Organisms

There is no “normal flora” in the uterus of the cycling mare. If culture technique is good, any organism isolated from the uterus is a potential cause of inflammation and infertility. Organisms most often isolated from mares with uterine infection include Streptococcus zooepidemicus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa,⁵ and very rarely the microaerophilic venereal pathogen Taylorella equigenitalis.⁶ Anaerobic bacteria such as Bacteroides fragilis also appear to be significant causes of endometritis, especially in postpartum and foal heat mares.⁷ Causative organisms are usually found in the caudal genital tract of the healthy mare, with numbers decreasing as one proceeds past the vaginovestibular sphincter into the vagina and uterus.⁸ The clitoral sinus is an important reservoir for these bacteria and for introducing them into the uterus during breeding or vaginal procedures.⁸ This is especially true of T. equigenitalis and anaerobic bacteria.^7,9 Outbreaks of T. equigenitalis and early recognition of infection are of great concern to the equine industry and animal health regulators.^9–11T. equigenitalis is a reportable disease in the Northern Hemisphere and a notifiable disease in the United Kingdom (UK) and most other countries.

Types of Infection

Bacterial uterine infections usually appear as either venereal, chronic, or postbreeding infections.¹² Venereal infections are caused by a stallion spreading a reproductive pathogen between mares. The classic example of such a pathogen is T. equigenitalis,⁶ the causative agent of contagious equine metritis (CEM). Klebsiella¹³ and Pseudomonas¹⁴ are also capable of inducing venereal infections. Chronic infections are probably caused by a combination of potentially virulent bacteria and weakened reproductive defenses, especially in mares predisposed to self-contamination.¹² These may appear as persistent infections with gram-negative bacteria in older mares with poor perineal conformation and are frequently difficult to treat. Postbreeding infections occur following deposition of semen in the uterus, following either natural mating or artificial insemination. Isolated organisms are commensals of the mare, differentiating the condition from a venereal infection. Postbreeding infections are usually transient, with rapid expulsion of semen and bacterial contaminants within hours of breeding,^2,15 but in susceptible mares they can become persistent due to failure of the uterus to expel fluid.¹⁶ Persistent postbreeding infections are a major cause of infertility, with S. zooepidemicus being a commonly isolated organism.¹⁷

Uterine Defense in Health and Disease

When discussing therapy and prevention of bacterial uterine infections it is valuable to understand how reproductive defenses against infection function in health and disease. Reproductive defenses include physical barriers to contamination and uterine defenses.

The physical barriers of the reproductive tract are the first line of defense against infection. These barriers include the vulva, vaginovestibular sphincter, and cervix. They prevent feces, air, and environmental bacteria from entering the reproductive tract. Feces tends to enter because of the proximity of the anus; air tends to enter as a result of negative pressure in the cranial vagina. In young mares the vulva lies mostly below the level of the pelvic brim. This orientation allows fecal material to fall past without adhering and requires upward movement to penetrate the vestibule, providing an effective barrier to feces and air. In older mares the perineal conformation may shift, pulling the vulva onto the pelvic floor.¹⁸ Feces falls directly onto the vulva, predisposing the mare to fecal contamination and facilitating the entry of air.

The vaginovestibular sphincter forms a second physical barrier. Parting of the vulval lips should not elicit a “sucking sound,” because the vaginovestibular sphincter prevents entry of air into the cranial vagina. Caslick’s procedure allows one to increase the effectiveness of the vulval seal or to compensate for an ineffective vaginovestibular sphincter.¹⁹

The cervix is the final physical barrier to infection of the uterus. It is a dynamic seal, opening in estrus and closing in diestrus and pregnancy. When open it allows semen into and fluid out of the uterus; when closed it forms a seal against the inward passage of microorganisms or the outward passage of the embryo.²⁰ The more tenacious cervical mucus of diestrus and pregnancy, in comparison with estrus, probably improves the cervical seal. Mares can remain fertile with anatomic imperfections in either the vulva or vaginovestibular sphincter, but less so the cervix. Damage to the cervix, such as from dystocia, can impair the ability of a mare to conceive or carry a foal, especially in cases of complete cervical lacerations. However, some mares with incomplete cervical lacerations may successfully carry foals to term. If the cervical seal is broken, the mare becomes vulnerable to inflammation by ascending organisms or air. If the cervical lumen is blocked by an adhesion or failure to dilate, pyometra may result from the inability to drain fluid. Surgical repair is an option in mares with cervical lacerations.²¹

An array of uterine defenses protects the uterus when the physical barriers to infection are breached. Physical barriers are always breached during breeding, when either the stallion ejaculates forcefully into the uterus or the artificial inseminator deliberately penetrates the cervix to deposit semen in the uterine body or even the uterine horns. Other intrauterine procedures, including culture, cytology, biopsy, endoscopy, lavage, infusion, embryo transfer, etc., are breaches of the physical barriers and may induce inflammation and even infection.⁸ Researchers have experimentally breached the physical barriers of mares with infusions of bacteria, irritants, or semen to determine how uterine defenses fight infection.

From these studies it is clear that the young, healthy mare is highly resistant to infection and quickly eliminates irritants from the uterus; the uterus of a healthy estrous mare can eliminate experimental infection with good subsequent fertility.^3,4 The sequence of events appears to be as follows: immediately following introduction of bacteria there is an acute uterine response, peaking at about 6 hours, in which plasma proteins and neutrophils move into the uterine lumen. Uterine contractions propel any inflammatory fluid retrograde through the cervix, and after about 12 to 24 hours uterine cultures are usually negative.¹⁵ During the acute response, especially within the first 6 hours following inoculation, serum complement and antibody facilitate the phagocytosis of bacteria by neutrophils.²² Meanwhile, uterine contractions facilitate removal of fluid through the cervix, helping to clear the uterus of debris.¹⁶ The postbreeding response of mares to spermatozoa is identical.^2,23 In both situations the rapid, aggressive response observed would be necessary to eliminate exponentially growing bacteria. However, in some mares, although bacterial numbers decline during the acute uterine response, they rebound 12 to 24 hours later, and a more persistent inflammation develops.¹⁵ Thus a failure to eliminate all remaining bacteria allows infection to persist.

Failure to eliminate uterine fluid by uterine contraction appears to be related to persistent uterine infections.^24,25 It appears that a defect in the myometrium of susceptible mares is responsible for this failure to expel fluid,^16,26 although hypersecretion may contribute to fluid accumulation.²⁷ If fluid accumulates in the uterus, hypothesized uterine defenses may be disrupted.²⁸ Inflammatory fluid that is retained in the uterus appears to lose its ability to support phagocytosis.²⁹ Furthermore, it has been known since the 1920s that in free fluid, neutrophils and bacteria will stratify to different levels of suspension, allowing bacteria an opportunity to escape.³⁰ Lymphatic drainage has been proposed to play an important role in uterine clearance³¹ but is disrupted when fluid accumulates in the uterine lumen. Similarly, the cleansing of the uterine surface by a mucus blanket propelled by cilia³² would be rendered ineffective in the presence of a large volume of free fluid. Any accumulations of uterine fluid cause major disruptions to proposed uterine defenses, whether the uterine defense be phagocytosis, lymphatic drainage, or mucociliary clearance.

The stage of the reproductive cycle greatly influences uterine defenses. During estrus the ability to clear infection is greater than in diestrus.³³ This is probably due to the open cervix,²⁰ expulsive myometrial contractions,^34,35 more active phagocytosis,^36–38 and by more frequent posturing to urinate. During diestrus the ability to clear infection is significantly reduced. Mares maintained on high progesterone often establish infection.³⁹ Uterine contamination during diestrus is usually less than in estrus due to the tight cervix, thick cervical mucus, sexual refusal, and less iatrogenic introduction. Because mares may develop large follicles during diestrus, artificial insemination of mares with large follicles, but no other signs of heat, should be avoided.

Microbial Pathogenesis

Although many uterine infections arise from defects in the mare’s uterine defenses, bacteria themselves play significant roles in causing disease. The pathogenesis of bacteria during uterine infections in horses have not been well studied. In the 1920s, workers in Kentucky first noted that exudates in streptococcal mare uterine infections are watery, whereas those in Klebsiella infections are tenacious,^13,40 providing an interesting insight into potential differences in pathogenesis of different bacteria. More recent studies in other species and in horses have provided more detail. Some bacteria tenaciously adhere to epithelial surfaces, preventing their physical removal. This is well known in colonization of the genitourinary tract in humans⁴¹ and probably true in many persistent equine uterine infections. It appears that S. zooepidemicus has adhesive ability,^42–44 probably mediated in part by fibronectin binding⁴⁵ and the hyaluronic capsule.⁴⁶ Similarly, both pili and capsule on K. pneumoniae appear to mediate attachment to epithelial cells.⁴⁷ Furthermore, biochemically complex biofilms secreted by several genera of bacteria, especially P. aeruginosa, provide an adhesive matrix contributing to persistent, opportunistic infections with tenacious microcolonies.^48,49 Biofilms also provide inherent resistance to antibiotics and both cellular and humoral immune defenses.^50,51 The persistence of T. equigenitalis in the equine genital tract may also be due in part to secretion of extracellular material.^52,53

Streptococci may disrupt uterine defense by promoting the inflow of uterine fluid. Superantigens,^54,55 streptokinases,^56–58 and other toxins released by streptococci may cause hyperirritation of the endometrium, contributing to hyperemia and the in-rush of fluid. Superantigens probably play a significant role in the pathogenesis of strangles, though their role in uterine infections is less clear.⁵⁹ If fluid can be eliminated, for example by saline lavage, then streptococci appear to become vulnerable to uterine defenses.⁶⁰

Resistance to phagocytosis is an important property of many uterine pathogens, including S. zooepidemicus^46,61 and K. pneumoniae.⁶² This is probably mediated with S. zooepidemicus by a combination of antigenic variation,⁶³ antiphagocytic M-like proteins,⁶⁴ the hyaluronic capsule,⁴⁶ and Fc receptors.^65,66 Furthermore, because Fc receptors on the streptococcal cell wall disrupt the complement cascade, they may provide opportunities for gram-negative bacteria to colonize the uterus in mixed infections.⁶⁷ Resistance of K. pneumoniae to phagocytosis is mediated in part by the polysaccharide capsule.⁶²

History

The classic history of a susceptible mare is one who has foaled successfully for several consecutive breeding seasons but now is consistently failing to conceive or carry a foal. These mares are usually in their teens when the condition becomes apparent. A common presenting complaint is that she has failed to conceive despite repeated breedings on multiple cycles to a fertile stallion.¹⁷ A history of repeated bouts of uterine infections is an excellent predictor of susceptibility to infection.⁶⁸ To construct a useful reproductive history can be challenging because infertile mares tend to change hands frequently. Where possible, it is helpful to obtain from the owner a brief year-by-year account of the mare’s production (i.e., In what years did she foal? Who was she bred to? In those years that she did not foal, had she been bred and failed to carry the pregnancy or had the owners chosen to leave her open?) Did the mare have any previously diagnosed uterine infections? Identifying past years when the mare had dystocia or abortion is particularly important because it can indicate trauma to the cervix. This information can usually be obtained quickly over the phone or immediately before examining the mare.

Having obtained an overall picture of the mare’s reproductive life, more detailed questions concerning the most recent season can be asked. On how many cycles has the mare been bred and roughly how many breedings per cycle? What is the quality of the stallion’s semen, and what is the breeding method (i.e., live cover or artificial insemination with fresh, chilled, or frozen semen)? When was the first breeding of the year, and how was it determined that the mare was cycling? Commonly mares are bred during spring transition; the contamination of the uterus that results from wasted breedings associated with lack of ovulation may significantly retard the mare’s fertility during subsequent cycles. Were cycles of normal length, or did she have short or long interestrous intervals, or was the mare short cycling? The inflammation of an infected/inflamed uterus can induce the release of prostaglandin, causing luteolysis and thus shortening the estrous cycle.⁶⁹ It is also useful to review management of the mare to determine how closely the mare was monitored. Was fluid detected in the uterus before or after breeding, and, if so, how was it managed? Was ovulation date determined? What were the results of uterine cultures, cytologic examinations, or other diagnostic work if they were performed? What previous treatment has the mare received, and what was the result? From these types of questions it will be possible to determine the nature, severity, and duration of the infertility, uncover management problems, and help identify uterine infection as a possible cause of infertility.

Physical Examination

Physical examination of the mare may provide clues to potential causes of infertility. A forward-tilted pelvis, poor perineal conformation, tendency to suck air into the vagina, and overall health of the mare can quickly be determined. A forward tilt to the pelvis may indicate impaired drainage of the reproductive tract or a tendency to pool urine in the cranial vagina. Although urine pooling is not usually associated with uterine infections, the sterile inflammation is sufficient to compromise fertility. Mares in poor condition may be more predisposed to infection overall and may tend to have poorer perineal conformation. Mares who have failed to shed their hair coat are probably not cycling,⁷⁰ and this may indicate contamination due to wasted breedings if presented for current infertility.

Palpation, Ultrasonography, and Scintigraphy

Transrectal palpation and ultrasonography of the reproductive tract and ovaries are powerful aids in diagnosing uterine infections and identifying susceptible mares, especially in the first 12 hours post breeding. Through ultrasonography one can quickly determine whether the mare is cycling or not, determine the stage of cycle, whether there is fluid in the uterus, the nature of the fluid, the number and size of endometrial cysts, confirm that the mare is not pregnant, and detect uterine inflammation.⁷¹ Accumulation of fluid in the uterus is one of the cardinal signs of a mare susceptible to infection and may indicate an ongoing infection. In a field setting, mares predisposed to uterine infection tend to have ultrasonographically more fluid in the uterus than normal mares over the course of an estrous cycle.²⁴ Whereas normal mares will have little or no fluid in the uterus (a trace of fluid is common during estrus), susceptible mares may accumulate significant volumes of fluid in the uterus during estrus that distend the lumen. In the postbreeding period, susceptible mares will retain fluid in the uterus, whereas normal mares should have little or no uterine fluid 6 to 12 hours after breeding. Accumulations of uterine fluid during the ovulatory period are consistently associated with decreased pregnancy rates.^72,73

Ultrasonographic examination of the uterus 24 hours post breeding is a valuable diagnostic procedure to diagnose and treat mares susceptible to uterine infections. Examination at 6 to 12 hours is even better⁷⁴ but may not always be possible. It is at this early postmating stage that the differences between healthy and susceptible mares are most apparent, and there is still time to institute effective therapy to evacuate the uterus.

Measurement of clearance of radiocolloid by scintigraphy is a powerful technique for diagnosing mares with a delay in uterine clearance, especially in a research setting,²⁵ but it is a technique currently unavailable to many practitioners. The technique involves intrauterine infusion of 10 mCi of technetium 99m-albumin colloid and monitoring rate of clearance using a large field-of-view gamma camera. Reproductively normal mares clear >50% of radiocolloid within 2 hours of infusion.

Through ultrasonography one can gain valuable insight into the nature of the uterine fluid during a uterine infection.⁷¹ If it is homogeneous and appears dark or black on the ultrasound screen, this usually indicates that the fluid is probably a mucoid transudate, suggesting that oxytocin alone may be appropriate for its evacuation. If it appears heterogeneous and flocculent, gray with white specks floating in it, this indicates cellular debris of an active infection; uterine lavage with saline may be necessary for most effective cleansing. Ultrasonography is particularly useful for detecting the presence of air in the uterus. The air-fluid interface is highly echogenic and appears as white sparkles within the uterus or vagina. Although a normal finding after intrauterine procedures, presence of air indicates that the mare’s external barriers to contamination may be failing.

Determining the exact day of ovulation by ultrasonography is also important in management of uterine infections. The embryo enters the uterus on day 6 (∼144 hours) post ovulation, allowing a short window of opportunity to cleanse the uterus. Monitoring day of ovulation allows one to institute intrauterine therapy with confidence before ovulation and up to about day 2 or at the most 3 post ovulation.⁷⁵ As the cervix tightens after ovulation, intrauterine therapy becomes less effective and more hazardous because evacuation of fluid by oxytocin is more difficult through the tighter cervix; the risk of introducing infection by intrauterine procedures increases as progesterone rises; the transient inflammation that intrauterine procedures induce may not subside before the embryo enters the uterus.

Vaginal Speculum Examination

Vaginal speculum examination is a useful way to detect mucoid or purulent exudates, urine accumulation, or inflammation of the reproductive tract.⁷⁶ The cervix of mares with uterine infections frequently appears “fire engine” red due to hyperemia. The anterior vagina may also be red and congested. Discharges from the uterus may pool on the floor of the anterior vagina and occasionally be seen dripping from the vaginal lips. Mucoid exudates resembling watery skim milk can sometimes be difficult to see on the floor of the vagina if the light is dim. More tenacious exudates may be seen clinging to the folds of the cervix. Urine pooling in the anterior vagina may sometimes appear as a thick yellow mucoid fluid, which can be mistaken for a suppurative exudate. Distinction between urine and suppurative exudate can be made by microscopic examination or by a reagent strip used to estimate blood urea nitrogen (Azostix).⁷⁷ Similarly, serum transudates may flow out of the uterus and pool on the vaginal floor, with a straw color very similar to urine. Distinction between serum and urine can also be made by using Azostix. Fluids may be obtained from the anterior vagina by tipping the speculum so fluid flows back into a specimen cup, or using an infusion pipette through the speculum attached to a syringe. Odor may also be helpful in determining the identity of the different fluids found in the mare’s vagina but is subjective.

Uterine Culture and Cytologic Examination

Uterine culture can provide much useful information in the diagnosis of uterine infections if used in conjunction with uterine cytologic examination. Uterine culture will provide misleading results if uterine cytologic examination is neglected.⁷⁸ Uterine cytologic examination provides direct evidence of uterine inflammation, usually in the form of large numbers of neutrophils relative to endometrial cells, adding significance to the culture results. If the examination is negative for neutrophils, then isolation of a few organisms on culture indicates a contaminated swab and should be regarded as a negative culture. Typical contaminants may include alpha-hemolytic streptococci, Enterobacter spp, and Staphylococcus epidermidis.⁷⁵ If cytologic examination results are positive, isolation of bacteria is probably significant, especially if in high numbers and in pure culture. Organisms such as S. zooepidemicus, E. coli, Klebsiella

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