Transmissible venereal tumour (TVT) is most common in tropical and subtropical area containing large populations of free-roaming and sexually active dogs. (4,9). Tumour affects the vagina and external genitalia of the bitch and the penis of the Anatolian Shepherd Dog. Transmission of the tumour occurs at coitus when infected male Anatolian Shepherd Dog. Moreover, autotransmission to the nasal and oral mucosa may occur by licking of the tumour. The lesions are the often friable and multilobulated. Although TVT is known benign tumours, metastases and malignancy risk may be very highly in some cases. Expression of the tumour is controlled by the immune system, with more rapid growth and metastasis occurring in immune-suppressed animals and pediatrics.
Metastasis of TVT to the skin, regional lymph nodes, eyes, brain, spleen, liver, lungs, pituitary, nose, tongue, lips, mammary region and thoracic and abdominal viscera has been reported (4,5). Diagnosis is based on the history, clinical findings and cytological examination. Exfoliative cytology proved to be safe and easy method for the diagnosis of TVT. Microscopically the tumour cells have poorly defined and lightly staining cytoplasm; are large and round and oval; and are uniform in size, with occasional large, hyperchromatic nuclei.
It is known that the surface of cancer cells differs in many structural and functional characteristics from those of normal cells. Neoplastic transformation of a variety of cell types is associated with changes in the compositon of membrane glycoproteins. Sialic acids, a group of sugars, have a strong electronegative charge and located in the outer cell membrane glycoconjugates in higher organisms. A small portion of the total sialic acid (TSA) is free in tissues and body fluids; most is bound to glycoprotein and glycolipid . They participate of the many biological and pathological processes including metastatic spread and tumor antigenity. Increased concentration of sialic acid at the surface of malignant cells in animal and human systems has been related to potential malignancy and changes in immunogenicity.
Although not much is known about the reasons for the increased sialylation and sialyltransferase activity observed in many cancer cells, often tumour cells seem to be efficiently protected from immune defense by highly sialylated surfaces; this may contribute to the spread of the tumour. In many types of neoplasia, serum TSA and LSA levels are diagnostic and prognostic indicator and more suitable for monitoring disease extent and anticancer therapy, Thougaard et al. reported that TSA might also be useful as a tumour marker in the dog. The aim of this study was to find out whether there is any correlation between the levels of sialic acid in serum and the canine TVT for using in diagnosis.
Seven bitches, with naturally occurring TVT admitted to University of Adnan Menderes, Faculty of Veterinary Medicine, Clinic of Obstetrics and Reproductive Diseases, were included in the study group. All of the bitches in this group were Turkish Anatolian Shepherd dogs. The mean age of the bitches was 6.71±2.54 years (1-17 years) and mean body weight was 25.28±1.77 kg (21-33 kg). The diagnosis for the tumour was based on clinical findings and cytological examination. Vaginal smears were stained using Papanicolaou method and examined microscopically. The duration of illness was between 1 and 7 months prior to the chemotherapy. All clinical signs were serosanguineous or bloody vaginal discharge, protrusion of a mass from the rima vulva, licking and odor. In the radiographic and ultrasonographic examination, no metastatic focuses could be detected.
Eight healthy female dogs between the ages of served as the control group. All of them in this group were also Turkish Anatolian Shepherd dogs. The mean age of the bitches was 5.00±1.77 years (1-14 years) and mean body weight was 25.12±2.62 kg (22-30 kg). After bitches in control group were examined generally, it was confirmed that the individuals were not suffering from any inflammatoric and metabolic disease. The absence of TVT cells was verified by microscopical examining in Papanicolau-stained vaginal smears. For the cytological diagnosis of the tumour, exfoliative cytology was used. Cotton swabs were wetted with saline and inserting into vagina. After fixation in ether-ethanol, smears were stained by using Papanicolau method. TVT cells were defined as the round to ovoid cells with distinct cytoplasmic borders, round nuclei with one or two prominent nucleoli and moderate amount of faintly light violet cytoplasm in all cytological slides. In addition, the dogs with tumours were staged at clinical according to the World Health Organization staging system after cytologic confirmation. T: Primary tumour size; N: Regional lymph nodes (inguinal) and M: Distant metastasis detected clinical, radiographic and ultrasonographic examination. It was examined their skin, eyes, nose, tongue, lips clinically, genital tract and abdominal organs ultrasonographically and lungs radiographicaly, respectively.
Serum samples for sialic acid and lipid bound sialic acid levels determination were obtained from venous blood after 12 h fasting by centrifugation of the clotted specimen within 30 min. Sera were separated and stored at -200C until analyses. The bitches did not receive chemotherapy (vincristine sulfate) before blood samples were collected Analyzing were performed in Adnan Menderes University, Faculty of Veterinary Medicine, Department of Biochemistry. Estimation of total sialic acid: Serum total sialic acid levels were detected as described by Sydow et al. Briefly, 400 μl of serum were treated with 3 ml of 5% perchloric acid for 5 min at 100ºC and centrifuged at 1400 g for 4 min. 2 ml of supernatant were mixed with 400 μl of Echrlich reagent (5g p-dimethylaminobenzaldehyde/ 50ml HCl/ 50ml distilled water). After incubation at 100ºC for 15 min, 2 ml of distilled water were added on sample and a spectrophotometer (Shimadzu, UV-1601) was used to read the optical density at 525 nm.
Estimation of lipid bound sialic acid: Serum LSA levels were measured as described. Briefly, 50μl of serum was extracted with chloroform-methanol (2:1 v/v) maintained at 4 0C. The lipid extract was separated with 0.5 ml of distilled water. The aqueous layer was precipitated with phosphotungostic acid. The precipitates were re-suspended in 1 ml of distilled water and lipid-bound sialic acid in suspension was determined with resorcinol reagent. The TSA and LSA contents were calculated using standard curves obtained for various concentration of N-acetyl neuraminic acid (Sigma, A-0812).
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