Contagious equine metritis--outbreak of the disease in Kentucky and laboratory methods for diagnosing the disease.

Contagious Equine Metritis (CEM) was initially reported during the 1977 breeding season in England (Crowhurst, 1977) and Ireland (Timoney, Ward & Kelly, 1977. The disease has also been diagnosed in France and Australia (Huges, Bryden & MacDonald, 1978). The first occurrence of CEM in the United States followed the importation or 2 stallions from France late in 1977 which resulted in an outbreak early in the 1978 breeding season (Swerczek, 1978). Mares usually develop clinical signs of CEM 8--10 days after being covered by an infected stallion, when a copious, greyish discharge is seen. Other mares may not show any outward signs of disease, but may have a shortened dioestrous period. Many mares recover spontaneously from the disease, but a small proportion become carriers of the CEM organism. The stallion does not show any clinical signs of disease, but remains a carrier. In this paper we recommend various laboratory procedures for the diagnosis of CEM in mares and stallions.     

Diagnosis and treatment of four stallions, carriers of the contagious metritis organism--case report

Contagious Equine Metritis (CEM), a venereal disease of horses caused by the bacterium Taylorella equigenitalis, was first diagnosed in 1977 and subsequently spread to many nations [Proc 24th AM Assoc Equine Pract (1979) 287]. The disease was confirmed in the United States in 1978 [Proc Am Assoc Equine Pract (1983) 295]. Specific regulatory procedures for this disease have been established in the United States and 37 other countries. From 1999 through 2001, four of 120 imported European stallions tested positive for CEM at a quarantine facility in Darlington, MD, USA. Two stallions were identified by positive bacterial cultures for T. equigenitalis on arrival. The other two positive stallions were negative on initial bacterial cultures, but were identified as CEM carriers when test mares (that they had mated) were culture-positive for T. equigenitalis. Since T. equigenitalis, is a fastidious slow-growing coccobacillus, additional sets of samples taken over a interval might be required to ensure positive stallions are detected before mating test mares. Likewise, additional sets of samples taken over a long interval after treatment of a stallion for CEM might be required to ensure that positive stallions treated for CEM are detected before mating test mares. Aggressive systemic antibiotic therapy accompanied by routine topical therapy might be required to treat some CEM-positive stallions.     

Epidemiological observations on contagious equine metritis in Kentucky, 1978.

Contagious equine metritis, introduced by importation of 2 comtaminated stallions from France, affected 54 Thoroughbred brood mares during the 1978 breeding season in Kentucky. The infection was diagnosed bacteriologically and by the use of a complement fixation test. Although lateral spread to stallions, and probably to a few mares, occurred through human agency in the breeding sheds of 2 stud farms, control measures instituted early in the epidemic confined the disease to brood mares bred by stallion on only these farms.     

Contagious equine metritis in Australia.

Contagious equine metritis (CEM) was first diagnosed in Australia in August 1977 and it has since been found on 6 farms in 3 states, having been isolated from about 24 mares and 2 stallions. Details are given of the epidemiology and control procedures used to combat CEM on one farm. Difficulty was experience in successfully treating one infected stallion; this was thought to be associated with inadequate cleaning and treating of the diverticulum of the urethral fossa. Introduction of the disease has had far-reaching consequences and may well result in the adoption of routine bacteriological tests on stallions and mares of unknown or dubious breeding history and other measures to minimize the possibility of spread between farms.     

Epidemiologic aspects of Taylorella equigenitalis

Contagious equine metritis (CEM) is a sexually transmissible disease in mares. Although the disease is commonly diagnosed by culturing the causative bacterium Taylorella equigenitalis (T. equigenitalis) . false negative results do occur. A recently developed Polymerase Chain Reaction (PCR) assay, however, appeared to be much more sensitive, with initial results indicating an unexpected high incidence of the agent in selected horses. In this study, samples from 107 randomly selected mares with no clinical signs of CEM submitted for conventional culture were all negative for T. equigenitalis . but in the PCR-assay 54 (49%) were positive for Taylorella -DNA. Positives in the PCR-assay were found in all breeds tested, even in horses imported from the isolated population in Iceland. These findings suggest that T. equigenitalis was present long before it was first isolated in 1977, The high incidence of Taylorella in horse populations without apparent clinical signs of CEM, the occurrence of incidental clinical case and the known variability between strains, all indicate that Taylorella is endemic in the horse population. In order to explore whether the organism is present in species other than the horse, we also used the PCR-assay on clinically health donkeys (n = 14), zebras (n = 15), Przewalski horses (n = 2) and cows (n = 21). All the animals showed negative results except one of the Przewalski horses, and one cow that was repeatedly found to give positive reaction. We also found that the fertility of 7 stallions with cultures positive for Taylorella (6 used in an AI-program and 1 by natural breeding) was not affected, as shown by the normal range of foaling rates in mares inseminated or bred by these stallions. The overall results may be interpreted to mean that Taylorella is of limited significance in horse breeding.     

An outbreak of contagious equine metritis in 1977 and its effect the following season.

An outbreak of contagious equine metritis occurred in Newmarket in 1977. This survey records the effect on fertility of 20 of the stallions which were infected. Swabbing of mares since then has detected 37 carrier mares harbouring the organism, most frequently in the clitoral area. This swabbing programme reduced the incidence of new cases in 1978 to 3 mares and 1 stallion.     

Spontaneous and oxytocin-induced uterine motility in cyclic and postpartum mares

Intrauterine pressure was measured in three cyclic and two postpartum mares. Pressure was recorded using a catheter tip pressure transducer. The transducer was passed transcervically into the uterus.. In cyclic mares recordings were started on Day 1 of estrus and continued daily until ovulation as well as on Days 1 and 8 of diestrus. In postpartum mares recordings were started within 48 h after foaling and continued until the mares ovulated. The intrauterine pressure changes in postpartum mares was also recorded on Days 1 and 8 of diestrus. Spontaneous uterine contractions were recorded in cyclic mares for 30 min and in postpartum mares for 10 min. Induced uterine motilities were recorded for 30 min in both groups after the administration of oxytocin (40 USP, i.v.). Total area under the contraction curve in a 10-min period was used as a uterine motility quantitating unit. All mares demonstrated uterine contractions during estrus and diestrus. All mares demonstrated significant responses to oxytocin during estrus and diestrus. It appears that estrogen priming is not necessary for a significant uterine response to oxytocin.     

Ovarian function in captive feral mares

Ovarian function was monitored for 33 mo in captive feral mares (Equus caballus) by following serum progesterone (P) levels. A P level greater than 2.0 ng/ml was considered indicative of ovulation. Feral mares were seasonally polyestrus with the majority of animals ovulating between May and October. During the first year after capture, none of the mares ovulated during the anestrous season. However, in subsequent years, approximately 10% of mares ovulated during the months of November, January and February. P levels during the luteal phase of the cycle ranged from 2.0 to 21.0 ng/ml which were similar to levels in domestic breeds of mares. The pattern of P concentrations during pregnancy was also similar to the pattern in domestic mares. These data confirmed the seasonality of ovulation in feral mares but indicated that this seasonality was not as rigid as previously believed. Captive feral mares were similar to domestic breeds in the percentage of mares ovulating all year and in the P levels achieved during the estrous cycle and pregnancy.     

Effect of administration of estradiol and progesterone,and bacterial contamination,on endometrial morphology of acyclic mares

Acyclic mares (free of uterine infection and with typical acyclic endometria on biopsy) received daily intramuscular injections in oil of either 1.5 mg estradiol benzoate (n - 4) or progesterone (n = 5). Estradiol and progesterone treatment induced biopsy changes consistent with estrus or diestrus respectively (Kenney, 1978). Subsequently, seven mares with typical acyclic endometria were given ten daily intra-uterine infusions of from 400 to 10 000 pg estradiol-17β. All mares developed Streptococcus zooepidemicus infections which masked any endometrial changes which might have been induced by estradiol infusion.     

Sexual behavior in ovariectomized and seasonally anovulatory pony mares (Equus caballus)

Ten ovariectomized (OVEX) and ten intact, but seasonally anovulatory (ANOV), pony mares were observed for sexual activity with five stallions, using a “harem group” social testing paradigm (two OVEX and two ANOV mares plus one stallion per group) for 15 consecutive daily tests lasting 20 min each. All mares in both conditions showed proceptive behavior in at least one test, all mares but one were mounted, and 14 of 20 mares received ejaculations. No statistical differences were found between the two conditions for any measure of proceptivity, copulatory activity, or days in estrus. The quality of estrus was judged to be equivalent to that displayed by periovulatory mares during their initial and terminal days of estrus, but less intense than that seen near ovulation. Mares in both groups were in estrus during approximately 60–70% of the tests and only 3 of the 20 mares were sexually refractory for more than five consecutive tests. Thus, the typical 2-week phase of sexual refractoriness seen in intact diestrous mares was absent in OVEX and ANOV mares, suggesting that the ovary plays a major role in actively suppressing estrous responses during the luteal phase of the cycle.     

Prostaglandin production by horse embryos and the effect of co-culture of embryos with endometrium from pregnant mares

Embryos, endometrial biopsies, and uterine lavage fluid were collected from pregnant and non-pregnant mares 14 days after ovulation. Embryos were cultured for 20.5 h with and without endometrial tissue from pregnant mares, and endometrial tissue was cultured alone. Endometrial content of PGF tended to be higher (P = 0.06) in non-pregnant than in pregnant mares, but the amount of PGF released from tissue during culture was similar for pregnant and non-pregnant mares. Lavage fluid from non-pregnant mares also tended (P = 0.08) to contain higher concentrations of PGF. Coincubation of embryos with endometrium from pregnant mares significantly (P = 0.01) lowered concentrations of PGF in medium. Tissue concentrations and release of PGE-2 and 6-keto-PGF-1 alpha were similar in endometrial samples from pregnant and non-pregnant mares and prostaglandin production was unaffected by the presence of an embryo during incubation. Horse embryos released all three prostaglandins during a 20.5-h incubation.     

Persistence of the luteal phase following ovulation during altrenogest treatment in mares

Two experiments were conducted to test the efficacy of altrenogest treatment in mares. The response to 15-d altrenogest treatment (Experiment 1) was characterized in 20 mares that were given 22 mg daily of altrenogest in oil (n = 10) or in gel (n = 10) from Day 10 to 25 after ovulation. In 17 mares, luteolysis occurred during altrenogest treatment (Day 17.7 +/- 0.5), while 2 mares retained their corpus luteum (CL), and 1 mare had a diestrous ovulation on Day 16, resulting in a prolonged luteal phase. Ten of the 17 mares in which the CL had spontaneously regressed returned to estrus after the end of treatment, and ovulated 5.7 +/- 0.8 d after the end of altrenogest treatment. Two of these 17 mares ovulated 2 and 3 d after the end of altrenogest treatment but ovulation was not accompanied by estrous behavior, and 5 mares ovulated during altrenogest treatment resulting in an interovulatory interval of 22.4 +/- 1.1 d (range: 20 to 25d). Five mares which ovulated during altrenogest treatment and 2 mares which ovulated during silent estrus after the end of altrenogest treatment failed to regress the CL around 14 d post ovulation, and had a prolonged luteal phase. In Experiment 2, the effect of altrenogest administered from luteolysis to ovulation on duration of the subsequent luteal period was analyzed. In 6 mares altrenogest was begun on Day 14 post ovulation and continued until the hCG-induced ovulation. The interval from ovulation during altrenogest treatment to spontaneous luteolysis was 45.6 +/- 2.4 d (range: 40 to 54d) in altrenogest-treated mares and was significantly longer than in 10 untreated control mares (14.5 +/- 0.3 d, range: 13 to 16d). The results suggest that the oil and gel altrenogest preparations are equally effective in modulating estrous behavior and time to estrus and ovulation. Altrenogest treatment started late in diestrus appears to result in a high incidence of ovulation during treatment and when luteolysis and ovulation occur during treatment; the subsequent luteal phase is frequently prolonged due to failure of regression of the CL.     

Effects of deslorelin or hCG administration on reproductive performance in first postpartum estrus mares

A tendency for deslorelin implants to suppress subsequent follicular growth and delay return to estrus following induced ovulation has been documented in nonlactating mares. To investigate this phenomenon in lactating mares, 22 broodmares in southeast Texas were administered either deslorelin or hCG to induce ovulation in the first postpartum estrus during February and March 2001. Mares were teased daily and examined twice weekly (Tuesdays and Thursdays) by transrectal ultrasonography. When a follicle >35 mm diameter was detected on Tuesday, mares were treated with either 2,500 U hCG administered intravenously or with one implant (2.1 mg) deslorelin administered subcutaneously. Mares were bred every other day until ovulation was detected or until they ceased behavioral estrus, and were examined 16 days after treatment to detect pregnancy. Follicular measurements were recorded for all mares during each examination, and interestrous intervals were recorded for mares not becoming pregnant. Treatment of mares with either hCG or deslorelin resulted in similar ovulatory responses and pregnancy rates. Deslorelin-treated mares had fewer ovarian follicles >20 mm in diameter 16 days after treatment than hCG-treated mares (P < 0.01). Interestrous intervals for mares failing to become pregnant on foal heat breeding were prolonged in deslorelin-treated compared to hCG-treated mares (P < 0.01). Date of treatment was negatively correlated with length of the interestrous interval in deslorelin-treated mares (P < 0.01), but was not correlated with length of interestrous interval in hCG-treated mares (P > 0.10). All mares failing to become pregnant from foal heat breedings became pregnant from later breedings, but the parturition to conception interval was prolonged in deslorelin-treated compared to hCG-treated mares that did not become pregnant on foal heat (P < 0.01).     

Effects of melatonin implants in pony mares. 1. Acute effects

The effects of melatonin implant treatment over a four week period on LH, estradiol (E2) and progesterone (P4) secretion during the breeding season were studied in ovary-intact and ovariectomized pony mares. Mares with melatonin implants had significantly higher daytime melatonin concentrations than mares with sharm implants (P = 0.0065). In ovariectomized mares, LH secretion did not differ between mares with melatonin and sham implants. In ovary-intact mares, melatonin implants altered the pattern of LH secretion (P = 0.0023) in such a way that an increase in LH secretion was observed during the periovulatory period. Estradiol and P4 secretion were unaffected by melatonin implants. These results suggest that constant administration of melatonin may enhance the secretion of LH during the periovulatory surge but does not adversely affect E2, P4 or basal LH secretion in mares during the breeding season.     

Natural outcome and ultrasonic identification of equine fetal twins

The natural outcome of bilateral twins (one in each uterine horn) that were viable on Day 40 was studied in 15 pony mares. Fetuses were monitored by transrectal ultrasonography until the outcome was determinable. One fetus (2 mares) or both fetuses (8 mares) died (cessation of heartbeats) during Months 2 and 3. Loss of fetuses was externally observed in 4 additional mares during the late fetal stage (Months 8 to 11); 2 mares lost 1 fetus and 2 lost both fetuses. Birth of 2 live foals occurred in only 1 of 15 mares (7%) First day of detected apposition between portions of the 2 allantochorions was studied daily on Days 40 to 51 in 7 of the 15 mares. Apposition was first detected on mean Day 46.3 (standard deviation: +/-3.1). In these 7 mares, death of 1 fetus (2 mares) or both fetuses (2 mares) occurred 1 or 2 days after the first day of apposition of allantochorions; no deaths occurred before the day of apposition. Each mare with loss of one fetus during this time gave birth to a living foal. The only structure indicative of twins that was consistently detected ultrasonically during all examinations was the common membrane resulting from the area of apposition between the 2 allantochorions. The common membrane is herein designated as the twin membrane because of its apparent diagnostic potential even in late pregnancy when the presence of the 2 fetuses may not be detected directly. Thickness of the twin membrane reached approximately 3 mm at Month 7 and thereafter gradually increased to 15 mm near the end of pregnancy. Two layers of the membrane (1 for each allantochorion) became distinct at Month 6 and Month 8 with 5.0 MHz and 3.5 MHz transducers, respectively; the 2 layers were separated by a hypoechogenic layer.     

Age and pasture effects on vernal transition in mares

The objectives of the present study were to determine if follicular activity was less in old than in young mares during the spring transition and if green pasture would hasten onset of the ovulatory season. Experiments were conducted over 2 sequential years using young mares (3 to 7 yr) and old mares (> or =14 yr). In Experiment 1, growth of the largest and second-largest follicles were compared for young mares (5 to 7 yr) and old mares (> or =14 yr) for 21 d prior to the first ovulation of the year. More follicular activity was noted in young than in old mares. Main effect of age was significant for diameter of the largest follicle, and interaction of day-by-age was significant for diameter of the second-largest follicle. Prior to the beginning of the breeding season, the mares were randomly divided into dry-lot and pasture groups. The interval from May 2 to ovulation was shorter (P < 0.005) for mares put on pasture on May 2 than for mares kept in dry lot (means +/- SEM, 14.5 +/- 2.7 and 21.3 +/- 3.2 d, respectively). In Experiment 2, follicular activity was compared among 3 age groups (3 to 7, 17 to 19, and > or =20 yr). The total number of follicles > or =10 mm was higher (P < 0.05) for young mares and lower (P < 0.05) for old mares than for mares of an intermediate age. Main effect of age and interaction of day-by-age were significant for diameter of largest and second-largest follicles, being smaller for mares > or =20 yr than for younger mares. The interval from development of a follicle > or =30 mm to ovulation was shorter (P < 0.05) for mares placed on pasture when a > or =30 mm follicle developed than the interval for mares kept in dry lot (5.7 +/- 0.7 and 8.2 +/- 0.9 d, respectively). In summary, less follicular activity occurred in old than in young mares during the transitional period, and mares pastured on green grass ovulated sooner in the spring than mares housed on dry lot and fed hay.     

Testosterone secretion during early pregnancy in mares

We have characterized the testosterone secretion pattern during the first 80 d of pregnancy in mares and determined the sources that contribute to circulating testosterone levels during this period. Ten untreated, pregnant mares (Group 1), 10 altrenogest-treated, pregnant mares (Group 2), and 10 altrenogest-treated, pregnant mares in which the CL was eliminated by administration of PGF-2alpha on Day 16 (Group 3) were used in this study. Complete luteolysis occurred following PGF-2alpha administration in all mares in Group 3. Six of the 10 mares in Group 3 did not have an active CL until after Day 60 of pregnancy (Group 3a) and were included in the analysis. The remaining four mares developed a new CL on Days 32, 40, 43 and 49 of pregnancy and were excluded from analysis. Mares without a functional CL (Group 3a) had significantly lower testosterone concentrations than mares with a functional CL (Groups 1 and 2), during the period before equine chorionic gonadotropin (eCG) secretion. At the onset of eCG secretion, testosterone concentrations increased rapidly but the rate of increase decreased with time in mares with a functional CL (Groups 1 and 2). In mares without a functional CL (Group 3a), testosterone concentrations did not increase at the onset of eCG secretion but increased at a gradually increasing rate after Day 50. The lower testosterone concentration in mares without a functional CL before eCG secretion suggests that the CL contributes significantly to the circulating testosterone concentration during the period before eCG secretion. The close time relationship between the onset of eCG secretion and the increase in testosterone secretion in mares with a functional CL and the lack of a testosterone increase in pregnant mares without a functional CL suggest that the increase in testosterone secretion after Day 35 of pregnancy is the result of eCG-stimulated, luteal testosterone synthesis.     

The effect of isosorbide dinitrate on uterine and ovarian blood flow in cycling and early pregnant mares: A pilot study

Poor uterine perfusion has been proposed as a cause of infertility in mares. The objective of this study was to investigate the effect of isosorbide dinitrate (ISDN), a nitric oxide donor, on uterine and ovarian blood flow resistance during diestrus and early pregnancy in mares. Six Trotter mares, aged 7 to 14 years, were examined daily during the first 11 days of three diestrous periods, and five of those mares were also examined during the first 11 days of two pregnancies. Six mares randomly received a placebo, a low dose (30 mg, ISDN30), or a high dose of ISDN (60 mg, ISDN60) through three nonconsecutive cycles. The treatments were administered orally, every 12 hours from Day 1 to 11 of the cycle (Day 0 = ovulation). Five of the 6 mares received a placebo or 60 mg of ISDN orally every 12 hours from Day 1 to 11 of pregnancy. The mares were short cycled on Day 12 of each trial. Transrectal color Doppler was used to determine blood flow resistance semiquantitatively and expressed as pulsatility index. Mean pulsatility index of both uterine arteries combined and of the dominant (ipsilateral to the CL) ovarian artery was lower (treatment effects: P ≤ 0.01; time effects: P ≤ 0.002) in mares receiving 30 mg or 60 mg of ISDN compared with placebo-treated mares. Blood flow resistance in the dominant ovarian artery was lower in ISDN-treated pregnant mares than in placebo-treated pregnant and cycling mares (treatment effect: P = 0.04; time effect: P = 0.003). Isosorbide dinitrate increases uterine and ovarian perfusion in cycling mares and ovarian perfusion in early pregnant mares. Further studies are needed to investigate these effects in relation to fertility of the mare.     

Seasonal variation and opioidergic regulation of growth hormone release in cyclic, ovariectomized, and pregnant pony mares

Modulation of reproductive functions is one of the multiple effects of growth hormone (GH). To investigate effects of reproductive functions on GH release in the horse, plasma GH concentrations in ovary-intact (n = 7) and ovariectomized (n = 8) mares during the anovulatory and breeding seasons and in pregnant mares (n = 6) at various stages of gestation were determined. To analyze an opioidergic regulation of GH release, repeated blood samples were taken over 3 h, and mares were injected with the opioid antagonist naloxone (0.5 mg/kg i.v.) or saline. GH was determined by RIA with an antiserum raised against porcine GH and equine GH as standard. In ovariectomized and ovary-intact, cyclic mares, GH concentrations were low and not different between the two groups in November and December. GH concentrations increased significantly (P < 0.05) in cyclic mares during May and June but were not affected by stage of the cycle and were low in ovariectomized mares. In pregnant mares, plasma GH concentrations remained high throughout pregnancy and did not decrease during winter but increased significantly (P < 0.05) postpartum. Naloxone induced a significant GH release in ovary-intact mares; this response was most pronounced (P < 0.05) during the breeding season. Naloxone did not affect GH in ovariectomized mares. During pregnancy, naloxone induced a significant release of GH around Day 280 (P < 0.05) but not at other times of pregnancy. In conclusion, GH release is influenced by season. The seasonal changes depend on ovarian factors, are absent in ovariectomized mares, and can be modulated by pregnancy. GH release is regulated at least in part by opioidergic pathways.     

Treatments resulting in pregnancy in nonovulating, hormone-treated oocyte recipient mares

Synchronization of follicle growth between oocyte donor and recipient mares is difficult. To avoid this, recipient mares in a clinical program were used during a period of low follicular activity, and were treated with estrogen before transfer and progesterone after transfer. Five pregnancies were established after oocyte transfer to nonovulating, hormone-treated recipient mares. One pregnancy was lost before 30 d gestation, and the other 4 foals were carried to term. One foal died at birth. Establishment and maintenance of pregnancy in these mares indicates that nonovulating, hormone-treated mares may offer an alternative to cyclic recipients in oocyte transfer programs.