Darko Marinković, Milan Aničić, Slobodanka Vakanjac, Svetlana Nedić and Vladimir Magaš
The estrus cycle of bitches is divided into four phases: proestrus, estrus, diestrus and anestrus, during which different morphological changes, and also cyclic changes of estrogen and progesterone receptors are present. Several pathological changes can be differentiated on the endometrium, but one of these is the most important - cystic endometrial hyperplasia, which frequently develops into pyometra. The aim of the present study was to describe morphological characteristics, and expression of estrogen and progesterone receptors on the endometrium of mixedbreed bitches during the different phases of the estrus cycle, cystic endometrial hyperplasia and pyometra. The uterus and ovaries of 36 mixed breed bitches in different phases of the estrus cycle and also with cystic endometrial hyperplasia (CEH) and chronic purulent endometritis - pyometra were examined macroscopically, histopathologically, and immunohistochemically for estrogen receptors (ΕR) and progesterone receptors (PR). During proestrus uterine cells showed a weak reaction for both estrogen and progesterone receptors, but during estrus a large number of uterine cells showed a strong reaction on estrogen receptors and moderate reaction on progesterone receptors. On the contrary, during diestrus the scores for the estrogen receptors decreased, while the progesterone receptors level increased - uterine cells expressed strong reaction for progesterone receptors, and moderate reaction for estrogen receptors. Uterine cells in cystic endometrial hyperplasia expressed a strong reaction for estrogen receptors, and moderate reaction for progesterone receptors, but on the other hand the uterine cells in the uterus with pyometra expressed a moderate to strong reaction for progesterone receptors, and a weak reaction for estrogen receptors. In further investigations it would be interesting to perform quantitative analysis for both estrogen and progesterone receptors during different phases of the estrus cycle and also in the uterus with cystic endometrial hyperplasia and pyometra.
Darko Marinković, Jòzsef Özvegy, Milan Aničić, Ivana Vučićević, Slađan Nešić and Vladimir Kukolj
Gastric dilatation and volvulus is a life-threatening condition characterized by rapid accumulation of food and gases that cause displacement and distension of the stomach. The large and giant, deep-chested breeds of dogs are at higher risk for developing the gastric dilatation and volvulus. Uncommonly, it can also develop in cats, but it is also described in free-range polar bears.
A case of gastric dilatation and volvulus in a brown bear (Ursus arctos) is described in this paper. This case was characterized by lack of any previous symptoms, sudden death, as well as macroscopic findings during necropsy - twisted distended stomach, congested displaced spleen and necrotic gastric wall. According to the available data this is the first described case report of the gastric dilatation and volvulus in brown bear (Ursus arctos).
Milan Aničić, Vladimir Kukolj and Darko Marinković
Equine glanders is a severe bacterial disease known since ancient times. Although eradicated in the most part of the world it is now considered re-emerging. Considering very scarce literature data, we used from formalin fixed collection material: nasal septum, lung and skin specimens from naturally infected horses. Tissues were grossly examined and photographed. Tissue samples, after standard processing, were stained with HE, Congo red and Groccot and microscopically examined. Gross changes include nodules and ulcers in the nasal mucosa with granulation and scarring, pyogranulomas in the lung tissue and nodules and ulcers of the skin. Microscopically marked inflammation of affected tissues with neutrophilic and mononuclear infiltration and fibrous tissue proliferation were seen. As a potent zoonotic agent it has been already used as a biological weapon in the past.
Darko Marinković, Natalija Milčić-Matić, Milan Jovanović, Ivana Vučićević, Slađan Nešić, Milan Aničić and Sanja Aleksić-Kovačević
Mast cell neoplasia in dogs can occur in two different forms: common as cutaneous tumor, or less common as a systemic form of neoplastic mast cell proliferation - systemic mastocytosis. The aim of this study was to compare the histological and cytological features, KIT receptor expression and presence of c-KIT proto-oncogene mutations in neoplastic cells of dogs with canine cutaneous mast cell tumor (CMCT) and systemic mastocytosis. Microscopical examination of the cytological smears obtained from all selected dogs revealed that cellular specimens were constituted mostly of round cells with a central nuclei and fine to coarse purple cytoplasmic granules. Histopathological examination of skin samples of dogs with CMCT and a dog with systemic mastocytosis showed proliferation of the neoplastic mast cells in the superficial and/or deep dermis. Similar findings were observed in tissue samples derived from lymph nodes, spleen, liver, myocardium and kidneys of a dog with systemic mastocytosis. Three dogs with high grade CMCT as well as one dog with systemic mastocytosis showed cytoplasmic CD117 expression, while 3 dogs with low grade CMCT, had membranous expression of CD117. Based on our study, histological features and cytoplasmic CD117 expression in neoplastic cells of dogs with systemic mastocytosis are similar to those in dogs with high grade CMCTs. Nevertheless, mutations of c-KIT proto-oncogene were not found in tumor samples either from dogs with CMCT or dog with systemic mastocytosis.
Jòzsef Özvegy, Darko Marinković, Miloš Vučićević, Bojan Gajić, Jevrosima Stevanović, Dejan Krnjaić and Sanja Aleksić-Kovačević
Blood smears stained with Diff Quick are the initial tool for cytological diagnosis of Haemogregarina spp. However, the development of sensitive and specific molecular methods enabled the detection and identification of parasites in the sample and to clarify the evolutionary relationships of adeleorinid parasites within the Apicomplexa.
The current study was attempted in order to perform cytological investigation and molecular identification of the hemoparasites in thirty European pond turtles (Emys orbicularis) from the quarantine section at Belgrade Zoo, which have been found in poor health condition with massive skin hemorrhages, based on intraerythrocytic parasitic forms on hematological smears and 18S rDNA sequence, respectively.
Different life cycle stages of the Haemogregarina sp. were noticed within the erythrocytes in the peripheral blood. Biochemical analysis indicated lower values of AST and iron in most of the infected turtles while hematological analysis showed a changed hematocrit value, a decrease in the number of red blood cells and low hemoglobin levels. Amplifications of the 18S rDNA sequence of Haemogregarina were detected in 30/30 (100%) turtles with clinical symptoms. The identity of PCR products was confirmed by direct DNA sequencing. Future research concerning H. stepanowi in Serbia should be applied to its definitive host-the leech.
Zoran Zorić, Olivera Lozanče, Darko Marinković, Miloš Blagojević, Ivana Nešić, Natalija Demus and Milena Đorđević
Hippopotamidae family is nowadays represented by two species within two different genera: pygmy hippopotamus (Choeropsis liberiensis) and common hippopotamus (Hippopotamus amphibius). The common hippopotamus has a very unique anatomy, and the shape of the body, especially the head is adapted for a semi-aquatic life style. The morphological examination and description of the gross anatomical features of the hippopotamus skull is described in this paper. The shape of the skull is adapted for the amphibian way of life. Their eyes, ears and nostrils are placed high on the roof of the skull which allows these organs to remain above the surface of the water while the animal is being submerged underwater. The skull is massive, but the brain case (neurocranium) is extremely small compared with the splanchnocranium and complete head. The dental formula of the common hippopotamus is: incisors (I) 2/2, canines (C) 1/1, premolars (P) 3-4/3-4 and molars (M) 3/3. Incisors and canine teeth are formed in the shape of tusks and are used for threat or “demonstration of power” among animals when vigorously fi ghting. Incisor teeth grow continuously and are twice bigger in males than in females.
Ivana Vučićević, Darko Marinković, Vladimir Kukolj, Miloš Vučićević, Milorad Mirilović, Slađan Nešić and Sanja Aleksić-Kovačević
Histopathological examination, grading, immunohistochemical staining and molecular genetic examinations are the proposed criteria that should be used for cutaneous mast cell tumors (CMCTs) classification. The presence of aberrant CD117 expression and mutations of the c-kit proto-oncogene could be an indicative parameter for final histological grading. Determination of the connection between the localization of KIT receptor expression and the histological grade of CMCTs without c-kit proto-oncogene mutations was the main goal of this study. The study included twenty four CMCTs and six control skin samples from 30 dogs of different ages, breed and sex. Formalinfixed and paraffin-embedded tissue samples were stained with hematoxylin-eosin and toluidine blue and immunohistochemically tested for CD117 expression. DNA was extracted from the same paraffin blocks and subsequent polymerase chain reaction amplification was performed using PE1 and PE2 primers. Degree of malignancy was determined based on the presence of mitotic figures, multinucleated cells, bizarre nuclei and karyomegaly in 10 high power fields. Based on histological features, fourteen of 24 CMCTs were of a high histological grade, while ten were classified as a lowgrade malignancy. CD117 cytoplasmic expression was observed in nine of fourteen high-grade malignancy CMCTs, which confirms the link between the aberrant CD117 expression and increased cell proliferation.
Ivana Vučićević, Darko Marinković, Vladimir Kukolj, Slađan Nešić, Milan Aničić, Biljana Đurđević and Sanja Aleksić-Kovačević
Peripheral nerve sheath tumors (PNSTs) comprise a heterogeneous group of neoplasms originating from the elements of the nerve sheath. They are divided into two forms: benign and malignant PNST. Both benign and malignant PNSTs are not very common in domestic animals but they are reported in different animal species. Histologically, PNSTs are composed predominantly of spindle cells arranged in bundles, whorls and sheets, with a different number of pleomorphic cells and mitotic figures.
The aim of this study was a reclassification of 17 dog tumor samples initially diagnosed with peripheral nerve sheath tumors using histopathological analysis. The main criterion for reclassification was immunohistochemical positivity for various antigens.
PNSTs are often histologically very similar to other spindle cell tumors and immunohistochemistry is required for differential diagnosis. PNSTs generally express vimentin, S-100 protein, glial fibrillary acidic protein (GFAP), collagen IV and laminin.
Four tumor samples were positive to muscular marker α-SMA and vimentin and negative for S-100 protein and desmin. The spindle cells whirling around some blood vessels were observed in these tumors so they were reclassified as perivascular wall tumors (PWTs). The other 13 tumors were S-100 protein and vimentin positive and α-SMA and desmin negative, thus classified as PNST.
The use of the immunohistochemical panel is necessary for distinguishing PNSTs from PWTs in routine diagnostics.
Nikola Vasković, Zoran Debeljak, Dejan Vidanović, Milanko Šekler, Kazimir Matović, Milan Aničić and Darko Marinković
Lumpy skin disease (LSD) is a disease of cattle and domestic buffaloes caused by Capripoxvirus which can lead to significant economic losses. Until several years ago it was limited to Africa and the Middle East, but recently it was reported in Turkey (2013), Greece (2015), and in 2016 it spread to Bulgaria, North Macedonia, Albania, Montenegro and Serbia. The aim of this study was to determine the microscopic lesions in the skin of naturally infected animals, highlight their features and compare them to the findings of other authors. Gross lesions in the skin of cattle infected with Lumpy skin disease virus (LSDV) were manifested in the form of skin nodules which were round, raised, clearly circumscribed, firm and randomly distributed in the whole skin including the vulva and udder. Histopathological lesions included degeneration of epithelial cells, hyperkeratosis, spongiosis, and acanthosis present in 85.88%, 81.18%, 84.71%, and 80.0% of samples, respectively. Intracytoplasmic inclusion bodies were noted in keratinocytes of the stratum spinosum in 56.47% of samples, and in hair follicle cells and sebaceous glands epithelial cells in 45.88% of samples. Cutaneous lesions were in the form of inflammatory infiltrate present in the dermis and subcutis in 97.65% of samples. Since there are only a few literature data in this field, this study expands the knowledge relating to morphological alterations in LSD. Based on the characteristic microscopic lesions described in the present study, histopathology should be considered as a very useful method for the diagnosis of LSD.
S. Đorđe Marjanović, Danica Bogunović, Mirjana Milovanović, Darko Marinković, Nemanja Zdravković, Vladimir Magaš and M. Saša Trailović
In the present study we tested the dose andh time dependence of the antinematodal effects of carvacrol and tyhmol on Caenorabditis elegans, and the efficacy of carvacrol, thymol, p-cymene and cinnamaldehyde,which were administrated in the drinking water of rats naturally infected with the pinworm Syphacia muris. The control treatment of the infected rats was carried out with piperazine. Thymol caused a dose and time-dependent mortality in adult C. elegans. The value of the Median Lethal Concentration (LC50) of thymol was 117.9nM after 24h and 62.89 nM after 48h of exposure. Carvacrol exhibited a higher antinematodal efficiency than thymol. The LC50 of carvacrol, after 24 hours of exposure, was 53.03 nM, while after 48 hours it was 33.83 nM. On the other hand, piperazine showed an extremely high efficacy against S. muris infection in rats. Piperazine, at a dose of 625 mg/kg bw, administered in drinking water continuously for 10 days, eliminates the infection completely. However, none of the investigated active ingredients of essential oils were effective against S. muris. The reason for the lack of efficiency may be due to their pharmacokinetic properties. A relatively low amount of, orally administered, active ingredients of essential oils reaches the distal segments of the gastrointestinal tract, where S. muris inhabits the gut (colon and cecum). The obtained results, on C. elegans, indicate a clear dose and time-dependent antinematodal effect of thymol and carvacrol. However, for clinical application, it is necessary to examine the efficacy of microencapsulated formulations with a controlled release of active ingredients of essential oils in certain parts of the gastrointestinal tract.