Age, sex and breed effect on laboratory parameters in natural Babesia canis infection.

We tested the hypothesis that age, breed, and sex are related to hematology, biochemistry, acute phase proteins (APPs), seroreactivity and level of parasitemia in dogs with an acute phase response (APR) due to Babesia canis infection. The study enrolled 61 privately owned dogs that naturally acquired B. canis infection. Groups were formed according to the age: young dogs less than one year, and adult dogs more than one year old. Moreover, the group of males was compared to females and purebred to mixed breed dogs. Seroreactivity was tested with immunofluorescence antibody test, level of parasitemia with real-time polymerase chain reaction (real-time PCR), hematology, and biochemistry with automatic analyzers, serum amyloid A with enzyme-linked immunosorbent assay, fibrinogen with heat precipitation and ceruloplasmin and paraoxonase-1 with manual spectrophotometric methods. For protein separation agarose gel electrophoresis was used. The main changes in the whole population of B. canis-infected dogs were fever, pancytopenia, and change in APPs level. One-third of young, and 96% of adult dogs were seropositive (P < 0.001). The level of parasitemia was higher in the young dogs (P < 0.001). Erythroid lineage parameters (P < 0.01), and leukocytes (P < 0.05) were lower in the young, when compared to the adult dogs. Young dogs had lower total globulins (P < 0.001), ß- and γ-globulins (P < 0.001), and higher α-globulins (P = 0.022) than adult dogs. Young dogs had higher concentrations of phosphate (P = 0.003) and cholesterol (P < 0.001) and lower amylase (P = 0.014) and lipase activity (P = 0.020) than adult ones. Male dogs had lower neutrophil count than females (P = 0.035), and purebred dogs had more band neutrophils than mixed breed dogs (P = 0.004). In conclusion, dogs with natural Babesia canis infection at a young age have more severe anemia and APR including leukopenia than adults. Male and purebred dogs might also have more severe APR than females and mix-breeds, as they have more pronounced changes related to the myeloid lineage.

Low serum levels of promatrix metalloproteinase-2 and -9 occur during acute Babesia canis infection in dogs.

Inflammation is a hallmark of the acute Babesia canis infection. Promatrix metalloproteinase (proMMP)-2 and -9 are involved in inflammation, but their levels have not been analyzed in canine babesiosis. We hypothesized that in dogs infected with B. canis, serum proMMP-2 and -9 levels change between presentation and recovery. Degree of the change differs if dogs develop systemic inflammatory response syndrome (SIRS). This study included 24 dogs with an acute B. canis infection, at presentation and after two weeks. We used routine hematology and biochemistry methods, spectrophotometry for the acute-phase proteins, microscopy for parasitemia and zymography for (pro)MMPs. In vitro endothelial cells and leukocyte short-term cultures, and platelet lysates were used to detect specific MMP activity. Statistical analyses included Wilcoxon test for paired samples, Mann-Whitney U test and Spearman's rank correlation. Our results showed that endothelial cells, leukocytes and platelets are the source of proMMP-2 and proMMP-9. Furthermore, both proMMPs were lower at presentation than after recovery (p < 0.001). At presentation, proMMP-9 levels correlated with parasitemia (rho = -0.616, p = 0.009), total leukocyte (rho = 0.704, p < 0.001) and neutrophil counts (rho = 0.741, p < 0.001). Extent of alterations in proMMP-2 levels between presentation and recovery was lower (p = 0.038) in dogs with SIRS than in non-SIRS dogs, while levels of proMMP-9 were comparable between these groups. Our conclusion is that during the acute B. canis infection, low serum levels of proMMP-2 and proMMP-9 at presentation reflect thrombocytopenia and leukopenia. Decreased proMMP-2 level could be associated with SIRS.

Systemic inflammatory response syndrome in dogs naturally infected with Babesia canis: Association with the parasite load and host factors.

The common signs of canine babesiosis caused by an infection with Babesia canis are fever, anorexia, lethargy, pulse alterations, anemia, and occasionally mild icterus. Dogs with these clinical signs can be divided into two groups: those with acute-phase reaction and those with systemic inflammatory response syndrome (SIRS). Factors associated with the occurrence of SIRS in canine babesiosis have not been thoroughly researched. This article outlines a cross-sectional study of 54 client-owned dogs with an acute B. canis infection, and evaluates the differences in age, gender, laboratory findings, parasite load, and seroreactivity against B. canis between the SIRS and the SIRS-free dogs. We have analyzed a complete blood count, serum biochemistry, serum amyloid A, ceruloplasmin, paraoxonase-1, serology, and PCR testing using standard methodologies. The frequency of SIRS among the investigated dogs reached 0.59. Male dogs and those seronegative against B. canis, were more frequent in the SIRS group, whilst age and parasite load could not be associated with the presence of SIRS. Dogs with SIRS had a lower count of total leukocytes, neutrophils, lymphocytes, and monocytes, and a lower concentration of iron and bilirubin compared with SIRS-free dogs. No significant differences in the concentration of acute-phase proteins have been noticed to exist between the groups of dogs. Further, the seronegative dogs had a lower count of lymphocytes and monocytes and a higher parasite load than the seroreactive dogs. Multivariate logistic regression analysis has identified leukopenia (<6 × 109/L) and monocytopenia (<0.2 × 109/L) as independent associates of SIRS in the investigated dogs, thus implying that these routine tests could be used as reliable markers for SIRS.

A short-term and long-term relationship between occurrence of acute canine babesiosis and meteorological parameters in Belgrade, Serbia.

Belgrade, the capital of the Republic of Serbia, is an endemic location for canine babesiosis caused by Babesia canis. This tick-borne disease occurs seasonally in regions with moderate continental climate. However, recent clinical data show that: 1) some cases of canine babesiosis have been recorded during the winter, and 2) canine babesiosis is spreading to the northern parts of Europe with a colder climate, which is a region previously free of this disease. Our study investigates the occurrence of canine babesiosis in different seasons over 2013-2016, and explores a short-term link between meteorological parameters and number of diagnosed cases of canine babesiosis in Belgrade. It also looks into possible long-term correlations that span one year before the onset of the disease. Based on 872 recorded cases over four years, our results show a bimodal seasonal distribution of canine babesiosis, with a pronounced peak in the spring, and a less conspicuous one in the autumn. Throughout the year, even over the coldest and warmest periods, there is a broad range of temperatures and relative humidities when the disease is recorded. Over one year prior to the spring and autumn onset of the disease, we found a noticeable impact of temperature and relative humidity, and to a lesser extent, of atmospheric pressure and cloud cover, on the number of diagnosed cases. These findings imply short-term and long-term relationships between occurrence of acute canine babesiosis and certain meteorological parameters, and they open further questions that need to be investigated in order to understand the epidemiology of this disease.

Association of acute Babesia canis infection and serum lipid, lipoprotein, and apoprotein concentrations in dogs.

BACKGROUND: Babesia canis infection induces a marked acute phase response (APR) that might be associated with alteration in lipid and lipoprotein metabolism and disease prognosis. HYPOTHESIS: Dogs with B. canis-induced APR develop dyslipidemia with altered lipoprotein concentration and morphology. ANIMALS: Twenty-nine client-owned dogs with acute B. canis infection and 10 clinically healthy control dogs. METHODS: Observational cross-sectional study. Serum amyloid A (SAA) was measured using ELISA. Cholesterol, phospholipids, and triglycerides were determined biochemically. Lipoproteins were separated using agarose gel electrophoresis. Lipoprotein diameter was assessed by polyacrylamide gradient gel electrophoresis; correlation with ApoA-1 (radioimmunoassay) and SAA was determined. RESULTS: Dogs with B. canis infection had a marked APR (median SAA, 168.3 µg/mL; range, 98.1-716.2 µg/mL) compared with controls (3.2 µg/mL, 2.0-4.2 µg/mL) (P < .001). Dogs with B. canis infection had significantly lower median cholesterol (4.79 mmol/L, 1.89-7.64 mmol/L versus 6.15 mmol/L, 4.2-7.4 mmol/L) (P = .02), phospholipid (4.64 mmol/L, 2.6-6.6 mmol/L versus 5.72 mmol/L, 4.68-7.0 mmol/L) (P = .02), and α-lipoproteins (77.5%, 27.7%-93.5% versus 89.2%, 75.1%-93.5%) (P = .04), and higher ApoA-1 (1.36 U, 0.8-2.56 U versus 0.95 U, 0.73-1.54 U) concentrations (P = .02). Serum amyloid A correlated with high-density lipoproteins (HDLs) diameter (rho = .43; P = .03) and ApoA-1 (rho = .63, P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Major changes associated with B. canis-induced APR in dogs are related to concentration, composition, and morphology of HDL particles pointing to an altered reverse cholesterol transport. Parallel ApoA-1 and SAA concentration increase is a unique still unexplained pathophysiological finding.

Molecular and Serological Prevalence of Anaplasma phagocytophilum, A. platys, Ehrlichia canis, E. chaffeenses, E. ewingii, Borrelia burgdorferi, Babesia canis, B. gibsoni and B. vogeli among Clinically Healthy Outdoor Dogs in Serbia.

Data concerning combined molecular and serological prevalence of emerging canine tick-borne pathogens in Serbia are lacking. A large population of outdoor living dogs in Belgrade, Serbia's' capital, present an excellent population for epidemiology study. Blood samples were collected from 111 dogs, including 46 shelter, 31 free roaming, and 34 hunting dogs. Species-specific real-time polymerase chain reaction (PCR) (IDEXX Laboratories, Inc., Westbrook Maine, USA) was applied for the molecular detection of Anaplasma phagocytophilum, A. platys, Ehrlichia canis, Babesia canis, B. gibsoni and B. vogeli. A research based SNAP assay (SNAP® M-A, IDEXX Laboratories, Inc., Westbrook Maine, USA) that uses genus and species-specific peptides was used to asses Anaplasma spp., A. phagocytophilum, A. platys, Ehrlichia spp., E. canis, E. chaffeensis, E. ewingii and Borrelia burgdorferi antibody status. B. canis, B. gibsoni and B. vogeli antibody status was assessed with an indirect immunofluorescence test (MegaCor Diagnostic, Horbranz, Austria). Anaplasma spp. and Ehrlichia spp. DNA was not amplified. One quarter of the dogs were A. phagocytophilum, one dog was A. platys, one was E. ewingii and two dogs were B. burgdorferi seroreactive with the SNAP® M-A. Babesia canis or B. gibsoni DNA was amplified by PCR from 16.2% of dogs, whereas 67.6% were seroreactive to one or more Babesia spp. Babesia vogeli was not PCR amplified. We conclude that outdoor dogs in this territory are reservoirs for B. canis and B. gibsoni and are frequently co-exposed to combinations of Anaplasma and Babesia spp.

Altered state of primordial follicles in neonatal and early infantile rats due to maternal hypothyroidism: Light and electron microscopy approach.

Thyroid hormones (TH) are one of the key factors for normal prenatal development in mammals. Previously, we showed that subclinical maternal hypothyroidism leads to premature atresia of ovarian follicles in female rat offspring in the pre-pubertal and pubertal periods. The influence of decreased concentration of TH on primordial follicles pool formation during neonatal and early infantile period of rat pups was not investigated previously. Maternal hypothyroidism during pregnancy has irreversible negative influence on primordial follicles pool formation and population of resting oocytes in female rat offspring. The study was done on neonatal and early infantile control (n-10) and hypothyroid (n-10) female rat pups derived from control (n-6) and propylthiouracil (PTU) treated pregnant dams (n-6), respectively. Ovaries of all pups were removed and processed for light and transmission electron microscopy (TEM). Number of nests, oogonia and oocytes per nest, primordial, primary, secondary and preantral follicles were determined. Screening for overall calcium presence in ovarian tissue was done using Alizarin red staining. Morphology and volume density of nucleus, mitochondria and smooth endoplasmic reticulum (sER) in the oocytes in primordial follicles was also assessed. Caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), both markers for apoptosis, and proliferating cell nuclear antigen (PCNA) for proliferation were determined in oocytes and granulosa cells in different type of follicles. In neonatal period, ovaries of hypothyroid pups had a decreased number of oogonia, oocytes and nests, an increased number of primordial follicles and a decreased number of primary and secondary follicles, while in early infantile period, increased number of primary, secondary and preantral follicles were found. Alizarin red staining was intense in hypothyroid neonatal rats that also had the highest content of dilated sER. Number of mitochondria with altered morphology in both groups of hypothyroid pups was increased. Apoptosis markers have not shown significant difference between groups but PCNA had an increased expression in the oocytes and granulosa cells in primordial follicles of hypothyroid rats. Light and electron microscopy analysis indicate that previously detected premature ovarian follicular atresia in pre-pubertal and pubertal hypothyroid rats is preceded with premature formation of primordial follicles followed by slight changes on sER and mitochondria in examined oocytes, and increased expression of PCNA.

N-Acetyl-l-cysteine enhances ex-vivo amplification of deciduous teeth dental pulp stem cells.

OBJECTIVE: Obtaining high number of stem cells is of interest for cell based therapies. N-Acetyl-l-cysteine (NAC) acts as a source of sulfhydryl groups and an anti-oxidative agent. The aim of this study was to test different NAC concentration on proliferation and differentiation of deciduous teeth dental pulp stem cells (DTSCs) in vitro as well as to define the possible underlining mechanism of its effect. DESIGN: Number of viable, apoptotic and senescent DTSCs was determined after addition of NAC (0.1mM, 1.0mM, 2.0mM). Also, cell cycle analysis, HIF1-α expression, LDH isoenzymes, superoxide-dismutase (SOD) and catalase (CAT) activity, sulfhydryl groups content, the level of lipids' and proteins' oxidative damage and differentiation capacity of NAC treated DTSCs was determined. RESULTS: DTSCs expressed HIF-1α in all conditions. The lowest NAC dose (0.1mM) increased the number of DTSCs by one fifth comparing to the control, most likely stimulating entry of cells into S phase of cell cycle and enhancing the activity of LDH5 isoenzyme. The highest NAC dose (2mM) inhibited DTSCs proliferation. Also, DTSCs had the lowest level of oxidative damage with 0.1mM NAC. All tested NAC concentrations enhanced DTSCs osteo-chondrogenesis. CONCLUSION: The lowest NAC dose exerted significant positive effect on DTSCs proliferation as well as antioxidative protection creating beneficial environment for stem cells in vitro cultivation especially when their clinical use is important for stimulation of osteo-chondrogenesis.

Hypoxia/hypercapnia-induced adaptation maintains functional capacity of cord blood stem and progenitor cells at 4°C.

We analyzed the effect of exposure to hypoxic/hypercapnic (HH) gas mixture (5% O2 /9% CO2 ) on the maintenance of functional cord blood CD34(+) hematopoietic stem and progenitor cells in severe hypothermia (4°C) employing the physiological and proteomic approaches. Ten-day exposure to HH maintained the Day 0 (D-0) level of hematopoietic stem cells as detected in vivo on the basis of hematopoietic repopulation of immunodeficient mice-short-term scid repopulating cells (SRC). Conversely, in the atmospheric air (20% O2 /0.05% CO2 ), usual condition used for cell storage at 4°C, stem cell activity was significantly decreased. Also, HH doubled the survival of CD34(+) cells and committed progenitors (CFCs) with respect to the atmospheric air (60% vs. 30%, respectively). Improved cell maintenance in HH was associated with higher proportion of aldehyde dehydrogenase (ALDH) positive cells. Cell-protective effects are associated with an improved maintenance of the plasma and mitochondrial membrane potential and with a conversion to the glycolytic energetic state. We also showed that HH decreased apoptosis, despite a sustained ROS production and a drop of ATP amount per viable cell. The proteomic study revealed that the global protein content was better preserved in HH. This analysis identified: (i) proteins sensitive or insensitive to hypothermia irrespective of the gas phase, and (ii) proteins related to the HH cell-protective effect. Among them are some protein families known to be implicated in the prolonged survival of hibernating animals in hypothermia. These findings suggest a way to optimize short-term cell conservation without freezing.

Serum amyloid A isoforms in serum and milk from cows with Staphylococcus aureus subclinical mastitis.

Serum amyloid A proteins (SAA) are very sensitive acute phase proteins, displaying multiple isoforms in plasma and different body fluids. They are currently under investigation as biomarkers of diseases. The aim of the present study was to compare the concentration and isoform expression of SAA in serum and milk of cows with bacteriologically negative milk (control group) and naturally occurring Staphylococcus aureus (S. aureus) subclinical mastitis (subclinical mastitis group). Somatic cell count (SCC) and bacteriological analyses were performed to establish the control and subclinical mastitis group. SAA concentration was evaluated using a commercial ELISA kit, while expression of different isoforms (serum A-SAA and milk M-SAA3 isoforms) was visualized by denaturing isoelectrical focusing and immunoblotting. The SAA concentrations in sera and milk of cows in the subclinical mastitis group were three and 100 times higher than in those from the control group of cows, respectively. Cows in the subclinical mastitis group had more acidic SAA isoforms in serum with the most prominent one at pI 5.5. This isoform was not detected in sera from the control group. Milk samples in the subclinical mastitis group contained abundant highly alkaline M-SAA3 isoforms and most of the serum isoforms, except for that at pI 5.5. In the subclinical mastitis group SAA isoforms with equivalent pI as serum isoforms accounted for 20% of the total SAA concentration in milk. There were significant differences in the concentrations and isoform patterns of SAA in serum and milk between the control and subclinical mastitis groups of cows. Also, we demonstrated that serum SAA isoforms were not transferred to milk proportion to their plasma content.

CD34+ cells obtained from "good mobilizers" are more activated and exhibit lower ex vivo expansion efficiency than their counterparts from "poor mobilizers".

BACKGROUND: The classification of patients into "good" or "poor" mobilizers is based on CD34+ cell count in their peripheral blood (PB) after granulocyte-colony-stimulating factor (G-CSF) injection. We hypothesized that, apart from their mobilization from marrow to the blood, the response to G-CSF of CD34+ cells also includes activation of proliferation, metabolic activity, and proliferative capacity. STUDY DESIGN AND METHODS: Mobilized PB CD34+ cells purified from samples obtained by cytapheresis of multiple myeloma or non-Hodgkin's lymphoma patients of both good (>50 CD34+ cells/microL) and poor (< or =50 CD34+ cells/microL) mobilizers were studied. The initial cell cycle state of CD34+ cells after selection and their kinetics of activation (exit from G(0) phase) during ex vivo culture were analyzed. Their proliferative capacity was estimated on the basis of ex vivo generation of total cells, CD34+ cells, and colony-forming cells (CFCs), in a standardized expansion culture. Indirect insight in metabolic activity was obtained on the basis of their survival (viability and apoptosis follow-up) during the 7-day-long conservation in hypothermia (4 degrees C) in the air or in atmosphere containing 3% O(2)/6% CO(2). RESULTS: CD34+ cells obtained from good mobilizers were in lower proportion in the G(0) phase, their activation in a cytokine-stimulated culture was accelerated, and they exhibited a lower ex vivo expansion efficiency than those from poor mobilizers. The resistance to hypothermia of good immobilizers' CD34+ cells is impaired. CONCLUSION: A good response to G-CSF mobilization treatment is associated with a higher degree of proliferative and metabolic activation of mobilized CD34+ cells with a decrease in their expansion capacity.

Low-oxygen and high-carbon-dioxide atmosphere improves the conservation of hematopoietic progenitors in hypothermia.

BACKGROUND: During short-term storage of hematopoietic cells (HCs) at 4°C a substantial decline in number and in functional capacity of progenitors occurs after 3 days. We hypothesized that physiologic O2 and CO2 concentrations of hematopoietic tissue microenvironment (approx. 3% O2 and approx. 6% CO2) could improve cell viability and functionality during storage at 4°C. STUDY DESIGN AND METHODS: Mobilized peripheral blood (PB) CD34+ cells from multiple myeloma or non-Hodgkin's lymphoma patients were stored in flasks containing air (approx. 20% O2 and approx. 0.05% CO2) or 3% O2/6% CO2 atmosphere, for 3, 5, and 7 days at 4°C. The total number of cells, the number of cells in G0 or G1 phase of cell cycle, and the apoptosis rate were determined. The functional capacity of stored cells was assessed by the capacity of progenitors to form colonies in methylcellulose (colony-forming cells [CFCs]) and of stem cells to repopulate the bone marrow (BM) of immunodeficient mice (SCI D-repopulating cell [SRC] assay). RESULTS: The total number of viable cells and cells in G1 phase as well as the number of total CFCs were significantly higher at 3% O2/6% CO2 than in air at all time points. Cells in G0 phase and SRC were equally preserved in both conditions. CONCLUSION: Atmosphere with low O2 and high CO2 concentration (3% O2/6% CO2) in hypothermia (+4°C) during 7 days of storage prevents cell damage and preserves a high number of functional HSCs and progenitors mobilized in PB by granulocyte-colony-stimulating factor.

Interleukin-6 (IL-6) and low O(2) concentration (1%) synergize to improve the maintenance of hematopoietic stem cells (pre-CFC).

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.