF piglets shared some similarity with breast-fed infant donors and differed from
that of HM-fed piglets. Densities of Lactobacillus, Clostridium cluster XIVa and Bacteroides fragilis group
were similar among all of the diet groups. Abundance of Clostridium cluster IV was higher in HM piglets in
comparison with FF, 2´-FL or LNnT piglets. Importantly, Bifidobacterium densities were highest in HM
group, intermediate in LNnT group and lowest in FF and 2´-FL groups. Thus, we have developed a
human microbiota-associated piglet model with which to investigate nutritional modulation of the
microbiota and host-microbe interactions. In this model, LNnT, but not 2´-FL, added to formula stimulated
the growth of Bifidobacterium, resulting in a bacterial microbiota resembling that of HIMA piglets fed HM.

PUBLICATIONS (not previously reported): 2012/10 TO 2014/09

Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Wang, M.,
Monaco, M.H., Cann, I.K., Mackie, R.I. and Donovan, S.M. Select human milk oligosaccharides modulate
the gut microbiota in a human microbiota-associated piglet model. 17th International Conference of the
Society for Research on Human Milk and Lactation, Kiawah Island, South Carolina, October 2014.