**5. Fungal endometritis**

These drugs may be ineffective in aged maiden mares in which the cervix often fails to dilate. In such cases, oxytocin therapy should be associated with topical application to the cervical epithelium of misoprostol, a synthetic PGE1 analogue, before breeding, or substituted by cloprostenol administration to sustain low-amplitude uterine contraction for 2 to 4 hours. Intramuscular administration of cloprostenol or PGF2α at a dosage of 250μgis recommended, before the ovulation or within 12 hours. However, administration post-ovulation is not advisable, since it decreases serum progesterone concentration and consequently the preg‐ nancy rates. The cloprostenol-induced contractions last longer; therefore, cloprostenol is more suitable for mares with lymphatic stasis. Side effects, such as sweating and abdominal

The best timing for ecbolic administration after uterine lavage associated to the antimicrobi‐ al infusion has not been established, yet. When treating anxious or excitable mares, seda‐ tives with α2-adrenergic agonist action, such as xylazine or detomidine, should be used. These sedatives optimize oxytocin effect and increase the intrauterine pressure, where as acepromazine, aα1-adrenergic antagonist, can cause a reduction of uterine contractions after

Higher levels of anti-inflammatory cytokines were observed in resistant mares compared with susceptible ones. The rationale for using immunomodulators is to equilibrate the expression of pro- and anti-inflammatory cytokines in susceptible mares and to rehabilitate the homeo‐

Glucocorticoids have been demonstrated to suppress the immune response, decreasing gene expressions of the pro-inflammatory cytokines, IL-1α, IL-6, IL-8, while enhancing defense mechanisms and stimulating a higher anti-inflammatory response. Thus, short-term steroid therapy maybe beneficial for treating post-mating endometritis in the mare [124]. An increase of pregnancy rates after single injection of dexamethasone within 1 hour post-mating, approximately 0. 1 mg⁄kgi. v. , associated with routine therapy, has been demonstrated in mares with history of fluid accumulation after breeding. Oral administration of 9-alphaprednisolone acetate at 0. 1 mg⁄kg twice daily for 4 days, beginning 48 hours before breed‐ ing, and the association of dexamethasone, administered within 1hour after mating, and prednisolone, administered once daily before and after mating, were confirmed to be efficacious only in susceptible mares, decreasing uterine edema, and reducing and clarify‐

Other immunomodulatory treatments have been tested to elicit a general increase in immune system activity by means of non-specific cell-mediated response and modulating the release of cytokines [127]. *Mycobacterium phlei* wall extractand *Propionibacterium acnes* were reported to be efficient in uterine infectioncaused by *S. equi* and *S. zooepidemicus* and in chronic endo‐ metritis, respectively [17, 93]. In 2014, mesenchymal stem cells and autologous conditioned serum were used to modulate successfully the uterine inflammatory response to spermatozoa in healthy mares [90]. However, the former seems to be more reliable than the latter, and additional studies are ongoing to determinate the effects of mesenchymal stem cells on

cramping, are minimal, transient, inconstant and dose-dependent [123].

oxytocin treatment [47].

310 Genital Infections and Infertility

**4. 5. Immunomodulators**

stasis of the local inflammatory response.

ing intra-uterine fluid [125–126].

Whereas a rich literature describes the etiopathogenesis of equine microbial endometritis, few papers are dedicated to the role of fungi as responsible for uterine inflammation in the mare. The incidence of fungal infection in mares affected by endometritis is probably significantly lower than the incidence of bacterial infection, but fungal endometritis is more challenging to treat and is associated to a guarded-to-poor prognosis for future fertility [129]. Fungal growth was demonstrated in different rates, ranging from 1 to 7%, considering either the total number of reproductive cultures submitted to laboratories or the percent of cultures positive for fungal compared to the total number of mares affected by endometritis [130–134]. The term "fungi" is referred to heterotrophic, aerobic or facultative anaerobic, non-motile organisms widely distributed in the environment [132]. They could have characteristics of both yeasts, round to oblong single-cell organisms, and molds, that are long, branched, filamentous structures [135]. The filamentous cells are also named hyphae and are often organized in small masses, called mycelia, sometimes isolated in fluid from uterine lavage. Cytological and histological speci‐ mens may demonstrate both yeasts, most adapted to grow in liquids, and hyphae, better suited to penetrate deeper inside the endometrial tissue [136].

#### **5. 1. Aetiology**

The most common organisms isolated during equine fungal endometritis are *Candida* spp. , *Aspergillus* spp. and *Mucor* spp. [137]. However, many other organisms have been reported (Table 3). Recently, *Cladophialophora bantiana* has been isolated in a 15-year-old Standard bred mare that displayed infertility and uterine fluid accumulation with numerous black, hairy granules. *C. bantiana* is an emerging pathogen, causing cerebral phaeohyphomycosis in humans, while animal cases are rare [138]. The inflammatory mechanisms associated with these types of endometritis have not been clarified until now, and probably endometritis involving fungus or yeast will be feared as long as the inflammatory mechanisms associated will not be studied [134].

The most common source of fungi causing reproductive disease in the mare is probably from skin or fecal origin and the primary reservoir for infectious agents is the caudal reproductive tract, vagina and external genitalia [132]. Fungal elements that cause reproductive disease are generally opportunistic and their growth is considered to be secondary to underlying factors impairing the local humoral and, particularly, cell-mediated immunity. Mares with uterine fungal infections may have predisposing conditions such as: anatomical defects, i. e. , an incompetent vulvar seal from multiple Caslick's procedure or trauma, or a tilted vulvar conformation; cervical trauma and inadequate closure of the cervix due to dystocia or fetotomy; immunosuppression; malnutrition; endocrine diseases; a history of antibiotic treatment; veterinary manipulations due to frequent artificial inseminations and intrauterine treatments [132, 137–139]. Even if fungal endometritis has been reported also in mares without the history of previous uterine therapies, this pathology has been more often documented in older multiparous mares, treated for bacterial endometritis in the past [136].

One of the most discussed predisposing factors is the prolonged use of antibiotics, which is believed to modify the normal flora of the caudal reproductive tract, disrupting the biological barriers and allowing yeast overgrowth. Alteration of the vaginal flora may also destroy organisms producing anti-fungal substances and/or competing for available nutrients, interfere with the local microbial synthesis of vitamins or change vaginal pH [132]. Intrauterine antibiotics reach the vagina when cleared by the increased myometrial tone or through passive leakage, affecting the vaginal flora and the fungal colonization. Repeated reproductive manipulations, such as mating, or inseminations, or infusions and irrigations during the estrus may transfer microorganism from the caudal reproductive tract into the uterus. In mares in which deficit of uterine clearance mechanisms exist, opportunistic fungi find an endometrial environment favorable to their proliferation [132]. Administration of progesterone may be another predisposing factor due to reduction of neutrophilic phagocytic ability, decreased myometrial activity and increased cervical tone. Furthermore, a moist environment, the exposure to large numbers of fungi and the presence of necrotic, ischemic or infected foci may contribute to the fungal infection [132]. Fungal endometritis due to *Cladophialophora bantiana* was diagnosed in an infertile mare with a history of dystocia, fetotomy and cervical laceration [138]. Nevertheless, subclinical fungal endometritis was also demonstrated in an 8-year-old Hanoverian mare at her first breeding, with no further reproductive available history [140].

Fungi have been isolated from urethral cultures (*Mucor* spp. ), fresh semen (*Absidia* spp. ) and extended semen (*Candida* spp. ) from stallions [141]. The transmission of fungal infection through the natural coitus or artificial insemination has not been demonstrated until now, in the horse. However, in humans, there is some evidence that the male may harbor fungi as asymptomatic carriers. This can cause the re-infection of the sexual partner. Thus, when multiple mares are diagnosed with fungal endometritis, cultures of the stallion's penis, prepuce and semen should be performed [136].

#### **5. 2. Diagnosis of fungal endometritis**

History of mares affected may reveal the presence of predisposing factors to fungal infections. Frequently, mares are presented with a history of infertility and of repeated use of intrauterine diagnostic and therapeutic procedures [132]. Clinical signs range from subclinical infection to profuse vaginal discharge [138]. Sometimes, at the physical examination, a gray vulvar discharge is evident [132]. The presence of mycelia, as granules, in the fluid from uterine lavage is strongly indicative. The diagnosis of fungal endometritis relies on endometrial cytology, uterine culture or uterine biopsy [136]. Microscopic examination of uterine fluid or uterine Endometritis and Infertility in the Mare – The Challenge in Equine Breeding Industry–A Review http://dx.doi.org/10. 5772/62461 313


**Table 3.** Most common fungi isolated from the mares' uterus

fungal infections may have predisposing conditions such as: anatomical defects, i. e. , an incompetent vulvar seal from multiple Caslick's procedure or trauma, or a tilted vulvar conformation; cervical trauma and inadequate closure of the cervix due to dystocia or fetotomy; immunosuppression; malnutrition; endocrine diseases; a history of antibiotic treatment; veterinary manipulations due to frequent artificial inseminations and intrauterine treatments [132, 137–139]. Even if fungal endometritis has been reported also in mares without the history of previous uterine therapies, this pathology has been more often documented in

One of the most discussed predisposing factors is the prolonged use of antibiotics, which is believed to modify the normal flora of the caudal reproductive tract, disrupting the biological barriers and allowing yeast overgrowth. Alteration of the vaginal flora may also destroy organisms producing anti-fungal substances and/or competing for available nutrients, interfere with the local microbial synthesis of vitamins or change vaginal pH [132]. Intrauterine antibiotics reach the vagina when cleared by the increased myometrial tone or through passive leakage, affecting the vaginal flora and the fungal colonization. Repeated reproductive manipulations, such as mating, or inseminations, or infusions and irrigations during the estrus may transfer microorganism from the caudal reproductive tract into the uterus. In mares in which deficit of uterine clearance mechanisms exist, opportunistic fungi find an endometrial environment favorable to their proliferation [132]. Administration of progesterone may be another predisposing factor due to reduction of neutrophilic phagocytic ability, decreased myometrial activity and increased cervical tone. Furthermore, a moist environment, the exposure to large numbers of fungi and the presence of necrotic, ischemic or infected foci may contribute to the fungal infection [132]. Fungal endometritis due to *Cladophialophora bantiana* was diagnosed in an infertile mare with a history of dystocia, fetotomy and cervical laceration [138]. Nevertheless, subclinical fungal endometritis was also demonstrated in an 8-year-old Hanoverian mare at her first breeding, with no further reproductive available history [140].

Fungi have been isolated from urethral cultures (*Mucor* spp. ), fresh semen (*Absidia* spp. ) and extended semen (*Candida* spp. ) from stallions [141]. The transmission of fungal infection through the natural coitus or artificial insemination has not been demonstrated until now, in the horse. However, in humans, there is some evidence that the male may harbor fungi as asymptomatic carriers. This can cause the re-infection of the sexual partner. Thus, when multiple mares are diagnosed with fungal endometritis, cultures of the stallion's penis, prepuce

History of mares affected may reveal the presence of predisposing factors to fungal infections. Frequently, mares are presented with a history of infertility and of repeated use of intrauterine diagnostic and therapeutic procedures [132]. Clinical signs range from subclinical infection to profuse vaginal discharge [138]. Sometimes, at the physical examination, a gray vulvar discharge is evident [132]. The presence of mycelia, as granules, in the fluid from uterine lavage is strongly indicative. The diagnosis of fungal endometritis relies on endometrial cytology, uterine culture or uterine biopsy [136]. Microscopic examination of uterine fluid or uterine

and semen should be performed [136].

312 Genital Infections and Infertility

**5. 2. Diagnosis of fungal endometritis**

older multiparous mares, treated for bacterial endometritis in the past [136].

swab cytology is an important diagnostic tool for fungal endometritis [138]. Cytological smears may show yeast or hyphae. Yeasts appear as 3 to 5 μm round-to oval single-cell organism and often have a capsule surrounding them that exclude dyes. This capsule is visible by altering the focal point when viewing the microscope slide and is considered a protective barrier against phagocytosis. Hyphae are less common and appear as subtle filamentous multicellular organisms [132, 136]. Yeasts are usually associated to superficial infections; on the contrary, hyphae are generally associated to deeper-seated fungal infections, requiring a longer treatment. Fungi should be always isolated and identified by cultures, to allow for antifungal drug sensitivities to be performed. They may be cultured initially on blood agar, and then passed on Sabaroud's agar [132]. Time for their growth is sensibly higher than bacteria and the laboratory should be aware that a search for fungal organism is requested. Otherwise, after the routine time of observation for microbial growth, the culture could be deemed negative, because of the early reporting of the results [142]. A microculture system, Dermatobac®, originally developed for the isolation of fungi in human medicine, was recently tested to evaluate its sensitivity in the diagnosis of equine fungal endometritis. Dermatobac® proved to be efficient, showing conclusive information after only 24 hours of culture [139]. A rapid, sensitive and repeatable quantitative polymerase chain reaction assay (qPCR) was also developed for the detection of fungal DNA from equine endometrial samples. The qPCR assay identified fungal DNA in samples from 12 of 17 mares suspected of having fungal endometritis and proved to be potentially useful as an adjunct to microbial culture and cytological exami‐ nation in clinical situations to provide identification of fungal organisms in a timely manner [143]. The clitoral fossa and vaginal mucosa should be cultured in addition to the uterine lumen because they could be the reservoir for fungal organism, allowing recontamination after successful uterine treatments [132].

Equine fungal endometritis is usually accompanied by bacteria, among which *β-hemolytic Streptococci* group C and *E. coli* are the most common species. The underlying factors impairing the local immunity, which promote the fungal growth, may favor the microbial growth as well [138]. Especially if bacteria are in low number, they can be missed at the culture, confounding the diagnosis. In these cases, after the treatment with antifungal drugs, in the absence of other concurrent microorganisms, a bacterial endometritis develops, and infertility persists. It is also possible that the microbial infection has its origin in the alteration of the vaginal microflora due to the antifungal treatments. For these reasons, it is important for early identification of mixed fungal and bacterial infection at either the cultural or the cytological examinations [136]. Endometrial biopsy may also be used in the diagnosis of fungal endometritis, when cytology is inconclusive or to confirm the successful treatment of uterine infection. Fungal elements may be observed either adherent to the luminal surface, within endometrial gland lumens or within the endometrium [132].

#### **5. 3. Therapy of fungal endometritis**

Many difficulties can prevent a successful treatment of mares affected by fungal endometritis, due to invasive forms that are not exposed to intrauterine therapies, poor uterine clearance mechanism, inadequate immune response or to recontamination from posterior reproductive tract sources, mixed or chronic infections. Therapy for fungal endometritis is made of a combination of predisposing factors correction, uterine lavage and intrauterine infusion of antifungal agents [137]. Any defect in the physical barriers to uterine infection, at the level of perineum, vulva, vulvo-vaginal folds and cervix, must be surgically corrected whenever possible [136]. All the tissue of the reproductive tract that may harbor the fungal elements should be treated. These include the clitoris, clitoral fossa, vagina, cervix and uterus.

Large volume uterine lavage may be used with the aim to decrease the overall fungal number, increase the infiltration of inflammatory cells and the myometrial tone and expose organism to antifungal solutions. Some substances and drugs are usually added to saline, which may interfere with the fungal growth by means of different mechanisms. The use of DMSO is reported at concentrations of 10–20%, because of its in vitro activity against the growth of *Candida albicans*[132]. A potential antifungal effect has been attributed to diluted iodine (0. 05– 1%) and to acetic acid (vinegar, 2% – 20 ml of white vinegar in 1 L of 0. 9% saline) [136]. Intrauterine iodine should be used with caution, because of the danger of tissue damage and adhesions in particularly sensitive mare, even if it was demonstrated that a 1% povidoneiodine infusion during days 0 and 2 post ovulation in healthy mares did not induce histological changes compatible with endometritis [96]. Ecbolic drugs, such as oxytocin (10-20 IU intra‐ muscularly or intravenously) or prostaglandins may be administered after the lavage to facilitate the expulsion of residual uterine fluid [136]. After the uterine lavage, the treatment should be completed with the intrauterine and/or systemic administration of an antifungal drug, ideally chosen on the basis of the results of cultural examination and susceptibility [136].

the laboratory should be aware that a search for fungal organism is requested. Otherwise, after the routine time of observation for microbial growth, the culture could be deemed negative, because of the early reporting of the results [142]. A microculture system, Dermatobac®, originally developed for the isolation of fungi in human medicine, was recently tested to evaluate its sensitivity in the diagnosis of equine fungal endometritis. Dermatobac® proved to be efficient, showing conclusive information after only 24 hours of culture [139]. A rapid, sensitive and repeatable quantitative polymerase chain reaction assay (qPCR) was also developed for the detection of fungal DNA from equine endometrial samples. The qPCR assay identified fungal DNA in samples from 12 of 17 mares suspected of having fungal endometritis and proved to be potentially useful as an adjunct to microbial culture and cytological exami‐ nation in clinical situations to provide identification of fungal organisms in a timely manner [143]. The clitoral fossa and vaginal mucosa should be cultured in addition to the uterine lumen because they could be the reservoir for fungal organism, allowing recontamination after

Equine fungal endometritis is usually accompanied by bacteria, among which *β-hemolytic Streptococci* group C and *E. coli* are the most common species. The underlying factors impairing the local immunity, which promote the fungal growth, may favor the microbial growth as well [138]. Especially if bacteria are in low number, they can be missed at the culture, confounding the diagnosis. In these cases, after the treatment with antifungal drugs, in the absence of other concurrent microorganisms, a bacterial endometritis develops, and infertility persists. It is also possible that the microbial infection has its origin in the alteration of the vaginal microflora due to the antifungal treatments. For these reasons, it is important for early identification of mixed fungal and bacterial infection at either the cultural or the cytological examinations [136]. Endometrial biopsy may also be used in the diagnosis of fungal endometritis, when cytology is inconclusive or to confirm the successful treatment of uterine infection. Fungal elements may be observed either adherent to the luminal surface, within endometrial gland lumens or

Many difficulties can prevent a successful treatment of mares affected by fungal endometritis, due to invasive forms that are not exposed to intrauterine therapies, poor uterine clearance mechanism, inadequate immune response or to recontamination from posterior reproductive tract sources, mixed or chronic infections. Therapy for fungal endometritis is made of a combination of predisposing factors correction, uterine lavage and intrauterine infusion of antifungal agents [137]. Any defect in the physical barriers to uterine infection, at the level of perineum, vulva, vulvo-vaginal folds and cervix, must be surgically corrected whenever possible [136]. All the tissue of the reproductive tract that may harbor the fungal elements

should be treated. These include the clitoris, clitoral fossa, vagina, cervix and uterus.

Large volume uterine lavage may be used with the aim to decrease the overall fungal number, increase the infiltration of inflammatory cells and the myometrial tone and expose organism to antifungal solutions. Some substances and drugs are usually added to saline, which may interfere with the fungal growth by means of different mechanisms. The use of DMSO is

successful uterine treatments [132].

314 Genital Infections and Infertility

within the endometrium [132].

**5. 3. Therapy of fungal endometritis**

The two main categories of antifungal agents are the polyene and the imidazole drugs, which interfere with the formation of fungal ergosterol, resulting in an increased permeability of the cell wall and eventual cell death. The polyene antibiotics include amphotericin B, nystatin and natamycin, whereas clotrimazole, econazole, ketoconazole, fluconazole and itraconazole are the most used azole derivatives [132]. Clotrimazole, miconazole and ketoconazole have two nitrogen molecules; fluconazole and itraconazole are called triazoles because of the presence of three nitrogen atoms in an azole ring [137]. Common antifungal agents used in the treatment of fungal endometritis are reported in Table 2. Amphotericin B (100–200 mg), clotrimazole (300–600 mg) and miconazole (500–700 mg) are active against *Candida* spp. , *Aspergillus* spp. and other dimorphic fungi, and are the antifungal drugs generally in use by intrauterine infusion. Nystatin (0. 5–2. 5 million UI) and fluconazole (100 mg) are active only against *Candida* spp. [97]. Although many antifungal agents are available for intrauterine therapy, few data are available about the disposition of antifungal drugs in endometrial tissue after oral admin‐ istration. A recent study showed that the oral administration of fluconazole (14 mg/kg once, followed by 5 mg/kg every 24 hours) resulted in mean plasma and endometrial tissue levels above the minimal inhibiting concentration for *Candida Albicans* for the entire treatment period, without potential sequel. Thus, oral fluconazole can be considered in a multimodal and multidrug treatment protocol to treat fungal endometritis in the mare [137]. Knowledge of commonly encountered fungi infecting the mare's reproductive tract and their respective drug susceptibilities should improve treatment efficacy in mares with fungal endometritis, when culture results are not available or incomplete. A study reports that the spectrum of fungal isolates from uterine samples from mares with reproductive problems includes 99 to 100% of organisms susceptible to the polyenes, while response to the azoles varied from 47 to 81%. Yeast isolates were 100% susceptible to the polyenes and least susceptible to miconazole (48%) while isolates of mold with septated hyphae were most susceptible to natamycin (100%) and least susceptible to fluconazole (0%). Results from this study suggest that polyenes are effective against uterine fungal isolates in vitro and may be the empiric treatment of choice for fungal endometritis, when cultural and susceptibilities examination are lacking. Furthermore, resistance to specific azoles was also observed increasing over time [129].

The study about the potential use of chitin inhibitors for the treatment of fungal infections in the horse gave contradictory results. These substances have insecticidal properties due to their ability to inhibit chitin synthesis, polymerization and deposition. It was supposed that they might interfere with the formation of new chitin in the cell wall, but have no effect on the fungal cell walls that have already formed. Thus, their potential effect may consist in the prevention of a new fungal growth and, thus, reinfection. Lufenuron is an insecticide chitin inhibitor that was evaluated as a treatment for endometrial fungal infections. Preliminary results were promising, and intrauterine lavages, performed with lufenuron suspended in sterile saline solution, were effective in eliminating fungal endometritis in four mares [144]. However, further studies did not confirm the efficacy against *Aspergillus* spp. or *Fusarium* spp. in vitro and indicated that very low concentrations in whole blood of horses were achieved after oral administration [145]. Further investigations about the efficacy of chitin inhibitors are necessary before they can be indicated for the treatment of fungal endometritis in the horse [136].

Another suggested protocol consists in morning lavage with 2% acetic acid solution followed by intrauterine irrigation with the specific antifungal antibiotic in the afternoon. The length of this therapy should be of 7–10 days. If uterine culture and cytology obtained in the first day of the next estrus are negative, the mare is bred with the minimum number of insemination and using sterile techniques and double-guarded pipette, to prevent accidental seeding with organisms from reservoir sources [136].

The prognosis for future fertility of mares affected by fungal endometritis is guarded to poor. The success of the therapy relies on the timely identification and treatment of delayed uterine clearance, poor perineal conformation, or fungal colonization of the more distal reproductive tract, the correct length of the treatment period, the appropriate choice and dosage of the antifungal [132]. The uterine biopsy for many of these mares indicates significant fibrosis, probably due to fungal infection, as well as to a previous bacterial infection and age-related changes. Once the fungal infection is resolved, a uterine biopsy may be advised to quantify the resulting uterine damage [136].
