Opsonization of bacteria by uterine secretions of cyclic mares.

Uterine flushings collected from mares before and after bacterial-induced inflammation were assayed for ability to opsonize Streptococcus zooepidemicus for phagocytosis by polymorphonuclear leukocytes. Opsonization was measured as the peak phagocytic rate of bacteria preincubated with uterine flushings relative to the peak phagocytic rate of unopsonized bacteria. Flushings from four mares with noninfected uteri were unable to opsonize bacteria regardless of whether uteri were flushed at estrus or on day 10 postovulation. In a second experiment, 7 X 10(9) live S. zooepidemicus were inoculated into the uterus of five mares during estrus. Uterine flushings collected at the estrus before inoculation or at the estrus after inoculation did not opsonize bacteria. Four of five flushings collected 6 hr post inoculation, however, were capable of opsonization. Based on heat inactivation at 56 degrees C, the opsonizing activity of one of four flushes was due to a complement protein. It was concluded that one aspect of the acute inflammatory response of the mare's uterus is accumulation of opsonins in the uterine lumen.

Uterine defense mechanisms in the mare: The use of intrauterine plasma in the management of endometritis.

Twenty-nine mares with either active or subclinical endometritis were treated with intrauterine infusions of their own plasma in one of two treatment schemes. Twenty-six mares actively infected, with an average of 2.4 years barren, were treated with combinations of saline irrigations and plasma infusions. Twenty-four showed clinical improvement and were bred, resulting in 15 pregnancies. Three mares with subclinical endometritis were treated post-breeding with a single plasma infusion and all became pregnant. The response to plasma infusions supports in-vitro observations which implicate serum-derived opsonins, particularly complement, as enhancers of phagocytic function.

A technique for long-term collection of uterine contents from mares.

A technique for collection of uterine contents from mares is described: it uses a purse-string suture of the cervix to retain a collecting device. Its use during collection of uterine fluids in various experiments is evaluated. The procedure was satisfactory for at least eight-hour collection periods.

Uterine defense mechanisms in the mare: Serum opsonins affecting phagocytosis of Streptococcus zooepidemicus by equine neutrophils.

The addition of serum to uterine secretions was shown to opsonize Streptococcus zooepidemicus and significantly enhance bacterial phagocytosis by equine neutrophils. Treatment of serum by heat inactivation at 56 degrees C, EDTA treatment, and C3 consumption reduced phagocytosis and therefore demonstrated that the process was complement-dependent. The amount of C3 present in uterine secretions was measured in a series of 14 mares infected with Streptococcus zooepidemicus . Ten of the 14 mares had detectable amounts of C3; however, the C3 had been cleaved and rendered nonfunctional. The importance of these findings in relationship to chronic uterine infections in the mare is discussed.

Reproductive function of mares given daily injections of prostaglandin F2alpha beginning at day 42 of pregnancy.

Mares at Day 42 of pregnancy received daily intramuscular (i.m.) injection of 5 mg of prostaglandin F2alpha (PGF2alpha) until the beginning of the first (Group I, n=3) or second estrous cycle (Group II, n=2). All mares aborted 3 to 4 d after the first injection; they displayed estrus 2 to 6 d after this injection. As determined by palpation per rectum and serum progesterone levels, each estrus was accompanied by an ovulation. Endometrial cups did not regress after PGF2alpha treatment since serum samples from the mares contained pregnant mare serum gonadotropin (PMSG) for at least 30 d after first injection, as determined by mare immunopregnancy test. After the first estrus, two of three mares in Group I displayed a prolonged diestrus (>25 d). In contrast, the first estrous cycle was short (8 to 12 d) for mares in Group II. Serum progesterone levels in the first 6 d postovulation were lower (P<0.05) for Group II than for Group I, indicating that formation of the corpus luteum was impaired by daily injections of PGF2. Results indicate that 1) daily injections of PGF2alpha can induce abortion in mares at Day 42 of pregnancy, 2) abortion is followed by estrus and ovulation, 3) the endometrial cups do not regress as a result of this treatment, and 4) daily injections of PGF2 can impair early corpus luteum development.

Immunoglobulins in uterine secretions of mares with differing resistance to endometritis.

The immunoglobulins IgA, IgG, IgG(T) and IgM were measured in uterine secretions from mares with normal uterine defense capability against bacterial contamination, and in mares with lowered resistance. Samples were collected for analysis at two stages of estrus and two stages of diestrus. All mares were then challenged with a pathogenic culture of Streptococci inoculated into the uterus. The immunoglobulins were quantitated on a similar schedule post-inoculation. Generally higher amounts of IgA, IgG and IgG(T) were found in the uterine secretions of mares which had an imparied resistance to endometritis than in mares with an efficient defense mechanisms. IgM was not detected in enough samples to suggest any differences.

Concentration of phenylbutazone (PBZ) and 13, 14-dihydro-15 keto PGF(2alpha) (PGFM) in blood and seminal plasma of stallions treated with PBZ.

Three stallions, 3 to 5 yr old and approximately 550 kg bodyweight, were used in a switchback experimental design to study the effect of daily, oral administration of 3g PBZ on the concentrations of PBZ and PGFM in blood (plasma) and seminal plasma (SP). Control and treatment periods were each 24 days' duration. Blood and semen samples were simultaneously collected every three days during these periods. Each stallion served consecutively as a control, treated, control, and treated subject for 24 days in each of the four periods. Concentrations of PBZ were obtained using HPLC and PGFM by specific RIA. Concentrations (mean +/- SE) of PBZ averaged 9.2 +/- 0.12 ug/ml in plasma but were undetectable in SP following the treatments. There was no significant difference in the plasma levels of PGFM between pre- and post- treatment values. However, there was a significant difference (P<0.001) in PGFM concentrations of seminal plasma before and after treatment. Results of this study suggest that daily, oral adminisration of 3g PBZ for 24 days to mature stallions can significantly decrease seminal plasma concentrations of PGFM. The physiological significance of this observation remains speculative.

Clearance of bacteria and non-antigenic markers following intra-uterine inoculation into maiden mares: Effect of steroid hormone environment.

Uterine response to inoculation with Streptococcus zooepidemicus organisms (antigenic markers) and 15-mum microspheres and charcoal (non - antigenic markers) was determined in seasonally acyclic maiden mares treated with either progesterone (P) n = 4, estradiol (E) n = 4 or oil vehicle (C) n = 4. At 3, 7 and 15 d after inoculation with bacteria and the 2 non - antigenic markers, uteri were flushed and the clearance of these materials, as well as the number of white blood cells and immunoglobulin concentration, determined. P-treated mares had higher numbers of bacteria and IgA and a greater volume of purulent fluid in the uterus than E- or C-treated mares at 7 d after inoculation. Clearance of inoculated materials began within 2 h in E-treated mares, and the non-antigenic markers were completely cleared in E- and C-, but not in P-treated mares, in 3 d. This suggests that in the P-dominated uterus, reduced physical clearance may contribute to an increased susceptibility to uterine infection.

Scintigraphic measurement of uterine clearance in normal mares and mares with recurrent endometritis.

The percentage of Technetium 99m-albumin colloid (99mTc-microAA), a radiocolloid, cleared from the uterine lumen within 4 h of intrauterine infusion, was measured in 15 mares during 2 consecutive cycles, on Day 3 of oestrus and 48 h after ovulation. Four nulliparous (Group 1) and 4 multiparous (Group 2) mares were classified as resistant and the remaining 7 multiparous mares were classified as susceptible (Group 3) to endometritis. Mares in Groups 1 and 2 cleared more 99mTc-microAA from their uteri than did mares in Group 3 during oestrus (P < 0.01) and 48 h after ovulation (P < 0.001). In the Group 1 + 2 mares, > 50% of the colloid was cleared in 7 and none in the remaining mare, apparently related to lack of cervical relaxation. Mean percentage of 99mTc-microAA cleared by Group 3 mares was negligible (< 5%), but some 99mTc-microAA was cleared by 3 of the 7 mares during 4 of the 6 studies conducted. Clearance of radiocolloid infused into the uterus of 3 reproductively normal mares during dioestrus was negligible. 99mTc-microAA infused into the uterus did not adversely affect endometrial integrity as determined by endometrial biopsy. Mares tolerated the procedures well. We conclude that scintigraphy can be used to detect impaired mechanical clearance of the uterus: reproductively normal mares clear > 50% 99mTc-microAA within 2 h of infusion whereas those susceptible to endometritis or mares with poor cervical dilatation may exhibit delayed uterine clearance.

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.

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.

Development and use of an enzyme-linked immunosorbent assay to monitor serum and urine acepromazine concentrations in thoroghbreds, and possible changes associated with exercise.

OBJECTIVES: To develop an ELISA that is sensitive and suitable for measurement of immunoreactive acepromazine (ACP) in horse serum and urine and to determine the acute effects of exercise on immunoreactive ACP values in Thoroughbreds. ANIMALS: 12 healthy Thoroughbreds (5 mares, 5 geldings, 2 stallions), aged 2 to 8 years. PROCEDURE: A commercially available antibody and a horseradish peroxidase-conjugated oxime derivative of immunoreactive ACP were used to develop a one-step ELISA. Horses were used in a crossover design study to evaluate possible effects of treadmill exercise on serum and urine ACP concentrations after a single (25 mg) IM injection of the drug. RESULTS: Immunoreactive ACP was detectable at concentrations as low as 50 pg/ml in serum and 100 pg/ml in urine, with intra- and interassay variabilities of 1.1 and 5.2%, respectively. The antibody had some cross-reactivity with a limited number of other phenothiazines. After drug administration, serum ACP immunoreactivity achieved a peak concentration (10.5 ng/ml) within 30 minutes and could be measured up to 48 hours in serum and 120 hours in urine. Although exercise had no significant effect on serum drug concentration, immunoreactive ACP disappeared more quickly (by 48 hours) from the urine of horses in the exercised group. CONCLUSIONS: This one-step ELISA provides a simple and sensitive means to measure immunoreactive ACP in equine serum and urine. The ability to detect drug several days after administration of a low dose of ACP should augment efforts to control illicit use of this drug in performance horses. Potential changes in ACP kinetics after exercise warrant further study.

Failure of superoxide dismutase to alter equine arachidonic acid-induced platelet aggregation, in vitro or ex vivo.

Superoxide dismutase (SOD), a free radical scavenger with anti-inflammatory activity, was administered IM to horses. Ex vivo platelet aggregation in response to arachidonic acid was monitored to determine whether exogenous SOD altered equine platelet prostaglandin metabolism. Preparations of platelet-rich plasma obtained before SOD administration were incubated with different concentrations of SOD and were aggregated with arachidonic acid. Superoxide dismutase did not exert a demonstrable effect, either ex vivo or in vitro. Aspirin abolished arachidonic acid-induced platelet aggregation in vitro. This indicates that SOD (in the resting state) does not exert an effect on platelet-derived free radicals that could alter the arachidonic acid pathway of equine platelets, that equine platelets do not release free radicals, or that equine platelets are insensitive to the products formed from free radicals by SOD.

Limitations in equine fetal electrocardiography.

Technical and interpretive limitations of equine fetal electrocardiography were evaluated in recordings obtained from 45 pregnant mares. Technical limitations were related to the small amplitude of the fetal electrocardiogram and the variability in the lead configuration providing the best recording. It was found that recording the fetal electrocardiogram at high sensitivity and high base-line fidelity in several different leads was necessary to obtain satisfactory tracings. Interpretive limitations were related in part to the small amplitude of the fetal electrocardiogram and to the marked variability in heart rate between fetuses. The great variability in normal fetal heart rate makes a diagnosis of fetal tachycardia or distress unless serial tracings are available.

XX male syndrome in a cryptorchid stallion.

A bilateral cryptorchid stallion with mild development of mammary glands was identified as an XX male by karyotyping. Necropsy revealed underdeveloped accessory sex organs and hypoplastic, inguinally located testes that were deficient of spermatogonia. Evaluation of routine hormonal profiles (without karyotyping) would have failed to diagnose this syndrome.

Medical evaluation of the reproductive system relevant to purchase.

Medical examination of the reproductive system of mares or stallions relevant to purchase is complex, imprecise, and potentially hazardous legally. In these ways it does not differ from other examinations of animals in which purchase is the objective. It is the responsibility of the veterinary profession to undertake these assignments and complete them as conscientiously as possible. After the examination is completed, communication with the principals and documentation of the procedures are essential to success and to the satisfaction of all involved.

Effects of susceptibility of mares to endometritis and stage of cycle on phagocytic activity of uterine-derived neutrophils.

Fourteen mares, 7 susceptible and 7 resistant to bacterial endometritis, were used to provide circulating and uterine-derived neutrophils. Uterine neutrophils were recruited by inoculating cell-free filtrates of Streptococcus zooepidemicus, or control vehicle. Mares were assigned to schedules for collection of neutrophils at oestrus or dioestrus. Phagocytic activity of circulating and uterine cells was evaluated by an assay for chemiluminescence after addition of opsonized streptococci. Chemiluminescence generated by circulating neutrophils was greater (P less than 0.05) for susceptible mares (28 +/- 4.9 V) than for resistant mares (13.4 +/- 2.8 V), but was unaffected by stage of cycle or by the interaction. Chemiluminescence by uterine-derived neutrophils from susceptible mares was greater (P less than 0.10) than for resistant mares. There was an interaction (P less than 0.05) with stage of oestrous cycle. Uterine cells from resistant mares in oestrus produced more chemiluminescence than did those from resistant mares in dioestrus (11.5 +/- 4.1 vs 7.1 +/- 2.1 V). The activity of uterine-derived cells of susceptible mares was unaffected by stage of cycle. Susceptibility to endometritis was not associated with a defect in the phagocytic function of uterine neutrophils. Also the function of uterine cells from resistant mares was greater during oestrus than dioestrus.

Species specificity of plasminogen activation and acquisition of surface-associated proteolytic activity by group C streptococci grown in plasma.

Our laboratory previously demonstrated that group C streptococcal isolates from humans and horses secrete streptokinases that preferentially activate plasminogens reflecting the origin of the isolates. To analyze the significance of these findings, series of streptokinase-producing Streptococcus equisimilis isolates recovered from humans and horses were examined. Southern blot analysis revealed that chromosomal DNA of the streptococcal isolates from humans reacted exclusively with a skc(hu) probe and that chromosomal DNA of streptococcal isolates from horses reacted preferentially with an skc(eq) probe in a distinct pattern. The streptococcal isolates were examined for the ability to acquire surface-bound plasmin-like activity when grown in the presence of human or equine plasma. Each of eight isolates from humans acquired significant enzymatic activity only when grown in the presence of human plasma, while each of eight isolates from horses acquired activity only when grown in the presence of equine plasma. Analysis of bacterial and host protein requirements indicated critical roles for streptokinase, activatable plasminogen, and fibrinogen. These requirements may explain why certain streptococcal isolates cause disease only in a limited number of mammalian hosts.