Veterinarni Medicina, 60, 2015 (11): 613–620

Original Paper

doi: 10.17221/8529-VETMED

Incidence of bacterial pathogens in equine uterine

swabs, their antibiotic resistance patterns, and selected
reproductive indices in English thoroughbred mares
during the foal heat cycle
T. Benko, M. Boldizar, F. Novotny, V. Hura, I. Valocky, K. Dudrikova,
M. Karamanova, V. Petrovic

Equine Clinic, University of Veterinary Medicine and Pharmacy, Kosice, Slovak Republic

ABSTRACT: Fertility problems of mares on a well-managed breeding farm with thoroughbred stallions have been
ascribed mostly to contamination of the reproductive apparatus of females with pathogens, particularly those of
bacterial origin. This study presents a summary of the frequency of bacterial pathogens isolated from 437 cervical
swabs which were collected from English thoroughbred mares intended for mating between 2008–2014, as well as of
resistance tests of these pathogens to seven commonly used antibiotics as follows: penicillin, gentamicin, tetracycline,
sulfisoxazole, cefotaxime, marbofloxacin and enrofloxacin. In addition, the study reports the changes in the levels of
plasma oestradiol and progesterone determined two to three days before and two to three days after the first post-
partum ovulation in mares with positive and dubious bacteriological findings and percentage of barren mares and
mares that conceived at first, second and third post-partum ovulations. It was observed that 21.5% of mares were
barren even after the third post-partum cycles. The oestradiol levels determined two to three days before the first
post-partum ovulation were significantly lower in mares positive for pathogenic microflora in their reproductive
apparatus compared to mares with the dubious findings (25.1 ± 5.8 pg/ml vs. 69.7 ± 18.3 pg/ml; P < 0.05), while the
mean progesterone levels did not differ significantly but displayed a rather wide range in positive mares (from 0.08
to 1.38 ng/ml) compared to dubious mares with only small variations (0.12 ± 0.03 ng/ml). Moreover, of the total
number of cervical swabs taken shortly before the first post-partum oestrus from all the mares intended for mating
as many as 69.7% were contaminated with pathogenic microflora (positive findings). Saprophytic microorganisms
only (the dubious findings) were isolated from 29.7% of swabs. From the 307 positive swabs, we could identify 40.4%
positive for β-haemolytic streptococci and 20.4% positive for Escherichia coli, the pathogens implicated in causing
reproductive disorders. Tests of antibiotic resistance of the investigated pathogens revealed that both Gram-positive
and Gram-negative bacteria showed high susceptibility to antibiotics such as cefotaxime, marbofloxacin and enro-
floxacin. On the other hand, both these bacterial groups showed high resistance to routinely used broad-spectrum
antibiotics, such as penicillin and tetracycline. Because further research is required for a full understanding of the
mechanism of pathogenesis of post-breeding endometritis, we can only hypothesise that uterine contamination with
pathogenic microflora, particularly with β-haemolytic streptococci and coliform bacteria, diagnosed before the first
post-partum ovulation, could negatively affect the hormonal regulation of oestrus and result in mare fertility problems.

Keywords: equine; endometritis; antibiotics; progesterone; oestrogen

One of the most serious problems encountered bacterial origin. These pathogens are introduced
in the reproduction of racehorses, associated with into the reproductive organs in several ways: (a)
mare fertility problems, is contamination of the most frequently soon after the parturition when
reproductive apparatus of these animals with an opened cervix allows the microorganisms pass
pathogenic microorganisms, particularly those of into the uterus from the vagina; moreover, retained
Supported by the Grant Agency for Science of the Slovak Republic, VEGA (Grant No. 1/0366/15).

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doi: 10.17221/8529-VETMED

lochia (amniotic fluid, portions of placenta) is a suit- ering of English thoroughbred mares. The health of
able substrate for multiplication of pathogens; (b) stallions is checked regularly and they are subjected
during oestrus, when the uterus may be contami- to serological testing for infectious equine anaemia
nated with smegma from the preputial sac of stal- and infectious metritis annually, before the onset
lions during natural mating of thoroughbreds. For of the mating season. On the other hand, it is dif-
example, LeBlanc (2003) reported that healthy mares ficult to obtain information on the health status of
can clear any excess fluid from the uterus within 8 h mares brought to the stud farm as they come from
following coitus while sub-fertile mares may retain various private farms. Therefore, after arrival at the
uterine fluid for more than two days after the cover- stud farm, the urogenital apparatus of each mare
ing. Unfortunately, the anatomy but also the physiol- should be regularly examined, as mentioned later.
ogy of the reproductive tract of mares delays uterine A cervical swab is taken and examined micro-
clearance and may lead to the development of endo- biologically from each mare intended for mating
metritis. Numerous studies performed in horses have at six to seven days post-partum and the entire
reported that the most common bacterial pathogens reproductive apparatus of the animal is subjected
isolated from uterine swabs include β-haemolytic to rectal and ultrasonographic examination in or-
streptococci, Escherichia coli, Staphylococcus au- der to detect the first approaching post-partum
reus, Klebsiella spp., Pseudomonas spp. and others oestrus by observation of the following signs: re-
(Dhingra and Sandhu 1987; Ricketts and Mackintosh laxed cervix with mild uterine tonus, oedematous
1987; Fodor et al. 1995; Langoni et al. 1997; LeBlanc endometrium, dominant ovarian follicle (diameter
1999; Albihn et al. 2003; Szeredi et al. 2003; Frontoso ≥ 30 mm), absence of uterine content (no cavity). If
et al. 2008). Infection of the reproductive tract with all these criteria are met the “healthy” mare is bred
these agents may result in endometritis and infertility. by one mount of the desired stallion. When, on the
For this reason it is very important for veterinarians to next day, the presence of the ovulation stigma is
detect mares positive for uterine contamination and not confirmed by ultrasonographic examination,
to initiate their antibiotic therapy. Because effective the mare is bred again every other day until the
antibiotic therapy should be based on microbiological ovulation is confirmed (but maximally up to three
cultivation and the subsequent testing of antibiotic mounts). In the case of retained uterine fluid the
resistance of relevant pathogens – a time-consuming following procedure is used: (a) if the cavity with
process even when performed in certified microbio- fluid is less than 2 cm in diameter, the mare is ad-
logical laboratories – veterinarians frequently choose ministered oxytocin i.m. at a dose of 10−20 IU;
a simple but flawed approach involving administra- in principle, if the fluid is still present after 6 h,
tion of broad-spectrum antibiotics and consider only oxytocin is administered again and the uterus is
the results of available relevant studies and their ex- examined after an additional 6 h; repeated posi-
periences. The careless preventive administration of tive finding indicates the 3rd (last) administration
“first-choice antibiotics”, practiced particularly be- of oxytocin; (b) if the cavity with more than 2 cm
tween the seventies and nineties of the past century, diameter persists, uterine lavage with saline so-
resulted in the development of resistance to broad- lution is performed and oxytocin is administered
spectrum antibiotics, such as penicillin and tetracy- immediately after using the procedure described
cline, in many microorganisms. previously. In general, the last administration of
The above-mentioned explains why a study of this oxytocin should take place within 48 h after ovu-
character is very important for the strategy of use of lation. Natural covering of a mare which had ex-
antibiotics in equine veterinary practice. This is par- cess fluid in the uterus is arranged according to
ticularly so because only a limited body of informa- a schedule described for a healthy mare but the
tion is available in this area and the national control natural cover must take place minimally 6 h before
programs focus only on food-producing animals. the therapeutic intervention.
All bacterial pathogens isolated from cervical
swabs were subjected to antibiotic resistance test-
MATERIAL AND METHODS ing and only on the basis of results (obtained no
later than on Day 3 following the sampling) effec-
The examined stud farm raises only English thor- tive antibiotic therapy of mares with persisting
oughbred stallions which are used for natural cov- positive uterine pathological findings was initiated.

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doi: 10.17221/8529-VETMED

Levels of oestradiol and progesterone were deter- The examined stud farm evaluated the success of
mined in plasma obtained by centrifugation of venous natural mating using ultrasonographic examination
blood withdrawn from v. jugularis into vacuum tubes on Days 15 (diagnosis of twins in the uterus), 27, 42
(Vacutest 5 ml sterile tube). Both oestradiol E2 and and 48 post-ovulation. Findings were confirmed us-
progesterone were determined using available com- ing ultrasonography. If the ultrasonographic exami-
mercial kits (Oestradiol E2 Enzyme Immunoassay nation performed on Day 15 post-ovulation failed
Kit and Progesterone Enzyme Immunoassay Test to confirm pregnancy, the mare was administered a
Kit) using the Enzyme Immunoassay method. synthetic analogue of prostaglandin F2α at a dose of
Before collecting the cervical swabs, the external 1 ml pro toto, and after three to five days examined
genitals of mares were rinsed twice with a 0.1% for the signs of oestrus.
solution of iodopovidone and dried. A sterile swab The number of mares that conceived at the first,
with double protection intended for sampling of second and third post-partum ovulation was ob-
biological material from the uterine cervix of mares tained from the evidence of the veterinarian re-
(EquiVet uterine culture swab, Kruuse, Denmark) sponsible for reproduction on the stud farm.
was introduced into the uterine cervix controlled Statistical procedures. The statistical software
by a hand protected by a rectal glove to minimise GraphPad Prism 2004 was used for the statistical
its contamination by vaginal microflora. analyses. The hormonal indexes were analysed us-
After inoculation of plates containing blood agar ing the unpaired Student’s t-test. These results are
and Endo agar with the content of swabs, the plates expressed as mean ± SD.
were incubated under aerobic conditions at 37 °C
for 24 h and observed for haemolytic reaction.
Proteus bacteria were identified on the basis of RESULTS
their characteristic “swarming pattern” of growth.
If staphylococcus colonies appeared on the plates During the investigated period more than one
the incubation lasted 48 h. All plates that did not fifth of all mares (21.5%) showed fertility problems
show haemolysis and contained no colonies char- after natural mating and remained barren even after
acteristic of the potential pathogenic bacteria were the third post-partum oestrus cycle. Of all mares
considered as dubious, containing only common that were pregnant at ultrasonographic examina-
saprophytic microflora. In contrast, each colony tion on Day 48 after the last ovulation, 68.5% con-
of haemolytic or potentially pathogenic bacteria ceived at the first, 24.0% at the second and 7.5% at
was inoculated onto a specific diagnostic agar. the third oestrous cycle after the parturition.
Antibiotic resistance was determined employing The level of oestradiol two to three days before the
Mueller-Hinton agar and in the case of staphylo- first post-partum ovulation was significantly lower
cocci, blood agar. The swabs were sent to an ac- in mares positive for potential pathogens in their
credited microbiological laboratory (Department reproductive apparatus compared to mares with du-
of Microbiology and Immunology, University of bious findings (25.1 ± 5.8 pg/ml vs. 69.7 ± 18.3 pg/ml,
Veterinary Medicine and Pharmacy in Kosice, P < 0.05), while the level of progesterone showed
Slovak Republic) and the results regarding antibi- no significant differences. However, in mares with
otic resistance of bacterial pathogens were obtained positive findings the range of progesterone levels was
on Day 3 following the sampling, or on Day 4 at much wider, from 0.08 up to 1.38 ng/ml, while in the
the latest. dubious mares the range was quite narrow (0.12 ±
Table 1. Levels of oestradiol and progesterone in plasma during first post-partum cycle in mares with dubious and
positive results in microbial cultivation

2−3 days before ovulation 2−3 days after ovulation

dubious finding positive finding dubious finding positive finding
Oestradiol (pg/ml) 69.7 ± 18.3a,b 25.1 ± 5.8b 20.9 ± 6.4a 18.2 ± 11.8
a a
Progesterone (ng/ml) 0.12 ± 0.03 0.41 ± 0.55 9.51 ± 5.98 5.52 ± 6.07

All results are presented as means ± SD

a,b
means with the same superscript in a row are statistically significant at the level P < 0.05

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doi: 10.17221/8529-VETMED

Table 2. Results of bacteriological examination of cervical swabs from mares and frequency of detection of individual
bacterial pathogens

Total number Total frequency Frequency

Micro-organisms
of isolates of isolates (%) of pathological isolates (%)
Positive findings
Staphylococcus aureus 27 6.3 8.8
Staphylococcus intermedius 6 1.4 2.0
Staphylococcus spp. 21 4.9 6.8
Non-haemolytic Escherichia coli 60 14.1 19.5
Haemolytic Escherichia coli 18 4.2 5.9
Pseudomonas aeruginosa 2 0.4 0.7
Pseudomonas spp. 11 2.5 3.6
β-haemolytic Streptococcus 124 29.0 40.4
Streptococcus spp. 33 7.7 10.7
Corynebacterium spp. 1 0.2 0.3
Serratia spp. 1 0.2 0.3
Proteus spp. 15 3.5 4.9
Bacillus spp. 1 0.2 0.3
Klebsiella spp. 5 1.2 1.6
Enterobacter spp. 1 0.2 0.3
Dubious findings
Saprophytic micro-organisms 130 30.3
Total number of swabs 427

0.03 ng/ml). Moreover, comparison of oestradiol high proportion of the tested bacteria of both types
levels before and after ovulation showed that oes- showed resistance to broad-spectrum antibiotics
tradiol levels in the dubious mares were significantly such as penicillin and tetracycline. For example, all
lower after ovulation while in the positive mares no six isolates of haemolytic Escherichia coli and 83.4%
significant change was observed (Table 1). of isolated non-haemolytic Escherichia coli were
Our investigations showed that 69.7% of cer- resistant to penicillin. Tetracycline inhibited the
vical swabs were positive for pathogenic micro- growth of 66.6% of haemolytic and 45.8% of non-
organisms; of those (307 positive findings) 40.4% haemolytic Escherichia coli, while other Escherichia
contained β-haemolytic streptococci and 20.4% coli isolates showed high or intermediate resistance
Escherichia coli (19.5% non-haemolytic, 5.9% to this antibiotic.
haemolytic). From 30 swabs we recovered more
than one pathogen by cultivation; for example
from eight swabs a combination of β-haemolytic DISCUSSION
streptococci and haemolytic Escherichia coli and
from four swabs β-haemolytic streptococci and Racehorse breeders strive to cover mares already
Staphylococcus aureus. On the other hand, com- during the first cycle, the so-called “foal heat”, or
mon saprophytic microflora alone was diagnosed during the second one at the latest. In this ideal
in 29.7% of all cervical swabs collected from mares case, the mare will come into heat in February and
(Table 2). the foal is born in January of the following year.
Table 3 presents the results of antibiotic resistance Such early covering is advantageous for the mare
testing of the isolated pathogens. It was revealed owner and the basic idea behind it is as follows:
that both Gram-positive and Gram-negative bac- (a) the first and second post-partum oestrus will
teria were highly susceptible to cefotaxime, mar- occur again in February, so the entire cycle will
bofloxacin and enrofloxacin. On the other hand, a be repeated regularly; (b) the foal born in January

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doi: 10.17221/8529-VETMED

Table 3. Susceptibility of isolated Gram-positive and Gram-negative bacterial pathogens from cervical swabs of
mares to common antibiotics

β-haemolytic Streptococcus Staphylococcus Staphylococcus Staphylococcus

Gram-positive
streptococci spp. aureus intermedius spp.
bacteria
(90 isolates) (27 isolates) (10 isolates) (2 isolates) (15 isolates)
Antibiotics tested S I R S I R S I R S I R S I R
Penicillin 93.3 – 6.7 85.2 3.7 11.1 90.0 – 10.0 100 – – 66.7 – 33.3
Gentamicin 8.9 8.9 82.2 44.4 7.4 48.2 100 – – 50 50 – 60.0 6.7 33.3
Tetracycline 44.4 16.7 38.9 40.7 – 59.3 100 – – 50 – 50 73.3 13.3 13.3
Sulfisoxazole 73.5 10.8 15.7 51.9 11.1 37.0 70.0 30.0 – 50 – 50 46.7 13.3 40.0
Cefotaxime 94.1 – 5.9 100 – – 100 – – 100 – – 88.9 11.1 –
Marbofloxacin 84.4 13.1 2.3 74.1 18.5 7.4 100 – – 100 – – 93.3 6.7 –
Enrofloxacin 83.1 11.3 5.6 58.3 33.3 8.4 100 – – – – – 100 – –
Haemolytic Non–haemolytic Psuedomonas
Gram-negative Proteus spp. Klebsiella spp.
Escherichia coli Escherichia coli spp.
bacteria (4 isolates) (5 isolates)
(6 isolates) (25 isolates) (1 isolate)
Antibiotics tested S I R S I R S I R S I R S I R
Penicillin – – 100 8.3 8.3 83.4 – – 100 25 – 75 – – 100
Gentamicin 83.3 – 16.7 54.2 4.2 41.6 100 – – 50 – 50 100 – –
Tetracycline 66.6 16.7 16.7 45.8 4.2 50.0 100 – – 25 – 75 60.0 – 40.0
Sulfisoxazole 16.7 – 83.3 25.0 8.3 66.7 – – 100 25 – 75 80.0 – 20.0
Cefotaxime 100 – – 81.8 8.3 8.3 – – – 100 – – – – –
Marbofloxacin 100 – – 91.8 4.1 4.1 100 – – 75 – 25 100 – –
Enrofloxacin 100 – – 81.8 18.2 – 100 – – – – – 100 – –

S = sensitive; I = intermediate; R = resistant

is “earlier” and will hold an advantage in perfor- the thickness of which is one of the decisive factors
mance tests (the tests are carried out only after in embryo implantation (Navot and Bergh 1991;
reaching the age of two years) compared to foals Michalas et al. 1996; Groothuis et al. 2007). This
which were born later. It is highly probable that fact can explain, on the one hand, our observations
the older foal will appear more mature in every as- of a higher than 20% infertility rate in live cover
pect compared to the younger one. Such a strategy, mares, and on the other hand, the fact that almost
however, faces some risks, because the anatomy 70% of cervical swabs from mares collected before
and physiology of mares makes it more difficult to the first oestrus cycle were positive for pathogenic
clear the uterus of all debris and fluid. Further, and microorganisms.
as mentioned previously, most fertility problems in As reviewed in Edwards (2005), progesterone and
mares are associated with the inability or delay of oestradiol have a number of important physiologi-
these animals to get rid of the contaminating micro- cal roles related to immunomodulation and inflam-
flora after natural mating (LeBlanc 2003). Recent mation. Although most of the relevant studies were
evidence showed that increased susceptibility of performed on human or laboratory female animals,
the urogenital apparatus of mares to infection is all data suggest that these sex steroid hormones
closely related to the level of oestrogen (Luthje et play a key role in the modulation of interactions
al. 2013), which corresponds to our observations between bacterial microorganisms and the host’s
of lower levels of oestrogen in mares positive for environment (Garcia-Gomez et al. 2013). This
pathogenic microflora in the uterus before the first bacterial-host communication can mediate the
post-partum ovulation in comparison with mares activation of virulence factors of microbial path-
with only common saprophytic microflora. In ad- ogens thus affecting the pathogenesis and prog-
dition, during the post-partum period, oestrogen nosis of infection in their host organism (Hughes
supports regeneration and growth of endometrium, and Sperandio 2008). From a patho-physiological

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doi: 10.17221/8529-VETMED

point of view, the aforementioned mechanisms are mares and it is well known that both these pathogens
only partially understood and require further re- are involved in reproduction disorders of females.
search. Based on our findings, we can state that Also other authors, such as Koskinen and Katila
the presence of pathological microflora in the (1987), Purswell et al. (1989), Waelchli et al. (1993),
genital apparatus of mares after the parturition Langoni et al. (1997) and Frontoso et al. (2008) ob-
seems to have a negative influence on the hormonal served that the dominant, most frequently detected
(oestradiol/progesterone) balance during oestrus. microorganisms cultivated from cervical swabs of
Furthermore, infection with same Gram-negative mares were β-haemolytic streptococci followed by
endotoxin-producing bacteria could also result in coliform bacteria. On the other hand, Albihn et al.
disruptions to hormonal responses (Salkowski and (2003) reported in their study that Escherichia coli
Vogel 1992; Barish et al. 2005). was isolated from 67% of swabs (of those 64% were
Similar studies conducted between the seventies non-haemolytic and 3% haemolytic Escherichia coli),
and nineties of the past century indicated that up while β-haemolytic streptococci were detected only
to 40% of mares that failed to conceive were posi- in 20% of cervical swabs of mares with fertility disor-
tive for pathogenic microflora in their reproduc- ders. This indicates that the microbiological findings
tive apparatus (Redaelli and Codazza 1977; Shin in the reproductive apparatus of mares vary consid-
et al. 1979; Ricketts et al. 1993). As mentioned erably and may be affected by multiple factors. One
previously, these were the years characterised by can assume that the presence of Escherichia coli in
“heedless” use of antibiotics mainly for preven- 20.4% of swabs may indicate a lower level of man-
tion of various infectious diseases which, however, agement (zoohygiene or inspection of health status)
resulted in development of resistance of bacterial in some private breeding units as many mares with
strains particularly to routinely used broad-spec- such findings were brought only for the purpose of
trum antibiotics. Moreover, studies published some natural cover by an English thoroughbred stallion
years later by Albihn et al. (2003), Baranski et al. kept on the investigated farm. Also the variability
(2003) and Frontoso et al. (2008) already reported in incidence of haemolytic Escherichia coli strains
that the incidence of bacterial infections in the re- (unambiguously associated with reproductive dis-
productive apparatus of mares ranged between 50% orders in females) in comparison with non-haemo-
to almost 70%. These results point to an increasing lytic strains, reported in studies by Barrelet (1995),
trend in the incidence of bacterial infection of the Albihn et al. (2003), Frontoso et al. (2008) as well
reproductive organs of mares and are in agreement as in our study indicate that both types of E. coli
with our results which showed that at least one bac- strains are important pathogens or potential patho-
terial pathogen was isolated from cervical swabs of gens populating the reproductive apparatus of mares
70% of the investigated mares. It is obvious that the after parturition. In agreement with other studies
principal cause of such a situation is a troublesome (Brown et al. 1979; Atherton and Pitt 1982; Frontoso
inheritance, namely, the high antibiotic resistance et al. 2008) our results showed that Klebsiella spp.
of bacteria, which has literally become a worldwide and Pseudomonas spp. constituted only a small
health problem. Moreover, it was observed that ani- proportion of pathogens isolated from the cervical
mals serve as a reservoir of bacterial resistance genes swabs of mares. For example, Staphylococcus au-
which can subsequently pass to bacteria circulating reus bacteria were recovered both from the uterus of
in the human population. The first scientific study healthy mares (Ricketts et al. 1993) as well as mares
that indicated the potential existence of a phenom- with reproductive disorders (Frontoso et al. 2008).
enon of transfer of genes resistant to antibiotics Many potentially pathogenic bacteria survive in the
which colonised the intestines of healthy dogs to reproductive apparatus and their multiplication is
bacteria causing enterococci infections in humans suppressed by common saprophytic bacteria but
was published by De Graef et al. in 2004. Thus, stud- when the immune responses are affected (e.g. after
ies involved in the monitoring of individual bacterial parturition) they can induce reproductive disorders.
pathogens and their resistance to antibiotics are very As mentioned previously, the antibiotic resis-
important also for human medicine. tance of some bacterial strains has become a se-
Our investigations showed that β-haemolytic rious health problem as resistance to multiple
streptococci and Escherichia coli were the patho- antibiotics has been commonly described (Cohen
gens most frequently isolated from cervical swabs of 1992; Gibbons 1992; Siu 2002; Frontoso et al. 2008)

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doi: 10.17221/8529-VETMED

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gentamicin, tetracycline and entrogloxacin were (2004): Antibiotic resistance among faecal indicator bac-
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Corresponding Author:
MVDr. Tomas Benko, University of Veterinary Medicine and Pharmacy, Equine Clinic, Komenskeho 73,
041 81 Kosice, Slovak Republic
E-mail: tomas.benko@uvlf.sk

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