Anoestrus is the physiological condition in which vaginal mucosa is quiescent, whereas sterilization causes the permanent loss of hormonal stimulation. The effects of the absence of oestrogen on the vaginal tract are well known in humans. Specifically, vaginal atrophy is a consequence of menopause, and alteration of the resident microbial community has been ascertained and investigated as a potential concurring cause of pathological conditions, such as vaginitis and urinary tract infections [16].
Notwithstanding the fact that follicular growth and oestrogen secretion occurs only twice yearly in dogs, the trophic effect of steroidal hormones on the lower uro-genital tract is well recognized in this species [1, 7].
Some complications are described as a consequence of sterilization. Urinary incontinence occurs due to lack of oestrogens and modification of the external urethral sphincter. In addition, modification of the vulva can lead to vulvar atrophy and cause perivulvar dermatitis [17]. In a study focused on verifying outcomes related to the timing of sterilization, the authors did not observe any difference in the incidence of the development of urinary incontinence for bitches sterilized in prepubertal age vs bitches undergoing sterilization at a later age in the 4 years after surgery [18].
In studies related to sterilization consequences, the effects of steroid hormone deprivation on vaginal mucosa conditions and bacteria colonization have not been investigated. This pilot study explores this rather complex field and applies methodologies, such as metataxonomic analysis, and previous comparable results are limited.
A limit of this investigation is the small number of animals and the possible inhomogeneity of the groups that included bitches of different breed, size and age. In addition, sterilized animals tended to be older than intact animals because elective surgery is not always performed at a very young age. Despite careful procedures to maintain sterility during sampling, we cannot exclude the possibility that bacterial contamination could have occurred in some conditions, especially in very small dogs or uncooperative animals.
Although cytological results suggested mucosal thinness and fragility in sterilized bitches, both metataxonomic investigation and culture techniques failed to discover strong differences in the microbial community colonizing the vaginal tract of either sterilized or intact bitches.
The lower number of different species isolated in sterilized bitches and the trend towards increased bacteria proliferation could be suggestive of an initial imbalance. However, in both groups of animals, the number of colony-forming units was consistently in the lower range, and this finding differs from that observed in clinical samples obtained from animals with vaginal exudates [13].
The metataxonomic approach highlighted a very high inter-individual variability, but no statistically significant difference between the different reproductive conditions were noted. Neither alpha nor beta measures were significantly different between the two groups; thus, no global characteristic could be identified that characterized these groups according to their condition.
At the phylum and class level, only Tenericutes and Mollicutes were significantly more abundant in anoestrus samples, and these microorganisms were only detected, by culture method, in one sample from the group of sterilized bitches.
Observing proportional abundance values, a discordance is noted between the mean and the median. This finding highlight that the identified prevalent genera were not evenly present in the samples in most cases. For example, Photobacterium had a considerable mean abundance among sterilized samples (14.03%), but this value was reduced when the median (3.42%) was used because this bacterium was found in very high percentages in only a few samples (e.g., S13 and S16) and in low quantity in all the other samples belonging to this group. On the other hand, Salmonella, Mycoplasma and Staphylococcus were present in moderate quantities in almost all the samples in both groups.
Many bacteria genera isolated in culture (Staphylococcus, Streptococcus, Enterococcus, Proteus) were also detected in the metataxonomic analysis; however, differences in relative abundance were noted. Mycoplasma was isolated from a single anoestrous bitch using culture techniques, whereas Mycoplasma resulted very widespread when genomic analysis was performed. Culture-independent methods allow for the identification of microorganism even in instances of minimal colonization or in cases that exhibited an inability or difficulty to grow in culture, so the results of the different techniques are not always comparable [19].
The genus Mycoplasma has special culture needs. Given the special organization and sample management procedures, the time that elapsed between the collection of samples and establishment of the culture could have compromised the viability of the Mycoplasma spp., thus preventing its detection in culture. In a previous work, the isolation of Mycoplasma from the vaginal tract did not vary between sterilized and intact bitches [20].
Quite surprisingly, the genus Salmonella was detected through sequencing in almost all the samples belonging to both groups of bitches. Salmonella is not part of the usual vaginal microbial flora of bitches [14, 20] and the genus was not even reported in previous investigations [15, 21]. In our study, bacteria belonging to the genus Salmonella did not grow in culture, the reason very likely being a very low microbial charge not sufficient to be detectable by culture methods, or a non-viable microorganism or even unsuitable culture conditions or last, the absence in culture of the ecological relationships that are typical of the microbial communities residing on biological samples [19].
Given that Escherichia and Shigella exhibit overlapping sequences [22], the metataxonomic analysis was unable to detect the genus Escherichia. Indeed, Greengenes resolves the issue of overlap leaving their sequences unclassified at levels below Enterobacteriaceae (https://greengenes.secondgenome.com/).
Culture-based techniques are believed to fail in detecting the majority of members of the resident microbial community: only viable bacteria that find appropriate growth conditions in culture can be isolated [15]. Our data from metataxonomic analysis do not confirm previous observations by Lyman et al. [15] who found two genera predominant in the bitch vagina, Hydrotalea and Ralstonia. It is difficult explain the presence of these two genera considering that the first group belongs to aquatic species, whereas the second is not commonly associated with animals [15]. In contrast to our investigation, the study was performed by amplifying the V4 region. This difference in techniques could explain the above-mentioned differences given that the choice of the hypervariable region has a well-known impact on metataxonomic results [23,24,25,26,27].
The relationship between the effect of time length of steroid hormone deprivation on the vaginal environment and the colonizing microorganisms deserves further investigations. In dogs, the absence of steroids stimulation is normal during anestrus, which lasts 3.5–4 months, so we expect that the effects of permanent hormonal deprivation would not be appreciable earlier than 3–4 months after sterilization. Gradual changes in the vaginal environment across the stages of menopausal transition are accompanied by modifications of the vaginal microbiota in women [6]. The vaginal bacteria communities of postmenopausal women, i.e. women without a menstrual cycle in the past 12 months [6, 16] show decreased proportions of lactobacilli, causing decreased lactic acid production and increased vaginal pH that possibly renders the vagina more susceptible to colonization by pathogenic bacteria and exacerbates the symptoms associated with vulvovaginal atrophy [16]. Indeed, lower relative abundance of Lactobacillus was also found in the bacterial community assemblage of postmenopausal women suffering from vulvovaginal atrophy, suggesting an alteration in vaginal microbiota homeostasis [6]. Bacteria of the genus Lactobacillus were detected in both groups of bitches by metataxonomic analysis regardless of their reproductive condition. Lactic acid-producing bacteria are the predominant colonizers of the vaginal tract of healthy women and are thought to be essential in maintaining urogenital health. A strong difference exists between the pH value of the vaginal tract of the bitch (approximately 7) and of women (approximately 4.5) [28]. This difference could explain why the microbiota composition is significantly different in the two species, and the role of lactic acid-producing bacteria is unclear in bitches [29].