Theriogenology Question of the Month

Jenny K. Boye William R. Pritchard Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Barbara A. Byrne Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Charles E. Alex Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Verena K. Affolter Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Bruce W. Christensen Department of Population Health and Reproduction, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Ghislaine A. Dujovne Department of Population Health and Reproduction, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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History

A pregnant (150 days of gestation) 6-year-old Friesian mare that had been imported from The Netherlands was transported to the Contagious Equine Metritis Quarantine Facility at the University of California-Davis Center for Equine Health. Initial examination revealed a healthy mare with a body condition score of 5/9 and vital parameters within reference limits. Results for a CBC included a WBC count of 8,160 cells/mL and no abnormalities in the differential cell count. Throughout her stay at the quarantine facility, the mare had a rectal temperature between 37.2° and 37.67°C (99.0° and 99.8°F), maintained a good appetite, and had no signs of discomfort. No vaginal discharge or increase in size of the mammary glands was detected during daily examinations performed by the quarantine staff.

On day 7 after arrival, a male fetus was found within its intact fetal membranes in the straw bedding of the stall. Gross examination of the fetus and fetal membranes revealed a fetus without obvious malformations but that was smaller than expected (836 g; crown-rump length, 20 cm; Figure 1). The fetus was submitted to the University of California-Davis Pathology Service.

Figure 1—
Figure 1—

Photograph of a male equine fetus aborted at 150 days of gestation and expelled in its intact membranes. The fetus weighed 836 g and had a crown-rump length of 20 cm.

Citation: Journal of the American Veterinary Medical Association 251, 6; 10.2460/javma.251.6.657

Examination of the mare on the day of abortion revealed vital parameters within reference limits and no signs of pain or discomfort. Perineal conformation was normal with good closure of the vulvar labia. No vaginal discharge was evident. Manual and speculum vaginal examinations revealed no evidence of inflammation, edema, tearing, or bruising of the vulva, vagina, or cervix. The cervix was open (grade 3/3) and relaxed. Transrectal ultrasonographic examination revealed mild uterine edema (grade 1/3). There was a small amount of hyperechoic fluid (1.5 cm) in the body of the uterus, and several medium-sized follicles were found on both ovaries.

An aseptic technique was used to obtain endometrial biopsy specimens. One specimen was submitted for bacterial and fungal culture, and a second specimen was fixed in neutral-buffered 10% formalin and submitted for histologic examination. An impression smear of the endometrial biopsy specimen was stained with Gram stain and examined, but no organisms were seen.

Uterine lavage was performed immediately after the biopsy was completed. The effluent was a light brown, cloudy fluid with an odor consistent with normal lochia; the effluent was discarded after gross inspection. The mare received 20 U of oxytocina IV after the lavage and was maintained on oxytocin (10 U, IM, q 8 h for 3 days).

The mare was reexamined on the day after abortion. Ultrasonographic examination revealed mild uterine edema (grade 1/3; unchanged from the preceding day) and a small amount of anechoic fluid (1 cm) in the body of the uterus.

Five days after the abortion, culture results for the endometrial biopsy specimen included growth of a small number of fungi that were typed as Aspergillus terreus. This fungal species has never been reported as a cause of abortion in mares; therefore, culture examination was repeated 6 days after the abortion. Transrectal ultrasonographic examination revealed no fluid in the uterine lumen. A low-volume lavage of the uterus was performed and recovered fluid was submitted for microbiological culture and cytologic examination. Examination of stained direct smears of the pellet revealed low numbers of gram-positive rods and gram-positive cocci. There was a moderate amount of cellular debris, and few (< 1%) polymorphonuclear neutrophilic leukocytes were identified.

Culture of the low-volume lavage fluid yielded growth of a small number of mixed colonies (1 colony of a sterile hyaline fungus and 1 colony of Aspergillus fumigatus). These were considered contaminants. There was no growth of A terreus. All samples submitted for routine surveillance during quarantine yielded negative results for Taylorella equigenitalis.

Question

What was the presumptive cause of the abortion?

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Answer

A presumptive diagnosis of fungal placentitis and infection of the fetus with A terreus was made.

Results

Histologic examination of the uterine biopsy sample obtained on the day of abortion revealed mild to moderate chronic multifocal neutrophilic, lymphoplasmacytic, and eosinophilic endometritis with edema and mild multifocal fibrosis of the lamina propria (Figure 2). It was graded IIA (Kenney and Doig scale) on the basis of moderate inflammation. Grade IIA predicts that a mare has a 50% to 80% chance of being able to carry a fetus to term. Periodic acid–Schiff and Gomori methenamine silver stains were used, but no fungal organisms were identified within the uterine tissues.

Figure 2—
Figure 2—

Photomicrographs of lower-magnification (A) and higher-magnification (B) views of endometrial tissues obtained from a mare that aborted at 150 days of gestation. H&E stain; bar = 25 and 100 mm for panels A and B, respectively.

Citation: Journal of the American Veterinary Medical Association 251, 6; 10.2460/javma.251.6.657

Examination of the fetus revealed tissues that were diffusely autolyzed, friable, and edematous. Fetal membranes were diffusely pink-gray and friable, and the amniotic sac was filled with red-brown watery opaque fluid with approximately 15 free-floating, 1- to 2-cm diameter, irregularly shaped fragments of soft gray-tan tissue. Sections of the fetal membranes, umbilical cord, gray-tan tissue fragments, heart, liver, lungs, spleen, kidneys, testes, stomach, and intestines were fixed in neutral-buffered 10% formalin for at least 24 hours. Samples were processed by routine methods, sectioned at a thickness of 5 mm, and stained with H&E and Gomori methenamine silver stains for microscopic examination. The gray-tan tissue fragments consisted of mats of fungal hyphae that had positive results when stained with Gomori methenamine silver stain; the hyphae were admixed with necrotic cellular debris. Hyphae were septate with a width of approximately 3 to 8 mm. They had thin, parallel walls and acute angle branching (Figure 3). Fetal membranes were overlaid by accumulations of hyphae and cellular debris. The chorionic surface was multifocally disrupted by small foci of necrosis with a few infiltrates of neutrophils admixed with karyorrhectic debris. Several allantoic arteries contained mural neutrophilic infiltrates and small amounts of necrotic debris. The amniotic epithelium was diffusely cuboidal to columnar (reactive). Fungal hyphae were abundant in the fetal gastrointestinal tract but were not associated with histologic evidence of tissue invasion, necrosis, or inflammation. There were no relevant histologic abnormalities in the fetal tissues or umbilicus. Samples of amniotic fluid and fetal liver, which had been frozen at −20°C (−4°F) at the time of necropsy and stored, were submitted for fungal culture following the identification of fungi during the histologic examination. Aspergillus terreus was identified by use of culture in both samples.

Figure 3—
Figure 3—

Photomicrograph of organisms obtained from the amniotic fluid of a mare that aborted at 150 days of gestation. A mat of fungal hyphae (3 to 8 mm in width) with thin parallel walls, regular septations, and some acute angle branching is evident. Approximately 15 of these fungal mats were found floating freely in the amniotic fluid. Gomori methenamine silver stain; bar = 100 mm.

Citation: Journal of the American Veterinary Medical Association 251, 6; 10.2460/javma.251.6.657

The mare was isolated from other horses until she left the quarantine facility 7 days after the abortion. Rectal temperature was recorded and physical examinations were performed twice each day while the mare was in the quarantine facility. All vital parameters and results of physical examinations were within anticipated limits at all times. All samples submitted during quarantine for routine surveillance to detect T equigenitalis yielded negative results.

A follow-up examination was performed 61 days after the abortion to ensure the mare had recovered. Transrectal ultrasonographic examination revealed medium-sized follicles (20 to 25 mm) on both ovaries, a corpus luteum on the left ovary, and no uterine edema. The cervix was tightly closed and toned. A uterine swab specimen was collected; cytologic examination of that swab specimen revealed normal endometrial cells, a few RBCs, and no signs of infectious pathogens or inflammation. Another low-volume lavage of the uterus was performed. Because the mare was in diestrus, a dose of cloprostenolb (250 mg, IM) was administered after the lavage procedure. The lavage fluid was submitted to the Colorado State Diagnostic Laboratory for identification of Aspergillus spp; however, PCR assay results were negative. Therefore, at that time, it was deemed that the endometritis after the abortion was resolved, as determined on the basis of the clinical findings and results for the swab specimen and PCR assay. Considering the mild findings for the original biopsy specimen and the lack of abnormal results for clinical and ultrasonographic examinations at 61 days after the abortion, it was decided that collection of a uterine biopsy specimen for evaluation was not warranted at that time.

Discussion

Fungal placentitis is generally considered a sporadic finding in mares. It accounts for < 5% of all diagnosed infectious abortions in horses.1–3 Aspergillus spp are the most common causative agents, and they are considered to have an ascending route for infection.3

Aspergillus spp are common environmental fungi found in soil and bedding and the stable environment. They typically cause noninvasive placentitis, which may acutely result in infection of the fetus. The fetus becomes infected by ingestion of infected amniotic fluid, via the umbilical vein, or through the skin. Fungal organisms were primarily found in the gastrointestinal tract of the fetus of the present report, which suggested that the infection most likely spread by ingestion of amniotic fluid by the fetus.

Pneumovagina, urovagina, retained fetal membranes, dystocia, difficulty in becoming pregnant, and a history of antibacterial treatment have been described as factors associated with ascending fungal endometritis.4 The pathogenesis of fungal endometritis and placentitis has not been established in horses, but both conditions will likely have the same pathogenesis. The described predisposing factors increase the likelihood that fungal organisms will cause an ascending endometritis or placentitis.

For the horse of the present report, there were no localized lesions found on the fetal membranes. Because the mare was never systemically ill and fungi were not detected during histologic examination of the endometrium, we speculated this was an ascending infection that spread to the fetus and fetal membranes. However, the mare had a normal vulvar conformation without signs of pneumovagina, and none of the aforementioned predisposing factors were evident.

The mare had been in the United States for only a short period at the time of abortion. Therefore, it was likely that the fungal infection had developed before entry to the United States and that the stress of transportation may have led to an exacerbation of the infection and subsequently to abortion. A detailed breeding history of the mare was not known; according to the owner, this was the first time that breeding of this mare had been attempted.

Aspergillus terreus has been described as an aggressive and systemically invasive fungus in humans and dogs. It is a pathogen found in human hospitals, and it affects immunosuppressed patients.5 Aspergillus terreus is characterized by a pronounced resistance to drugs and a high fatality rate in the form of disseminated aspergillosis in dogs6 and invasive aspergillosis in humans.5 To the authors’ knowledge, the information provided here was the first description of A terreus–associated abortion in a horse. The mare never had signs of systemic disease, and the endometrial infection resolved without treatment. Aspergillus terreus was not detected in the uterus 6 days after the abortion by use of microbial culture or 61 days after the abortion by use of PCR assay.

It must be stressed that the diagnosis of infection and abortion attributable to A terreus could easily have been missed if we had relied only on the results for the biopsy specimen and culture. There was no evidence of A terreus during histologic examination of the uterine tissues, and the organism was found in small numbers on the initial culture. The initial culture after the abortion was performed by use of a biopsy specimen. Cultures of endometrial biopsy specimens provide accurate results regarding susceptibility and positive predictive value for bacterial endometritis, when compared with results for culture of endometrial swab specimens or low-volume lavage fluid.7 Low-volume lavage fluid and biopsy specimens for culture of endometrial samples are more diagnostically sensitive than endometrial swab specimens.7 Low-volume lavage fluid may provide practitioners with a sample that represents the entire contents and surface of the uterus, and direct smears of centrifuged pellets of low-volume lavage fluid provide excellent material for cytologic examination. In the mare of the present report, the cervix was open and examinations were performed immediately after the abortion; thus, the risk of contamination was considered higher for low-volume lavage fluid than for an endometrial biopsy specimen. In the mare described here, the causative agent was cultured in extremely small numbers from a biopsy specimen.

The final diagnosis was made after identifying hyphae by use of special stains and on the basis of positive results for culture of fetal liver and amniotic fluid. The PCR assay for fungal DNA is a highly sensitive test and will detect both dead and live fungi.8 Testing by use of a PCR assay at 61 days after the abortion, accompanied by results of clinical examination, ultrasonographic examination of the reproductive tract, and cytologic examination of a uterine swab specimen, confirmed the absence of fungal elements.

The evaluation at 61 days after the abortion revealed no evidence of conformational or anatomic abnormalities, generalized pathological conditions, or signs of inflammation in the uterus. Negative results for the PCR assay and lack of predisposing factors for ascending fungal endometritis favored the prognosis for a full recovery and return to optimum fertility.

It is challenging to treat fungal endometritis, which has resulted in numerous treatment regimens, although the prognosis is considered guarded for complete recovery and return to fertility. A guarded prognosis should be emphasized when fungal endometritis is found in mares with concurrent problems or factors known to contribute to this condition. Therefore, treatment of fungal endometrial infections should primarily be aimed at removing predisposing factors (eg, use of a Caslick surgery to ensure optimal vulvar closure or treatment to remove fluid pooled in the uterus) and ensuring that concurrent antifungal treatment is based on a drug with the appropriate therapeutic spectrum, at an effective dose, and for a suitable duration.

Outcome

Follow-up evaluation of the mare at 61 days after the abortion confirmed that the fungal infection had resolved without antifungal treatment. Moreover, the mare conceived successfully during the following breeding season and was confirmed pregnant at 30 days after that breeding. At that time, ultrasonography revealed that the mare had a healthy-appearing fetus with a normal fetal heartbeat and fetal membranes.

Footnotes

a.

Vet One, Cambridge, ON, Canada.

b.

Estrumate, Schering-Plough Animal Health, Summit, NJ.

References

  • 1. Giles RC, Donahue JM, Hong CB, et al. Causes of abortion, stillbirth, and perinatal death in horses: 3,527 cases (1986–1991)? J Am Vet Med Assoc 1993; 203:11701175.

    • Search Google Scholar
    • Export Citation
  • 2. Hong CB, Donahue JM, Giles RC Jr, et al. Equine abortion and stillbirth in central Kentucky during 1988 and 1989 foaling seasons? J Vet Diagn Invest 1993; 5:560566.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Laugier C, Foucher N, Sevin C, et al. A 24-year retrospective study of equine abortion in Normandy (France)? J Equine Vet Sci 2011; 31:116123.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Stout TA. Fungal endometritis in the mare Pferdeheilkunde 2008; 24:8387.

  • 5. Steinbach WJ, Benjamin DK, Kontoyiannis DP, et al. Infections due to Aspergillus terreus a multicenter retrospective analysis of 83 cases Clin Infect Dis 2004; 39:192198.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Kelly SE, Shaw SE, Clark WT. Long-term survival of four dogs with disseminated Aspergillus terreus infection treated with itraconazole Aust Vet J 1995; 72:311313.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Nielsen JM. Endometritis in the mare: a diagnostic study comparing cultures from swab and biopsy Theriogenology 2005; 64:510518.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Ferris RA, Dern K, Veir JK, et al. Development of a broad-range quantitative polymerase chain reaction assay to detect and identify fungal DNA in equine endometrial samples? Am J Vet Res 2013; 74:161165.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Figure 1—

    Photograph of a male equine fetus aborted at 150 days of gestation and expelled in its intact membranes. The fetus weighed 836 g and had a crown-rump length of 20 cm.

  • Figure 2—

    Photomicrographs of lower-magnification (A) and higher-magnification (B) views of endometrial tissues obtained from a mare that aborted at 150 days of gestation. H&E stain; bar = 25 and 100 mm for panels A and B, respectively.

  • Figure 3—

    Photomicrograph of organisms obtained from the amniotic fluid of a mare that aborted at 150 days of gestation. A mat of fungal hyphae (3 to 8 mm in width) with thin parallel walls, regular septations, and some acute angle branching is evident. Approximately 15 of these fungal mats were found floating freely in the amniotic fluid. Gomori methenamine silver stain; bar = 100 mm.

  • 1. Giles RC, Donahue JM, Hong CB, et al. Causes of abortion, stillbirth, and perinatal death in horses: 3,527 cases (1986–1991)? J Am Vet Med Assoc 1993; 203:11701175.

    • Search Google Scholar
    • Export Citation
  • 2. Hong CB, Donahue JM, Giles RC Jr, et al. Equine abortion and stillbirth in central Kentucky during 1988 and 1989 foaling seasons? J Vet Diagn Invest 1993; 5:560566.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Laugier C, Foucher N, Sevin C, et al. A 24-year retrospective study of equine abortion in Normandy (France)? J Equine Vet Sci 2011; 31:116123.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Stout TA. Fungal endometritis in the mare Pferdeheilkunde 2008; 24:8387.

  • 5. Steinbach WJ, Benjamin DK, Kontoyiannis DP, et al. Infections due to Aspergillus terreus a multicenter retrospective analysis of 83 cases Clin Infect Dis 2004; 39:192198.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Kelly SE, Shaw SE, Clark WT. Long-term survival of four dogs with disseminated Aspergillus terreus infection treated with itraconazole Aust Vet J 1995; 72:311313.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Nielsen JM. Endometritis in the mare: a diagnostic study comparing cultures from swab and biopsy Theriogenology 2005; 64:510518.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Ferris RA, Dern K, Veir JK, et al. Development of a broad-range quantitative polymerase chain reaction assay to detect and identify fungal DNA in equine endometrial samples? Am J Vet Res 2013; 74:161165.

    • Crossref
    • Search Google Scholar
    • Export Citation

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