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.

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.

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.

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.

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.

Canine distemper virus and Toxoplasma gondii co-infection in dogs with neurological signs

O presente estudo relata a ocorrência de co-infecção entre o vírus da cinomose canina (CDV) e Toxoplama gondii em cães com sinais neurológicos. Amostras de soro e tecido nervoso (pos-mortem) de 21 cães, suspeitos de cinomose canina foram analisadas pela Reação de Imunofluorecência indireta (RIFI) para pesquisa de anticorpos contra T. gondii e N. caninum e por RT-PCR para CDV. Dezessete (80,9%) cães foram positivos para o CDV pela RT-PCR e 8 (38,1%) foram positivos para anticorpos contra T. gondii. Sete cães (41,1%) apresentaram-se positivos para ambos agentes, caracterizando processo de co-infecção. Somente 1 (4,7%) cão foi soropositivo para N. caninum (RIFI=100), entretanto este mesmo animal foi positivo para T. gondii (RIFI=4096) e para CDV (RT-PCR).

Consolidation of international guidelines for the management of canine populations in urban areas and proposal of performance indicators

Consolidation of international guidelines for the management of canine populations in urban areas and proposal of performance indicators The objective of this study is to propose a generic program for the management of urban canine populations with suggestion of performance indicators. The following international guidelines on canine population management were revised and consolidated: World Health Organization, World Organisation for Animal Health, World Society for the Protection of Animals, International Companion Animal Management Coalition, and the Food and Agriculture Organization. Management programs should cover: situation diagnosis, including estimates of population size; social participation with involvement of various sectors in the planning and execution of strategies; educational actions to promote humane values, animal welfare, community health, and responsible ownership (through purchase or adoption); environmental and waste management to eliminate sources of food and shelter; registration and identification of animals; animal health care, reproductive control; prevention and control of zoonoses; control of animal commerce; management of animal behavior and adequate solutions for abandoned animals; and laws regulating responsible ownership, prevention of abandonment and zoonoses. To monitor these actions, four groups of indicators are suggested: animal population indicators, human/animal interaction indicators, public service indicators, and zoonosis indicators. The management of stray canine populations requires political, sanitary, ethologic, ecologic, and humanitarian strategies that are socially acceptable and environmentally sustainable. Such measures must also include the control of zoonoses such as rabies and leishmaniasis, considering the concept of "one health," which benefits both the animals and people in the community.

Effects of volume resuscitation on splanchnic perfusion in canine model of severe sepsis induced by live Escherichia coli infusion

Abstract Introduction We conducted the present study to investigate whether early large-volume crystalloid infusion can restore gut mucosal blood flow and mesenteric oxygen metabolism in severe sepsis. Methods Anesthetized and mechanically ventilated male mongrel dogs were challenged with intravenous injection of live Escherichia coli (6 × 109 colony-forming units/ml per kg over 15 min). After 90 min they were randomly assigned to one of two groups – control (no fluids; n = 13) or lactated Ringer's solution (32 ml/kg per hour; n = 14) – and followed for 60 min. Cardiac index, mesenteric blood flow, mean arterial pressure, systemic and mesenteric oxygen-derived variables, blood lactate and gastric carbon dioxide tension (PCO2; by gas tonometry) were assessed throughout the study. Results E. coli infusion significantly decreased arterial pressure, cardiac index, mesenteric blood flow, and systemic and mesenteric oxygen delivery, and increased arterial and portal lactate, intramucosal PCO2, PCO2 gap (the difference between gastric mucosal and arterial PCO2), and systemic and mesenteric oxygen extraction ratio in both groups. The Ringer's solution group had significantly higher cardiac index and systemic oxygen delivery, and lower oxygen extraction ratio and PCO2 gap at 165 min as compared with control animals. However, infusion of lactated Ringer's solution was unable to restore the PCO2 gap. There were no significant differences between groups in mesenteric oxygen delivery, oxygen extraction ratio, or portal lactate at the end of study. Conclusion Significant disturbances occur in the systemic and mesenteric beds during bacteremic severe sepsis. Although large-volume infusion of lactated Ringer's solution restored systemic hemodynamic parameters, it was unable to correct gut mucosal PCO2 gap.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.