Finite Element Modeling for Development and Optimization of a Bone Plate for Mandibular Fracture in Dogs

This study aimed to develop a plate to treat fractures of the mandibular body in dogs and to validate the project using finite elements and biomechanical essays. Mandible prototypes were produced with 10 oblique ventrorostral fractures (favorable) and 10 oblique ventrocaudal fractures (unfavorable). Three groups were established for each fracture type. Osteosynthesis with a pure titanium plate of double-arch geometry and blocked monocortical screws offree angulanon were used. The mechanical resistance of the prototype with unfavorable fracture was lower than that of the fcworable fracture. In both fractures, the deflection increased and the relative stiffness decreased proportionally to the diminishing screw number The finite element analysis validated this plate study, since the maximum tension concentration observed on the plate was lower than the resistance limit tension admitted by the titanium. In conclusion, the double-arch geometry plate fixed with blocked monocortical screws has sufficient resistance to stabilize oblique,fractures, without compromising mandibular dental or neurovascular structures. J Vet Dent 24 (7); 212 - 221, 2010

Genotyping of Toxoplasma gondii isolates from free range chickens in the Pantanal area of Brazil

The aim of this paper was to genetically characterize Toxoplasma gondii isolates from free range chickens in regions of Brazilian territory in the state of Mato Grosso do Sul (MS) where T. gondii strains have never been studied. In total, T. gondii isolates from 22 free range chickens were included in this study. Fifty chickens from Eldorado, thirty from Rio Verde and ten from Aquidauana were sampled between January and April 2007. In relation to the genetic diversity of T. gondii isolates from chickens in MS, the magnitude of the diversity in the isolates sampled in this study was comparable to the overall diversity in a composite data set. These 22 isolates in MS revealed 11 genotypes, whereas the 321 isolates ever genotyped in Brazil have revealed 95 genotypes. The values of Simpson`s Diversity Index for the whole population of T. gondii isolates in Brazil, the whole population of T. gondii isolates from chickens in Brazil and the population surveyed in this study were 0.97, 0.95 and 0.90, respectively. Seven of the 11 genotypes revealed from chicken isolates from MS are newly described genotypes and six of them each have a single isolate. In conclusion, the results obtained from isolates in MS corroborate previous studies on T. gondii isolates in Brazil, thus confirming their diversity and atypicality. Nonetheless, the applicability of PCR-RFLP markers for epidemiological inferences remains controversial. (c) 2011 Elsevier B.V. All rights reserved.

Detection and molecular characterization of a canine piroplasm from Brazil

In the beginning of the 20th century, a new canine disease was reported in Brazil under the name ""nambiuvu"", whose etiological agent was called Rangelia vitalii, a distinct piroplasm that was shown to parasitize not only erythrocytes, but also leucocytes and endothelial cells. In this new century, more publications on R. vitalii were reported from Brazil, including an extensive study on its ultrastructural analysis, in addition to clinical, pathological, and epidemiological data on nambiuvu. However, a molecular analysis of R. vitalii has not been performed to date. In the present study, we performed molecular phylogenetic analyses of R. vitalii based on fragments of the genes 18S rRNA and the heat shock protein 70 (hsp70), amplified by PCR performed on blood samples derived from five clinical cases of dogs presumably infected with R. vitalii in southern Brazil. In addition, we examined Giemsa-stained thin blood smears from these same dogs. DNA sequences (604-bp) of the 18S rRNA gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (95%) with Babesia sp. China-BQ1. DNA sequences (1056-bp) of the hsp70 gene obtained from the five dogs were identical to each other, and by Blast analysis, this sequence shared the highest degree of sequence identity (87%) with Babesia bigemina. Phylogenetic analyses inferred from either of the two genes resulted in the newly genotype being placed in the Babesia spp.sensu stricto clade with very high bootstrap support (95-100%) in three analyses (Neighbor-Joining, Maximum parsimony, and Maximum likelihood). Giemsa-stained thin blood smears from the dogs were shown to contain piroplasm organisms within erythrocytes, monocytes and neutrophils (individual forms), and schizont-like forms within neutrophils, in accordance with literature reports of R. vitalii. Based on these results, we conclude that R. vitalii, the etiological agent of ""nambiuvu"" in southern Brazil, is a valid species of piroplasm. Further studies are required to evaluate the validity of the genus Rangelia. (C) 2011 Elsevier B.V. All rights reserved.

Experimental infection with Rangelia vitalii in dogs: Acute phase, parasitemia, biological cycle, clinical-pathological aspects and treatment

Recently we conducted the molecular characterization of Rangelia vitalii, a protozoan with high pathogenicity for young dogs in southern Brazil. To date, the descriptions of the disease have been restricted to natural infection cases. Therefore, this study aimed to evaluate the parasitemia, biological cycles and clinical-pathological findings in dogs experimentally infected with R. vitalii in the acute phase of disease, and also aimed to test a therapeutic protocol based on the diminazene aceturate. For this study, we used 12 young dogs (females), separated into two groups. Group A was composed of healthy dogs, not-infected (n = 5), and Group B consisted of animals infected with R. vitalii (n = 7). After infection, the animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite 5 days post-infection (PI). Parasitemia increased progressively in these animals and had the highest peak of circulating parasites between 9 and 11 days PI. Subsequently, the parasitemia reduced and the protozoan was seen inside the leukocytes in days 17, 19 and 21 PI. The most prominent clinical signs observed at the 20 day PI of experiment were lethargy, fever and anorexia. We observed a decrease of hematocrit of infected animals compared with not-infected dogs, featuring a moderate anemia. Pathological evaluation of one dog in Group B at day 21 PI revealed splenomegaly, hepatomegaly, lymphadenopathy, and hemorrhages at necropsy. Histological examination showed only follicular hyperplasia in the spleen and lymph nodes, and the etiologic agent in the vascular endothelium. At 21 days PI, it was performed the treatment of dogs in Group B (n = 6) with a single dose of diminazene aceturate, which showed a curative efficacy of 100% in cleaning R. vitalii from blood of infected dogs. (C) 2011 Elsevier Inc. All rights reserved.

Experimental infection of pregnant queens with two major Brazilian clonal lineages of Toxoplasma gondii

Toxoplasma gondii isolates from Brazil are biologically and genetically different from European and North America isolates. Recently, four genotypes were considered the common clonal lineages in Brazil and were designated as types BrI, BrII, BrIII, and BrIV. The pathogenicity of two major Brazilian lineages was investigated after oral inoculation of queens in the middle third of their pregnancies with T. gondii cysts. Twelve pregnant queens without T. gondii antibodies were distributed in group A (infected with a type BrI isolate); group 2 (infected with type BrIII isolate), and group 3 (non-infected control). Infection with type BrI isolate caused toxoplasmosis manifestations and abortion from one litter. Toxoplasmosis manifestations besides premature stillbirth of one litter were observed in queens infected with type BrIII isolate. Indirect fluorescence antibody test showed T. gondii antibodies in all eight infected queens at 30 days after inoculation. In two 10-day-old kittens of the same litter (group 1), titers of 16 and 64 were detected. At the same time, titers of 16, 32, and 32 were detected in three kittens from the same litter (group 2). Experimental infection with tissue cysts from a type BrI and type BrIII isolates of T. gondii developed similar reproductive disturbance in primary infected pregnant queens.

SURVEY OF FELINE LEUKEMIA VIRUS AND FELINE CORONAVIRUSES IN CAPTIVE NEOTROPICAL WILD FELIDS FROM SOUTHERN BRAZIL

A total of 57 captive neotropical felids (one Leopardus geoffroyi, 14 Leopardus pardalis, 17 Leopardus wiedii, 22 Leopardus tigrinus, and three Puma yagouaroundi) from the Itaipu Binacional Wildlife Research Center (Refugio Bela Vista, Southern Brazil) were anesthetized for blood collection. Feces samples were available for 44 animals, including one L. geoffroyi, eight L. pardalis, 14 L. wiedii, 20 L. tigrinus, and one P. yagouaroundi. Total DNA and RNA were extracted from blood and feces, respectively, using commercial kits. Blood DNA samples were evaluated by polymerase chain reaction (PCR) for feline leukemia virus (FeLV) proviral DNA, whereas reverse transcriptase-PCR was run on fecal samples for detection of coronavirus RNA. None of the samples were positive for coronaviruses. A male L. pardalis and a female L. tigrinus were positive for FeLV proviral DNA, and identities of PCR products were confirmed by sequencing. This is the first evidence of FeLV proviral DNA in these species in Southern Brazil.

Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 X 10(6) cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 degrees C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced. (C) 2010 Elsevier Inc. All rights reserved.

Serum Starvation and Full Confluency for Cell Cycle Synchronization of Domestic Cat (Felis catus) Foetal Fibroblasts

Nuclear transfer of domestic cat can be used as a tool to develop reproductive biotechnologies in wild felids. The importance of cell cycle phase during the nuclear transfer has been a matter of debate since the first mammalian clone was produced. The cell cycle phase of donor cells interferes on maintenance of correct ploidy and genetic reprogramming of the reconstructed embryo. The use of G0/G1 arrested donor cells has been shown to improve nuclear transfer efficiency. The present study was conducted to test the hypothesis that domestic cat foetal fibroblasts cultured up to the fifth passage and submitted to full confluency provide a higher percentage of cells at G0/G1 stage than fibroblasts cultured in serum starved media. Results demonstrated that serum starvation increased (p < 0.05) the percentage of G0/G1 fibroblasts when compared with control. Moreover, the combined protocol using confluency and serum starvation was more efficient (p < 0.05) synchronizing cells at G0/G1 stage than serum starvation or confluency alone for the first 3 days of treatment. In conclusion, serum starvation and full confluency act in a synergistic manner to improve domestic cat foetal fibroblast cell cycle synchronization at the G0/G1 stage.

Oestrogen and Progesterone Receptor Gene Expression in Canine Oocytes and Cumulus Cells Throughout the Oestrous Cycle

The aim of this research was to analyze oestrogen receptor-alpha (ER alpha), ER beta and progesterone receptor (PR) gene expression in the canine oocyte and cumulus cells throughout the oestrous cycle. Ovaries from 38 bitches were recovered after ovariohysterectomy and sliced. The phase of the oestrous cycle was determined by vaginal cytology, vaginoscopy and serum hormonal measurements. Oocytes were mechanically denuded by repeated pipetting. For each phase of the cycle, a sample was composed by a pool of 50 oocytes (sample number: prooestrus = 3, oestrus = 8, dioestrus = 5 and anoestrus = 5) or a pool of cumulus cells (prooestrus = 4, oestrus = 7, dioestrus = 4 and anoestrus = 6). Oocyte and cumulus cells` total RNA was isolated and reverse transcription was conducted to perform real-time PCR. Oestrogen receptor-alpha was expressed throughout the cycle in the oocyte (33.33%, 25.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively) and cumulus cells (50.0%, 47.14%, 25.0% and 66.67% for prooestrus, oestrus, dioestrus and anoestrus, respectively). In the oocyte, the ER beta was also expressed in all phases of the cycle (33.33%, 50.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively), whereas in cumulus cells, ER beta was only expressed during prooestrus (50%) and oestrus (14.29%). Interestingly, while the oocyte PR was not detected in any phase of the cycle, this receptor was expressed during prooestrus (50%), oestrus (42.86%) and anoestrus (16.67%) in cumulus cells. In conclusion, canine oocytes express ER alpha and ER beta throughout the oestrous cycle, however, there is a lack of PR expression in all these phases. Moreover, in cumulus cells, only ER alpha was expressed throughout the oestrous cycle.

Ultrastructure of Motor Nerve Terminals in the Anterior Third of Wistar Rat Tongue

The nerve terminals of intrinsic muscular fibers of the tongue of adult wistar rats was studied by using silver impregnation techniques, transmission electron microscopy (TEM), and high resolution scanning electron microscopy (HRSEM) to observe the nerve fibers and their terminals. Silver impregnation was done according to Winkelman and Schmit, 1957. For TEM, small blocks were fixed in modified Karnovsky solution, postfixed in 1% buffered osmium tetroxide solution, and embedded in Spurr resin. For HRSEM, the parts were fixed in 2% osmium tetroxide solution with 1/15 M sodium phosphate buffer (pH 7.4) at 4 degrees C for 2 h, according to the technique described by Tanaka, 1989. Thick myelinated nerve bundles were histologically observed among the muscular fibers. The intrafusal nerve fiber presented a tortuous pathway with punctiform terminal axons in clusters contacting the surface of sarcolemma. Several myelinated nerve fibers involved by collagen fibers of the endoneurium were observed in HRSEM in three-dimensional aspects. The concentric lamellae of the myelin sheath and the axoplasm containing neurofilaments interspersed among the mitochondria were also noted. In TEM, myofibrils, mitochondria, rough endoplasmic reticulum, Golgi`s apparatus, and glycogen granules were observed in sarcoplasm. It is also noted that the sarcomeres constituted by myofilaments with their A, I, and H bands and the electron dense Z lines. In areas adjacent to muscular fibers, there were myelinated and unmyelinated nerve fibers involved by endoneurium and perineurium. In the region of the neuromuscular junction, the contact with the sarcolemma of the muscular cell occurs forming several terminal buttons and showing numerous evaginations of the cell membrane. In the terminal button, mitochondria and numerous synaptic vesicles were observed. Microsc. Res. Tech. 72:464-470, 2009. (C) 2009 Wiley-Liss. Inc.

Accumulative copper poisoning in buffaloes

The main objective of this study was to evaluate the clinical and hematological alterations, and the hepatic concentration of Cu in buffaloes with chronic copper poisoning (CCP). Ten buffalo yearling steers were randomly distributed into two groups: one copper supplemented (BUFCu; n = 6) and another control group (BUF; n = 4). The group BUFCu received, by ruminal fistula, 2 mg Cu/kgBW (as CuSO4.5H2O) daily during one week; after which 2 mg Cu/kgBW was added during each additional week, until the end or the experiment (105(th) day). Three liver biopsies were realized during the experiment (day zero, 45(th), and 105(th) day) to determine the degree of copper accumulation. Alterations in body weight, clinical examination, and hematological values were monitored every 15 days. Two buffaloes supplemented with copper demonstrated clinical manifestations consistent with CCP, and died. Two distinct clinical manifestations were observed, one classical (n = 1) and another atypical (n = 1), characterized by remarkable high levels of liver copper, progressive hyporexia followed by anorexia, dehydration, severe apathy, decreased rumen movements, oliguria, and death. Some animals were resistant to CCP although high copper intake. In buffalos with clinical picture similar to the one described as atypical, CCP should be considered as a possible diagnostic even without presence of macroscopic hemoglobinuria.

Bispectral index in dogs at three intravenous infusion rates of propofol

Objective To establish the correlation between the bispectral index (BIS) and different rates of infusion of propofol in dogs. Study design Prospective experimental trial. Animals Eight adult dogs weighing 6-20 kg. Methods Eight animals underwent three treatments at intervals of 20 days. Propofol was used for induction of anesthesia (10 mg kg(-1) IV), followed by a continuous rate infusion (CRI) at 0.2 mg kg(-1) minute(-1) (P2), 0.4 mg kg(-1) minute(-1) (P4) or 0.8 mg kg(-1) minute(-1) (P8) for 55 minutes. The BIS values were measured at 10, 20, 30, 40, and 50 minutes (T10, T20, T30, T40, and T50, respectively) after the CRI of propofol was started. Numeric data were submitted to analysis of variance followed by Tukey test (p < 0.05). Results The BIS differed significantly among groups at T40, when P8 was lower than P2 and P4. At T50, P8 was lower than P2. The electromyographic activity (EMG) in P2 and P4 was higher than P8 at T40 and T50. Conclusion An increase in propofol infusion rates decreases the BIS values and EMG.

Prognostic impact of adenomatous polyposis coil gene expression in osteosarcoma of the extremities

Purpose: To evaluate the impact of adjuvant chemotherapy on the outcome of osteosarcoma of the extremities, and to identify prognostic factors using the expression of adenomatous polyposis coli (APC), cadherin and beta-catenin Wnt-signalling markers. Methods: The clinical, demographic, anatomic and pathological factors including a detailed analysis of the immunohistochemical expression of cadherin, B-catenin and APC were retrospectively examined in 97 patients with osteosarcoma of the extremities (metastatic and non-metastatic at diagnosis), treated with surgery and/or chemotherapy from 1985 to 2000. Results: APC immunoreactivity showed a statistically significant association with age and serum alkaline phosphatase levels (p = 0.025 and p = 0.038). When survival was the end-point of multivariate analysis, race segregated patients with poor survival as did lack of cadherin expression. For overall survival, cadherin immunoreactivity and the interaction between APC expression and response to adjuvant chemotherapy were significant (p = 0.012 and p < 0.001). No significant clinical association was evident with immunohistochemical expression of cadherin, beta-catenin. Conclusion: Lack of expression of cadherin was a significant variable to overall and disease-free survival. Significantly, positive APC immunoreactivity and adjuvant chemotherapy were associated with a favourable treatment response. Studies using newer immunohistochemical markers within the Wnt-signalling pathway may guide the development of more appropriate therapeutic targets for future individualised treatment. (C) 2010 Elsevier Ltd. All rights reserved.

Downregulation of CD9 protein expression is associated with aggressive behavior of oral squamous cell carcinoma

Squamous cell carcinoma of the oral cavity (OSCC) is a malignancy characterized by a high degree of local aggression and metastasis to cervical lymph nodes. Tetraspanins are proteins with functional roles in a wide array of cellular processes and are reported to be associated with tumor progression. The present study investigated the expression of the CD9, CD37, CD63, CD81 and CD82 tetraspanins in OSCC using immunohistochemistry (IHC) and quantitative Real Time-PCR (qRT-PCR). Tissue microarray (TMA) analysis of samples from 179 cases of OSCC and 10 normal samples oral mucosa were evaluated immunomorphologically. We analyzed CD9 and CD82 expression by qRT-PCR in 66 OSCC cases and 4 normal samples of oral mucosa. Expression of CD63, CD37 and CD81 was not detected in the samples studied. CD82 was downregulated or negative in 127 of 179 (80%) specimens; no correlation was observed between CD82 expression, clinicopathological parameters, disease-free survival and 5-year overall survival. CD9 expression was downregulated or negative in 75 of 129 (42%) OSCC samples. Loss of CD9 expression in OSCC samples correlated with the incidence of lymph node metastasis (p = 0.017). Disease-free survival and the 5-year overall survival of patients with downregulated or negative CD9 expression were significantly lower than in patients with positive CD9 expression (p = 0.010 and p = 0.071, respectively). No correlation was found between CD9 or CD82 expression and clinicopathological parameters by qRT-PCR. Our results suggest that the downregulation or lack of expression of the CD9 protein might indicate a more aggressive of OSCC. (C) 2009 Elsevier Ltd. All rights reserved.

Glial fibrillary acidic protein in tumor types with cartilaginous differentiation

Glial fibrillary acidic protein (GFAP) is a member of the intermediary filament protein family. It is an important component of astrocytes and a known diagnostic marker of glial differentiation. GFAP is expressed in other neural tumors and pleomorphic adenoma and, less frequently, in cartilage tumors, chordomas, and soft tissue myoepitheliomas. The aim of this study was to evaluate the role of GFAP and its reliability in nonglial tumors as an immunohistochemical marker. We evaluated GFAP gene and protein expression using Q-PCR and immunohistochemistry, respectively, in 81 and 387 cases of soft tissue, bone tumors, and salivary pleomorphic adenomas. Immunohistochemistry staining for GFAP was observed in all osteosarcomas (8 cases), all pleomorphic adenomas (7 cases), in 5 of 6 soft tissue myoepitheliomas, and in 21 of 76 chondrosarcomas. By Q-PCR, GFAP was highly expressed in pleomorphic adenomas and, to a lesser extent, chondrosarcomas, soft tissue myoepitheliomas, and chondroblastic osteosarcomas. The results that we obtained by immunohistochemistry and Q-PCR were well correlated. GFAP is a potential marker for tumors with cartilaginous differentiation, supported by evidence that GFAP is expressed in certain cases of myoepithelial tumors by immunohistochemistry, including soft tissue myoepitheliomas, which are related to cartilaginous differentiation. These findings contribute significantly to the diagnosis of soft tissue myoepitheliomas with cartilaginous differentiation and chondroblastic osteosarcoma in mesenchymal tumors. Modern Pathology ( 2009) 22, 1321-1327; doi: 10.1038/modpathol.2009.99; published online 7 August 2009