In February 2013, a female German Shepherd dog, 11 years of age, body weight 40 kg, was referred to the Small Animal Clinic of the Faculty of Veterinary Medicine, Trakia University – Stara Zagora. The owner reported a one-week history of a purulent discharge from the external genitalia, fastidious appetite and depression. According to owner observation, the last estrus was 6 months ago, the dog had given birth six times, without complications. Until the referral to the clinic, the dog had been treated with procain benzylpenicillin and dihydrostreptomycin sulphate (Intramicine, Ceva Sante Animale, France) for 5 days and two subcutaneous aglepristone (Alizin, Virbac, France) injections, but in the owner’s view – without effect. The rectal body temperature was 38.2°C, the heart rate 86/min, the respiratory rate 25/min, and visible mucous coats – pink-red. The abdomen was slightly enlarged, soft and not painful on palpation. The vulva was oedematous, with purulent discharge.

Ultrasonography was done with the ultrasound apparatus DC-6 Vet (Mindray, China), equipped with 6.5 MHz convex transducer using the transabdominal approach. The findings were documented on a Mitsubishi P91E (Tokyo, Japan) printer. During the examination, caudally to the kidney, a large well differentiated hypoechoic mass 7.18 × 6.65 cm in size was visualized in the region of the left ovary, with clearly distinguished several small cavities (Fig. 1-A). The examination of the uterus showed an extremely enlarged anechoic fluid-filled lumen and a thickened wall of the uterus (Fig. 1-B).

A vaginal swab was collected and a cytovaginal smear was prepared and stained (Hemacolor, Merck). Light microscopy (magnification 40×) of the smear showed about 50% superficial cornified epithelial cells and a lot of segmented neutrophils (Fig. 2).

Complete blood cell counts were assayed on an automated haematological analyser BC-2800 Vet (Mindray, China), and blood biochemical parameters – on an automated biochemical analyser BS 120 (Mindray, China). Blood laboratory analysis (Table 1) showed a marked leukocytosis and mild anaemia.

Blood serum progesterone was assayed using a Progesterone ELISA kit (Dialab, Austria), and estradiol – with Estradiol ELISA kit (Human, Germany). The tests use a competitive sandwich ELISA for quantitation of progesterone and estradiol in biological fluids. The sample optical densities were calculated from respective standard curves in pg/ml (for estradiol) and ng/ml (for progesterone). Thus, the blood progesterone of the patient was 3.96 ng/ml, and the blood estradiol – 86 pg/ml.

After aseptic preparation of the abdominal region, the bitch was premedicated with 0.04 mg/kg atropine sulfate (Atropinum sulfuricum; Sopharma; Bulgaria), applied subcutaneously. Fifteen minutes later, anaesthesia was induced by intravenous administration of 0.4 mg/kg diazepam (Diazepam; Sopharma; Bulgaria) and 10 mg/kg ketamine (Anaket 10%; Richter Pharma Ag; Austria). After endotracheal intubation, the anaesthesia was maintained with isoflurane (Terrell™; Minrad Inc.; USA). The median laparotomy was performed. After opening the peritoneum, an enlarged uterus and a considerably altered left ovary, the size of a grapefruit, were observed (Fig. 3), while the right ovary had normal structure and size. There were no tumor metastases in the other abdominal organs. Complete ovariohysterectomy was then performed. Specimens for histology were collected from the excised tumor mass. The abdominal cavity was flushed twice with sterile saline and closed with cross stitch pattern using USP 1/0 polyglycolic acid absorbable sutures (Marlin; Catgut GmbH; Markneukirchen), and the skin was sutured with simple interrupted non-absorbable sutures USP 0 (Vitalon; Dr Hammer & Co. GmbH; Hamburg). Postoperative treatment included antibiotic – 8.75 mg/kg amoxicillin clavulanic acid (Synulox RTU; Zoetis; USA) intramuscularly for 5 days and a NSAID – 2 mg/kg ketoprofen (Ketofen 1%; Merial; France), intramuscularly for 3 days. An Elizabethan collar was placed. Skin sutures were removed after 10 days. The post-operative examination showed that the patient’s recovery was good.

The specimens for histological examination were fixed in 10% neutral formalin. Samples were cut on a paraffin microtome with 4 μm thickness. The cross sections were processed using the standard histological techniques (4) and stained with haematoxylin/eosin. Neoplastic cells were diffusely located (Fig. 4) and resembled primitive germ cells. Their nuclei were vesicular and prominent nucleoli were frequently seen. Mitotic figures were observed in some areas. In regression zones, polynuclear cells and lymphocytes were observed. The tumor stroma was sparse (Fig. 5).