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Haematologic abnormalities possibly associated with exposure to vector-borne pathogens are rarely reported in clinically healthy outdoor dogs. Therefore, we analysed changes in the complete blood count (CBC) of clinically healthy outdoor dogs seroreactive to Anaplasma spp. and Babesia spp., with or without microfilariosis. Stray, shelter and hunting dogs, 81 in total, that were polymerase chain reaction negative for Anaplasma spp. and Babesia spp. were divided into groups according to their seroreactive status and results of a modified Knott’s test: seronegative to both Anaplasma spp. and Babesia spp. SN (N=26); seroreactive to A. phagocythophilum SR-A (N=12); seroreactive to B. canis, B. gibsoni and/or B. vogeli SR-B (N=25); and seroreactive to both of the pathogens SR-AB (N=8). These four groups were negative to microfilariosis, unlike the fifth group, seroreactive to either or to both of the pathogens and with microfilariosis SR-M (N=10). The frequencies of CBC alterations among all analysed dogs were: 0.35 – leucocytosis, 0.44 – granulocytosis, 0.28 – anaemia, 0.74 – microcytosis, 0.37 – increased mean cell haemoglobin concentration (MCHC) and 0.33 – thrombocytopenia. The frequency of alterations did not differ across the groups. An exception was the SR-M group wherein increased MCHC peaked with a frequency of 0.80, while in the other four groups, the frequency ranged between 0.10 and 0.50. Clinically healthy outdoor dogs have multiple CBC abnormalities, consistent with stress and low-level chronic inflammation, but not associated with a previous exposure to Anaplasma spp. or Babesia spp. The presence of microfilaria increases haemolysis in vitro.

Introduction. Analysis of canine lipoprotein fractions after agarose gel electrophoresis (agEF) separation could be an important diagnostic tool in primary and secondary dyslipidemia diagnosis. The aim of this study was to measure concentrations of triglycerides and cholesterol and to analyze lipoprotein fractions in dogs after basal (12 hours) and short-term (24 and 36 hours) fasting, i.e., frequent conditions in clinical practice. Materials and Methods. Blood samples were collected from six lean dogs of both sexes and different breeds, after 12, 24 and 36 hours of fasting. Concentrations of glucose, triglycerides and cholesterol were determined on an automated wet biochemistry analyzer, lipoprotein fractions were separated by agEF and leukocyte numbers were assessed on an automated hematology analyzer. Results and Conclusions. Results showed there was no significant change in glucose, triglyceride and cholesterol concentrations nor in leukocyte numbers during dog fasting. Moreover, there was no change in α1- and α2-fractions, but there was a significant decrease in preβ- and β-fraction of lipoproteins. It is know that high density lipoproteins (HDL) have α-mobility and very low density (VLDL) and low density lipoproteins (LDL) have preβ- and β-mobility. Thus, it is possible that reverse cholesterol transport maintained by HDL is not affected during short-term fasting. On the contrary, synthesis of VLDL and formation of LDL are probably decreased because endogenous synthesis of triglycerides is decreased or their clearance is increased.

Introduction. The ovarian surface epithelium (OSE) undergoes intensive regeneration and remodeling after each ovulation during the whole reproductive period. This process increases the risk of one of the most common ovarian tumors in women and the female dog. Considering the fact that maternal hypothyroidism highly impacts cell proliferation and cell death during folliculogenesis in the early neonatal period, we aimed to analyze its effect on OSE morphology and dynamics. Materials and Methods. The study was performed on newborn (24-h-old) and neonatal (4-day-old) female rats, a randomized trial between the control and hypothyroid groups, born under controlled circumstances and hypothyroid mothers, respectively. Their ovaries were analyzed histologically and processed to determine the OSE cell height as an average value of four measurement points. Also, the immunopositivity of the proliferating cell nuclear antigen (PCNA) and caspase-3 were assessed semiquantitatively. Results and Conclusions. No major structural differences of OSE were found between groups within the given ages except for a slight increment of OSE cell height and incompleteness of apical cell membrane with cytoplasmic projections in hypothyroid animals. PCNA immunopositivity of the OSE cells was higher in ovaries of hypothyroid animals of both ages in comparison to the controls. Moreover, only scarce OSE cells were caspase-3 positive in both groups and ages, with no difference in immunopositivity. Our study confirms the impact of hypothyroidism in the early postnatal period on morphology and proliferation rate of OSE cells, with no effect on caspase-3 dependent cell removal, which may serve as a premise for future investigation of potential carcinogenesis, in terms of prevention and treatment of ovarian cancer. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 175061]

The aim of this study was to investigate whether prepartal leukogram in cows with retained placenta could indicate the presence of subclinical systemic inflammatory response before the onset of disease. After calving, sixteen highly pregnant Holstein cows, aged 3 to 9 years, without clinical signs of the disease prior to calving were divided into two groups: the first group (n=9) were animals without retained placenta, or any visible inflammation after birth; the second group (n=7) were cows with retained placenta. Blood was sampled three times before parturition, at intervals of one week, and once 24 hours after birth. The number of total leukocytes, segmented and non segmented neutrophilic granulocytes (NG), lymphocytes and monocytes were determined by standard laboratory techniques. The results have shown that in the group of cows with retained placenta the number of mature neutrophils was slightly elevated in the third, second and last week before calving, and equal number of non segmented neutrophils in regard to the group with no retention. The results have also shown that, in both groups of cows, 24 hours after calving, the number of total leukocytes and the number of segmented neutrophils decreased, but the number of the non segmented neutrophils increased. Based on this, we can conclude that cows with retained placenta had no systemic inflammatory response during three weeks prepartal period, but 24 hours after calving, systemic inflammatory response was documented in all the cows. Moreover, the intensity of inflammatory response in cows with retained placenta was not more pronounced in comparison to cows without retained placenta. [Projekat Ministarstva nauke Republike Srbije, br. 175061]

Background/Aim. The last decade has been profoundly marked by persistent attempts to use ex vivo expanded and manipulated mesenchymal stem cells (MSCs), as a tool in different types of regenerative therapy. In the present study we described immunophenotype and the proliferative and differentiation potential of cells isolated from pulp remnants of exfoliated deciduous teeth in the final phase of root resorption. Methods. The initial adherent cell population from five donors was obtained by the outgrowth method. Colony forming unit-fibroblast (CFU-F) assay was performed in passage one. Cell expansion was performed until passage three and all tests were done until passage eight. Cells were labeled for early mesenchymal stem cells markers and analysis have been done using flow cytometry. The proliferative potential was assessed by cell counting in defined time points and population doubling time was calculated. Commercial media were used to induce osteoblastic, chondrogenic and adipogenic differentiation. Cytology and histology methods were used for analysis of differentiated cell morphology and extracellular matrix characteristics. Results. According to immunophenotype analyses all undifferentiated cells were positive for the mesenchymal stem cell markers: CD29 and CD73. Some cells expressed CD146 and CD106. The hematopoietic cell marker, CD34, was not detected. In passage one, incidence of CFU-F was 4.7 ± 0.5/100. Population doubling time did not change significantly during cell subcultivation and was in average 25 h. After induction of differentiation, the multicolony derived cell population had a tri-lineage differentiation potential, since mineralized matrix, cartilage-like tissue and adipocytes were successfully formed after three weeks of incubation. Conclusion. Altogether, these data suggest that remnants of deciduous teeth dental pulp contained cell populations with mesenchymal stem cell-like features, with a high proliferation and trilineage differentiation potential and that these cultures are suitable for further in vitro evaluation of cell based therapies. [Projekat Ministarstva nauke Republike Srbije, br. 175061]

Diseases of the lower urinary pathways with urethral obstruction are frequent urological conditions demanding urgent treatment that lead to the occurrence of post renal azotemia due to difficult or disabled urine elimination. The aim of these investigations was to establish the most frequent causes for the occurrence of obstructions of the lower urinary pathways in cats, the most frequent clinical signs, as well as the changes in the hematological and biochemical blood and urine parameters. The obtained results indicate that the most frequent cause of urethral obstruction in cats is the presence of urethra stones, sand, and urethra plugs caused by the aggregation of struvite crystals on organic matrix. The laboratory findings showed that all animals were dehydrated, azotemia was established in half the animals, and signs of uremia were evident in 10% cats. Over 90% cats had disrupted tubular function, and only 50% had clear laboratory signs of inflammation. It can be concluded from the presented results that all cats with urethra obstruction exhibit a certain degree of disrupted kidney function, which is, in most cases, a reversible pathological change when treated with timely and adequate therapy. [Projekat Ministarstva nauke Republike Srbije, br. 175061]

Histological evaluations of soft tissue reactions in implanted biomaterials is a long established method of investigating their biocompatibility. It is a very important procedure convenient for various biomaterials, as appropriate for soft as for hard tissue, but at the same time this method is not sufficient for a prediction of their clinic performance. The wide spread practice of this method is in the simplicity of its use in subcutaneous or intramuscular implantations on the animal model, and consequently exploring principal and surrounding tissue is very simple with screening tests for esimating the irritaton index and interaction between tissue and biomaterials. The screening tests (evaluation tests) involve implantation of biomaterials in various locations in the body, different tissue treatment as immunohistochemical tissue analysis, transmissional, scanning and confocal microscopy. Interactions among soft tissue with bioceramics and the evaluation of their cellular and tissue performance in inflammation and immunological response are the first step in the estimation of biocompatibility. [Projekat Ministarstva nauke Republike Srbije, br. 175061]

The paper presents the results of investigations of the effect of toluene on bone marrow cells of female Wistar rats treated intraperitoneally with toluene for 8 or 11 days, in doses of 0.602 μg/200 g body mass. Cytogenetic analyses were performed on the metaphase figure of chromosomes in order to determine the frequency of structural aberrations – breaks and gaps. The values of the mitotic index and number of poliploid cells were determined. No significant increase was determined in the frequency of breaks and gaps in chromosomes of treated animals in comparison with the controls, which means that, under the experimental conditions, toluene did not exhibit a definite genotoxic effect. However, it has been determined that there was a significant increase in the value of the mitotic index, as well as a significant increase in the number of poliploid cells in both groups of treated rats in comparison with controls.

Numerous discoveries in medicine are results of experiments on different animal species. The most frequently used animals in hematopoiesis investigations are laboratory mice and rats, but so-called big animals, such as pigs, sheep, cats, dogs, and monkeys, evolution-wise closer to humans have a place in experimental hematology as well. The specific problematics of a certain animal specie can lead to fundamental knowledge on certain aspects of the process of hematopoiesis end the biology of stem cells in hematopoiesis. Furthermore, comparative investigations of certain phenomena in different species help in the recognition of the general rules in the living world. In the area f preclinicalinvesti- gations, animal models are an inevitable step in studies of transplantation biology of stem cells in hematopoiesis, as well as in studies of biologically active molecules which have an effect on the hematopoietic system. Knowledge acquired on animal models is applied in both human and veterinary medicine.

The aims of this study were to define age-related histological changes in the articular cartilage of the stifle joint in non-chondrodystrophic dogs and to determine whether physical activity has a positive impact on preservation of cartilage structure during ageing. Twenty-eight German shepherd dogs were included in the study. These dogs had no evidence of joint inflammation as defined by clinical assessment, radiology and synovial fluid analysis (specifically absence of synovial fluid serum amyloid A). The dogs were grouped as young working (n ¼ 4), young non-working (n ¼ 5), aged working (n ¼ 13) and aged non-working (n ¼ 6) animals. Gross changes in the stifle joints were recorded and biopsy samples of femoral and tibial articular cartilage were evaluated for thickness; chondrocyte number, density, surface area and morphology; isogenous group morphology; tidemark integrity; subchondral bone structure; presence of proteoglycans/ glycosaminoglycans; and expression of type I, II and X collagens. The major age-related changes, not related to type of physical activity, included elevated chondrocyte density and thinning of tibial cartilage and increased chondrocyte surface area in the superficial and intermediate zone of the femoral cartilage. There was also expression of type X collagen in the femoral and tibial calcified and non-calcified cartilage; however, type X collagen was not detected in the superficial zone of old working dogs. Therefore, ageing, with or without physical activity, leads to slight cartilage degeneration, while physical activity modulates the synthesis of type X collagen in the superficial cartilage zone, partially preserving the structure of hyaline cartilage., (2014 Elsevier Ltd. All rights reserved.)

Low O(2) concentration (1%) favors the self-renewal of hematopoietic stem cells and inhibits committed progenitors (CFC). Since IL-6 influences both stem cells and committed progenitors at 20% O(2), we studied its effects in cultures at 1% O(2). The pre-CFC activity in Lin- population of mouse bone marrow was analyzed following 10 days of serum-free culture in medium (LC1) supplemented with IL-3 with and without IL-6, at 20 and 1% O(2) and phenotypic differentiation and proliferative history monitored. The IL-6 receptor expression and initiation of VEGF-A synthesis were also investigated. At 20% O(2), the effects of IL-6 on pre-CFC were negligible but effects on CFC were apparent; conversely, at 1% O(2), the IL-6 enhances activity of pre-CFC but not of CFC. Unlike at 20% O(2), at 1% O(2) a subpopulation of cells remained Lin- in spite of extensive proliferation. However, the absolute number of Lin- cells, did not correlate with pre-CFC activity. A relative increase in VEGF transcripts at 1% O(2) in presence of IL-3 alone was enhanced by the addition of IL-6. IL-6 enhanced pre-CFC activity at 1% O(2) and this was correlated to the induction of VEGF. These data reinforce the concept that physiologically low oxygenation of bone marrow is a regulator of stem cell maintenance. Since the 20% O(2) does not exist in tissues in vivo, further studies in vitro at lower O(2) concentrations should revise our knowledge relating to cytokine effects on stem and progenitor cells.