Dorota Różańska

Mitochondrial D-loop mutations and polymorphisms are connected with canine malignant cancers

Abstract The aim of the conducted investigations was to identify differences in the D-loop nucleotide sequence between neoplastic tissue, normal tissue, and blood and to determine their correlation with the type of cancer in dogs. In 62.5% of the analyzed tumors of epithelial origin and 25% tumors of mesenchymal origin, substitution was detected within the D-loop sequence between the neoplastic tissue, normal tissue, and blood. Two mutations occurring in the carcinogenic process in position T15620C have been identified in epithelioma glandulae sebacei and carcinoma planoepithelialae keratodes. Blood and cancer heteroplasmy was diagnosed for carcinoma planoepithelialae keratodes and “Comedo” carcinoma. The results of the study indicate that polymorphic changes in the D-loop sequence promote carcinogenesis in dogs. Heteroplasmy diagnosed in blood and tumor cells and absence thereof in normal tissue may imply mtDNA recombination. High prevalence of mtDNA mutations in canine tumors may suggest mtDNA genetic instability, which is likely to play a role in carcinogenesis.

Mitochondrial DNA polymorphism in genes encoding ND1, COI and CYTB in canine malignant cancers

Abstract The aim of the study was to identify DNA changes in mitochondrial gene fragments: NADH dehydrogenase subunit 1 (ND1), cytochrome c oxidase subunit I (COI) and cytochrome b (CYTB) in tumor tissue, normal tissue and blood, and to define their association with the tumor type in dogs. Molecular analysis included 144 tests in total. A functional effect of the non-synonymous protein coding SNP was predicted. The presence of polymorphisms in all tested gene fragments in individual tissues of dogs was observed. Heteroplasmic changes were found in ND1 and CYTB in epithelioma glandulae sebacei and in CYTB in lymphoma centroblasticum. The results of in silico analysis show the impact of these alleles (COI: 507, ND1: 450, 216, CYTB: 748) on the functioning of proteins and thus their potential role in carcinogenesis. The possible harmful effects of changes in polypeptides in positions T193N, V98M, V118M and H196P were evaluated. It seems that polymorphisms occurring in cells can have a negative impact on functioning of proteins. This promotes disorders of the energy level in cells.

Current status of prevalence of yeast-like fungi in the environment of horses bred in Poland

Abstract This study presents an analysis of the yeast-like fungi population in the healthy horses’ living environment. The study material was sampled from various elements and equipment of a stable. We compared the yeast-like fungi population from the breeding environment of five groups of horses (English Thoroughbred horses, Arabian horses, Polish Half-Bred horses, cold-blooded horses, and Hucul horses and Highland ponies). The samples were collected in the summer and winter period. The laboratory examination involved 260 samples and resulted in identification of yeast-like fungi belonging to 13 species. Over 50% of the samples collected from the horse environment in wintertime and fewer than 18% of the summer samples displayed fungal growth. The largest amounts of yeast fungi were isolated from samples collected from water and feed troughs. The laboratory analysis demonstrated qualitative diversity of the yeast-like fungi population depending on the breeding environment of the individual horse groups. Quantitative differences of isolates were additionally related to the season of the year. Streszczenie Niniejsza praca przedstawia analizę występowania grzybów drożdżopodobnych w środowisku życia zdrowych koni. Materiał do badań pobrano z różnych elementów stajni i wyposażenia. W pracy porównano częstość izolacji grzybów z próbek pochodzących ze środowiska hodowlanego 5 grup koni (pełnej krwi angielskiej, czystej krwi arabskiej, małopolskich i zimnokrwistych oraz koni rasy huculskiej i Highland ponies). Próby pobierano w okresach letnim i zimowym. Łącznie przebadano laboratoryjnie 260 prób, z których wyhodowano i zidentyfikowano grzyby drożdżopodobne należące do 13 gatunków. Wykazano zróżnicowanie częstości izolacji różnych gatunków w zależności od środowiska hodowlanego poszczególnych grup koni. Różnice te związane były ponadto z porą roku. Z prób pobranych ze środowiska koni w okresie zimowym w ponad połowie przypadków wystąpił wzrost grzybów, natomiast w okresie letnim - w niespełna 18%. Najwięcej drożdżaków wyizolowano z prób pobranych z poideł i żłobów.

Mitochondrial D-loop mutations can be detected in sporadic malignant tumours in dogs

Abstract The aim of this study was to identify mutations in the D-loop region of mtDNA in head, neck, and limb tumours in dogs, and determination of their relationship with the process of neoplastic transformation. Blood and tumour tissue samples from 19 dogs with diagnosed sporadic malignant tumours were analysed. DNA extraction, amplification, and sequencing of the mtDNA D-loop, and bioinformatic analyses were performed. Five mutations and 19 polymorphisms were observed in 68.42% of all tumours. Polymorphic variants were noted in 42.86% of the head and neck tumours and in 58.33% of the limb tumours. Mutations were observed in 21.05% of dogs. The mutations were found in 28.57% of the head and neck tumours and in 16.66% of the limb tumours. The mutations were identified in 50% of the studied epithelial cancers. In the mesenchymal tumours, no mutations in the D-loop region were observed. Mitochondrial haplotype A17 was found in over 40% cases of limb tumours. No association between the age, breed, sex, type of tumour, and detected polymorphic variants were observed. Different mutational changes in the D-loop sequences of mtDNA identified in the blood and tumour tissues may indicate a relationship between the type of tumour and individual changes in the D-loop nucleotide sequences of mtDNA.

Testicular disorder of sex development in four cats with a male karyotype (38,XY; SRY-positive)

The molecular background of disorders of sex development (DSD) in cats is poorly recognized. In this study we present cytogenetic, molecular and histological analyses of four cats subjected for the analysis due to ambiguous external genitalia. Three cases, with rudimentary penises and an abnormal position of the urethral orifice, represented different types of hypospadias. The fourth case had a normal penis, a blind vulva and spermatogenetically active testes. Histological studies showed structures typical of testes, but spermatogenic activity was observed in two cats only. All the cats had a normal male chromosome complement (38,XY) and the Y-chromosome linked genes (SRY and ZFY) were also detected. Fluorescent in situ hybridization (FISH), with the use of the feline BAC probe harboring the SRY gene, excluded the possibility of chromosome translocation of the Y chromosome fragment carrying the SRY gene onto another chromosome. Sequencing of four candidate genes (SRY--sex determining region Y; AR--androgen receptor; SRD5A2--steroid-5-alfa reductase 2 and MAMLD1--mastermind-like domain containing (1) revealed one SNP in the SRY gene, one common polymorphism in exon 1 of the AR gene (tandem repeat of a tri-nucleotide motif--CAG), six polymorphisms (5 SNPs and 1 indel) in the SRD5A2 gene and one SNP in the MAMLD1 gene. Molecular studies of the candidate genes showed no association with the identified polymorphisms, thus molecular background of the studied DSD phenotypes remains unknown.

Mycological profile of the integumentary system in felinski ponies

Abstract Yeast-like microorganisms are frequently isolated from horses. There is a serious interpretation problem with assessment of the health status in the diagnostics of infectious diseases, which is associated with the insufficient knowledge of the composition of the natural flora in the horses’ integumentary system. The aim of the investigations was to analyse the yeast-like flora isolated from healthy Felin ponies. The study involved 44 horses aged from 2 to 12 years. The analysed group comprised 28 mares and 16 stallions. The animals were kept in an outdoor rearing system with a possibility of periodic use of a wooden/brick shed. The investigations were conducted in an annual cycle divided into 4 periods corresponding to the seasons of the year. Smears were sampled from horses’ nostrils, mouth, ear, back, groin, vagina or foreskin, and collateral groove. Microbiological analysis of the yeast-like strains was performed after plating 1015 samples of material taken from healthy horses. The identification of fungal species was based on their culture characteristics and biochemical profile. Fourteen yeast species were isolated and identified from the sampled material. Candida guilliermondii and Candida albicans 1 were the most prevalent fungi. The study has shown seasonal variability of the composition of the integumentary system microflora in Felin ponies. The greatest number of isolates was obtained from the material sampled from the mouth.

Penile hypoplasia and rudimentary prepuce in a dog (78,XY; SRY-positive): a case report.

This is the first report in the literature concerning penile hypoplasia and rudimentary prepuce in a 78,XY; SRY-positive male that received a successful surgical treatment. A six-month-old male mixed-breed dog with body weight of 3.5 kg and with symptoms of prolonged stranguria and abnormalities of the external genitalia were presented by the owner at the University Surgical Clinic. Clinical, biochemical, radiological, pathological, and genetic examinations of the dog were carried out. Penile hypoplasia and rudimentary prepuce were diagnosed. Early diagnosis of penile hypoplasia and rudimentary prepuce in small animals requires a high level of vigilance and is based on clinical and ultrasonographical findings. The radiography revealed a fan-shaped widening of the caudal part and shortening of the os penis. A hyperechogenic os penis with a wide posterior part and a slightly curved, smooth anterior end was imaged. No normal prepuce structures were observed. The endocrinological examination showed a substantially decreased testosterone level. Fast surgical intervention is preferable and confirms the diagnosis. In the presented case report enlargement of the preputial orifice was applied in order to prevent urinary retention and recurrent urinary tract infections. Orchiectomy was also performed. After the surgery, immediate clinical improvement was noted. The testicular atrophy diagnosed in the histological analysis explains the low level of the hormone. The cytogenetic analysis revealed a normal male set of chromosomes – 78,XY. The molecular analysis showed presence of the SRY gene as well as the ZFY gene, which reside on the Y chromosome and the ZFX, which is X-linked. The successful amplification of the SRY, ZFX, and ZFY genes confirmed the presence of both X and Y chromosomes. Sequencing and comparison with the reference sequence of the canine SRY gene indicated a normal sequence in the examined dog. Given the absence of polymorphisms and mutations in the coding sequence of the SRY gene, it can be assumed that it is not associated with the observed phenotype. The studied case was classified as testicular XY (SRY-positive) disorder of sex development (DSD), the aetiology of which remains unknown.

Unilateral flank ovariohysterectomy in guinea pigs (Cavia porcellus)

Abstract AIMS: To describe a simple, minimally invasive method of ovariohysterectomy via a unilateral flank approach in guinea pigs, for use in routine desexing of healthy female guinea pigs or treatment of ovarian cysts. METHODS: The subjects of this retrospective study were 41 client-owned guinea pigs submitted for routine desexing or treatment of ovarian cysts. They included 16 healthy female guinea pigs aged 8–12 months (Group 1), and 15 females aged from 9 months to 3 years (Group 2), and 10 females aged from 3 to 7 years (Group 3) with different-sized ovarian cysts. Prior to surgery, the animals received clinical examination, blood testing (complete blood count and serum biochemistry profile) and examination of the abdomen using ultrasonography, to assess the condition of the reproductive tract and ensure the guinea pigs were fit for surgery. Ovariohysterectomy was performed via a unilateral flank incision made close to the erector spinae muscle starting approximately 1 cm caudal to the last rib. Both ovaries, uterine horns, and the uterine cervix were localised, ligated, and dissected through this unilateral retroperitoneal incision. RESULTS: Ovariohysterectomy was successfully completed via a single flank incision in 38/41 (93%) guinea pigs. Three guinea pigs with ovarian cysts from Group 3, which were >6 years old died during surgery due to circulatory and respiratory failure under anaesthesia. In the remaining 38 cases, surgery proceeded without complications. A further two guinea pigs from Group 3 were reluctant to move or eat for the first 3 days after surgery but recovered after provision of supportive care. All 38 animals fully recovered and wound healing was normal. CONCLUSIONS: This is the first report of ovariohysterectomy via a unilateral flank incision in guinea pigs. This approach is a simple, minimally invasive and safe alternative to the midline or bilateral flank approaches currently used for surgery of the reproductive tract in guinea pigs.