In Silico Screening of Putative Corynebacterium pseudotuberculosis Antigens and Serological Diagnosis for Caseous Lymphadenitis in Sheep by Enzyme-Linked Immunosorbent Assay.

Corynebacterium pseudotuberculosis is the etiologic agent of Caseous Lymphadenitis (CLA), a disease leading to severe damage in sheep and goats farming due to the lack of serological diagnosis, treatment, and effective prophylaxis. In this context, several strategies in an attempt to discover new antigens to compose diagnosis assays or vaccines are fundamental. Therefore, this study aimed to use bioinformatics software to evaluate the critical chemical characteristics of unknown proteins of C. pseudotuberculosis by selecting them for heterologous expression in Escherichia coli. For this purpose, six protein sequences of ascorbate transporter subunit, UPF protein, MMPL family transporter, Ribonuclease, Iron ABC transporter domain-containing permease, and fimbrial subunit were obtained. In silico analyses were performed using amino acid sequences to access immunodominant epitopes and their antigenic and allergenic potential and physicochemical characterization. The expressed proteins were used as an antigen for serological diagnosis by ELISA. All proteins showed distinct immunodominant epitopes and potential antigenic characteristics. The only proteins expressed were PTS and Ribonuclease. In parallel, we expressed CP40 and all were used with ELISA antigen in 49 CLA positive sera and 26 CLA negative sera. The proteins alone showed 100% sensitivity and 96.2%, 92.3%, and 88.5% specificity for rPTS, rRibonuclease, and rCP40, respectively. When proteins were combined, they showed 100% sensitivity and 84.6%, 92.3%, 88.5%, and 92.3% specificity for rPTS/rCp40, rRibonuclease/rCP40, rPTS/rRibonuclease, and rPTS/rRibonuclease/rCP40, respectively. The results of this study show an excellent correlation of sensitivity and specificity with all proteins. None of the specificity values preclude the potential of rPTS, rRibonuclease, or rCP40 for use in ELISA diagnostic assays since the results of this work are superior to those of other studies on CLA diagnosis described in the literature.

Effects of cashew gum and nanoparticles on cooled stallion semen.

BACKGROUND: Cryopreservation of stallion spermatozoa tends to cause plasma membrane damage due to the low ratio of cholesterol to phospholipids. Gums have been suggested as an alternative cryoprotectant to glycerol for stallion spermatozoa. Therefore, the present experiment was designed to verify whether the effect of addition of cashew gum (CG), or nanoparticles (NP) containing CG, to the extender before cooling on sperm quality in stallion semen. Ejaculates from 6 stallions were extended and split between six treatment groups (control, a-tocopherol [TOC], CG1, CG0.5, NP1 and NP0.5), stored in cryotubes at 4 °C. RESULTS: Aliquots were analysed by computer-assisted sperm motility analysis on the day of collection, and after 24 h and 48 h of cold storage. After 48 h, the total motility with NP1 (78.53 + 6.31%) was similar to control 85.79 + 6.31% at 0 h. The same pattern was observed for progressive motility. Membrane integrity assessed by flow cytometer was similar between control, TOC and G1 at all storage times. The DNA fragmentation in the control group increased at all time points, whereas chromatin integrity was maintained after 24 h in TOC and NP0.5 compared to 0 h. There was no increase in the proportion of live spermatozoa producing hydrogen peroxide, but there was a tendency for an increased proportion of spermatozoa in the live superoxide category in CG1 after 24 h cooled storage. CONCLUSIONS: The addition of CG or CG-derived NP to extender for stallion semen was not harmful to the sperm cells.

Histological analysis of the periodontal ligament and alveolar bone during dental movement in diabetic rats subjected to low-level laser therapy.

OBJECTIVE: The purpose of this research was to evaluate the histological changes of the periodontal ligament and alveolar bone during dental movement in diabetic rats subjected to low level laser therapy (LLLT). METHODS: The movement of the upper molar was performed in 60 male Wistar rats divided into four groups (n=15): CTR (control), DBT (diabetic), CTR/LT (irradiated control) and DBT/LT (irradiated diabetic). Diabetes was induced with alloxan (150 mg/kg, i.p.). LLLT was applied with GaAlAs laser at 780 nm (35 J/cm(2)). After 7, 13 and 19 days, the periodontal ligament and alveolar bone were histologically analyzed. RESULTS: The mean of osteoblasts (p<0.01) and blood vessels (p<0.05) were significantly decreased in DBT compared with CTR at 7 days, whereas the mean of osteoclasts was lower at 7 (p<0.001) and 13 days (p<0.05). In DBT/LT, only the mean of osteoclasts was lower than in CTR (p<0.05) at 7 days, but no difference was observed at 13 and 19 days (p>0.05). The collagenization of the periodontal ligament was impaired in DBT, whereas DBT/LLT showed density/disposition of the collagen fibers similar to those observed in CTR. CONCLUSIONS: LLLT improved the periodontal ligament and alveolar bone remodeling activity in diabetic rats during dental movement.

Leukemia inhibitory factor stimulates the transition of primordial to primary follicle and supports the goat primordial follicle viability in vitro.

The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF) on the activation and survival of preantral follicles cultured in vitro enclosed in ovarian fragments (in situ). Goat ovarian cortex was divided into fragments to be used in this study. One fragment was immediately fixed (fresh control - FC) and the remaining fragments were cultured in supplemented minimum essential medium (MEM) without (cultured control - CC) or with different concentrations of LIF (1, 10, 50, 100 or 200 ng/ml) for 1 or 7 days, at 39°C in air with 5% CO2. Fresh control, CC and treated ovarian fragments were processed for histological and fluorescence analysis. The percentage of histological normal preantral follicles cultured for 7 days with 1 ng/ml (49.3%), 10 ng/ml (58.6%) and 50 ng/ml (58%) of LIF was higher than in the CC (32.6%; p < 0.05). After 7 days of culture, the percentage of primordial follicles in situ cultured with LIF decreased and primary follicles increased in all LIF concentrations compared with FC and CC (p < 0.05). In conclusion, LIF induced primordial follicle activation and supported preantral follicle viability of goat ovarian tissues cultured for 7 days.

Bone morphogenetic protein-6 (BMP-6) induces atresia in goat primordial follicles cultured in vitro / A proteína morfogenética óssea-6 (BMP-6) induz a atresia em folículos primordiais caprinos cultivados in vitro

This study investigated the effects of bone morphogenetic protein 6 (BMP-6) on in vitro primordial follicle development in goats. Samples of goat ovarian cortex were cultured in vitro for 1 or 7 days in Minimum Essential Medium (control medium) supplemented with different concentrations of BMP-6. Follicle survival, activation and growth were evaluated through histology and transmission electron microscopy (TEM). After 7 days of culture, histological analysis demonstrated that BMP-6 enhanced the percentages of atretic primordial follicles when compared to fresh control (day 0). Nevertheless, BMP-6 increased follicular and oocyte diameter during both culture periods. As the culture period progressed from day 1 to day 7, a significant increase in follicle diameter was observed with 1 or 50ng/ml BMP-6. However, on the contrary to that observed with the control medium TEM revealed that follicles cultured for up to 7 days with 1 or 50ng/ml BMP-6 had evident signs of atresia. In conclusion, this study demonstrated that BMP-6 negatively affects the survival and ultrastructure of goat primordial follicles.

O presente estudo investigou os efeitos da proteína morfogenética óssea-6 (BMP-6) no desenvolvimento in vitro de folículos primordiais caprinos. Amostras de córtex ovariano de cabras foram cultivados por 1 ou 7 dias em Meio Essencial Mínimo (meio controle) suplementado com diferentes concentrações de BMP-6. As taxas de sobrevivência, ativação e crescimento foram avaliadas por histologia clássica e microscopia eletrônica de transmissão (MET). Após 7 dias de cultivo, a análise histológica demonstrou que a BMP-6 aumentou o percentual de folículos primordiais degenerados no dia 7 quando comparados ao controle fresco (D0). Além disso, houve um aumento significativo do diâmetro folicular e oocitário em ambos os períodos de cultivo em todos os tratamentos na presença de BMP-6. Com a progressão do cultivo do dia 1 para o dia 7, nos tratamentos com 1 ou 50ng/ml de BMP-6, foi observado um aumento significativo no diâmetro folicular. Entretanto, contrário ao observado no meio controle, a MET revelou que os folículos cultivados nesses tratamentos apresentavam sinais evidentes de atresia. Em conclusão, esse estudo demonstrou que a BMP-6 afeta negativamente a sobrevivência e a ultra-estrutura de folículos primordiais caprinos.