• FINE FOCUS, VOL. 3 (2)

THE COLONIZATION 
AND ESTABLISHMENT 
OF THE NEONATAL 
MAMMALIAN 
MICROBIOME

VICTORIA A. KOURITZIN & LELUO GUAN *
DEPARTMENT OF AGRICULTURAL, FOOD AND 
NUTRITIONAL SCIENCE, UNIVERSITY OF ALBERTA, 
EDMONTON, ALBERTA, CANADA

MANUSCRIPT RECEIVED 13 APRIL 2017; 
ACCEPTED 06 JULY 2017

NEONATAL MAMMALIAN MICROBIOME • 89

Copyright 2017, Fine Focus. All rights reserved.

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    • FINE FOCUS, VOL. 3 (2) NEONATAL MAMMALIAN MICROBIOME • 91

In current agriculture practices, such as the dairy 
industry, the use of antibiotics is being discouraged 
due to the occurrence of antibiotic resistant bacteria. 
However, antibiotics are used commonly to treat 
calf diarrhea, which is a serious issue that negatively 
influences calf health, growth, and development. 
Recent research highlights the gut microbiota as a 
potential source to improve the gut health of a calf, 
which could minimize the antibiotic use. However, 
limited knowledge is available for the early life gut 
microbiota and its relationship with calf’s performance. 
It is known that the microbiota has an influence on 
immune system development, as well as behavioral 
development, and metabolic development. Further, an 
atypical microbial population, or a microbial shift, has 
been linked to autoimmune, anxiety and metabolic 
disorders. The process of microbial and host interactions 
starts at birth, suggesting that mammals are initially 
colonized by microbes immediately following and 
during birth. Differing modes of delivery, caesarian or 
vaginal delivery, and possibly the length of time of the 
birthing process, may determine initial colonization of 
the infant. Further, the establishment of the microbiota 
can be influenced by host genetics, diet, and maternal 
environment. Therefore, this review aims to summarize 
the current understanding of the neonatal mammalian 
microbiota obtained from human and mice studies, 
and to outline future research directions on microbial 
colonization and possible manipulation strategies that 
can be used to manipulate the gut microbiota in dairy 
calves. By understanding the process of how mammals 
and microbes interact it is possible to better target future 
research in order to solve the problem of calf diarrhea.  

ABSTRACT

CORRESPONDING 
AUTHOR
LeLuo Guan
410 Agriculture/Forestry 
Centre, University of Alberta, 
Edmonton, AB, Canada T6G2P5; 
Tel, 780-492-2480
Fax, 780-492-4265
lguan@ualberta.ca

KEYWORDS
• Early-life Microbiome 
• Calf
• Colonization  
• Establishment 

Calf diarrhea can cause more of a financial 
loss than any other calf ailment (15). Diarrhea in 
the young animal prevents absorption of fluids 
and nutrients, inhibiting growth and health (1). 
Further, without immediate intervention, calf 
diarrhea can be transmitted between calves 
and can result in high mortality. Currently, 
the common practice to prevent calf diarrhea 
is the use of antibiotics. Yet, industry is 
encouraged to reduce the use of preventative 
antibiotics due to the increasing prevalence of 
antibiotic resistance. Therefore, an alternative 
to antibiotic prevention for calf diarrhea is 
needed. Recent research highlights the gut 
microbiota as an important factor in immune 
function development and maintaining 
neonate gut health, making the gut microbiota 
a potential source to improve the gut health 
of calves to reduce the prevalence of diarrhea. 
However, limited knowledge on the calf 
gut microbiota is available. Additionally, the 
mechanisms of how the microbiota influences 
calf gut development and health are still 
largely unknown and undefined. Therefore, 
this review aims to summarize the current 
understanding of the neonatal mammalian 
microbiota based on the findings from human 
and mice studies from three aspects including 
initial colonization of the neonatal mammals, 
the impact of the microbiota on gut immune 
function of the developing neonate mammal, 
and factors affecting microbial establishment 
during early life.  Further, this review aims 
to identify the knowledge gap of the calf 
microbiota in order to direct future research 
towards identifying potential ways to reduce 
the incidence of, and possibly prevent, calf 
diarrhea through the manipulation of gut 
microbiota.

MAMMALIAN 
DEVELOPMENT 
AFFECTED BY     
MICROBIOTA 

It was hypothesized by Louis Pasteur 
that the microbiota has an important and 
necessary influence on mammalian life (21). 
Mammals have a rich and diverse microbial 
population that interacts with, and influences 
the development of biological processes in 
mammals like immune function and metabolic 
systems (21). Additionally, the microbiota causes 
long-term impacts on emotional systems 
(4, 23). The following section of this review 
will outline specific changes in microbiota 
and how they can affect the immune system 
development, anxiety and depression, and 
digestive and metabolic functions.

IMPACT OF THE 
MICROBIOTA ON IMMUNE 
SYSTEM DEVELOPMENT

The interaction between host and microbes 
is essential for proper immune function 
development. Young animals enter a critical 
period soon after birth when exposure 
to antigens is imperative for immune 
development. If the exposure is delayed, 
immune development can be impacted. This 
can be explained by an experiment using 
germ free mice (21). Firstly, it is important 
to note that the authors demonstrated that 
germ free mice have an atypical cytokine 
response to orally treated lipopolysaccharide 
(LPS) when compared to conventional mice. 
It is important to note that LPS is a large 
molecule found on the outer membrane of 
Gram-negative bacteria, and the previously 

INTRODUCTION

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Figure 1. The relationship between the colonization and establishment of the gut microbiota on the early-life 
calf’s immune system, mood and metabolism, shown in blue, and the impact of the calf’s early diet, genetics 
and internal environment on the gut microbiota, shown in purple. Also shown is the maternal impact on the 
calf by genetics and maternal environment, and the maternal impacts on the microbial colonization of the 
calf, shown in red.

described atypical response differs from a 
normal response in that the atypical response 
is delayed and exaggerated. Further, these 
authors noted that the juvenile mice can 
resume a normal cytokine response, and 
behave normally, normal being immediate 
and moderate, when treated with probiotic 
Bifidobacterium infantis. However, when adult 
germ free mice were treated with the same 
probiotics they were unable to be converted 
back to the normal response. Additionally, 
these effects on the immune system can have 
long-term consequences, which are still being 
investigated (10, 19). It has been found that 
antibiotic use in early life influenced fecal 
bacterial composition and can be linked with 
development of intestinal diseases later in life 
(17). However, there is convincing research 
that interruptions in the establishment of 
the microbiome during early life can result 
in allergies, asthma and other autoimmune 
diseases in adults (10). Additionally, it was 
found that the development of eczema can 
be minimized if infants were treated with 
probiotics (10). 

After birth, the gut microbiota may 
contribute to mammalian immune system 
development through interactions between 
hosts. The interaction between microbe and 
host immune development is important, which 
has been demonstrated by germ free animals 
as described above. Germ free animals do 
have immunities; however, they cannot cope 
with pathogens (22). This reveals that without 
the proper microbial exposure in early life, 
the immune system will not properly develop 
and can increase risk of disease in later life. 
Further, a more diverse intestinal microbiota 
during the first week of life is associated with 
a reduced risk of subsequent eczema in infants 
(10). Additionally, interventions that enhance 
microbial diversity in early life may provide 
an effective means for the prevention of 
eczema in high risk infants (10). 

IMPACT OF MICROBIOTA 
ON ANXIETY AND 
DEPRESSION

Initially, the evidence of microbial 
impact on host behavior was gained from 
comparisons of germ free and bacterial 
colonized mice. In order to test the idea that 
postnatal microbial colonization may affect the 
development of brain plasticity, researchers 
compared the hypothalamic-pituitary response 
to differing levels of stress restraint using 
genetically identical germ free and specific 
pathogen free mice (23). In this study, the 
individual mice were placed into a 50 ml 
conical tube for 1 hour, or into a glass container 
lined with ether soaked absorbent paper for 2.5 
minutes. The authors found exaggerated stress 
response in germ free mice when compared 
to pathogen free mice (23). Further, the same 
authors observed a reduction in stress response 
in germ free mice after administration of 
probiotic Bifidobacterium infantis. These results 
have demonstrated the differences in brain 
function between germ free and colonized 
mice, suggesting the potential relationship 
between gut microbiota and animal behavior.  
However, other researchers found that 
administration of antimicrobials to colonized 
mice reduced the microbial population, and 
increased exploratory behavior in mice (4). 
They also found that the same antimicrobials 
given to germ free mice had no effect on their 
behavior. Furthermore, they reported that 
administering the microbial population from 
colonized mice to germ free mice reduced 
exploratory behavior in the germ free mice. 
Such change of exploratory behavior suggests 
a higher activity of mice when treated with 
antimicrobials. Though increased exploratory 
behavior proposes a contrast to a greater fear 
response, they may actually be complementary 
depending on the motivation of the 
exploration (23). However, it is clear that more 

research needs to be done to determine the 
impact of postnatal microbial colonization on 
the development of neural systems that induce 
stress in animals.

IMPACT OF THE GUT 
MICROBIOTA ON 
DIGESTION AND 
METABOLIC FUNCTIONS

Recent studies have found that a shift in 
microbiota during early life can cause obesity, 
as well as other metabolic diseases like diabetes 
in later life (19). The study on fecal microbiome 
of 298 stool samples from 22 children with 
type 1 diabetes, and 22 normal children of the 
same age found a significant difference in host-
microbial interactions between two groups 
(6). There was a higher interaction between 
host and Enterococcus, Sarcina, Prevotella, 
and Corynebacterium in the children with 
type 1 diabetes (7). Relatedly, the authors also 

found less interaction between Enterococcus, 
Sarcina, Prevotella, and Corynebacterium 
and host in children without type 1 diabetes 
(7). This correlation of host and bacterial 
interaction, may suggest that metabolism could 
be affected by the gut microbiota. To further 
identify the causal effect of microbiota on host 
metabolic dysfunction, genetically deficient 
Toll-like receptor 5 (TLR5) mice were used 
to induce obesity, and then the obese TLR5 
mice microbiota was transplanted to wild type 
germ free mice (24). Found in the gut mucosa, 
Toll-Like receptor 5 is a class of receptor 
involved in innate immune responses, which 
plays a role in satiety. Without TLR5, the 
mice exhibited significant hyperphagia, and 
developed metabolic disease (24). Next, the 
germ free wild type mice treated with the gut 
microbiota derived from the TLR5 deficient 
mice, immediately showing distinct microbial 
changes, and later becoming obese (24). The 
change in body composition of germ free mice 

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from normal to obese after colonized with a 
specific microbial population, suggests that the 
gut microbiota may impact body composition 
through affecting the host metabolic functions.

INITIAL 
COLONIZATION

Mammals are currently believed to 
be sterile when in utero and microbial 
colonization can be observed minutes after 
birth (18). Initial colonization can be defined as 
the period during, and immediately following 
birth when microbes first interact with and 
colonize the sterile infant. Because microbial 
colonization of the mammalian neonate 
happens so quickly it is assumed that initial 
colonization happens from the birth canal or 
from the first exposure to the environment in 
neonates delivered by caesarean section (18).

MODE OF DELIVERY: 
VAGINAL OR CAESAREAN 
SECTION

Since the mode of initial colonization is 
thought to happen during the birthing process, 
it can be assumed that the two different modes 
of birth, caesarean section or vaginal birth, 
would result in different infant microbial 
populations.  Indeed, mode of delivery 
impacts microbial population initially, but 
the differences minimize with aging (18). 
Specifically, caesarean section was associated 
with both lower abundance and diversity of 
the phyla Actinobacteria and Bacteroidetes, and 
higher abundance and diversity of Firmicutes 
from birth to three months of life. It was also 
found that Bifidobacterium and Bacteroides 
genera seems to be more frequent in vaginally 
delivered infants compared with caesarean 
section delivered ones (18). However, after 

six months of age, the microbial differences 
minimized. Furthermore, it was found that 
delivery mode was not significantly associated 
with childhood development of obesity 
through a longitudinal study from infancy to 
7 years of age (2). Though they noted initial 
differences in microbial population with the 
different mode of delivery, the differences 
did not make a lasting impact and by 7 years 
of age the mode of delivery no longer had an 
impact on microbial population.

LENGTH OF DELIVERY
Currently, there is no research on the impact 

of length of delivery on microbial population. 
The length of delivery can be hypothesized to 
impact initial microbial colonization because 
a longer birth would mean the infant spends 
longer in the birth canal, meaning more 
exposure to birth canal microbiota. This could 
be the case, especially in the birthing process 
of calves. For example, heifers generally have 
a longer delivery than cows because it is their 
first time through the birthing process. It is 
possible that having calves born from heifer 
or cow may impact the exposure to birth 
canal microbiota thus impact initial microbial 
colonization. However, it can be assumed 
that length of delivery would be similar to 
type of delivery in that though it may be 
initially significant, the differences in microbial 
population may not last into adulthood. 
However, if there is a difference in length of 
delivery, it is important to determine if those 
differences last into adulthood.

FACTORS 
AFFECTING 
MICROBIAL 
ESTABLISHMENT

The establishment of the microbiota is a 
dynamic process following initial colonization, 
which depends on factors such as host genetics, 
diet, maternal environment, early antibiotic 
exposure, and highly sterile environments (9, 
12, 13).

HOST GENETICS
The genetics of the host determine the 

microenvironment of the gut thus affecting 
the suitability of the internal environment 
for microbial colonization.  An early study in 
mice found host genetics had a 12% impact on 
microbial variation in the gut (25). Additionally, 
different researchers found a significant 
link between a mutation in human genes 
and a corresponding shift in gut microbiota 
characterized by a depletion of total numbers 
of bacteria, loss of diversity, and major 
shifts in bacterial populations within the 
Bacteroidetes, Firmicutes and Proteobacteria 
phyla (11). Moreover, another different research 
group was able to identify a core measurable 
microbiota of 64 conserved taxonomic 
groups using quantitative pyrosequencing of 
the microbiota and individual host genotype 
had a measurable contribution to microbial 
variation which can be explained by litter and 
cohort effects (3). Through further statistical 
analysis they found suggestive genome-wide 
linkage with relative abundances of specific 
microbial taxa, providing clear evidence for 
the importance of host genetic control in 
shaping individual microbiome diversity in 
mammals (3). Although there have been recent 
linkages between host genetics and microbial 
population, and microbial population and 
body composition (2, 3, 17), there has not yet 
been a genetic connection made between gut 
microbiota and long-term growth performance 
such as weight gain.

DIET DURING EARLY LIFE
Differences in microbial population have 

been found with different diets during 

early life (9, 12). Breast-fed infants have a 
gut microbiota dominated by Bifidobacteria, 
whereas formula fed infants have a more 
heterogeneous composition and less 
Bifidobacteria (9). Furthermore, formula 
with the more closely resembles maternal 
milk results in a microbial population 
that is dominated by Bifidobacteria, like 
breast-fed babies (9, 12, 14, 20). These results 
suggest that the diet of infants can change 
the gut microbiota population through the 
establishment process. 

MATERNAL ENVIRONMENT
Like diet, prenatal maternal stress can 

influence the composition of the infant’s gut 
microbiota. Maternal stress is hypothesized to 
change the maternal physiological environment, 
including increased heart rate and introduction 
of stress hormones (2). Additionally, stress 
can also change the maternal environment 
behaviorally by increasing or decreasing the 
mother’s appetite, activity level, and potentially 
changing the mother’s diet. Such change in 
maternal environment can affect the mother’s 
microbial population, and can potentially 
influence initial colonization of offspring at 
birth (8, 26). Maternal stress can cause increase 
in cytokine concentration and inflammation, 
possibly influencing the developing fetus. 
This maternal immune response creates a 
different environment for the developing fetus 
potentially influencing, or changing their 
immune function development. In addition 
to maternal stress, other maternal states, 
like maternal fitness, can impact the infant’s 
microbial population. Additionally, researchers 
have found a high body mass index (BMI) of 
the pre-pregnancy mother had a significant 
impact on the development of obesity in the 
offspring (2). Further, the same study reported 
that if those children of a high BMI mother 
were treated with antibiotics early in life, it 
reduced obesity prevalence in these children 
(2). Contrastingly, children born to normal 

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BMI mothers and treated to early antibiotics 
were seen to have an increased risk of 
becoming obese later in life (2). Such influence 
of the maternal body composition on the 
fetal microbiome can potentially be due to a 
change in the maternal microbial population 
influencing initial colonization of the fetus. 
Further, because when children born from 
a mother with a high BMI had a decreased 
chance of developing obesity when they were 
treated with antibiotics early in life, suggests 
a potential cause for microbial influence (2). 
Understanding, and acknowledging the impact 
of the maternal microbiota on the offspring’s 
microbiota is important when it comes to 
management and treatment because it is clear 
that maternal environment has an effect on the 
infant’s microbiota. 

KNOWLEDGE 
GAPS IN 
STUDYING 
EARLY LIFE: 
MICROBIOTA IN 
CALVES

The microbial colonization in cattle, 
especially in neonates, has profound impacts 
on nutrition, health, animal physiology and 
productivity (6). Recent research outlining the 
importance of the microbiome in maintaining 
neonate health makes the microbiota a 
potential source of treatment for calf diarrhea 
(1). Nevertheless, information on the calf 
microbiota is still limited and more research is 

necessary before the microbiota can be used 
to limit instances, and possibly even prevent, 
calf diarrhea. Additionally, food safety (E. 
coli O157 colonizes the young claves) may 
be another area of possible research into the 
importance of the microbiome of beef cattle 
(6). By understanding the process of how 
neonatal mammals and microbes interact, it 
is possible to better target future research in 
order to solve the problem of calf diarrhea, 
or other related concerns. Two interesting 
effects of the microbiota on dairy calves 
worth noting are the effects of antibiotic 
treatment on calf behaviour, and the maternal 
control on the calf`s microbiota. Also, another 
important area of investigation is the long term 
effects of microbial on weight gain and body 
composition of cattle.

The effect of the microbiota on behaviour 
is important to highlight in calves because 
when calves are treated with antibiotics for 
calf diarrhea, the antibiotics may reduce their 
microbial population, which may lead to 
exaggerated stress response. Therefore, it is 
important to investigate if these results can be 
replicated in calves, and potentially investigate 
a probiotic for calves to reduce their stress 
response. Especially a link between stress 
and immune efficiency has also been noted 
(5). The implications of antimicrobials and 
probiotics on calf behavior may be important 
for calf management. Based on these findings, 
producers may have the options to give calves 
the probiotics after antibiotic treatment of calf 
diarrhea.

Another important aspect is the effect of 
the mother on the calf microbiota colonization, 
including maternal genetics and maternal 
environment. The relationship of host genetics 
to microbial population is important for 
calves because it suggests that potentially 
genetic selection, together with a probiotic, 
could be the solution to treat calf diarrhea. 
Also, maternal environment is important to 
note when investigating potential treatments 
to calf diarrhea because potentially a change 

in management of pregnant animals may 
reduce instances of calf diarrhea. However, 
more research needs to be done to see the 
influence of the mother’s environment, and 
body composition, on the microbial population 
of the calf. In addition, it is a common practice 
to remove dairy calves from their mothers at 
a very young age, often increasing their risk 
of microbial infection because of this high 
stress period.  There are many gaps in the 
knowledge of the calf microbiota that need to 
be addressed through research before microbial 
manipulation can be a potential treatment of 
calf diarrhea. To date, the understanding of the 
early life colonization in dairy claves is very 
limited and it is therefore important to research 
the role of calf gut microbiome in immune 
system development. 

Since beef cattle are raised in less intensive 
production systems than dairy, there have 
been different areas of focus on each of them 
(15). While microbial investigation of dairy 
cattle has focused mostly on calf diarrhea, and 
mastitis, or bacterial colonization of the utter, 
the microbial focus of beef cattle has been on 
weight gain and feed efficiency. Though dairy 
and beef cattle are different in production 
systems, and physiology, each bovine sector 
may benefit from collaboration. Therefore, it 
is important to outline some recent advances 
in the beef industry. Firstly, rumen variation 
between beef cattle has been noted between 
animals housed in the same environment 
and fed the same diet (6). Thus, the genetic 
component, or possibly another undiscovered 
factor, influence the establishment of the 
gut microbiota. However, more research is 
needed to explain the observed individualized 
microbial variation. Further, a significant 
relationship between weight gain and rumen 
microbiome was discovered when comparing 
the most and least efficient animals (17). 
Regardless, more research needs to be done 
to investigate long term impacts of microbial 
manipulation on gut health to prevent calf 
diarrhea,  and growth for better weight gain. 

CONCLUSION 
Due to the financial impacts of calf diarrhea 

and the increased occurrence of antibiotic 
resistance, it is important to find way to 
treat calf diarrhea that does not include the 
use of preventative antimicrobials (14).  It is 
common for calves in the dairy industry to 
be given preventative antimicrobial during 
early life. Recent research has identified the 
gut microbiota as a potential tool in reducing 
the incidence of calf diarrhea. However, 
research on the calf microbiome is limited. 
Consequently, this review summarized the 
current understanding of neonatal mice and 
human microbiota to outline the importance 
of the microbiota, as well as microbial 
colonization and establishment. In addition, 
the knowledge gaps in calves have been 
identified. However, it was determined that 
future research is needed to investigate the 
relationship between early life microbiota and 
calf immune system, behavior, and metabolism 
(Figure 1). Additionally, further investigation 
is needed to identify the response of the calf 
microbiome to a change in host genetics, 
birth length, diet, and maternal environment. 
Though the understanding of mammalian 
microbiota has had many recent advances, 
specific information on the calf microbiome 
is needed before microbial manipulation can 
be a potential treatment to calf diarrhea. More 
research is needed in order to understand 
how the calf gut microbiota may respond to 
manipulation. 

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