Cellular and humoral defence mechanisms in mares susceptible and resistant to persistent endometritis

Both random and directional migration of blood neutrophils from 9 mares susceptible to persistent endometritis were significantly less (p less than 0.05) than neutrophils from 8 resistant mares. Serum from susceptible mares had significantly more (p less than 0.01) chemotactic activity than serum from resistant mares. Although phagocytosis of yeast blastospores by blood neutrophils from 4 resistant and 3 susceptible mares was similar, uterine neutrophils from susceptible mares were significantly worse (p less than 0.01) at phagocytosis than uterine neutrophils from resistant mares. Uterine washings from 17 susceptible mares were significantly better at opsonising yeast blastospores than washings from 14 resistant mares; however, washings from both groups had a similar ability to promote killing of S. zooepidemicus by neutrophils. When an immunologically non-specific endometritis was induced, washings from 3 susceptible mares were significantly worse at promoting bactericidal activity by 144 h than washings from 4 resistant mares (p less than 0.01). Haemolytic complement activity was significantly greater (p less than 0.001) in washings from 17 susceptible mares than from 14 resistant mares. Induction of acute endometritis resulted in high levels of haemolytic complement activity in 2 of 3 susceptible mares at 24 and 144 h, but only in small increases in 4 resistant mares. Thus, some abnormalities in neutrophil function were detected and a possible defect in promotion of neutrophil bactericidal activity by uterine secretions from susceptible mares but there was no evidence for any deficiency in haemolytic complement activity.     

Comparison of uterine protein content and distribution of bacteria in the reproductive tract of mares after intrauterine inoculation of Haemophilus equigenitalis or Pseudomonas aeruginosa

Two groups of 3 mares were inoculated with Haemophilus equigenitalis or Pseudomonas aeruginosa on the 1st day of estrus. Uterine flushing samples were recovered on day 3 of estrus and day 8 after ovulation for each cycle. Mares were killed 22, 25, and 30 days after inoculation with P aeruginosa and 45, 46, and 49 days after inoculation with H equigenitalis. Pseudomonas aeruginosa was recovered from the uterus of 2 mares 48 hours after inoculation. Although the initial flushing sample of 1 of these 2 mares had an increased total protein concentration, there appeared to be little difference between protein concentrations of other uterine flushing samples. Haemophilus equigenitalis was recovered from the uterus of each of the 3 mares at postmortem. One mare had a slight, purulent discharge from the vulva. Total protein values were not increased in flushing samples from this mare after inoculation with H equigenitalis. Total protein values decreased in the last flushing sample of each of the 2 remaining mares. Swabbing the uterus was more effective than was homogenizing the uterine mucosa in isolating H equigenitalis.     

Opsonins of Streptococcus in uterine flushings of mares susceptible and resistant to endometritis: control of secretion and partial characterization

The release of opsonins into the uterine lumen of mares susceptible or resistant to endometritis was examined after intrauterine inoculation of a filtrate of Streptococcus culture fluid or vehicle. Uterine flushings were collected at 0.5 hour before and 2, 4, 6, 8, and 24 hours after inoculation on day 2 or 3 of estrus and on day 7 or 8 after ovulation. Amounts of opsonins in flushings were quantified as the H2O2 produced by leukocytes incubated with flushings-opsonized bacteria, compared with H2O2 produced by leukocytes incubated with nonopsonized bacteria. Opsonin values in flushings increased (P less than 0.025) in all mares after inoculation of filtrate or vehicle. For mares resistant to endometritis, opsonin values were greater at diestrus than at estrus. The opposite was true for mares susceptible to endometritis, resulting in a status (susceptible vs resistant) X stage of cycle interaction (P less than 0.025). Overall, opsonins were higher (P less than 0.05) in flushings of mares susceptible to endometritis than in flushings of mares resistant to endometritis, but this difference was only apparent at estrus. Preliminary characterization of opsonins in uterine secretions by ammonium sulfate fractionation and gel filtration indicated that opsonins were mainly associated with an ammonium sulfate-soluble fraction of high molecular weight (greater than 4 X 10(6] and an ammonium sulfate-precipitable fraction that was associated with immunoglobulin G.     

An Investigation of Uterine Nitric Oxide Production in Mares Susceptible and Resistant to Persistent Breeding‐Induced Endometritis and the Effects of Immunomodulation

The first objective of this study was to evaluate intrauterine nitric oxide (NO) and endometrial inducible NO synthase (iNOS) in mares susceptible or resistant to persistent breeding‐induced endometritis (PBIE) within 24 h after breeding. Mares susceptible (n = 6) or resistant (n = 6) to PBIE were inseminated over five cycles, and uterine secretions and endometrial biopsies were collected before and 2, 6, 12 and 24 h after insemination. Uterine secretions were analysed for NO and biopsies were analyzed for iNOS expression. A second experiment evaluated the effect of treatment with dexamethasone or mycobacterial cell wall extract (MCWE) on uterine NO production and endometrial iNOS mRNA expression. Six susceptible mares were inseminated over three cycles with (i) killed spermatozoa without treatment (control), (ii) killed spermatozoa with 50 mg of dexamethasone IV or (iii) MCWE IV 24 h prior to insemination with killed spermatozoa. Six resistant mares were inseminated with killed spermatozoa as a control. Six hours after breeding, uterine biopsies and secretions were collected and evaluated for NO and iNOS mRNA. In Experiment 1, resistant mares had an increase in iNOS mRNA expression 2 h post‐breeding compared to baseline (p = 0.045), 12 h (p = 0.014) and 24 h (p = 0.001). Susceptible mares had higher expression 2 h compared to 6 h (p = 0.046). No differences were observed in mRNA or protein expression of iNOS between resistant and susceptible mares. Resistant mares had a relatively steady amount of total intrauterine NO over 24 h, while susceptible mares had an increase over time, with a significantly higher increase in total NO than resistant mares at 6 (p = 0.04) and 12 h (p = 0.032). In Experiment 2, no differences were observed for iNOS mRNA expression. Susceptible mares had increased NO when compared to resistant mares (p = 0.008) and MCWE decreased NO (p = 0.047).     

Uterine clearance mechanisms during the early postovulatory period in mares

Uterine response to inoculation with Streptococcus zooepidemicus organisms, 51Cr-labeled 15-microns microspheres, and charcoal was evaluated in 9 mares (4 resistant and 5 susceptible to endometritis) to determine mechanical and cellular clearance rates during the early postovulatory period. Mares were inoculated at estrus prior to ovulation during estrous cycles 1, 3, and 5. Uterine swab specimens for aerobic and anaerobic bacteriologic culture and serum for progesterone determination were obtained on postovulation day 3 during estrous cycle 1, on the day of ovulation during estrous cycle 3, and on postovulation day 5 during estrous cycle 5. Immediately thereafter, the uterus was irrigated with 50 ml of sterile physiologic saline solution containing tracer amounts of 125I-labeled human serum albumin. Streptococcus zooepidemicus was isolated from 10 of 15 (67%) uterine specimens collected from susceptible mares and incubated aerobically. Escherichia coli also was isolated from 2 of the 10 specimens incubated aerobically. Anaerobic bacteriologic culture of specimens from all mares yielded no growth. Chromium-labeled microspheres were recovered twice from 2 susceptible mares, on day 0 and day 5. Charcoal was retained in 5 specimens collected from 3 susceptible mares. Bacteriologic culture of specimens from resistant mares did not yield growth. On day 0, chromium-labeled microspheres and charcoal were recovered once from 1 resistant mare. Mares susceptible to endometritis accumulated more fluid within the uterine lumen after ovulation than did resistant mares (mean +/- SEM, 52.73 +/- 15.22 ml and 7.41 +/- 1.96 ml, respectively; P less than 0.01). From this study, it appeared that uterine cellular and bactericidal mechanisms are dysfunctional during the early postovulatory period. However, there appeared to be no disruption of the mechanisms responsible for mechanical clearance of materials inoculated in the uterus.     

Comparison of peripheral blood and uterine-derived polymorphonuclear leukocytes from mares resistant and susceptible to chronic endometritis: chemotactic and cell elastimetry analysis

The functional competence of peripheral blood and uterine-derived polymorphonuclear leukocytes (PMN) from 12 mares were analyzed for chemotactic responsiveness using a chemotactic chamber (filter) assay and for deformability by cell elastimetry analysis. Peripheral blood PMN obtained from control mares and from 8 mares experimentally inoculated via the uterus with 1 x 10(9) Streptococcus zooepidemicus had similar normal chemotactic responsiveness and were highly deformable before and at 12 hours after inoculation. Uterine PMN obtained 12 hours after uterine inoculation with S zooepidemicus from resistant mares were not as deformable as peripheral blood PMN, but were within normal functional limits. The chemotactic responsiveness of uterine PMN from these mares was normal. Uterine PMN obtained from mares considered susceptible to endometritis 12 hours after uterine infection did not have chemotactic responsiveness and were nondeformable. The results indicated profound differences in the functional competence of uterine PMN between mares considered resistant and susceptible to chronic endometritis.     

Chemotactic properties and protein of equine uterine fluid

Forty uterine fluid samples were obtained from 4 mares classified as resistant to uterine bacterial infection. The uterus of each mare was flushed with 50 ml of saline solution during estrus and diestrus of successive estrous cycles. Bacteria or fungi were isolated from 4 samples, and 7 additional samples were obtained from a mare with active intrauterine infection. Fluid volumes obtained during estrus (means = 40.3 +/- 11 ml) tended to be greater than those recovered during diestrus (means = 36.8 +/- 7.9 ml), but the difference was not significant. Concentrations and yields of protein in recovered fluid did not vary significantly with the day of the estrous cycle. Protein concentration was significantly increased in samples from the infected uterus (P = less than 0.001). The ability of uterine fluid samples to attract neutrophils was measured using chemotactic chambers. There was no significant difference between distances migrated by neutrophils toward fluids obtained during estrus or diestrus. Chemotaxis scores tended to be higher with samples from the infected uterus, and the difference was significant for samples from 2 mares. Chemotaxis was neither significantly correlated with protein concentration of uterine fluid, nor with serum estrogens or progesterone.     

X-ray Microanalysis of the Secretory Epithelium of the Endometrial Glands and Intraluminal Uterine Fluid in Oestrus Mares

X‐ray microanalysis was used to determine the occurrence of transudation as a source of intrauterine fluid in mares during oestrus. The content of some selected elements was studied in secretory vesicles of the epithelium of endometrial glands and in intraluminal fluid of healthy mares of varying age and parity. An in situ cotton tampon was used to absorb intrauterine fluid from 23 gynaecologically healthy mares during oestrus. Immediately after tampon withdrawal, endometrial tissue for biopsy was removed from the uterine body/horn junction while videoendoscopy was performed. The tissue specimens were frozen ultra‐rapidly against a copper surface chilled with liquid N₂. X‐ray microanalysis of frozen fully hydrated endometrial samples and of microdroplets of intraluminal fluid (n = 20) was performed, using a SEM microscope equipped with a cryo‐stage and an X‐ray microanalysis system. The concentrations of sodium (Na), potassium (K), chlorine (Cl), sulphur (S), phosphorus (P) and calcium (Ca) in the epithelium of endometrial glands and in intraluminal fluid were compared with those in serum. There was a significant difference in the concentrations of all analysed elements between the secretory gland epithelium and the intraluminal fluid apart from S. The major elements in glandular epithelium were K and P. Although there was a significant difference in the concentrations of Na, Cl, S and Ca between the uterine fluid and serum, there was still a conspicuous resemblance in the relationship between the different elements in uterine fluid and serum, with Na and Cl being the dominant elements. Sulphur and especially Ca were present only in small amounts throughout. Neither age nor parity affected the elemental concentrations of Na, K, Cl, S, P and Ca in gland epithelium, uterine fluid or serum. It was concluded that serum transudation contributes to a great extent to the composition of uterine fluid, having a diluting effect upon the specific secretion of the uterine glands.     

Making sense of equine uterine infections: the many faces of physical clearance

Equine uterine infections inflict major losses on the equine industry. Persistent inflammation of the oviduct and uterus leads to loss of the conceptus and mares susceptible to infection have weakened uterine defences partly due to retention of inflammatory exudate. Bacteria may trigger inflammation, resist phagocytosis, or adhere to the endometrium and types of infection range from genital commensals in susceptible mares to reproductive pathogens in normal mares. Uterine infections are diagnosed by history, detection of uterine inflammation, and isolation of typical organisms and susceptible mares may be identified by detection of intrauterine fluid during oestrus, or at 6-48 h post-breeding. Therapy includes oxytocin, uterine lavage, antibiotics, and prostaglandin analogues and clinical studies indicate additive benefits of oxytocin and antibiotics. Improved conception rates have been associated with autologous, intrauterine plasma, despite controversy about its bactericidal efficacy. Because of the potential for endometrial damage, intrauterine antiseptics require caution.     

Effect of exogenous ovarian steroids on the uterine luminal prostaglandins in ovariectomised mares with experimental endometritis

Prostaglandins (PGs) F and E2 were measured in lavage fluid from the uterus of ovariectomised mares after experimental induction of uterine inflammation. Treatment with progesterone alone, or progesterone followed by oestradiol, significantly increased the concentrations of these PGs in the lavage compared with mares treated with oestradiol or control mares. Ovarian steroids, therefore, influenced uterine PG synthesis in response to an inflammatory stimulus. To determine whether the uterine lavage procedure might contribute to the concentrations of prostaglandins in the lavage, the procedure was also performed on six intact mares. With the exception of washings obtained at luteolysis, uterine concentrations of PGF (measured as the plasma metabolite 15-keto-13,14-dihydro PGF2 alpha) had returned to prewashing levels within 30 minutes of the start of uterine lavage. Lavage was therefore unlikely to have influenced the concentrations of prostaglandins in the lavage fluid.     

Effect of intrauterine infusion of Escherichia coli endotoxin in postpartum pony mares

Fifteen pony mares were assigned to 1 of 3 treatment groups after foaling: Group 1, 35 ml of sterile saline solution was infused into the uterine lumen within 24 hours after parturition (6 mares); group 2, 300 mg of Escherichia coli endotoxin was infused into the uterine lumen within 24 hours after parturition (6 mares); and group 3, 300 mg of E coli endotoxin was infused into the uterine lumen between 72 and 96 hours after parturition (3 mares). Rectal temperatures were taken at -1, -0.5, 0, 0.5, 1, 1.5, 2, 3, 4, and 5 hours after treatment. Venous blood samples were also taken at these times for routine WBC counts. Data were analyzed as a repeated measurement design with linear and quadratic orthogonal contrasts performed where significant time and interaction with time occurred. Pretreatment averages of total WBC and neutrophil counts were compared with their nadir posttreatment averages by a t test when treatment-by-time interaction was significant for the parameter. Rectal temperature (37.9 +/- 0.1 C) remained stable and did not vary among treatment groups after intrauterine infusions. In contrast, total WBC and neutrophil counts did vary among treatment groups across time. However, for treatment groups 1 and 3, neither blood total WBC count nor neutrophil count after intrauterine infusions was different from pretreatment observations. In group 2, total WBC count decreased (P less than 0.10) from a pretreatment average of 11.5 +/- 0.4 X 10(3) cells/mm3 to a nadir concentration of 10.0 +/- 0.6 X 10(3) cells/mm3 by 60 minutes after infusion of endotoxin into the uterus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endometrial nitric oxide synthase activity in mares susceptible or resistant to persistent breeding‐induced endometritis and the effect of a specific iNOS inhibitor in vitro

Emerging research suggests that the nitric oxide system may play a role in persistent breeding‐induced endometritis (PBIE) in the mare. Differences in uterine nitric oxide (NO) levels between mares susceptible or resistant to PBIE and a dose‐dependent inhibitory effect of NO on uterine contractility have been demonstrated. The objectives of this study were to investigate the difference in total nitric oxide synthase (NOS) activity of the endometrium between susceptible and resistant mares and the effect of a specific inducible nitric oxide synthase (iNOS) inhibitor on the endometrial NOS activity in vitro. Six susceptible and six resistant mares were selected based on preset criteria and the results of an intrauterine challenge with killed spermatozoa during oestrus. Endometrial biopsy samples were collected 24 hr post‐challenge and cultured at 37°C for 24 hr in L‐arginine supplemented minimum essential medium with or without a specific iNOS inhibitor (1,400 W dihydrochloride, 1 mM). The medium and the cultured endometrial tissue were collected after 24 hr of culture and assayed for NO and total protein, respectively. Total NO content of the medium, normalized to endometrial tissue wet weight or total protein, was used as a measure of endometrial NOS activity. Non‐parametric tests were applied for statistical analysis. Susceptible mares had significantly greater endometrial NOS activity than resistant mares. The iNOS inhibitor treatment significantly reduced NOS activity in endometrial samples derived from susceptible and resistant mares. These findings provide a basis for in vivo testing of specific iNOS inhibitors as preventative or therapeutic options for PBIE in mares.     

SCINTIGRAPHIC MEASUREMENT OF UTERINE CLEARANCE IN MARES

Uterine clearance of technetium 99m-albumin colloid (^99mTc-μAA) was qualitatively and quantitatively measured in 5 reproductively normal mares and 5 mares susceptible to endometritis (infertile). The percentage of 370 MBq ^99mTc-μAA cleared from the uterine lumen within 2 hr of intrauterine infusion was measured in 10 mares on day 3 of estrus and 48 hr after ovulation. The procedure was repeated 3 times on day 3 of estrus in 6 mares to determine repeatability. Six mares were infused with 1110 MBq ^99mTc-μAA on day 3 of estrus to evaluate the effect of increasing the dose to reduce the imaging time. There was no statistically significant difference in the mean percentage of radiocolloid cleared from the uterus during day 3 of estrus or 48 hr after ovulation or in the percent cleared when the studies were repeated in individual mares. There was no statistically significant difference in uterine clearance between the 370 and 1110 MBq dose studies in each mare from 15 to 120 min. Reproductively normal mares cleared approximately 50% of the radiocolloid from the uterus by 120 min while susceptible mares cleared less than 15%.     

Concentrations of uterine luminal prostaglandins in mares with acute and persistent endometritis

Intrauterine infusion of 1 per cent oyster glycogen solution was used to induce acute endometritis in four genitally normal mares. Numbers of viable neutrophils recovered in uterine washings had increased by 1 h after infusion and remained elevated for at least 72 h. There was a significant correlation between numbers of viable neutrophils and total protein concentrations and between prostaglandin (PG)F and PGE2 concentrations in washings. There was also a significant relationship between concentrations of 15-keto-13, 14-dihydro PGF2 alpha in plasma and PGF in washings. Intrauterine concentrations of PGF were influenced by cycle stage and in turn the induced acute endometritis interfered with normal ovarian function. Mares with persistent endometritis had significantly higher concentrations of PGF and total protein and percentage of neutrophils and mononuclear cells in washings than normal mares. White blood cells from mares were capable of producing PGF and PGE2 in vitro.     

Concentrations of immunoreactive leukotriene B4 in uterine lavage fluid from mares with experimentally induced and naturally occurring endometritis

Acute endometritis was induced in ovariectomized pony mares by infusion of a 1% solution of oyster glycogen. Maximum concentrations of immunoreactive leukotriene B4 in uterine washings coincided with the greatest rate of infiltration of neutrophils into the uterine lumen. Concentrations of immunoreactive leukotriene B4 decreased to basal levels 6 h after infusion and were unaffected by administration of ovarian steroids to ovariectomized mares. Uterine washings from mares with persistent endometritis did not contain significantly different concentrations of leukotriene B4 from genitally normal mares.     

A study of Klebsiella pneumoniae infection in the uterus of the mare.

Two experiments incorporating 13 mares were conducted for the purpose of producing and monitoring intrauterine infection with Klebsiella pneumoniae. In the pilot study, the infection was produced with strains of K pneumoniae type 68 and type 10 isolated from the genital tract of stallions with a history of breeding problems. In the principal study, K pneumoniae type 68 was used to produce the infection. Tampons and guarded culture swabs were used to obtain uterine samples in the pilot study. In comparing the efficacies of isolation of K pneumoniae with the tampons and isolation with standard guarded culture swab, the tampon proved to be a more reliable means with which to isolate K pneumoniae and was used in the principal study. In both studies, inoculated mares became infected and remained infected at least until the postinoculation estrous cycle was initiated or was completed. Some of the inoculated mares remained infected through more than one estrous cycle. The numbers of K pneumoniae decreased in the uterus of mares after completing the estrous cycle after inoculation. Klebsiella pneumoniae was not demonstrable in frozen tissue sections of uterine biopsy specimens stained by fluorescent antibody technique. Postinoculation sera antibody titers to K pneumoniae, as determined, using the capsule swelling technique, were no higher than 1:8.     

Quantitation of the immunoglobulins in reproductive tract secretions of the mare

IgG, IgA, IgM and albumin concentrations were measured in serum, follicular fluid and oviductal, uterine and intestinal secretions of the horse. Follicular protein concentrations were found to be dependent on serum concentration and molecular size. Of the immunoglobulins only IgG was detectable in oviductal secretions, but IgG:albumin ratios did not differ significantly from those in serum. IgG, IgA and IgM were measured in uterine secretions, with IgG predominant. Serum transudation into uterine secretions was minimal. In intestinal secretions, IgA levels were slightly higher than IgG, with albumin and IgM at low levels. In five mares with histories of chronic metritis, IgG, IgA and albumin concentrations were significantly elevated in uterine secretions.     

Antibody response in the bovine genital tract to intrauterine infusion of Actinomyces pyogenes

Antibody titres and immunoglobulin concentrations were measured in serum and in uterine and vaginal secretions from five heifers after intrauterine infusion of Actinomyces pyogenes. Infection stimulated an increase in uterine antibody titres of IgG2 and IgA, which approached significance. There was no increase in antibody titres in vaginal secretions or in serum. For each class of immunoglobulin, the ratio of specific antibody to immunoglobulin tended to increase in uterine secretions after infection, whereas no change was detected in the serum. These results indicate that at least a portion of the antibody was derived from local uterine synthesis.     

Endometritis in the mare caused by a Coryneform organism--a case report and experimental studies

Persistent purulent endometritis in a mare was attributed to an unclassified species of Corynebacterium. Following intrauterine infusions of 20% betadine for 5 days the purulent vulval discharge ceased and the mare appeared clinically normal. Based on histological examination of endometrial biopsy samples, the severe acute inflammatory reaction had largely resolved 2 days after therapy. Three maiden mares considered resistant to bacterial endometritis received single intrauterine inoculations of 1.8 X 10(9) colony-forming units of the Corynebacterium species. The uterine response was followed by vaginal speculum examinations, uterine cultures and cytology, and endometrial histology. After an acute inflammatory reaction, each mare had recovered completely within 2 weeks. Most rapid recovery occurred in the mare in estrus at the time of inoculation. Subsequent secondary infections were detected in two mares. The uncertainty of correlations between results obtained by various diagnostic techniques emphasized the problems associated with each. This report illustrates the concept that endometritis in individual mares may relate more to as yet unidentified "mare factors" controlling uterine defense than to primary invasion by bacteria.     

Use of a cervical stent for long‐term treatment of pyometra in the mare: A report of three cases

An effective long‐term treatment is necessary for mares with pyometra, because the condition tends to recur. In many affected animals, several conformational or anatomical anomalies contribute to impaired uterine clearance. Ovariohysterectomy is the surgical procedure of choice. Conservative therapy consists of draining and flushing the uterus, and systemic anti‐inflammatory and antimicrobial treatment. Uterine secretions tend to accumulate again after local treatment, especially in mares with poor vaginal conformation or cervical adhesions. Herein, we describe three cases in which a cervical stent was used in mares after mechanical or manual dilation of the cervix to achieve permanent draining of the uterus. The mares remained symptom‐free for up to 6 years and exhibited good clinical progress and good performance in competitions. Potential complications of the procedure include loss of the stent and obstruction caused by viscous secretion. A cervical stent is a relatively easy and low‐cost option for the long‐term treatment of pyometra in mares, particularly in cases where excessive costs of surgery and risks of a general anaesthesia are to be avoided.