Chitosan-Based Films with 2-Aminothiophene Derivative: Formulation,Characterization and Potential Antifungal Activity

In this study, films of chitosan and 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (6CN), a 2-aminothiophene derivative with great pharmacological potential, were prepared as a system for a topical formulation. 6CN-chitosan films were characterized by physicochemical analyses, such as Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), X-ray diffraction (XRD), and scanning electronic microscopy (SEM). Additionally, the antifungal potential of the films was evaluated in vitro against three species of Candida (C. albicans, C. tropicalis, and C. parapsilosis). The results of the FTIR and thermal analysis showed the incorporation of 6CN in the polymer matrix. In the diffractogram, the 6CN-chitosan films exhibited diffraction halos that were characteristic of amorphous structures, while the micrographs showed that 6CN particles were dispersed in the chitosan matrix, exhibiting pores and cracks on the film surface. In addition, the results of antifungal investigation demonstrated that 6CN-chitosan films were effective against Candida species showing potential for application as a new antifungal drug.     

Effect of dietary carbohydrate-to-lipid ratios on growth, lipid deposition and metabolic hepatic enzymes in juvenile Senegalese sole (Solea senegalensis, Kaup)

A study was undertaken to determine the effect of various dietary carbohydrate‐to‐lipid ratios on growth performance, whole‐body composition and tissue lipid content in Senegalese sole (Solea senegalensis) juveniles. Data on the dietary regulation of key hepatic enzymes of the lipogenic and glycolytic pathways (glucose‐6‐phosphate dehydrogenase, G6PD; malic enzyme, ME; fatty acid synthetase, FAS; pyruvate kinase, PK and glucokinase, GK) were also generated. Four isonitrogenous (crude protein: 52% dry matter (DM)) diets were formulated to contain one of two lipid levels (11% and 21% DM). Within each dietary lipid level, the nature of the carbohydrate fraction (raw or extruded peas) was varied. Triplicate groups of 54 sole (initial body weight: 23.6±1.2 g) were grown in recirculated seawater over 67 days. Fish were fed using automated feeders. At the end of the study, whole‐body, liver, viscera and muscle samples were withdrawn for analyses. During the experimental period, the mean fish weight about doubled in all treatments. No significant differences were found in growth performance (ranging from 1.1% to 1.4% body weight day^?1) among dietary treatments. High‐fat diets increased whole‐body fat content. Similarly, daily fat gain ranged from 0.54 to 0.78 g kg^?1 day^?1 and highest values were found in fish fed high‐lipid diets. Dietary treatments also affected tissue lipid content (liver, viscera and muscle), with highest values in fish fed high‐fat diets. The nature of dietary carbohydrates had little influence on performance criteria, but affected tissue lipid deposition. The activities of G6PD, ME and FAS were depressed by elevated levels of dietary lipid, confirming the inhibitory effect of dietary fats on lipid biosynthesis. At both dietary lipid levels, ME and FAS activities were little affected by dietary carbohydrate. Activities of PK and GK were not affected by the starch level of the diets. In Senegalese sole juveniles, the lipogenic pathway is more susceptible to modulation by dietary means (particularly through lipid intake) than the glycolytic pathway.     

Associations between morphometric variables and weight and yields carcass in Pirapitinga Piaractus brachypomus

The objective of this work was to evaluate the association between morphometric variables and carcass characteristics in Pirapitinga. We used a thousand specimens of Pirapitinga with an average weight of 1,200 g, which were stunned, slaughtered, weighed, measured, and processed for morphometric and processing yield analysis, to obtain weights, carcass and fillet yields. Initially, the linearity of the variables was verified. Pearson's simple and partial correlation tests were performed between all metrics. Track analysis was performed considering the weights and yields of carcass and filet as dependent variables and the others as independent variables. Ridge regression models were used to eliminate the effects of multicollinearity among the independent variables. Observations showed that the simple correlations between body weights and yields were superior to the partial ones in terms of magnitude. The length and circumference of the specimens were the measures most linearly associated with weight, carcass weight and fillet with rib weight. As for carcass yield and fillet yield, linear correlation coefficients were low and not significant when associated with body weights. With the path analysis, we could observe significant positive correlations between the morphometric measurements for weight, carcass weight and fillet with rib weight. The standard length, body circumference and body circumference/body height ratio are the most correlated. The latter are the most important variables in the evaluation of body weights in Pirapitinga fish. They can serve as criteria for indirect selection in searching for fish with better carcass characteristics. As for fillet and carcass yields, the morphometric variables have not shown to be efficient for indirect selection.     

In Vitro Effect of the Reproductive Hormones on the Oxidative Burst Activity of Polymorphonuclear Leucocytes from Cows: A Flow Cytometric Study

In this study, the effect of reproductive hormones and substances with hormonal activity on the oxidative burst activity of blood polymorphonuclear leucocytes (PMN) high yielding dairy cows was evaluated. Different concentrations of: progesterone, oestradiol 17β, FSH, LH, GnRH, cortisol and PGF2α were incubated in vitro for 4 h with PMN of seven high milk yielding cows, during the period of anoestrous postpartum. Controls were run in parallel in which each hormone was replaced by its solvent. After incubation with hormones the competence of PMN to generate H₂O₂ was monitored by flow cytometry. A down‐regulation on the oxidative burst activity of PMA‐stimulated PMN was observed when cells were incubated with progesterone. Significant (p ≤ 0.001) differences between control and progesterone incubated cells were observed from 6.56 μg/ml. The same predisposition was observed when PMNs were incubated with cortisol. Besides for all concentrations employed, a decrease in the burst activity was observed, only beyond 0.19 mg/ml, statistical differences between the results obtained by the control and the cortisol incubated cells were obtained. Concerning oestradiol 17β, an increase on H₂O₂‐production was observed when PMN were incubated with 15 pg/ml and 45 pg/ml of this steroid (p ≤ 0.05), followed by a depression of the cell’s activity when unphysiological concentrations were employed. Significant (p ≤ 0.05) differences between the obtained with the control and oestradiol 17β incubated cells were observed only in the highest concentration of oestradiol. No statistical differences were observed in the metabolic burst activity of PMN incubated with FSH, GnRH and LH when compared with the results obtained by the control.     

Intra-articular pressure profiles of the cadaveric equine fetlock joint in motion

The study of the influence of motion and initial intra-articular pressure (IAP) on intra-articular pressure profiles in equine cadaver metatarsophalangeal (MTP) joints was undertaken as a prelude to in vivo studies. Eleven equine cadaver MTP joints were submitted to 2 motion frequencies of 5 and 10 cycles/min of flexion and extension, simulating the condition of lower and higher (double) rates of passive motion. These frequencies were applied and pressure profiles generated with initial normal intra-articular pressure (-5 mmHg) and subsequently 30 mmHg intra-articular pressure obtained by injection of previously harvested synovial fluid. The 4 trials performed were 1) normal IAP; 5 cyles/min; 2) normal IAP; 10 cycles/min; 3) IAP at 30 mmHg; 5 cycles/min and 4) IAP at 30 mmHg; 10 cycles/min. The range of joint motion applied (mean +/- s.e.) was 67.6+/-1.61 degrees with an excursion from 12.2+/-1.2 degrees in extension to 56.2+/-2.6 degrees in flexion. Mean pressure recorded in mmHg for the first and last min of each trial, respectively, were 1) -5.7+/-0.9 and -6.3+/-1.1; 2) -5.3+/-1.1 and -6.2+/-1.1; 3) 58.8+/-8.0 and 42.3+/-7.2; 4) 56.6+/-3.7 and 40.3+/-4.6. Statistical analyses showed a trend for difference between the values for the first and last minute in trial 3 (0.05>P<0.1) with P = 0.1 and significant difference (P = 0.02) between the mean IAP of the first and last min in trial 4. The loss of intra-articular pressure associated with time and motion was 10.5, 16.9, 28.1 and 28.9% for trials 1-4, respectively. As initial intraarticular pressure and motion increased, the percent loss of intra-articular pressure increased. The angle of lowest pressure was 12.2+/-1.2 degrees (mean +/- s.e.) in extension in trials 1 and 2. In trials 3 and 4, the lowest pressures were obtained in flexion with the joints at 18.5+/-2.0 degrees (mean +/- s.e.). This demonstrated that the joint angle of least pressure changed as the initial intra-articular pressure changed and there would not be a single angle of least pressure for a given joint. The volume of synovial fluid recovered from the MTP joints in trial 3 compared to 4 (trials in which fluid was injected to attain IAP of 30 mmHg) was not significantly different, supporting a soft tissue compliance change as a cause for the significant loss of intra-articular pressure during the 15 min of trial 4. The pressure profiles generated correlate well with in vivo values and demonstrated consistent pressure profiles. Our conclusions are summarised as follows: 1. Clinically normal equine MTP joints which were frozen and then later thawed were found to have mostly negative baseline intra-articular pressures, as would be expected in living subjects. 2. Alternate pressure profiles of the dorsal and plantar pouch at baseline intra-articular pressure document the presence of pressure forces that would support 'back and forth' fluid movement between joint compartments. This should result in movement of joint fluid during motion, assisting in lubrication and nutrition of articular cartilage. 3. If joint pressure was initially greater than normal (30 mmHg), as occurs in diseased equine MTP joints, joint motion further increased joint capsule relaxation (compliance) and, therefore, reduced intra-articular pressure. 4. Peak intra-articular pressures reached extremely high values (often >100 mmHg) in flexion when initial pressure was 30 mmHg. Joint effusion pressures recorded for clinical MCP joints are frequently 30 mmHg. These IAP values are expected to produce intermittent synovial ischaemia in clinical cases during joint flexion. 5. Additional in vivo studies are necessary to confirm our conclusions from this study and to identify the contributions of fluid absorption and the presence of ischaemia in a vascularised joint.     

Relation between Physical Properties of the Zona Pellucida and Viability of Bovine Embryos after Slow-freezing and Vitrification

In vitro-produced bovine morulae/blastocyst embryos (n = 119) were slow-frozen and vitrified and the physical alterations of the zona pellucida (ZP) was observed by scanning electron microscopy (SEM) to find an explanation for the loss of developmental capacity of the embryos after freezing/thawing. A control group was provided, in which embryos (n = 38) were neither frozen nor vitrified. Embryos were in vitro-cultured in a standard CO2 Heraeus incubator and their viability was assessed 24 and 48 h after the start of culture, evaluating their morphological aspect. After 24 h of culture, embryo survival rate for slow-freezing/thawed (n = 23), vitrified/thawed (n = 20) and control embryos (n = 20) was 39, 27 and 90%, and 35, 14 and 65% after 48 h of culture, respectively. For evaluation of physical changes occurring in ZP, 20 embryos were slow-frozen, 18 were vitrified and 18 were used as control. All embryos were fixed, dried and examined under an SEM. Embryo's diameter, as well as the number of pores and their diameter was measured in squares of 6.4 microm width. We observed that, on average, the diameter of the embryos (92.26 +/- 10.15 microm) did not differ significantly among all embryos. As far as the diameter of the pores in the outer surface of the ZP is concerned, the results revealed a significant difference (P < 0.05) between control (0.48 +/- 0.0025 microm), slow-frozen (0.34 +/- 0.0007 microm) and vitrified (0.27 +/- 0.0006 microm) embryos. For the number of pores, statistical differences (p < 0.05) were observed between control and vitrified embryos (45.4 +/- 7.3 vs 38.2 +/- 8.2). It is possible that ZP functions as a barrier which is positive when dealing with pathogens, but is harmful when nutrients were supplied from the outside, especially at 48 h of culture. Results indicate that the steps of cryopreservation cause alterations in ZP, with irreversible damage on the further developmental competence of bovine embryos.     

Coagulase gene typing of Staphylococcus aureus isolated from cows with mastitis in southeastern Brazil

A typing procedure based on polymorphism of the coagulase gene (coa) was used to discriminate Staphylococcus aureus isolated from Minas Gerais dairy cows with mastitis. Amplification of the gene from the 64 S. aureus isolates produced 27 different polymerase chain reaction (PCR) products; 60 isolates showed only 1 amplicon, and 4 showed 2 amplicons. The isolates were grouped into 49 types by analyzing the restriction fragment length polymorphism (RFLP) of the coa gene; the 10 most common types accounted for 39% of the isolates. The results demonstrate that many variants of the coa gene are present in the studied region, although only a few predominate.     

Characterization of a plasmid-encoded adhesin of an avian pathogenic Escherichia coli (APEC) strain isolated from a case of swollen head syndrome (SHS)

An avian pathogenic Escherichia coli (APEC) strain designated SHS4, isolated from a chicken with clinical signs of swollen head syndrome (SHS), adhered to but did not invade Hep-2 and tracheal epithelial cells. The PCR amplified fimA, csgA and tsh gene sequences. It produced Ia, Ib, E1, E3, K, and B colicins, but not colicin V and aerobactin. It harboured two plasmids of 60 and 98MDa and was resistant to streptomycin and tetracycline. Conjugation with a nalidixic acid (Na) resistant K-12 recipient strain (MS101) showed that the 98MDa plasmid did not transfer, whereas transfer of the 60MDa plasmid resulted in concomitant transfer of adhesion to Hep-2 and tracheal epithelial cells, production of the colicins Ia, E1, E3, and K, and the tsh-related DNA sequence. Transposon (TnphoA) mutagenesis of strain TR4 gave rise to strain Mut23, which lost its adhesive capacities, but was still able to express the same colicins as did strain TR4. PCR was able to amplify the tsh-related DNA sequence in this strain and a molecular probe based on transposon TnphoA indicated that the transposon was inserted in the 60MDa plasmid. Based on these results, we suggest that the 60MDa plasmid have adhesion genes, which may be responsible for the initial colonization of the upper respiratory tract of chickens.     

Virulence factors of avian pathogenic Escherichia coli isolated from broilers from the south of Brazil

Sixty-three Escherichia coli strains isolated from broilers with respiratory problems were examined for virulence factors, hemolysin synthesis ability, motility, hemagglutination capacity, operon pap presence, colicin production, and serum resistance. The capacity to hemagglutinate guinea pig erythrocytes was found in 53 (84.1%) of the samples, but only 30 (47.6%) agglutinated chicken erythrocytes. D-mannose-sensitive hemagglutination against guinea pig erythrocytes was found in 19 (30.2%) samples and against chicken erythrocytes, in 15 (23.8%) samples, whereas the D-mannose-resistant hemagglutination with guinea pig erythrocytes was found in 34 (54%) samples, and 13 of these (20.6%) showed this characteristic against chicken erythrocytes. Operon pap, P fimbria codifier, was detected in 26 samples in a total of 34 D-mannose-resistant samples. Colicin production was observed in 55 (87.3%) of the strains, and 41.8% presented V colicin production. Of the samples analyzed, 56 (88.9%) presented serum resistance, six (9.5%) were intermediate, and only one (1.6%) was sensitive to the action of the complement. The diversity of virulence profiles detected in the samples in this study explains in part the multifactorial characteristics of avian colibacillosis.     

First Portuguese isolate of Neospora caninum from an aborted fetus from a dairy herd with endemic neosporosis

Neospora caninum was isolated from the brain of an aborted 4-month-old fetus from a dairy cow herd with endemic neosporosis in Porto, Portugal. The fetal brain homogenate was inoculated interperitoneally first into outbred Swiss Webster mice given dexamethasone and then the peritoneal exudates from these mice was co-inoculated with mouse sarcoma cells in the peritoneal cavity of mice given dexamethasone. N. caninum tachyzoites were seen in peritoneal exudate of the second passage. Tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies and contained N. caninum specific DNA. This Portuguese isolate of N. caninum has been successfully maintained in cell culture. The dam of the aborted fetus had an antibody titer of 1:10240 in the Neospora agglutination test (NAT). Antibodies to N. caninum were found in 76 of 106 cows from this herd in titers of 1:40 in 31, 1:80 in 22, > or =1:160 or more in 23 in the Neospora agglutination test. This is the first isolation of a viable N. caninum-like parasite from any host in Portugal.