Theriogenology 152 (2020) 18e28

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Theriogenology
journal homepage: www.theriojournal.com

Do newborn puppies have their own microbiota at birth? Influence of

type of birth on newborn puppy microbiota
Maja Zako
sek Pipan a, *, Leonida Kajdi

c b, Anja Kalin b, c, Tanja Plavec c, d, Irena Zdovc b
a
Clinic for Reproduction and Large Animals, Veterinary Faculty, University of Ljubljana, Ljubljana, Slovenia
b
Institute of Microbiology in Parasitology, Veterinary Faculty, University of Ljubljana, Ljubljana, Slovenia
c
Small Animal Clinic, Veterinary Faculty, University of Ljubljana, Ljubljana, Slovenia
d
Small Animal Veterinary Hospital Hofheim, Hofheim am Taunus, Germany

a r t i c l e i n f o a b s t r a c t

Article history: With recent research in humans, a hypothesis known as the sterile womb paradigm has been challenged.
Received 21 December 2019 The objectives of this study were to determine the presence of placental and fetal microbiomes in dogs,
Received in revised form the effect of different types of parturition on the fetal microbiome, and the effect that the fetal micro-
11 March 2020
biome has on early puppy development.
Accepted 11 April 2020
Available online 21 April 2020
A total of 96 newborn puppies from 17 dams were included in the study. Puppies were divided into
two groups depending on the type of parturition (vaginal birth (VB) or cesarean section (CS)). Imme-
diately after birth, swabs of the placenta and meconium were taken. Swabs of the oral and vaginal
Keywords:
Neonatology
mucosa of the dam were taken in the second half of the pregnancy and just before parturition. All
Birth samples were analyzed with a classical bacteriological examination, and bacterial colonies were iden-
Puppies tified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
Microbiota The weight gain of each puppy was tracked daily in the first 7 days postpartum.
Weight gain Bacteria from several different genera were isolated from 86.5% of meconium samples and 57% of
placenta samples. While the meconium microbiota resembled bacteria from the maternal vagina in VB
puppies, the meconium microbiota of puppies born by CS indicated a relative resemblance to maternal
oral and vaginal microbiota. A statistically significant difference in the relative growth rate between
puppies born by VB and CS was found (p < 0.05), with puppies born by VB gaining weight faster
compared to the CS group. This difference was even more noticeable when VB puppies were compared to
puppies born by elective CS. Puppies born without a detectable meconium or placental microbiota
showed a slower growth rate than those with a meconium microbiota, regardless of the type of
parturition (p < 0.05).
The findings of this study provide new information about the placental microbiome in healthy
pregnant dams and suggest intrauterine colonization of the fetus in dogs. It seems that the type of
delivery and bacterial colonization might be an important consideration for the weight gain in puppies in
the first few days of life.
Published by Elsevier Inc.

1. Introduction
Until the second half of the 19th century, it was believed that a
fetus develops in a sterile environment and that the initial bacterial
colonization of the newborn occurs while traveling through the
maternal birth canal. This hypothesis is also known as the sterile
* Corresponding author. Clinic for Reproduction and Large Animals, Veterinary
Faculty, University of Ljubljana, Gerbi

ceva 60, 1000, Ljubljana, Slovenia.
E-mail address: maja.zakosekpipan@vf.uni-lj.si (M. Zako
sek Pipan).
womb paradigm and has remained dogma for more than a century
[1]. However, many recent studies employing modern sequencing
technologies in humans suggest that the uterus, placenta, amniotic
fluid, meconium and umbilical blood from healthy pregnancies
contain bacterial communities [2e13]. These studies suggest that
the placental microbiome may initiate colonization within the fetus
[2e4] and that bacterial transmission through the placental barrier
is a part of the normal developmental process of the fetus [1].
Several studies have shown the influence of delivery type on the
composition of the gut microbiota of newborn babies. The gut
microbiota of vaginally born (VB) infants is composed of bacteria

https://doi.org/10.1016/j.theriogenology.2020.04.014
0093-691X/Published by Elsevier Inc.
 M. Zako
sek Pipan et al. / Theriogenology 152 (2020) 18e28
present in the vagina of the mother but also includes bacteria
present in the mother’s gut [14,15]. In contrast, the gut microbiota
of infants born by cesarean section (CS) resembles maternal skin
and oral microbiota [16,17]. However, it needs to be mentioned that
in these studies, the samples of the infants’ gut microbiota were
obtained a few days after birth and not immediately after
parturition.
The presence of an endometrial microbiome has been reported
in healthy cows [18e21], giant pandas [22] and dogs [23]. Microbes
have also been found in foal amniotic fluid [24], newborn calf
meconium [25] and pregnant bovine uterus [26]. Recently, a
transplacental translocation of Staphylococcus aureus through the
maternal/fetal barrier to the fetus was reported in a pregnant ewe
[27].
While a layer of maternal endothelium separates canine fetal
chorion from maternal blood (endotheliochorial placenta), in
humans, maternal blood comes into direct contact with the fetal
chorion (hemochorial placenta). This direct connection in humans
allows for a much better exchange of substances, including im-
munoglobulins, between the mother and the offspring. While hu-
man babies are born with high levels of immunoglobulins, in dogs,
only 5%e10% of immunoglobulins are transferred through the
endotheliochorial placenta from the mother to her offspring [28]. In
dogs, the transplacental exchange of antibodies is very limited and
mainly occurs during the last third of gestation in a specific
placental region with a hemochorial organization [29]. This
different structure of the canine placenta could minimize the
transmission of bacteria from the endometrium and blood to the
placenta and fetus and thereby prevent the formation of the
placental microbiome and early intrauterine microbial colonization
of the puppy.
There is a paucity of knowledge about the placental and fetal
microbiomes in dogs. The objectives of this study were to deter-
mine the presence of placental and fetal microbiomes in dogs and
evaluate the effect of different types of parturition (VB vs. CS) on the
fetal microbiome and early puppy development.
2. Materials and methods
2.1. Animals
The study was approved by the Faculty Ethics Committee. All
animals included in the study were client-owned. Owners vol-
unteered their dogs for the purpose of this research, and they
signed a consent form allowing collection of samples from the dam
and the puppies. The number of puppies ranged from two to nine
per litter. The types of parturition were vaginal birth (VB) and
caesarian section (CS). Each newborn puppy was weighed, assessed
using a modified Apgar scale and examined for the presence of
congenital malformation (congenital oronasal fistula, cleft palate,
atresia ani). If necessary, resuscitation was performed: rubbing to
stimulate breathing, cleaning mucous from the upper airways,
oxygen supplementation, heating, glucose supplementation, and,
on one occasion, external heart massage. In cases when the dam did
not have colostrum at the time of birth, the puppies were given a
drop of 40% glucose under the tongue. Each puppy was marked
immediately after birth with a collar of a different color for the
purposes of identification of the puppies and marking of the
samples.
2.2. Cesarean section (CS)
Emergency CS (EM-CS) and elective CS (EL-CS) were included in
the CS group. In the case of EL-CS, the day of surgical delivery was
determined using the average 63-day gestation from ovulation
19
guidelines, coupled with the information collected from the fetal
ultrasonographic measurements of both the inner chorionic cavity
and the biparietal diameter, and by serial blood progesterone
concentration monitoring. CS was always performed using the
same protocol for anesthesia to minimize the effect of the drugs on
newborns. Briefly, the preoperative health status of the dam was
determined with clinical examination, complete blood count
(ADVIA® 120, Siemens; München, Germany) and blood gas analysis
(VetScan i-STAT® 1, Abaxis; Union City, California). Blood samples
were collected from a peripheral vein. A cephalic vein was used for
intravenous cannulation. Animals were then preoxygenated with
100% oxygen for 10 min before the induction of anesthesia. In the
meantime, the abdomen was shaved, washed with mild soap
(Aniosgel 85 NPC, Ecolab Deutschland GmbH, Monheim am Rhein,
Germany), rinsed with water, dried and disinfected with propan-2-
ol and benzalkonium chloride (Cutasept F, Bode, Germany) first
with a spray and 3 min later with the paint technique. For induction
to anesthesia, propofol (4e7 mg/kg of body weight) was given
intravenously (Propoven; Fresenius Kabi Ltd.; Runcorn; Great
Britain). Orotracheal intubation was performed after induction
using a cuffed endotracheal tube of the appropriate size. Metha-
done (Comfortan; Dechra; Northwich; Great Britain) was admin-
istered to the dams subcutaneously (after the skin incision) at a
dose of 0.2 mg/kg on body weight. General anesthesia was main-
tained with the inhalation anesthetic sevoflurane (Sevoflurane;
Dra€ger; Lübeck; Germany) at a concentration of 1.5e3%. During
general anesthesia, the animals received a balanced isotonic crys-
talloid solution (Hartmann solution; B. Braun; Melsungen; Ger-
many) intravenously at a rate of 5e10 ml/kg/hour. CS was
performed with a midline incision of the abdomen from the pubis
to the umbilicus and a ventral incision of the uterine body to enable
rapid removal of the fetuses. The most caudal fetus and its placenta
were removed first, followed by fetuses and placentas from the left
and right uterine horn alternately.
The uterus was sutured with one layer of a simple continuous
suture pattern using 3/0 or 4/0 glycomer 631 (Biosyn; Covidien;
Dublin; Ireland). At the end, 0.25e1 IE of oxytocin (Oxytocin; veyx-
Pharma Gmbh; Schwarzenborn; Germany) was injected into the
uterus to promote uterine contractions and cleaning. The abdom-
inal cavity was thoroughly inspected and rinsed with 0.9% sodium
chloride (NaCl; B Braun; Melsungen, Germany) warmed to body
temperature before the abdominal wall was sutured. The abdom-
inal wall was closed with a simple continuous suture pattern with
2/0 or 3/0 glycomer 631 (Biosyn; Covidien; Dublin; Ireland). The
subcutaneous tissues and skin were closed with a continuous
subcutaneous and intradermal suture with 3/0 or 4/0 polyglytone
6211 suture (Caprosyn; Covidien; Dublin; Ireland).
Another dose of methadone (0.2 mg/kg per body weight) was
given subcutaneously 4 h after the first dose. Postoperative anal-
gesia was provided with tramadol chloride (Tramal; Stada; Bad
Vilbel; Germany) at a dose of 3 mg/kg orally every 12 h, as neces-
sary, for a maximum of three days after the procedure.
2.3. Vaginal birth (VB)
All of the vaginal deliveries took place in the home environment
of the owner. Immediately after birth, the newborn puppies were
released from their amniotic sacs if they were born with them. The
process of acquiring placental samples was made as clean as
possible. Placental samples in VB puppies were taken only when
the placenta was delivered with the puppies and when we were
able to intercept the placenta before it fell on the bedding or was
licked by the dam. People handling the puppies and placentas wore
sterile gloves.
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sek Pipan et al. / Theriogenology 152 (2020) 18e28

Table 1
Number and proportion (%) of puppies according to the type of parturition, sex and survival of newborns.

Number of puppies (n) Proportion of puppies [%]

Type of parturition Vaginal birth 26 27.1
Cesarean section Elective 48 50.0
Emergency 22 22.9
Sex of the puppies Females 43 44.8
Males 53 55.2
Survival Born alive 91 94.8
Stillborn puppies 5 5.2

2.4. Sample collection and handling
Vaginal and oral swabs were taken from the dams on two
separate occasions: in the second half of the pregnancy (between
days 30e45) and immediately before the CS or the VB.
2.4.1. Vaginal swabs
Vaginal swabs were taken after cleaning the vulva with a mild
chlorhexidine solution (Skinsept mucosa dermal solution; Ecolab;
Saint Paul; Minnesota). The labia were gently opened using sterile
gloves, and a speculum was inserted into the vestibulum vaginae. A
sterile swab (Deltalab; Rubi; Spain) was then carefully inserted into
the vagina in a craniodorsal direction and gently rotated for 30 s.
2.4.2. Oral samples
Oral samples were taken from the buccal mucosa of the oral
cavity. The lips were lifted, and the mucosa was swabbed a few
times. Care was taken to avoid the teeth.
2.4.3. Placental samples
In puppies born by VB, placental swabs were taken when the
third stage of labor followed immediately after the birth of the
puppy. Samples were taken with a sterile swab from the side of the
placenta facing the chorioallantoic sac. In the CS group, sterile
placental swabs were taken immediately after placenta removal;
the side of the placenta facing the chorioallantoic sac was sampled.
2.4.4. Meconium samples
Circular movements with sterile cotton swabs soaked in warm
sterile water were performed around the perineum region of the
newborn puppies immediately after first colostrum intake to
stimulate defecation. To prevent contamination of the meconium
samples with maternal oral microflora, dams were not allowed to
lick their puppies before meconium sampling. Meconium samples
were taken with a sterile swab immediately after defecation.
2.4.5. Colostrum samples
Nipples were disinfected with propan-2-ol and benzalkonium
chloride before sampling. Samples were taken from the caudal pair
of nipples due to the increased production of colostrum in this part
of the mammary gland.
Colostrum samples were taken immediately after all of the
puppies were born and 4 h after the parturition in the VB group. In
the CS group, colostrum samples were taken immediately after the
surgical procedure and 4 h after the procedure.
Nipples were wiped with a sterile ringer lactate after sampling
to clean them before allowing the puppies to suckle.
2.4.6. Bacteriological examination
All samples were collected in a sterile way under field condi-
tions and then immediately inoculated onto nutrient agar plates
(Oxoid, UK) supplemented with 5% of sheep blood. Each sample
was inoculated onto two separate plates, one of which was incu-
bated in aerobic conditions and the other in anaerobic conditions
(GENbag, GENbox; bioMe rieux, Inc.; Durham; North Carolina). The
cultures were incubated at 37
C for two to five days.
2.4.7. Plate evaluation and bacterial identification by mass
spectrometry
After incubation, the plates were checked for microbial growth,
and all morphological types of the colonies were subcultured to
obtain a pure culture. Aerobic and anaerobic subcultures were

Table 2
Number and proportion (%) of different breeds included in the study.

Breed size Breed Number and proportion of Number and proportion of Number and proportion of female Number and proportion of male
dams [%] puppies [%] puppies [%] puppies [%]

Miniature Schnauzer 1 (5.0%) 5 (5.2%) 2 (4.7%) 3 (5.7%)

Yorkshire Terrier 1 (5.0%) 4 (4.2%) 1 (2.3%) 3 (5.7%)
Jack Russell Terrier 1 (5.0%) 4 (4.2%) 1 (2.3%) 3 (5.7%)
Pomeranian 1 (5.0%) 3 (3.1%) 1 (2.3%) 2 (3.8%)
Miniature Poodle 1 (5.0%) 2 (2.1%) 1 (2.3%) 1 (1.9%)
Boston Terrier 4 (20.0%) 16 (16.7%) 11 (25.6%) 5 (9.4%)
Whippet 1 (5.0%) 7 (7.3%) 4 (9.3%) 3 (5.7%)
Total small breeds 10 (50.0%) 41 (42.7%) 21 (48.8%) 20 (37.7%)
Pembroke Welsh 3 (15.0%) 17 (17.7%) 7 (16.3%) 10 (18.9%)
Corgi
English Bulldog 1 (5.0%) 4 (4.2%) 2 (4.7%) 2 (3.8%)
French Bulldog 2 (10.0%) 13 (13.5%) 5 (11.6%) 8 (15.1%)
Total medium to 6 (30.0%) 34 (35.4%) 14 (32.6%) 20 (37.7%)
large breeds
Greater Swiss 1 (5.0%) 6 (6.3%) 1 (2.3%) 5 (9.4%)
Mountain Dog
Newfoundland 1 (5.0%) 9 (9.4%) 4 (9.3%) 5 (9.4%)
Labrador Retriever 1 (5.0%) 4 (4.2%) 2 (4.7%) 2 (3.8%)
German Shepherd 1 (5.0%) 2 (2.1%) 1 (2.3%) 1 (1.9%)
Total giant breeds 4 (20.0%) 21 (21.9%) 8 (18.6%) 13 (24.5%)
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Table 3
Bacterial species isolated from the oral and vaginal mucosa of the dams and the placenta and meconium of the puppies.

Gram positive bacteria Oral mucosa Vaginal mucosa Placenta Meconium

Staphylococcus
S. epidermidis þ þ þ þ
S. pseudintermedius þ þ þ
S. hominis þ þ
S. aureus þ þ þ
S. haemolyticus þ þ þ
S. warneri þ þ þ
S. hominis þ þ
S. capitis þ
S. lugdunensis þ
S. sanguinis þ
S. pettenkoferi þ
S. caprae þ
S. auricularis þ
S. saccharolyticus þ
S. arlettae þ þ
Staphylococcus spp.

Streptococcus
S. minor þ
S. salivarius þ þ þ
S. hyovaginalis þ
S. oralis þ þ
S. pluranimalium þ þ þ þ
S. pyogenes þ
S. canis þ
S. equi subsp. Zooepidemicus þ
S. mitis þ þ
S. sanguinis þ
S. parasanguinis þ
S. pneumoniae þ
Streptococcus spp. þ þ þ

Enterococcus
E. faecalis þ þ
E. canintestini þ

Actinomyces
A. canis þ þ
A. oris þ
A. odontolyticum þ

Arthrobacter spp. þ þ þ

Corynebacterium
C. xerosis þ
Corynebacterium spp. þ þ

Canibacter oris þ

Rothia
R. nasimurium þ þ
R. dentocariosa þ
R. mucilaginosa þ

Lactobacillus
L. lactis
L. johnsonii þ
Lactobacillus spp. þ þ þ

Micrococcus
M. lylae þ
M. caseolyticus þ

Cutibacterium (Propionibacterium)
C. acnes þ þ þ
C. granulosum þ

Eubacterium tenue þ

Rhodococcus rhodochrous þ

Clostridium
C. perfringens þ
Clostriridum spp. þ

Bacillus
B. cereus þ
B. licheniformis þ
B. flexus þ
B. megaterium þ
B. pumilus þ
(continued on next page)
Table 3 (continued )

Gram positive bacteria Oral mucosa Vaginal mucosa Placenta Meconium

B. thuringiensis þ
B. simplex þ þ
Bacillus spp.

Arcanobacterium canis þ

Kocuria
K. kristinae þ
Kocuria spp. þ þ

Fingoldia magna þ þ

Streptomyces spp. þ

Lactococcus lactis þ

Gram negative bacteria

Neisseria
N. weaveri þ þ
N. zoodegmatis þ þ þ
N. canis þ
N. meningitidis þ
N. animaloris þ

Pasteurella
P. canis þ þ
P. dagmatis þ þ
P. stomatis þ þ
P. multocida þ þ
Pasteurella spp. þ þ þ

Haemophilus
H. haemoglobinophilus þ þ þ
H. sputorum þ þ
Haemophilus spp. þ þ þ

Pseudomonas
P. aeruginosa þ þ
P. stutzeri þ

Moraxella
M. canis þ
M. olsloensis þ þ
Moraxella spp. þ þ

Prevotella heparinolytica þ

Bacteroides pyogenes þ

Bergeyella zoohelcum þ þ

Streptobacillus
S. moniliformis þ
Streptobacillus spp. þ

Acinetobacter
A. schindleri þ
A. junni
A .lwoffii

Klebsiella
K. pneumoniae þ
K. oxytoca þ
Klebsiella spp.

Paracoccus versutus þ

Escherichia coli þ þ þ

Pantoea.
P. calida þ
Pantoea spp. þ

Brevundimonas aurantiaca þ

Exceptionsa

Mycoplasma
M. canis þ þ
Mycoplasma spp. þ

Gardnerella
G. vaginalis þ
a
In addition to gram-positive and gram-negative bacteria, we also isolated bacteria from two genera that, due to their special morphological characteristics, could not be
classified into the first two groups. These bacteria belong to the genus Mycoplasma (due to the absence of the cell wall, they do not have the ability of Gram stain) and to the
genus Gardnerella (gram-variable).
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sek Pipan et al. / Theriogenology 152 (2020) 18e28 23

Table 4
The proportion of births in which we isolated the same bacterial species from the mothers and their puppies.

BACTERIAL MATCHING - DIRECTION AND PROPORTION CS (%) VB (%)

EL-CS (%) EM-CS (%)

vaginal mucosa / placenta 11.1 33.3 40

vaginal mucosa / meconium 55.5 0 80
oral mucosa / placenta 22.2 33.3 40
oral mucosa / meconium 33.3 16.6 80

incubated at 37
C for another 24 and 48 h, respectively. Bacterial
colonies were determined by matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry (MALDI-TOF MS)
(Microflex LT system; Bruker Daltonics, USA) employing MALDI
Biotyper 3.1 software (Bruker Daltonics) according to the manu-
facturer’s instructions. A small amount of bacterial colony was
transferred as a thin film directly onto an MSP 96 polished steel BC
target and left for approximately 1 min to dry at room temperature.
The sample was then overlaid with 1 ml of HCCA (a-cyano-4-
hydroxycinnamic acid) matrix solution (Bruker Daltonics) and
subjected to MALDI-TOF MS analysis. The process was performed
by a direct-transfer method. The confidence scores for successful
identification were 2.0 for the species level and 1.7 for the genus
level according to the manufacturer’s database. The extended
direct-transfer method with 70% formic acid was performed when
isolates with a score cut-off values below 1.7 were obtained. Iso-
lated colonies were further examined.
2.4.8. Growth rate of newborn puppies
The growth rate of the puppies was followed until they were
eight weeks old. The body weight of the puppies was measured
each morning in the first week. Later, the body weight was taken
once weekly in a 7-day interval. The day of the parturition was
determined as Day 0.
The relative growth was calculated as the relative change in the
body weight compared to the puppy’s initial birth weight.
2.4.9. Statistical analysis
The data are presented as the means ± standard deviation (SD)
and median. Normal distribution of the data was tested by the
Shapiro-Wilk test. In cases of normal distribution, parametric tests
were performed, and in cases of non-normally distributed data,
non-parametric tests were performed.
The statistical analyses were performed using the Statistical
programming language R 3.5.0 (http://www.R-project.org), and
P < 0.05 was considered significant.
The differences in the relative weight gain between puppies
born by CS and puppies born by VB were tested by the parametric t-
test for independent samples. The same test was used to investigate
the association between the puppies’ weight gain and whether
they were born with sterile meconium/placenta or bacterial cul-
tures present in the meconium or in the placenta.
3. Results
3.1. Basic information
Samples were collected from December 2016 to July 2018.
During this time, 96 puppies born to 17 dams (3 dams whelped
twice: two Boston Terriers and one Welsh Corgi) were included in
our research. The mean age of the dams was 4.8 ± 1.6 years (range:
1.8e7 years). Regarding the type of birth, there were five VBs, nine
EL-CSs and six EM-CSs. The stillborn puppies (n ¼ 5) were excluded
from the statistical data analysis. Three stillborn puppies were born
by EM-CS and 2 by EL-CS. The data are presented in Table 1.
The dams were of 14 different breeds and are presented in
Table 2.
3.1.1. Isolated bacteria
3.1.1.1. Basic information on the isolated bacteria. In the second half
of the pregnancy, swabs of the oral and vaginal mucosa were not
acquired in 40% of the dams because the owners decided to
participate in our research just before the VB or EM-CS was per-
formed. Placental samples from 12 puppies were not collected
because the dams ate the placenta before we could stop them.
Meconium samples from five puppies were not obtained. Bacterial
species isolated from the oral and vaginal mucosa of the dams are
presented in Table 3. The bacterial species that were isolated from
the placenta and the meconium of the puppies are presented in the
same table.
3.1.1.2. Bacteria isolated from samples of meconium and placenta.
Placenta samples were collected from 79 (82.3%) puppies. We were
not able to obtain placentas from all of the puppies in the vaginal
deliveries due to a variety of causes (i.e., unable to identify to which
puppy the placenta belonged, the dam ate or licked the placenta

Table 5
Proportion of puppies in which the same bacterial species were isolated from samples of oral mucosa and/or vaginal mucosa and samples of meconium and/or placentas and
whether the same bacterial species were isolated from the oral and vaginal mucosa in the dams.

VAGINAL BIRTH CESAREAN SECTION

EL- CS EM- CS

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

VM-PL 40 0 57 0 0 50 0 0 0 0 0 0 0 0 0 0 0 0 11 11
VM-M 100 0 14.3 16.6 25 50 10 0 33.3 0 28.5 0 0 10 0 0 0 0 0 0
OM-PL 0 25 14.2 0 0 0 0 25 0 0 0 0 25 0 0 0 0 0 11 11
OM-M 0 75 71 33.3 75 0 10 75 0 0 0 0 50 0 16.6 0 0 0 0 0
VM-OM ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ X X ✓ ✓ X X ✓ X X ✓ ✓ ✓

Legend: N ¼ parturition number, VM ¼ vaginal mucosa, OM ¼ oral mucosa, PL ¼ placenta, M ¼ meconium, ✓- the same bacterial species were isolated from the vaginal and
oral mucosa in the dam, X - the same bacterial species were not isolated from the vaginal and oral mucosa in the dam.
24 M. Zako
sek Pipan et al. / Theriogenology 152 (2020) 18e28

Fig. 1. The puppies’ relative growth rate in the 1st week (the lines represent weight
gain average according to parturition type).

Fig. 3. A comparison of the relative weight increase on the 3rd day in puppies born by
before sampling). No bacteria were isolated from 34 (43%) of the caesarian section and vaginal parturition (p < 0.01).
placental samples. Meconium samples were obtained from 86
puppies. No bacteria were isolated from 12 (13.5%) of the meco-
nium samples. The same bacterial species were isolated from the oral mucosa
samples and from the placental samples of the puppies in 30% of
3.1.1.3. Bacteria isolated from oral and vaginal mucosa samples. the parturitions, and in 25% of the parturitions, the same bacterial
species were isolated from the vaginal mucosa samples of the dam
The bacteria isolated from the mothers’ oral mucosa most often
belonged to the genera Neisseria (13.85%), Streptococcus (13.85%), and placental samples of the puppies. The same bacterial species
were isolated in the oral mucosa samples of the dam and in the
Pasteurella (10.77%), Haemophilus (6.15%), Staphylococcus (6.15%),
meconium samples of the puppies in 40% of the parturitions. In 45%
Actinomyces (5.13%) and Moraxella (4.62%).
of the parturitions, the same bacterial species were isolated from
The bacteria isolated from mothers’ vaginal mucosa most often
the vaginal mucosa samples of the mother and meconium samples
belonged to genera Staphylococcus (22.41%), Streptococcus (15.52%),
of the puppies. The proportion of births in which we isolated the
Haemophilus (10.34%), Enterococcus (8.62%), Escherichia (4.31%) and
same bacterial species from the mothers and their puppies is pre-
Neisseria (4.31%).
sented in Table 4.
The meconium microbiota of vaginally born puppies was mostly
3.1.2. Comparison between dam and puppy microbiota
colonized by bacteria from the maternal vagina. However, the
Major overlapping of the isolated bacterial species was found in
meconium microbiota of puppies born by CS indicated a relative
66.6% of the CS puppies and in all of the VB puppies (100%) when
resemblance to the maternal oral and vaginal microbiota (Table 5).
comparing dam and puppy microbiota.

Fig. 2. A comparison of the relative weight increase on the 2nd day in puppies born by Fig. 4. A comparison of the relative weight increase on the 4th day in puppies born by
caesarian section and vaginal parturition (p < 0.01). caesarian section and vaginal parturition (p < 0.01).
 M. Zako
sek Pipan et al. / Theriogenology 152 (2020) 18e28
Fig. 5. A comparison of the relative weight increase on the 3rd and 4th days in puppies that had their own bacterial microbiota in the meconium at birth and puppies with sterile
meconium (p < 0.05).
Fig. 6. A comparison of the relative weight increase on the 3rd and 4th days in puppies
that had their own bacterial microbiota in the meconium at birth and puppies with
sterile meconium (p < 0.05).
The same bacterial species were isolated from the vaginal mu-
cosa samples and the colostrum samples in 30% of the parturitions.
In 20% of parturitions, the same bacterial species were isolated from
the oral mucosa samples and the colostrum samples.
3.1.2.1. The correlation of the dams’ oral mucosa and meconium with
the placenta of the puppies. Bacteria belonging to 11 different
genera were isolated from the samples that were collected from the
dams’ oral mucosa and from the placenta or the meconium of the
puppies from the same birth. These genera were Staphylococcus,
Streptococcus, Cutibacterium, Moraxella, Neisseria, Lactobacillus,
Haemophilus, Actinomyces, Arthrobacter, Klebsiella and Bergeyella.
3.1.2.2. The correlation of the dams’ vaginal mucosa and meconium
with the placenta of the puppies. Bacteria belonging to 4 different
genera were isolated from samples collected from dams’ vaginal
mucosa and from the placenta or meconium of the puppies from
the same birth. These genera were Staphylococcus, Streptococcus,
25
Fig. 7. A comparison of the relative weight increase on the 2nd day in puppies that had
their own bacterial microbiota in the placenta at birth and puppies with placenta
without microbiota (p < 0.05).
Haemophilus and Cutibacterium.
3.1.2.3. Prevalent bacterial genera isolated from placental and
meconium samples. Fifty-nine different bacterial species belonging
to 23 different genera were isolated from meconium samples, and
29 different bacterial species belonging to 15 different genera were
isolated from placental samples.
Bacterial colonies were isolated from 86.5% of the meconium
samples that we collected and mainly belonged to the genera
Staphylococcus (49.2%), Streptococcus (7.25%), Cutibacterium (7.73%),
Neisseria (5.32%), Bacillus (3.86%), Pasteurella (2.42%), Rothia (1.45%),
Acinetobacter (1.45%) and Haemophilus (1.45%). The prevalent bac-
terial species isolated from the meconium samples were S. warneri,
S. epidermidis, S. pseudintermedius, C. acnes and N. zoodegmatis.
Individual bacterial colonies were isolated from 57% of the
placenta samples and mainly belonged to the genera Staphylococcus
(43.75%), Streptococcus (10.0%), Cutibacterium (8.75%), Lactobacillus
(5.0%), Bacillus (2.5%) and Pasteurella (2.5%).
26 M. Zako
sek Pipan et al. / Theriogenology 152 (2020) 18e28
3.1.3. Relative growth rate
Puppies born by VB had the highest relative growth rate, and
puppies born by EL-CS had the lowest relative growth rate (Fig. 1).
Puppies born by VB had a significantly better relative growth
rate compared to the CS group on the second (p < 0.01) (Fig. 2),
third (p < 0.05) (Fig. 3) and fourth day (p < 0.05) (Fig. 4). The growth
rate of the puppies was followed until they were eight weeks old,
but a statistically significant difference in the relative growth rate
between puppies born by VB and the CS was not observed later in
their lives (p > 0.05).
3.1.4. Puppy survival in the first weeks of life
The survival rate of the puppies in the first eight weeks of their
lives was 96.7%, not including the stillborn puppies. Within the first
week after parturition, 3.3% of the puppies died.
3.1.5. Relative growth and presence of bacteria
3.1.5.1. The comparison of the relative growth rate between puppies
that had meconium without a microbiota and puppies that had
bacteria in their meconium. Puppies born without a culturable
bacterial microbiota in their meconium had a slower growth rate
than puppies with a culturable bacterial microbiota in their
meconium. Even though there was no statistically significant dif-
ference on the first and the second day of life (p > 0.05), there was a
statistically significant difference in the growth rate on the third
(p < 0.05) (Fig. 5) and fourth day (p < 0.05) (Fig. 6). A difference in
the growth rate was also evident on the fifth and sixth days, but it
was on the verge of statistical significance (p ¼ 0.06; p ¼ 0.07). After
this period, there was no statistically significant difference in the
growth rate between puppies with and without a culturable
meconium microbiota present at birth (p > 0.05).
3.1.5.2. The comparison of the relative growth rate between puppies
that had placenta without a microbiota and puppies that had bacteria
in their placenta. Puppies born without a culturable microbiota in
the placenta had a slower growth rate in the first days after
parturition than puppies with a culturable bacterial microbiota in
their placentas. We could not detect a statistically significant dif-
ference on the first day after parturition (p > 0.05). On the second
day, the growth rate of the puppies with bacteria isolated from their
placentas had a significantly (p < 0.05) higher growth rate (Fig. 7).
From the second day on, there was no statistically significant dif-
ference in the growth rate between the groups (p > 0.05).
4. Discussion
The present study confirms that the placenta and meconium of
newborn puppies are not always sterile and may have their own
bacterial microbiota. Neisseria zoodegmatis, Actinomyces canis and
Streptococcus spp. were the predominant bacteria in the oral cavity
of the dam, and Staphylococcus spp. and Streptococcus spp. were
predominant bacteria in the vagina. Similar to the oral cavity and
the vagina, Staphylococcus spp., Streptococcus spp. and Neisseria
zoodegmatis were the most common bacteria isolated from the
placenta and meconium. Some of these results are in accordance
with earlier studies, where a similar pattern has been described,
and the predominant bacteria of 7-week-old German Shepherd
puppies were the same as in the pregnant and lactating dams [30].
Previous research suggests that there is a large animal to animal
variation in the microbiome [23] and that the variability of the
puppies’ microbiota depends on the breed, age, diet and environ-
ment. Moreover, Hand et al. [31] described that closely related dogs
had a more similar microbiota profile compared to unrelated dogs
within the same breed [31]. In dogs, data on vertical and horizontal
transmission of bacteria and their transition into the oral cavity and
colonization of the oral cavity are lacking. It is known that
S. pseudintermedius clones are transmitted from the dam to the
puppies around birth and may persist in the offspring for long
periods of time [32]. Similarly, S. pseudintermedius can be isolated
from puppies within 8 h of birth, with the mouth being colonized in
78% of cases [33]. In the present study, S. pseudintermedius was
isolated from the placenta and meconium of the puppies, as well as
from the mouth and vagina of the dams. Since this bacterium was
also isolated from placental samples from the puppies born by CS,
intrauterine transmission is plausible. In the present study, how-
ever, no bacteria were isolated from 43% of the placental samples
taken from the side of the placenta facing the chorioallantoic sac. In
humans, it was suggested that the maternal basal plate of the
placenta may serve as a protective environment for the placental
microbes, and most of the microorganisms were isolated from that
part [34]. Since we did not strive to take placental samples from the
maternal basal plate, this could be the reason why no bacteria were
isolated in 43% of the placental samples.
Our findings suggest that the colonization pattern of the
puppies’ microbiome at birth was associated with the type of de-
livery. In 66.6% of the CS deliveries and in 100% of the VB deliveries,
the same bacterial isolates were found in the puppies and their
mothers. In 40% of all the deliveries, the same bacterial species
were present in the mothers’ oral microbiota and their puppies’
meconium, while in 45% of the deliveries, the same bacteria were
present in dams’ vaginal mucosa and meconium. Until recently,
meconium in humans was considered to be sterile [35]. However,
several studies have shown that bacteria are present in the meco-
nium of infants [11,36]. In dogs, this is the first study in which the
meconium of puppies was examined for the presence of a bacterial
microbiota. The meconium microbiota of newborn puppies deliv-
ered by CS slightly differed from those delivered vaginally. While
the meconium microbiota of VB puppies mostly contained bacteria
from the maternal vagina, the meconium microbiota of CS puppies
showed a relative resemblance to the vaginal microbiota but also
contained some bacteria from maternal oral microbiota. In humans,
infant meconium shares more than 0% of bacteria with the
maternal oral microbiota. The same study also reported that
meconium microbiota had 58% bacterial similarity with the vaginal
samples from the mother regardless of the delivery mode [37]. In
our study, the predominant bacteria isolated in meconium were the
same regardless of the type of delivery, but there were more
potentially pathological bacteria found in the meconium of CS
puppies. Moreover, puppies delivered by CS had lower total
meconium microbiota diversity compared to vaginally delivered
puppies. The same observation was found in most human studies
that were reviewed by Rutayisire et al. [38]. This could be explained
by the normal colonization of the neonate meconium during
vaginal birth. However, despite the careful sampling of the meco-
nium in puppies (i.e., cleaning of the anal area before sampling and
wearing sterile gloves), bacterial contamination of the meconium
samples with vaginal microflora in the VB puppies cannot be
completely excluded. The effect of the delivery mode on the
meconium microbiota is still not fully explained in animals and
humans, since there are contradictory conclusions between
different studies [15,39]. The observed controversies might be the
result of a small sample size, sample timing, sample collection and
storage, different techniques of bacterial culturing and DNA sam-
pling methods, experimental contamination and other factors.
Additionally, in our study, puppies were allowed to have colostrum
before sampling. Since we collected meconium immediately after
colostrum intake, meconium contamination with colostrum bac-
teria is possible but less likely.
On the other hand, most of the studies are in agreement that the
mode of delivery has a great impact on infants’ gut microbiome in
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sek Pipan et al. / Theriogenology 152 (2020) 18e28
the first few months of life [40,41]. Biasucci et al. [41] reported that
the delivery mode in newborn infants is important for gut devel-
opment and that infants delivered by CS were found to have an
unusual gut microbiota. Recently, a clear association between
mother and infant gut colonization by bifidobacteria was observed
[40]. Unfortunately, the gut microbiota of the dams was not the
subject of our study, and this should be evaluated in further studies.
In puppies born vaginally, the relative weight gains on days 2, 3
and 4 were higher compared to the puppies born with CS. While
this could be the effect of the puppies’ maturity at birth, the better
weight gain could also be the result of the observed increased
meconium microbiota diversity in vaginally delivered puppies and,
consequently, better nutrition uptake from the gut.
Interesting results were obtained in our study when looking at
puppies’ microbiota and their weight gain in the first days of life.
Namely, puppies with a bacterial microbiome in the meconium or
placenta gained more weight than puppies born with a sterile
meconium or placenta. The weight gain difference became signif-
icant on the 3rd and 4th days. Even though the difference was not
significant on day one, from a clinical point of view, puppies with a
sterile placenta lost an average of 1.9% of their body weight, while
those with a placental microbiota gained an average of 0.06% of
their body weight. This is important since canine perinatal mor-
tality is known to be relatively high in the first week of life [42]. In
our study, the mortality rate of puppies in their first week of life
was 3.3%, which is lower than that reported in the literature. This
could be explained by the constant monitoring of the dams and
puppies during whelping and in the first week after parturition.
The puzzling question is why were we able to isolate bacteria
from the placenta and/or meconium of some puppies but not
others? This could be influenced by many factors, and the placenta
unit itself is one of them. Endotheliochorial placenta in dogs is less
permeable to immunoglobulins, and individual variabilities in the
placental structure might play a role in the intrauterine bacterial
colonization of the canine fetus. Researchers in human medicine
have noted that microbial colonization that begins in utero is
largely influenced by the maternal microbiome. Additional colo-
nization occurs during the birthing process, with differing micro-
bial compositions noted at various neonatal body sites based on the
method of delivery [43]. It is also known that maternal health has
an influence on offspring gut microbial colonization [44], and
treating women with vancomycin during pregnancy had a signifi-
cant negative impact on neonates’ intestinal microbiota at birth
[45]. Despite the normal clinical examination and normal blood-
work results in all the dams included in our study, subclinical al-
terations in their health status, potentially affecting the
transmission of a microbiome to the puppies, cannot be completely
ruled out.
The findings of the present study provide us with a better un-
derstanding of the canine microbiota immediately after parturition.
Specifically, it describes the meconium microbiota of newborn
puppies and evaluates the effect that different types of parturition
have on immediate postpartum puppy growth. According to the
results, we can conclude that there is early colonization of the fetus
with bacteria, and the placenta and meconium are not always
sterile, as previously suggested. Likewise, it seems that the micro-
biota of newborn puppies depends on the mode of delivery.
Moreover, the mode of delivery and the bacterial colonization of
the placenta or meconium affected early weight gain in puppies but
did not affect their survival. This study provided us with a large
amount of data that are useful for further research on the rela-
tionship between the beneficial and detrimental microbiome in-
fluences at birth and their roles during pregnancy, parturition and
in early development of puppies, since microbial interactions dur-
ing these times may have clinical relevance in the growth and
27
survival of newborn puppies. In addition, it should be borne in
mind that in our research, we investigated only bacteria that could
be cultivated by conventional microbiological conditions. Other
bacteria that could be of importance for normal puppy develop-
ment were potentially missed. Another weakness of this study is
the fact that smaller and brachiocephalic breeds represented the
majority of puppies in the CS group, while there were larger breeds
in the VB group. Although data on microbiota in different breeds of
dogs are lacking, this variability, if present, could have an impact on
the microbiome of newborn puppies.
Continued research into how the microbiome is developed in
puppies and what factors influence its transmission is essential.
Ideally, puppies would be born via vaginal birth. However, this is
not always feasible. Likewise, further insight into the relationships
between the puppies’ microbiome and healthy development in the
postpartum period is needed, since we might be able to modulate
the early gut microbiome with particular beneficial bacterial spe-
cies and potentially minimize the occurrence of fading puppy
syndrome.
Funding
The authors acknowledge the financial support received from
the Slovenian Research Agency (research core funding No. P4-0092,
No. P4-0053 and project No. J4-8224).
Declaration of Competing interest
The authors declare that they have no competing interests.
CRediT authorship contribution statement
Maja Zako
sek Pipan: Conceptualization, Investigation, Meth-
odology, Writing - original draft. Leonida Kajdi
c: Writing - review
& editing, Investigation, Methodology, Project administration, Re-
sources. Anja Kalin: Writing - review & editing, Investigation,
Methodology, Project administration, Resources. Tanja Plavec:
Funding acquisition, Formal analysis, Investigation, Methodology,
Writing - review & editing. Irena Zdovc: Data curation, Funding
acquisition, Supervision, Validation, Writing - review & editing.
Acknowledgements
The authors thank Barbara Lukanc PhD for performing anes-
thesiology for dogs going through cesarean section and to the dog
owners for participating in the study. The authors also thank Maja
Lepen and Olga Kosar for their invaluable help with bacteriological
tests.
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