Many different bacterial organisms have been isolated from genital tract of
healthy bitches (Holzmann et al., 1979; Allen
and Dagnall, 1982; Doig et al., 1991; Janowski
et al., 2008b). The microorganisms most commonly isolated are E.
coli, Streptococcus sp., Staphylococcus sp., Pasteurella sp.,
Proteus sp., Bacillus sp., Corynebacterium sp., Pseudomonas
sp. and Micrococcus sp., Nisseria sp., Klebsiella sp.
and Moraxella sp. (Hirsh and Wiger, 1977; Olson
and Mather, 1978; Allen and Dagnall, 1982; Olson,
1986). Some of these organisms have been cultured from healthy as well as
infertile bitches (Feldman and Nelson, 2004). All normal
bitches have bacterial flora present in the anterior vagina and similar types
of aerobic bacteria are present in the vaginal vaults of infertile bitches (Bjurström
and Linde-Forsberg, 1992). The canine vaginal bacteria flora is not sterile
and a large of contaminant or normal flora area routinely from caudal vagina.
The vaginal flora contains a large variety of bacteria species, including both
aerobic and anaerobic organisms (Zunin et al., 1981).
The canine vaginal bacterial flora varies relation to breed, stage of oestrus
cycle and season (Holzmann et al., 1979; Doig
et al., 1991; Bjurström and Linde-Forsberg,
Anaerobic bacteria and mycoplasmas were not considered normal flora in the
canine uterus (Watts et al., 1996). But Mycoplasma
and Ureaplasma are organisms commonly cultured from the vaginal tract of normal
However, a syndrome of vaginitis, poor conception, early embryonic death, embryonal
and fetal resorption, abortion, stillborn pups, weak newborns and/or neonatal
death has been suggested to be caused by these smallest of free-living microorganisms
(Feldman and Nelson, 2004). A stage of oestrus cycle may
influence the bacterial flora. Presence of bacteria in uterus during proestrus
and oestrus was not uncommon; these bacteria are members of the vaginal flora
(Olson and Mather, 1978; Watts et
al., 1998). Bacteria were rarely present in the normal bitches during
other stage of the oestrus cycle (Watts et al., 1996;
Schultheiss et al., 1999). Noguchi
et al. (2003) cultured a large number of microorganisms during oestrus
than during metoestrus and anoestrus. In another study, the number and species
of bacteria isolated did vary with the stage of the ovarian cycle (Bjurström
and Linde-Forsberg, 1992).
A large number of organisms are cultured during oestrus than diestrus or anestrus
and these are retrieved from bitches with reproductive tract disease from normal
bitches (Hirsh and Wiger, 1977). It has been also reported
that It has been also reported that the type of bacteria isolated do not appear
to vary with the different stages of the oestrus cycle but increased number
of organisms appear to be present during proestrus and oestrus (Allen
and Dagnall, 1982; Baba et al., 1983). According
to some researchers, uterine microflora reflects the bacterial flora of the
vagina and cervix (Baba et al., 1983; Watts
et al., 1996).
The purpose of this study was to investigate vaginal, cervical and uterine bacterial flora in clinically normal ovariohysterectomized bitches of various breeds during the different stages of the oestrus cycle.
MATERIALS AND METHODS
Animals: Fifty-five clinically healthy mixed-bred bitches, aged 1.5-3 years, weighing between 15 and 30 kg were used in this study. The stages of the sexual cycle were determined based on history, physical examination and vaginal cytology. Bitches were clinically examined at the time of sample collection for swelling of vulva and presence of discharge. All the animals were normal and there were no gross lesions in the reproductive tract.
Ovariohysterectomy operation: The bitches were anaesthetised by an injection
of xylazine (2mg kg-1 b.w., Alfazyne-Alfasan, the Netherlands) and
ketamin HCI (15 mg kg-1 b.w., Alfamine-Alfasan, the Netherlands).
Ovariohysterectomy were performed by medial laparotomy, according to the routine
methods (Stone, 1995).
Vaginal cytology: Vaginal cytology samples obtained daily throughout
the study period by sterile cotton swabs. Thereafter, the smears were stained
using Giemsa stain and observed under light microscopy and percentage of cell
types was determined and interpreted as previously described by Olson
Microbiological culture: Samples for microbiological culture were collected from the vagina, cervix and uterus at all stage of the reproductive cycle. Vaginal swabs were collected before the operation, while uterine and cervical swabs were taken during the operation. Steril plastic swabs were used for sampling. The outer surface of vagina was disinfected using alcohol 90% before sampling. After sampling, each swab was put into a tube containing 3 mL sterile 0.9% NaCl solution and vortexed in order to release whole collected material from the swabs to the aqueous phase. These materials in tubes were centrifuged 3000 g for 10 min.
Sediments were used as inocula. Sediment was inoculated onto 5% ovine blood
agar (Oxoid), Mac Conkey (Oxoid) agar plates were incubated in aerobic conditions
for 24-48 h at 37°C. In addition, sediment was also inoculated to Hayflicks
Medium Broth tubes and agar plates were incubated in 5% CO2 for 5-7
days at 37°C for Mycoplasma isolation. After incubation period, a single
colony was subcultured and identificated by biochemical tests, results were
interpreted in accordance with the recommendations of Barrow
and Feltham (1995) and Poveda (1998).
RESULTS AND DISCUSSION
Bacteria were found in the vagina during the oestrus cycle. No bacteria were
isolated from the cervix and the uterus in all bitches during all stages of
sexual cycle. From a total 55 bitches in different stages of reproductive cycle
(Anoestrus = 11, Proestrus = 16, Oestrus = 13, Metoestrus = 9 and Pregnant =
6), 61 bacterial organisms were isolated. Details on the vaginal culture in
55 bitches at the different stages of sexual cycle are shown in Table
1. The commonest isolates were Yeast (45.4%) and E. coli (30.9%).
These organisms including Proteus sp. (20%), Streptococcus sp.
(12.7%) and Enterococcus sp. (1.8%) were isolated from all bitches. The
highest rate of isolation of bacteria per dog was recorded among the proestrus
(1.4), followed by oestrus (1.2), pregnant (1.0), metoestrus (0.9) and anoestrus
(0.8). It was confirmed that different bacteria could grow as well as the same
bacteria at the different stages of oestrus cycle and vaginal flora of mixed
character was isolated (Günay et al., 2004;
Laurusevicius et al., 2008).
According to some researchers, uterine microflora reflects the bacterial flora
of the vagina and cervix (Osbaldiston, 1978; Baba
et al., 1983; Watts et al., 1996).
Similarly, Zdunczyk et al. (2006) and Janowski
et al. (2008a, b) reported that similar bacterial
floras were present in the vagina and uterus of bitches. In contrast, other
researchers have found the uterus devoid of microflora (Olson
and Mather, 1978; Dhaliwal et al., 1998).
In the study, no bacteria were isolates from the uterus. The findings were in
agreement with data obtained by Olson and Mather (1978)
and Dhaliwal et al. (1998).
In the present study, no mycoplasmas were cultured samples from the vagina,
cervix and uterus in this study. The findings were similar to those of Watts
et al. (1996), who found no mycoplasmas in the vagina and uterus
of healthy bitches. On the contrary, Bjurström and
Linde-Forsberg (1992) found mycoplasma from the vagina of normal bitches.
Some researchers (Baba et al., 1983; Wadas
et al., 1996) reported that mycoplasmas have also been isolated from
both healthy uteri and pyometra infections. However, in some cases no microorganisms
at all could be isolated from diseased uteri (Osbaldiston,
1 978; Wadas et al., 1996). The reason of
these differences could be related to variation in composition of canine vaginal
flora in different breeds.
||Bacteria isolated from the vagina of bitches at the different stages of
the oestrus cycle
A larger of bacteria is cultured during oestrus than metoestrus and anoestrus
(Noguchi et al., 2003) and the highest bacterial
count was obtained in proestrus (Janowski et al., 2008a;
Laurusevicius et al., 2008) and oestrus than other
stages of sexual cycle (Baba et al., 1983; Allen
and Dagnall, 1982).
The lowest isolation was recorded in proestrus and oestrus bitches (Holst,
1986; Mshelia et al., 2001). The highest
bacterial count was recorded in the proestrus and oestrus bitches in the study.
The results were in agreement with the findings of Noguchi
et al. (2003), Allen and Dagnall (1982),
Baba et al. (1983), Janowski
et al. (2008a) and Laurusevicius et al.
(2008) but not with the findings of Holst (1986)
and Mshelia et al. (2001). In the study, lower
rates of bacteria were isolated from metoestrus, anoestrus and pregnant animals.
These findings were in agreement with results reported by Schultheiss
et al. (1999). The stage of the cycle did not alter the types of
bacteria isolated but increased numbers were present in proestrus and oestrus
(Allen and Dagnall, 1982; Baba et
The results of this study showed that, an increased number of bacteria were appeared during proestrus and oestrus in vaginal flora. These organisms could be considered as a part of the normal canine vaginal flora. The results of the present study have indicated that no bacteria and mycoplasma were isolated in cervix and uterus in healthy bitches during the different stages of reproductive cycle.
The study showed that uterus was sterile and the uterine microflora did not reflect the vaginal and cervical microflora. It can be said that bacterial isolation in healthy bitches is important because bacteria can become pathogenic and it could have an influence on the future fertility.
The researchers would like to thank Vet. Surg. Bulent ÇIFTCI for his assistance and providing the animals.