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Vaginal and uterine bacteria of healthy bitches during different stages of their
reproductive cycle

Article  in  The Veterinary record · August 2012

DOI: 10.1136/vr.100886 · Source: PubMed

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Short Communications

Short Communications
Vaginal and uterine
Inoculation was performed on blood agar, MacConkey’s and
­bromocresol purple agar. Uterine swabs were also inoculated into
nutrient and trypticase soy broths. All plates and broths were incubat-

bacteria of healthy ed aerobically for 24–48 hours at 37°C. Positive broth cultures were
subcultured onto the corresponding solid media. Standard bacteriolog-
ical procedures were used for identification (Quinn and others 2000).
bitches during different Tissue samples from the uterine body and horns were routinely
processed and stained for histological examination. Statistical evalua-

stages of their
tions were made using a chi-squared test and Fisher’s exact test.
Bacteria were isolated from the vagina of 33 bitches (82 per cent).
The results were similar to those of other authors (Hirsh and Wiger

reproductive cycle
A. Maksimović, Z. Maksimović, S. Filipović,
H. Beširović, M. Rifatbegović
Bacteria commonly isolated from the vagina of healthy bitch-
es include: Escherichia coli, Streptococcus canis, Pasteurella multocida,
Staphylococcus aureus, S taphylococcus pseudintermedius and haemolytic
Streptococcus species (Olson and Mather 1978, Allen and Dagnall
1982, Baba and others 1983). Some researchers advocate bacterial
isolation in pure or 2-isolate mixed culture as an aid in the diagnosis
of reproductive diseases (Hirsh and Wiger 1977, Bjurström 1993).
However, there is limited data reporting pure or mixed bacterial
cultures isolated during specific stages of the reproductive cycle in
healthy bitches, which should complicate the interpretation of bac-
teriological findings. The true bacteriological status of the uterus
remains unclear. Results of previous studies (Baba and others 1983,
Watts and others 1996, Schultheiss and others 1999) differed on
b­ acteria isolated, frequency of isolation and stage of reproductive
cycle examined.
The aim of this study was to investigate vaginal and uterine bac-
terial flora in clinically healthy bitches during different stages of the
reproductive cycle, and to correlate culture findings with gross and
histologic lesions of the uterus.
Vaginal swabs, uterine swabs and uterine tissue samples from 40
clinically healthy bitches were collected for bacteriological, cytologi-
cal and histological examination. Bitches of different breeds, ages and
stages of the reproductive cycle were separated according to whether
they were domestic (n=20) or feral (n=20).
The stage of the reproductive cycle was determined by history,
physical examination and vaginal cytology. Sampling, staining
(Giemsa and Diff-Quick stains) and interpretation of the smears for
vaginal cytology were performed in accordance with Holst (1986).
Vaginal bacterial specimens were obtained by inserting a sterile swab
into the cranial portion of the vagina. A swab of the mucosa from the
uterine horns was taken for bacteriology immediately after ovariohys-
terectomy (OHE).
1977, Olson and Mather 1978, Allen and Dagnall, [b1]1982). The
most frequent isolates were Streptococcus species (Table 1), frequently in
pure culture (46.6 per cent). The bacterial isolates did not vary signifi-
cantly during the various stages of the oestrus cycle, which is in agree-
ment with previous studies (Olson and Mather 1978, Baba and others
1983). Bacterial flora is usually mixed in clinically healthy bitches,
and the isolation of bacteria in pure culture suggests pathogens have
overgrown normal flora (Olson and others 1986, Duijkeren 1992).
In our study, bacteria were isolated more frequently in pure culture
during pro-oestrus and anoestrus (P=0.028), while mixed bacterial cul-
tures were found during oestrus and dioestrus (P=0.041). These results
suggest that isolation of pure bacterial culture does not necessarily
indicate the presence of infection.
Only one (2.5 per cent) uterine sample yielded a positive bacte-
rial culture using solid media for primary isolation. The isolate was
identified as Arcanobacterium haemolyticum. Similar results were reported
by Olson and Mather (1978), where bacteria were recovered in pure
culture from one of 22 uteri collected at OHE. By contrast, Baba and
others (1983) recovered bacteria frequently (62 per cent) from uteri
obtained postmortem. In our study, the frequency of isolation of bac-
teria from the uterus was enhanced by the use of nutrient or trypticase
soy broths. Bacteria were isolated in pure culture from the uteri of 25
bitches (62.5 per cent) (Table 2). Schultheiss and others (1999) also
reported isolation of pure bacterial cultures from all positive uterine
samples.
Significant differences were found between domestic bitches and
feral bitches in the numbers of uterine samples from which S. pseud-
intermedius and coagulase-negative Staphylococcus species were isolated
(Table 2). This result could be explained perhaps by the divergent
immune status of the two observed categories. To our knowledge,
there is no report that can support these findings.
The stage of the reproductive cycle affects the viability of bacte-
ria in the uterus of clinically healthy bitches (Watts and others 1996,
Schultheiss and others 1999), but the results of current studies dif-
fer as to the stages where bacteria are present. During our study,
TABLE 1: The frequency of bacteria isolated from the vagina
of 33 bitches
% of total number % of positive % of the
Isolates of samples samples isolates

Staphylococcus spp. 32.5 39 22

Veterinary Record (2012) doi: 10.1136/vr.100886 (coagulase negative)
S. pseudintermedius 5 6 3.4
A. Maksimović, DVM, MVSc, H. Beširović, DVM, MVSc, PhD Staphylococcus aureus 2.5 3 1.7
S. Filipović, DVM, MVSc, PhD, Department of Pathology, Veterinary Streptococcus spp. 20 24 13.5
Department of Surgery, Veterinary Faculty, Zmaja od Bosne 90, Sarajevo (β haemolytic)
Faculty, Zmaja od Bosne 90, 71000, Bosnia and Herzegovina Streptococcus spp. 12.5 15 8.5
Sarajevo 71000, Bosnia and (α haemolytic)
Herzegovina E-mail for correspondence: Streptococcus spp. 12.5 15 8.5
alan.maksimovic@vfs.unsa.ba, (nonhemolytic)
M. Rifatbegović, DVM, MVSc, PhD, Escherichia coli 25 30 17
Z. Maksimović, DVM, MVSc, alanmaksimovic@yahoo.com Proteus spp. 15 18 10.2
Department of Microbiology and Provenance: not commissioned;
Corynebacterium spp. 10 12 6.8
Infectious Diseases, Veterinary Faculty, Pasteurella spp. 7.5 9 5
externally peer reviewed Enterococcus spp. 2.5 3 1.7
Zmaja od Bosne 90, Sarajevo 71000,
Neisseria spp. 2.5 3 1.7
Bosnia and Herzegovina Accepted July 16, 2012

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Short Communications

TABLE 2: Bacteria isolated from uterine samples using nutrient reproductive cycle. Pure bacterial cultures can be isolated from the
and trypticase soy broths vagina of healthy bitches during pro-oestrus and anoestrus, while
mixed bacterial cultures are found during oestrus and dioestrus. Usage
Isolates Total domestic feral P of pre-enrichment broths significantly increases the likelihood of iso-
Staphylococcus spp. 7 0 7 0.006 lation of bacteria from the uterus of healthy bitches. Isolates obtained
(coagulase negative) in this manner probably represent commensals in light growth,
S. pseudintermedius 7 6 1 0.026 which makes their isolation difficult by direct agar plating methods.
Micrococcus spp. 5 2 3 1.000
Bacteria can be detected in the uterus during all stages of the reproduc-
Bacillus spp. 2 2 0 0.203
Corynebacterium spp. 1 1 0 0.461 tive cycle, and most frequently during dioestrus. These generally do
E coli (β haemolytic) 1 1 0 0.461 not reflect vaginal flora.
Streptococcus spp. 1 0 1 1.000
(α haemolytic) Acknowledgements
Streptococcus spp. 1 0 1 1.000
The authors wish to thank Professor Čazim Crnkić for help on statis-
(β haemolytic)
tical analysis, Professor Nazif Varatanović for support, Scott Cornwell
for English language revision of this article, Muamer Obhod̄aš and
Aida Dolovac for technical support.
most of the bacteria were detected in the uterus during dioestrus
(46 per cent), where the frequency of isolation was 100 per cent. References
Similarly, Schultheiss and others (1999) frequently isolated uterine ALLEN, W. F. & DAGNALL, G. J. R. (1982) Some observations on the aerobic bacte-
bacteria during dioestrus. Curiously, the most clinically important rial flora of the genital tract of the dog and bitch. Journal of Small Animal Practice 23,
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commonly during this phase (Dhaliwal and others 1998). This flora of Adult dogs. American Journal of Veterinary Research 44, 606–609
implies that uterine resistance to bacterial infections decreases dur- BJURSTRÖM, L. (1993) Aerobic bacteria in the vagina of bitches with reproductive
ing dioestrus when comparing this with other stages of the oestrus disorders. Acta Veterinaria Scandinavica 34, 29–34
cycle. Lactoferrin is a non-specific antimicrobial agent whose highest DHALIWAL, G. K., WRAY, C. & NOAKES, D. E. (1998) Uterine bacterial flora and
uterine lesions in bitches with cystic endometrial hyperplasia (pyometra). Veterinary
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dioestrus, and remain low until the subsequent pro-oestrus (Kida and Veterinary Record 131, 367–369
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­ estrus and oestrus, but they rarely found growth during other stages KIDA, K., BABA, E., TORII, R., KAWATE, N., HATOYA, S., WIJEWARDANA, V.,
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in the vagina and uterus of the same animals. Similarly, Olson and OLSON, P. N., JONES, R. L. & MARTHA, E. C. (1986) The use and misuse of vagi-
Mather (1978) reported different bacteria species in the cranial and nal cultures in diagnosing reproductive diseases in the bitch. In Current Therapy in
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lesions. From a total of 69 uterine samples Schultheiss and others SCHULTHEISS, P. C., JONES, R. L., KESEL, M. L. & OLSON, P. N. (1999) Normal
(1999) detected histologic lesions in two samples with positive bacte- bacterial flora in canine and feline uteri. Journal of Veterinary Diagnostic Investigation 11,
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Vaginal and uterine bacteria of healthy

bitches during different stages of their
reproductive cycle
A. Maksimovic, S. Filipovic, M. Rifatbegovic, et al.

Veterinary Record published online August 17, 2012

doi: 10.1136/vr.100886

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References This article cites 10 articles, 2 of which can be accessed free at:
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