A case of Candida guilliermondii abortion in an Arab mare

Abstract

Ascending infections of equine uterus frequently result in placentitis and abortions; most of these infections are bacterial and are less commonly due to fungi. This report describes an abortion case in an Arab mare due to Candida guilliermondii that was diagnosed via cytological, histological, cultural and biomolecular assays. The histological lesions found were severe necrotizing placentitis associated with fetal pneumonia. To our knowledge this is the first case of C. guilliermondii abortion reported in equine species.

1. Introduction

Abortion, stillbirth and neonatal deaths are an important source of economic loss for an equine industry. As reported in previous studies, the causes of equine abortion vary with geographic area and can change over time [1]. In early studies, infectious agents were reported as major causes of equine reproductive loss [2] whereas later reports indicated that the loss from noninfectious causes, e.g., twinning and stillbirth, surpassed the prevalence of those caused by infectious agents [3]. In mares, most bacterial and fungal infections of the pregnant uterus that result in abortion are attributable to ascending infections via the cervix and vagina following contamination of the external genitalia [4]. Undoubtedly, there are several risk factors such as poor uterine contractility, anatomical malformations and inefficient uterine defense mechanism. This may explain why fungal endometritis is commonly associated with a history of uncorrected anatomical defects that lead to pneumovagina, recurrent persistent post-breeding endometritis and frequent intrauterine antibiotic therapy [5]. The most common source of fungi causing reproductive disease in the mare is probably represented from skin or fecal origin. There are a variety of fungi that have been identified from uterine cultures with Candida spp. and Aspergillus spp, isolated as the most common yeast and mold, respectively. Although early reports primarily identified Candida albicans as the fungal pathogen infecting the reproductive tract of the mare, more recent literature demonstrates that many other Candida species and fungal agents such as Cryptococcus and Aspergillus, can be responsible for equine fungal endometritis or abortion [4,6]. Yeasts of the genus Candida are widely distributed in the vegetative parts of plants and soil, as well as external mucosal surfaces (oral cavity, external genitalia) and intestine of domestic animals where they compose the normal microbial flora and are eliminated by excreta [7]. However, despite being saprobes, there have been many reports of disease caused by this microorganism in horse, which can present with different clinical manifestations, related to osteomyelitis [8], ulcerative keratitis [9], arthritis [10] and endometritis [11]. However, while fungal endometritis is well documented in the scientific literature, little is known about the ability of Candida to cause placentitis and abortion in mares: only single report was found in literature about an intrauterine inoculation of Candida parapsilopsis which resulted in embryonic loss in pony mares [12]. The purpose of this case report is to describe the diagnostic procedures applied in a rare case of placentitis and the subsequent abortion in an Arab mare due to Candida guilliermondii. The newly assigned teleomorph species name of C. guilliermondii is now Meyerozyma guilliermondii according to the recent work in 2010 by Kurtzman and Suzuki, but for clarity the species name Candida guilliermondii is used throughout this report [13].

2. Case

A 10-year-old Arab mare had failed to conceive for two consecutive years (2010–2011). In 2011, the mare was twice treated locally with ampicillin for an endometritis due to Streptococcus equi subsp. zooepidemicus. In July 2012, she was presented for routine transrectal ultrasonographic examination of the reproductive tract before planned breeding with frozen semen. The mare had poor perineal conformation with 50% of the vulva dorsal to the ischiatic arch. A reproductive examination was carried out, including bacteriological and mycological tests of a uterine swab and low volume uterine lavage. No inflammatory cells or fungal organisms were seen on cytology and no bacterial growth was detected after a 48 h aerobic culture. In August 2012, the mare underwent artificial insemination with frozen semen and pregnancy was confirmed 17 days after breeding via transrectal ultrasonographic examination. Because the mare had a poor vulvar conformation, a Caslick׳s procedure was performed. During the first months of gestation, on physical examination the mare appeared bright, was alert and was in good body condition but at approximately the sixth month of gestation premature udder development was observed and interpreted as a warning sign of placentitis and abortion. Transrectal ultrasound revealed increased thickness of the combined uterus and placenta with evidence of chorioallantoic edema but no placental separation. Abortion occurred the day after presentation, on 18th February 2013 (day 0). The aborted fetus was in good condition. The placenta appeared thickened with a gritty appearance and covered by a dense brown exudate and necrotic material, mostly around the cervical star (Fig. 1). A direct swab from the placental surface was carried out and cytological slides immediately air dried. No gross lesions in the fetus were visible at necropsy. Smears from uterine exudates were stained with May Gruenwald–Giemsa (MGG) and observed under a light microscope. The samples were highly cellular with blue proteinaceous ground and a mixed inflammatory population composed of degenerate and hypersegmented neutrophils and large macrophages. The cytoplasm of the macrophages was filled by dozens of 3–6 μm wide round to oval bodies with thin body walls and bluish cores; some yeasts were in active budding with occasional pseudohyphae formation (Fig. 2). The organisms were consistent with yeast-like fungus. Immediately, samples from the placenta and other fetal organs were submitted to the diagnostic laboratory for evaluation. Tissues were fixed in buffered 10% formalin for approximately 48 h before preparing histological sections which were subsequently stained (day +2). Histologically, the placenta was markedly thickened with edema of the lamina propria and diffuses infiltration by lymphocytes, plasma cells, and macrophages. The villous surface of the allantochorion was covered by acellular eosinophilic necrotic material containing bluish nuclear debris and colonies composed of myriads of round to oval yeast, 4–5 μ wide, pale staining, with thin body walls (blastospheres) occasionally seen in the cytoplasm of macrophages and neutrophils. Occasional yeast showed peripheral budding with rare short chains of blastospheres (pseudohyphae) (Fig. 3A and B). The yeast was strongly stained by Periodic-Acid–Schiff (PAS) and Grocott׳s silver stain. In fetal tissues, a lymphocytic and macrophagic infiltrate was seen in the interalveolar septa in the lung and the periportal areas in the liver but no fungal bodies were seen histologically. The other organs were unremarkable. The same day of abortion, the placenta, lung, liver, spleen, kidney, and stomach contents were cultured for aerobic bacteria and fungi on blood agar, MacConkey agar, mannitol salt agar and Sabouraud dextrose agar+chloramphenicol at 37 °C in 5–10% CO₂ for 3 days. A heavy growth of yeast with the morphology of Candida was isolated from all our samples and non-pathogenic bacterial growth was obtained from these organs (day +3). Identification of the yeast was based on its morphology on Sabouraud dextrose agar: the colonies were flat, glossy, smooth-edged and cream-colored (Fig. 4). A lactophenol cotton blue preparation from the cultured material was performed and the microscopic examination of this isolate revealed oval and elongated yeast cells of various sizes. Candida cells formed clusters of yeast cells with relatively few short pseudohyphae having small groups of blastoconidia at the septa. No true hyphae were produced. Moreover, a biochemical profile was obtained using a commercial yeast identification kit API-ID 32C system and Vitek-2 system was performed, in accordance with the manufacturer׳s instructions (bioMérieux, S.P.A.). These two biochemical identification methods were not able to discriminate between Candida famata and C. guilliermondii, giving for each species a probability score of 50%. Another technique used for a rapid and reliable identification of yeast was the Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI–TOF MS) (day +7) which resulted in confirming C. guilliermondii with a score of 2.303. For the PCR assay (day +7), DNA was extracted from the yeast colony using a commercial kit (DNA Mini kit, Qiagen) in accordance with the manufacturer׳s instructions. The DNA extracted from the Candida spp. isolate was subjected to PCR amplification. The primer pair NL1 and NL4 was used for the amplification of the D1/D2 domain of the 26S rRNA gene [14]. Initial experiments determined the optimum PCR conditions to be as follows: a reaction volume of 50 μl contained 0.25 mM each deoxynucleoside triphosphate, 1.5 mM magnesium chloride, 0.5 μM primer forward (NL1 5-GCA TAT CAA TAA GCG GAG GAA AAG-3), 0.5 μM primer reverse (NL4 5-GGT CCG TGT TTC AAG ACG G-3), Taq buffer 1× and 2.5 U of Taq polymerase (Promega, Southampton, UK), and 1.5 μl of Candida DNA as template. The reaction mix was kept in the GeneAmp PCR System 2400 thermocycler (Applied Biosystems, Foster City, CA). The PCR amplification was carried out after initial DNA denaturation at 95 °C for 10 min, then 35 cycles of: a denaturation step at 94 °C for 1 min, an annealing step at 57 °C for 1 min and an extension step at 72 °C for 2 min. The PCR products were analyzed by electrophoresis on 1.5% agarose gel, stained with ethidium bromide (0.5 μg/mL) and visualized under UV light. The PCR products were purified using Wizard SV Gel and PCR Clean-up System (Promega Corporation, Madison, WI, USA) in accordance with the manufacturer׳s recommended protocol and subjected to direct sequencing. All gel-purified PCR products were sequenced using gene specific primers. The final sequences were submitted to a BLAST analysis to verify specific amplification. After alignments in the EMBL GenBank database, the sequences were found to be identical to M. guilliermondii (GenBank accession: JQ686902.1), confirming that this species of Candida was the agent of the abortion. The final diagnosis was severe diffuse necrotizing placentitis with intralesional yeast, identified as C. guilliermondii, associated with fetal pneumonia and hepatitis. The uterus of the mare was infused (day +20) with 500 mg of miconazole suspended in 60 mL of sterile saline solution for 5 days, a treatment which was repeated for two consecutive heats. A further uterine lavage was performed (day +34), with 3 L of sterile saline solution, and samples were obtained for cytological evaluation. Although there were no yeast organisms seen at that time, the mare was not rebred during that breeding season.

Fig. 1.

Macroscopic placenta appearance. Chorionic surface of the placenta is covered by a thick brown exudates and yellowish necrotic areas.

Fig. 2.

Cytological appearance of the direct placental swab. Non-degenerated neutrophils and macrophages containing many round to oval, 4–6 µ wide, bluish yeasts with thin body walls and occasional narrow base budding (May-Grünwald–Giemsa, bar: 10 µ).

Fig. 3.

Histological appearance of the placenta. (A) Chorionic villi showing diffuse coagulative necrosis with scattered aggregates of PAS positive fungi (arrows) associated with focal infiltrates of inflammatory cells (arrowheads) and diffuse infiltration of chorionic lamina propria (asterisk). (Periodic Acid–Schiff, bar: 20 µ). (B) Infiltration of neutrophils and macrophages around clusters of PAS positive yeasts, some of which are intracytoplasmic. Inset: detail of previous picture showing pseudohyphae composed of a short chain of blastospores. (Periodic Acid–Schiff, bar: 20 µ; inset: bar: 10 µ).

Fig. 4.

Colonies׳ appearance on Sabouraud dextrose agar after 48 h of incubation at 37 °C. Candida appears as yellowish-white colonies.

3. Discussion

C. guilliermondii is a saprophytic opportunistic pathogenic yeast considered to have low virulence. Limited information is found in the literature concerning the epidemiology, ecological niches, and frequency of occurrence in the clinical and environmental settings of non-albicans Candida species, particularly C. guilliermondii. It is interesting to note that the organism is widely distributed in nature and frequently isolated from soil, plants, insects, seawater, the atmosphere, exudates of several trees, and processed foods besides being a part of saprophyte human and animal microflora on the skin and mucosal surfaces. However, it is considered to be an uncommon causative agent of disease [15]. Virulence of these non-albicans Candida species is strictly related to the decrease in the efficiency of local or systemic host immune system. In this case, a systemic immunodepression was excluded as the mare was otherwise healthy and recovered quickly from the abortion and a strong inflammatory reaction was visible on the histological sections. Most probably the disease resulted from massive contamination of the uterus during the gynecological procedures of artificial insemination resulting in an ascending infection through cervix, placentitis and abortion. In cattle with Candida abortion, the pathogenesis of uterine infection is assumed to be hematogenous [16]. In the present case, however, the lesions were most severe around the cervical star, as happens in ascending bacterial infections causing abortion in mares, and the affected area was very thick and leathery. In these cases, the cervical star often becomes too thick to rupture during foaling and causes the placenta to break at the junction of the body and horns. In this particular case, it was not possible to determine if the contamination occurred during artificial insemination or later, after the implant of the conceptus. In women, Candida abortion occurs rarely but it is often associated with the presence of an Intra Uterine Device (IUD) that can carry infection from contaminated external genitalia into a relatively sensitive organ, the uterus [17]; the same could have happened in the mare. Poor perineal conformation could have represented a predisposing factor as it increased the fecal-born contamination of external genitalia by Candida species. It may also be possible that there were compromised uterine defense mechanisms associated with delayed uterine clearance resulting in a decreased ability to clear fungi from her uterus. The use of antibiotics is commonly incriminated as being a predisposition to fungal infections; however, reports also occur where no intra-uterine therapy was undertaken [6]. The mechanism involved in fungal contamination and colonization of the pregnant uteri of mares is still not well understood and studies on the pathogenesis are scarce; however, the authors agree in attributing the abortion to an ascending infection through the vagina. Several studies in humans and non-human primates suggest that proinflammatory cytokines play a key role in the pathogenesis of infection-associated preterm delivery or abortion [18]. Candida abortion in mares is very rare [12]. Ball et al. [12] reported an intrauterine inoculation of C. parapsilopsis which induced embryonic loss in pony mares. Embryonic losses could have been due to (1) luteolysis secondary to the induced endometritis, (2) a direct effect of the inoculated fungal pathogen, (3) an effect of the induced inflammatory response, or (4) a combination of these factors. They conclude that the rapid decline in serum progesterone concentrations after Candida inoculation in experimentally infected mares suggests that a uterine-induced luteolysis is the cause of embryonic loss. In the current case, abortion was due to severe diffuse necrotic placentitis and invasion of the fetus by Candida as demonstrated by isolation of the yeast from several fetal tissues. Another interesting consideration derives from a recent paper about the microbial quality of frozen equine semen. The authors analyzed the prevalence of yeasts in frozen equine semen and show that Candida was found in 57.1% of cases (Candida incospicua, 42.8% and C. guilliermondii, 14.3%). This data is very interesting because the microbial surveillance of frozen semen is quite important to verify the contamination and thus the chance of transmission of the pathology to the mare by artificial insemination [19]. In men, there is some evidence that the male may harbor fungi that can result in re-infection of their sexual partner. Fungi have been isolated from the penis and from seminal fluid in human males, suggesting that infection of urethra or seminal vesicles may occur. Males thus infected remain asymptomatic carriers [20]. To our knowledge, the case presented here is the first report on equine abortion caused by C. guilliermondii.

Conflict of interest statement

There are none.