Post-thaw viability of in vivo-produced canine blastocysts cryopreserved by slow freezing

The objectives were to evaluate the reexpansion blastocoele rate, post-thaw viability, and in vitro development of canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol (GLY) or 1.5 m ethylene glycol (EG). Fifty-one in vivo-produced canine blastocysts were randomly allocated in two groups: GLY (n = 26) and EG (n = 25). After thawing, embryos from MO were immediately stained with the fluorescent probes propidium iodide and Hoechst 33 342 to evaluate cellular viability. Frozen-thawed embryos from M3 and M6 were cultured in SOFaa medium + 10% FCS at 38.5 degrees C under an atmosphere of 5% CO2 with maximum humidity, for 3 and 6 days, respectively, and similarly stained. The blastocoele reexpansion rate (24 h after in vitro culture) did not differ between GLY (76.5%) and EG (68.8%). Post-thaw viable cells rate were not significantly different between GLY and EG (66.5 +/- 4.8 and 57.3 +/- 4.8, respectively, mean +/- SEM), or among MO (62.3 +/- 5.7%), M3 (56.9 +/- 6.0%), and M6 (66.5 +/- 6.0%). In conclusion, canine blastocysts cryopreserved by slow freezing in 1.0 m glycerol or 1.5 m ethylene glycol, had satisfactory blastocoele reexpansion rates, similar post-thawing viability, and remained viable for up to 6 days of in vitro culture. (C) 2012 Elsevier Inc. All rights reserved.

Solanum americanum as a reservoir of natural enemies of the tomato red spider mite, Tetranychus evansi (Acari: Tetranychidae)

Brazilian populations of the pathogenic fungus Neozygites floridana (Weiser & Muma) Remaudiere & S. Keller (Entomophthoromycotina: Entomophthorales) and the predatory mite Phytoseiulus longipes Evans (Acari: Phytoseiidae) are potential candidates for introduction into Africa for classical biological control of the tomato red spider mite, Tetranychus evansi Baker & Pritchard (Acari: Tetranychidae). The efficiency of these natural enemies against T. evansi has been demonstrated under laboratory conditions, but little is known about their performance on native Solanaceae in the field. The American nightshade, Solanum americanum Mill., is native to the Americas and may serve as an alternative host plant for T. evansi and its natural enemies in the absence of tomato plants. In this work, we studied the population dynamics of T. evansi and its natural enemies on S. americanum in a screen house, semi-field and field plots in Recife, Pernambuco, Brazil, to evaluate the potential of natural enemies for controlling T. evansi. Of the four natural enemies found in association with T. evansi, only N. floridana and P. longipes were clearly associated with the reduction of the populations of T. evansi. Neozygites floridana was observed in a screen house, semi-field and field plots, but P. longipes was only detected in the semi-field plots. Increases in the population of T. evansi were always followed by increases in the density of one of these natural enemies, suggesting that they were important factors regulating T. evansi populations on S. americanum. The presence of this host plant near the release sites in Africa thus might increase the chances for the permanent establishment of these natural enemies.

Invasion of the dinoflagellate Ceratium furcoides (Levander) Langhans 1925 at tropical reservoir and its relation to environmental variables

Dinoflagellates of the genus Ceratium are chiefly marine but there are rare occurrences in freshwater. In this study we analyze the invasion and progressive establishment of Ceratium furcoides, an exotic species, in the Furnas Reservoir. Samples were taken at 36 points in the reservoir, during the months of March, June, September and December, 2007. Measurements of some physical and chemical variables were simultaneously performed at each site. The occurrence of C. furcoides was registered at 20 sites, with densities varying between 0.57 and 28,564,913.0 ind.m(-3). Blooms of this species were recorded in points which were classified as mesotrophic, coinciding with the places receiving high amounts of untreated domestic sewage. C. furcoides density was correlated with temperature, nutrients (nitrate and nitrite) and water electric conductivity. The highest density was recorded in June when temperature was low. The presence of Ceratium furcoides in the reservoir apparently has not yet affected the reservoir water quality or other plankton communities. However, if it becomes fully established it could perhaps become a problem in the reservoir or even to spread out to other reservoirs in Rio Grande basin.

Hematological alterations during experimental canine infection by Trypanosoma cruzi

To confirm that Beagle dogs are a good experimental model for Chagas disease, we evaluated hematological alterations during the acute and chronic phases in Beagle dogs infected with the Y, Berenice-78 (Be-78) and ABC strains of Trypanosoma cruzi, correlating clinical signs with the parasitemia curve. We demonstrate that the acute phase of infection was marked by lethargy and loss of appetite. Simultaneously, we observed anemia, leukocytosis and lymphocytosis. Also,we describe hematological alterations and clinical signs that were positively correlated with the parasitemia during the experimental infection with the three strains of T cruzi, and demonstrate that experimental infection of Beagle is a trustworthy model for Chagas disease.

Morphological characterization of the progenitor blood cells in canine and feline umbilical cord

The umbilical cord blood (UCB) is an important source of hematopoietic stem cells with great deal of interest in regenerative medicine. The UCB cells have been extensively studied as an alternative to the bone marrow transplants. The challenge is to define specific methods to purify and characterize these cells in different animal species. This study is aimed at morphological characterization of progenitor cells derived from UCB highlighting relevant differences with peripheral blood of adult in dog and cats. Therefore, blood was collected from 18 dogs and 5 cats' umbilical cords from fetus in various developmental stages. The mononuclear cells were separated using the gradient of density Histopaque-1077. Characterization of CD34+ cells was performed by flow cytometric analysis and transmission electron microscopy. Granulocytes (ancestry of the basophiles, eosinophiles, and neutrophiles) and agranulocytes (represented by immature lymphocytes) were identified. We showed for the first time the ultrastructural features of cat UCB cells. Microsc. Res. Tech. 75:766770, 2012. (C) 2011 Wiley Periodicals, Inc.

Seasonal dynamics of cyanobacteria in a eutrophic reservoir (Arcoverde) in a semi-arid region of Brazil

Environmental conditions favor the predominance of dense populations of cyanobacteria in reservoirs in northeastern Brazil. The aim of this study was to understand cyanobacterial population dynamics in the rainy and dry seasons at two depths in the Arcoverde reservoir. Microalgae and cyanobacteria samples were collected during 24 hours with intervals of 4 hours (nycthemeral) at sub-surface and 10 m using a van Dorn bottle and a determined biomass. Physical and chemical variables were obtained and the data were analyzed using the principal component analysis (PCA). No nycthemeral variations in the taxonomic composition or distribution of the populations of cyanobacteria were found between the different times of day in either the rainy or dry season. In both seasons, the greatest biomass of the phytoplankton community was made up of cyanobacteria at two depths and all times of the day. Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju was dominant at all times of the day on both the surface and at the bottom. In the rainy season, the differences in cyanobacterial biomass between the surface and bottom were less significant than in the dry season. The differences in cyanobacterial biomass between surface and bottom were less pronounced than those found in the dry season. We concluded that a) physical variables better explain the alterations of species in the phytoplankton community in an environment dominated by cyanobacteria throughout the year; b) seasonal climatic factors associated to periods of stratification and de-stratification are important for alterations in the community and variations in biomass and, c) the turbidity caused by rainfall favored the emergence and establishment of other cyanobacteria, especially Planktothrix agardhii (Gomont) Anagnostidis & Komarek.

Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells

Background: The bone morphogenetic proteins (BMPs) belong to a unique group of proteins that includes the growth factor TGF-beta. BMPs play important roles in cell differentiation, cell proliferation, and inhibition of cell growth. They also participate in the maturation of several cell types, depending on the microenvironment and interactions with other regulatory factors. Depending on their concentration gradient, the BMPs can attract various types of cells and act as chemotactic, mitogenic, or differentiation agents. BMPs can interfere with cell proliferation and the formation of cartilage and bone. In addition, BMPs can induce the differentiation of mesenchymal progenitor cells into various cell types, including chondroblasts and osteoblasts. The aim of this study was to analyze the effects of treatment with rhBMP-2 on the proliferation of canine mesenchymal stem cells (cMSCs) and the tumor suppression properties of rhBMP-2 in canine osteocarcoma (OST) cells. Osteosarcoma cell lines were isolated from biopsies and excisions of animals with osteosarcoma and were characterized by the Laboratory of Biochemistry and Biophysics, Butantan Institute. The mesenchymal stem cells were derived from the bone marrow of canine fetuses (cMSCs) and belong to the University of Sao Paulo, College of Veterinary Medicine (FMVZ-USP) stem cell bank. After expansion, the cells were cultured in a 12-well Transwell system; cells were treated with bone marrow mesenchymal stem cells associated with rhBMP2. Expression of the intracytoplasmic and nuclear markers such as Caspase-3, Bax, Bad, Bcl-2, Ki-67, p53, Oct3/4, Nanog, Stro-1 were performed by flow citometry. Results: We evaluated the regenerative potential of in vitro treatment with rhBMP-2 and found that both osteogenic induction and tumor regression occur in stem cells from canine bone marrow. rhBMP-2 inhibits the proliferation capacity of OST cells by mechanisms of apoptosis and tumor suppression mediated by p53. Conclusion: We propose that rhBMP-2 has great therapeutic potential in bone marrow cells by serving as a tumor suppressor to increase p53 and the pro-apoptotic proteins Bad and Bax, as well as by increasing the activity of phosphorylated caspase 3. Study design: Canine bone marrow mesenchymal stem cells associated with rhBMP2 in canine osteosarcoma treatment: "in vitro" study

Complications of misdiagnosis of maxillary canine ectopic eruption

Ectopic eruption of maxillary canines can be associated with root resorption of adjacent teeth. This case report describes and discusses an interesting case of a 15-year-old girl with a Class III malocclusion and an impacted maxillary canine. Because of the unfavorable position of the ectopic canine and the severe root resorption of the maxillary left central and lateral incisors, the treatment options included extraction of the maxillary permanent canines. The mandibular first premolars were extracted to compensate for the Class III malocclusion. A panoramic radiograph taken earlier in the mixed dentition already indicated a possible eruption disturbance of the maxillary left permanent canine. The importance of early diagnosis of maxillary canine ectopic eruption is highlighted in this case report. The early identification of radiographic signs of an ectopic pathway of eruption should be followed by deciduous canine extraction to prevent canine retention and maxillary incisor root resorption. (Am J Orthod Dentofacial Orthop 2012;142:256-63)

The Value of Immunohistochemical Expression of BAX in Formulating a Prognosis for Canine Cutaneous Mast Cell Tumours

Irnmunohistochcmical expression of BAX was evaluated in 24 canine cutaneous mast cell tumours in order to verify the relationship of this expression to the histopathological grade of the lesions and its prognostic value for clinical outcome. BAX expression increased with higher histopathological grades (P = 0.0148; P < 0.05 between grades I and III). Animals with high levels of BAX expression were 4.25 times more likely to die from the disease and had shorter post-surgical survival times (P = 0.0009). These results suggest that alterations in BAX expression may be related to the aggressiveness of canine cutaneous mast cell tumours, indicating that immunohistochemical detection of BAX may be predictive of clinical outcome. (C) 2011 Elsevier Ltd. All rights reserved.

Derivation and characterization of progenitor stem cells from canine allantois and amniotic fluids at the third trimester of gestation

Fetal tissues are frequently discarded before (amniocentesis) or after birth, which both facilitates stem cell access and helps to overcome ethical concerns. In the present study, we aimed to isolate and characterize stem cells from the allantoic and amniotic fluids (ALF; AMF) of third trimester canine fetuses. This gestation age has not been previously explored for stem cells isolation. The gestational age, cell culture conditions and method of isolation used in this study allowed for the establishment and efficient expansion of ALF and AMF cells. We showed that the majority of ALF and ALF cells express the stem cell markers, such as vimentin, nestin and cytokeratin 18 (CK18). Under appropriate culture conditions AMF derived cells can undergo differentiation into osteogenic, adipogenic, chondrogenic and neuron-like lineages. ALF derived cells showed adipogenic, and chondrogenic potential. Therefore, ALF and AMF cells derived at the third gestation trimester can be qualified as progenitor stem cells, accordingly referred as (alantoic fluid progenitor/stem) ALF PS cells and (amniotic fluid progenitor/stem) AMF PS cells. (C) 2012 Elsevier Ltd. All rights reserved.

Calcium nitrate addition to control the internal load of phosphorus from sediments of a tropical eutrophic reservoir: Microcosm experiments

The main objective of this study was to perform laboratory experiments on calcium nitrate addition to sediments of a tropical eutrophic urban reservoir (Ibirite reservoir, SE Brazil) to immobilize the reactive soluble phosphorus (RSP) and to evaluate possible geochemical changes and toxic effects caused by this treatment. Reductions of 75 and 89% in the concentration of RSP were observed in the water column and interstitial water, respectively, after 145 days of nitrate addition. The nitrate application increased the rate of autotrophic denitrification, causing a consumption of 98% of the added nitrate and oxidation of 99% of the acid volatile sulfide. As a consequence, there were increases in the sulfate and iron (II) concentrations in the sediment interstitial water and water column, as well as changes in the copper speciation in the sediments. Toxicity tests initially indicated that the high concentrations of nitrate and nitrite in the sediment interstitial water (up to 2300 mg L-1 and 260 mg L-1, respectively) were the major cause of mortality of Ceriodaphnia silvestrii and Chironomus xanthus. However, at the end of the experiment, the sediment toxicity was completely removed and a reduction in the 48 h-EC50 of the water was also observed. Based on these results we can say that calcium nitrate treatment proved to be a valuable tool in remediation of eutrophic aquatic ecosystems leading to conditions that can support a great diversity of organisms after a restoration period. (C) 2012 Elsevier Ltd. All rights reserved.

A cross-sectional study on canine Leishmania (L.) infantum chagasi infection in Amazonian Brazil ratifies a higher prevalence of specific IgG-antibody response than delayed-type hypersensitivity in symptomatic and asymptomatic dogs

This was a cross-sectional study which analyzed the prevalence and the clinical and immunological spectrum of canine Leishmania (L.) infantum chagasi infection in a cohort of 320 mongrel dogs living in an endemic area of American visceral leishmaniasis in the Amazonian Brazil by using, mainly, the indirect fluorescence antibody test (IFAT-IgG) and the delayed-type hypersensitivity (DTH), and the parasite research by the popliteal lymph node aspiration. The IFAT and DTH reactivity recognized three different immune response profiles: (1) IFAT((+))/DTH(-) (107 dogs), (2) IFAT((-))/DTH(+) (18 dogs), and (3) IFAT((+))/DTH(+) (13 dogs), providing an overall prevalence of infection of 43 % (138/320). Thus, the specific prevalence of IFAT ((+)) /DTH ((-)) 33.4 % (107/320) was higher than those of IFAT ((-)) /DTH ((+)) 5.6 % (18/320) and IFAT ((+)) /DTH ((+)) 4.0 % (13/320). Moreover, the frequency of these profiles among 138 infected dogs showed that the IFAT ((+)) /DTH ((-)) rate of 77.5 % (107/138) was also higher than those of 13.0 % (18/138) of IFAT ((-)) /DTH ((+)) and 9.5 % (13/138) of IFAT ((+)) /DTH ((+)) rates. The frequency of asymptomatic dogs (76 %-105) was higher than those of symptomatic (16.6 %-23) and oligosymptomatic ones (7.4 %-10). A total of 16 (11.6 %) L. (L.) i. chagasi isolates were obtained from infected dogs, all from the IFAT ((+)) /DTH ((-)) profile: 41 % (9/22) from symptomatic, 33.3 % (3/9) from oligosymptomatic, and 5.2 % (4/76) from asymptomatic dogs. These findings strongly suggested that despite the higher frequency of asymptomatic dogs (76 %-105), the majority (72.4 %-76) was characterized by the IFAT ((+)) /DTH ((-)) profile with a doubtful immunogenetic resistance against infection.

E-cadherin in canine mast cell tumors: Decreased expression and altered subcellular localization in Grade 3 tumors

Mast cell tumors (MCTs) are the most frequent round cell tumors in dogs and comprise approximately 21% of all canine cutaneous tumors. MCTs are highly invasive and metastatic corresponding to the histological grade. E-cadherin is an adhesion molecule expressed in epithelial cells and although it is an epithelial cellular marker, studies have shown expression of E-cadherin in canine round cell tumors. To better characterize the expression pattern of E-cadherin in several different histological grades of MCTs in dogs, the expression and localization of the adhesion molecule was investigated using immunohistochemistry. For this purpose, 18 cutaneous MCTs were classified into three histological grades, 1, 2 or 3. Clinical history and follow-up data were available for all of the dogs. Cytoplasmic and nuclear expressions of E-cadherin in all three types of tumors were verified by immunostaining using two different antibodies. There was decreased E-cadherin expression in the more aggressive MCTs (Grade 3), suggesting an association between E-cadherin and tumor aggressiveness. Additionally, the loss of E-cadherin expression in either the cytoplasm or nucleus in more aggressive and undifferentiated tumor types confirmed the importance of cellular adhesion in tumor behavior. (C) 2012 Published by Elsevier Ltd.

Paleolimnologically inferred eutrophication of a shallow, tropical, urban reservoir in southeast Brazil

We studied the eutrophication history of a tropical shallow reservoir in the So Paulo metropolitan region, southeast Brazil. We analyzed grain size, geochemistry, diatom assemblages, and land-use records in a sediment core from the reservoir to infer its trophic state history during the last similar to 110 years (1894-2005). Eighty diatom species were observed in the core and shifts in the relative abundances of planktonic and benthic taxa indicate major limnological changes associated with complex interactions between hydrologic factors and eutrophication. Discostella stelligera was associated with deforestation and water physical changes whereas Aulacoseira granulata, a species abundant throughout the core, was mostly associated with high flux conditions and erosion events, regardless of trophic state. Eutrophication was triggered by construction of the city zoo (1958) and installation of the So Paulo State Department of Agriculture (1975) within the Gar double dagger as watershed, and increasing loads of untreated sewage from these institutions. The data suggest that deterioration in water quality began after similar to 1975 and markedly accelerated after similar to 1990. The reservoir has been hypereutrophic since 1999. Steady increases in geochemical proxies for trophic state, along with a decrease in C/N ratios, indicated higher nutrient concentrations and the prevalence of autochthonous production towards the core top. Appearance of Achnanthidium catenatum similar to 1993 highlighted the onset of a marked eutrophication phase. The subsequent dominance of Planothidium rostratum and Cyclotella meneghiniana suggested a sharp shift to a hypereutrophic state since 1999. Land-use history proved valuable for validating the chronology and interpreting anthropogenic impacts. Multi-proxy analysis of the sediment record provided an effective tool for tracking ecological shifts in the reservoir ecosystem. This study provides the first reconstruction of lake eutrophication history in Brazil and highlights the importance of hydrological/physical changes as drivers of diatom assemblage shifts in reservoirs, which may confound trophic state inferences based on shifts in the planktonic/benthic diatom ratio.

Cortisol Profile and Clinical Evaluation of Canine Neonates Exposed Antenatally to Maternal Corticosteroid Treatment

Contents The effects of glucocorticoids on both foetal canine lung and endogenous serum cortisol concentration have not been clearly delineated. Therefore, we aimed to investigate whether maternal corticosteroid treatment can alter maternal and neonatal cortisol profile and improve neonatal vitality. We allocated six bitches of different breeds and their neonates into two groups: control group (CONT) maternal administration of saline solution at 55days post-ovulation (n=3); and betamethasone group (BETA) administration of a single dose of 0.5mg/kg betamethasone (Celestone Soluspan(R)) at 55days post-ovulation (n=3). Caesarean sections were scheduled for day 63 after ovulation. However, BETA group dams showed precocious signs of labour, and c-sections were performed at 58days post-ovulation. Maternal and neonatal evaluations were performed periodically between betamethasone administration and birth, respectively. Neonates from both groups presented unsatisfactory (<5) Apgar score at birth. However, in spite of an earlier improvement on vitality found on CONT group and the premature delivery on BETA group, both groups showed acceptable Apgar score 120min after birth. Neonatal cortisol concentrations were higher on CONT group compared to BETA group at birth. In addition, a gradual decrease on maternal cortisol concentrations was observed in the BETA group from treatment until parturition. These findings suggest that despite the down-regulation on the hypothalamic-pituitary-adrenal axis and the induction of premature delivery, betamethasone treatment was able to provide similar vitality when compared to the untreated neonates born at term.