Pheno and genotyping of Staphylococcus aureus, isolated from bovine milk samples from São Paulo State, Brazil

In the present study, 87 Staphylococcus aureus isolates obtained from milk samples of 87 cows with mastitis in 6 different municipal districts of 2 regions of São Paulo State, Brazil, were compared pheno and genotypically. Pulsed-field gel electrophoresis (PFGE) analysis of the strains was performed, and PCR was carried out to detect genes for a number of staphylococcal cell surface proteins, exoproteins, and 3 classes of agr genes. Nine distinct S. aureus lineages (LA-LI) were identified by PFGE. The lineages LA and LE, which accounted together for 63 strains (72.2%), were prevalent and had been collected from all of the 6 municipal districts, indicating a broad geographic distribution of these lineages; LB, LC, LD, LF, LG, LH, and LI, however, were isolated sporadically and accounted for 24 strains (27.8%). Some characteristics, like penicillin resistance and the presence of cap8 and agr class II genes, were associated with the prevalent lineages (LA and LE), and penicillin susceptibility and the presence of cna and cap5 genes were associated with sporadic lineages. According to the present results, some S. aureus lineages possess a combination of genes that confer the propensity to cause and disseminate infection, and only a limited number of clones are responsible for the cases of bovine mastitis on the various farms. © 2004 NRC Canada.

Comparative analysis of noncoding sequences of orthologous bovine and human gene pairs

Genomic sequence comparison across species has enabled the elucidation of important coding and regulatory sequences encoded within DNA. Of particular interest are the noncoding regulatory sequences, which influence gene transcriptional and posttranscriptional processes. A phylogenetic footprinting strategy was employed to identify noncoding conservation patterns of 39 human and bovine orthologous genes. Seventy-three conserved noncoding sequences were identified that shared greater than 70% identity over at least 100 bp. Thirteen of these conserved sequences were also identified in the mouse genome. Evolutionary conservation of noncoding sequences across diverse species may have functional significance, and these conserved sequences may be good candidates for regulatory elements.

The effect of adhesive systems on the pullout strength of a fiberglass-reinforced composite post system in bovine teeth

Purpose: To evaluate the pullout strength of a glass fiber-reinforced composite post (glass FRC) cemented with three different adhesive systems and one resin cement. The null hypothesis was that pullout strengths yielded by the adhesive systems are similar. Materials and Methods: Thirty bovine teeth were selected. The size of the specimens was standardized at 16 mm by sectioning off the coronal portion and part of the root. The specimens were divided into three groups, according to the adhesive system, which were applied following the manufacturers' instructions: G1, ScotchBond Multi-Purpose Plus; G2, Single Bond; G3, Tyrian SPE/One-Step Plus. The glass FRCs (Reforpost) were etched with 37% H3PO4 for 1 min and silanized (Porcelain Primer). Thereafter, they were cemented with the dual resin cement En-Force. The specimens were stored for 24 h, attached to an adapted device, and submitted to the pullout test in a universal testing machine (1 mm/min). The data were submitted to the one-way ANOVA and Tukey's test (α = 0.05). Results: G1 (30.2 ± 5.8 Kgf) displayed the highest pullout strength (p < 0.001) when compared to G2 (18.6 ± 5.8 Kgf) and G3 (14.3 ± 5.8 Kgf), which were statistically similar. Analysis of the specimens revealed that all failures occurred between the adhesive system and the root dentin (pullout of the post cement), regardless of group. Conclusion: The multiple-bottle, total-etch adhesive system provided higher pullout strength of the glass FRC when compared to the single-bottle, total-etch, and single-step self-etching adhesive systems. The null hypothesis was rejected (p < 0.001).

Bovine serum albumin displays luciferase-like activity in presence of luciferyl adenylate: Insights on the origin of protoluciferase activity and bioluminescence colours

Luciferyl adenylate, the key intermediate in beetle bioluminescence, is produced through adenylation of D-luciferin by beetle luciferases and also by mealworm luciferase-like enzymes which produce a weak red chemiluminescence. However, luciferyl adenylate is only weakly chemiluminescent in water at physiological pH and it is unclear how efficient bioluminescence evolved from its weak chemiluminescent properties. We found that bovine serum albumin (BSA) and neutral detergents enhance luciferyl adenylate chemiluminescence by three orders of magnitude, simulating the mealworm luciferase-like enzyme chemiluminescence properties. These results suggest that the beetle protoluciferase activity arose as an enhanced luciferyl adenylate chemiluminescence in the protein environment of the ancestral AMP-ligase. The predominance of luciferyl adenylate chemiluminescence in the red region under most conditions suggests that red luminescence is a more primitive condition that characterized the original stages of protobioluminescence, whereas yellow-green bioluminescence may have evolved later through the development of a more structured and hydrophobic active site. Copyright © 2006 John Wiley & Sons, Ltd.

Bio-safety technology in production of bovine herpesvirus type 5 (BoHV-5) using an alternative serum-free medium

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Toxoplasma gondii in semen of experimentally infected swine

Eight reproductive boars were divided into three groups and inoculated with Toxoplasma gondii [GI (n=3) 1.5×104 oocysts strain P; GII (n=3) 1.0×106 tachyzoites strain RH; and GIII (n=2) non-inoculated control]. Clinical, hematological, parasitemia and serological tests and studies of the parasite in the semen through bioassay and PCR, and in reproductive organs (Bioassay and immunohistochemical analyses) were conducted to evaluate the toxoplasmic infection. Blood and semen were collected on day -2, -1, 1, 3, 5, 7, 9, 11, 14 and weekly up to 84 days post-inoculation (DPI). No clinical or hematimetric alteration was observed in the boars. Parasitemia was detected in one boar inoculated with oocysts at the 7th DPI and in another boar infected with tachyzoites (GII) at the 3rd and 49 th DPI. Serological tests revealed antibodies against T. gondii in animals inoculated with oocysts or tachyzoites at the 7th DPI with dilutions of 1:256 and 1:64, which reached peaks of 1:4096 at day 11 and 9, respectively. The bioassays revealed the presence of the parasite in semen samples of a boar inoculated with oocysts (GI) at 3, 49 and 56 DPI and from two boars infected with tachyzoites (GII), one animal at 5 and two animals at 49 days DPI. Mice inoculated with semen from the control group (GIII) remained serologically negative. PCR analysis showed T. gondii DNA in the semen of Boar 1 and Boar 3 inoculated with tachyzoites and oocysts, respectively. The immunohistochemical tests showed T. gondii in the reproductive organs of Boar 1 and Boar 2, inoculated with tachyzoites and oocysts, respectively. These findings suggest the possible occurrence of venereal transmission of T. gondii in swine.

Fatigue resistance of bovine teeth restored with resin-bonded fiber posts: Effect of post surface conditioning

This study evaluated the effect of post surface conditioning on the fatigue resistance of bovine teeth restored with resin-bonded fiber-reinforced composite (FRC). Root canals of 20 single-rooted bovine teeth (16 mm long) were prepared to 12 mm using a preparation drill of a double-tapered fiber post system. Using acrylic resin, each specimen was embedded (up to 3.0 mm from the cervical part of the specimen) in a PVC cylinder and allocated into one of two groups (n = 10) based on the post surface conditioning method: acid etching plus silanization or tribochemical silica coating (30 μm SiOx + silanization). The root canal dentin was etched (H2PO3 for 30 seconds), rinsed, and dried. A multi-step adhesive system was applied to the root dentin and the fiber posts were cemented with resin cement. The specimens were submitted to one million fatigue cycles. After fatigue testing, a score was given based on the number of fatigue cycles until fracture. All of the specimens were resistant to fatigue. No fracture of the root or the post and no loss of retention of the post were observed. The methodology and the results of this study indicate that tribochemical silica coating and acid etching performed equally well when dynamic mechanical loading was used.

Report of a chimeric origin of transposable elements in a bovine-coding gene

Despite the wide distribution of transposable elements (TEs) in mammalian genomes, part of their evolutionary significance remains to be discovered. Today there is a substantial amount of evidence showing that TEs are involved in the generation of new exons in different species. In the present study, we searched 22,805 genes and reported the occurrence of TE-cassettes in coding sequences of 542 cow genes using the RepeatMasker program. Despite the significant number (542) of genes with TE insertions in exons only 14 (2.6%) of them were translated into protein, which we characterized as chimeric genes. From these chimeric genes, only the FAST kinase domains 3 (FASTKD3) gene, present on chromosome BTA 20, is a functional gene and showed evidence of the exaptation event. The genome sequence analysis showed that the last exon coding sequence of bovine FASTKD3 is ∼85% similar to the ART2A retrotransposon sequence. In addition, comparison among FASTKD3 proteins shows that the last exon is very divergent from those of Homo sapiens, Pan troglodytes and Canis familiares. We suggest that the gene structure of bovine FASTKD3 gene could have originated by several ectopic recombinations between TE copies. Additionally, the absence of TE sequences in all other species analyzed suggests that the TE insertion is clade-specific, mainly in the ruminant lineage. ©FUNPEC-RP.

A scanning electronic microscopy study of the action of different desensitizing agents on bovine dentin

This study sought to use scanning electronic microscopy (SEM) to evaluate the dentinal tubule occlusion potential of different desensitizing agents. Ten slices of bovine dentin were divided into six fragments, cleaned (using ultrasound), and etched for 15 seconds with a 35% phosphoric acid solution. All but one of the groups received a different desensitizing agent; the sixth group served as a control and received no additional treatment. After the agents were applied, the dentin specimens were analyzed by SEM and scores were assigned based on the extent of tubular obliteration. Only three agents demonstrated tubular sealing that was significantly different from that of the control group.

Scanning electron microscopic study of the in situ effect of salivary stimulation on erosion and abrasion in human and bovine enamel

This in situ study investigated, using scanning electron microscopy, the effect of stimulated saliva on the enamel surface of bovine and human substrates submitted to erosion followed by brushing abrasion immediately or after one hour. During 2 experimental 7-day crossover phases, 9 previously selected volunteers wore intraoral palatal devices, with 12 enamel specimens (6 human and 6 bovine). In the first phase, the volunteers immersed the device for 5 minutes in 150 ml of a cola drink, 4 times a day (8h00, 12h00, 16h00 and 20h00). Immediately after the immersions, no treatment was performed in 4 specimens (ERO), 4 other specimens were immediately brushed (0 min) using a fluoride dentifrice and the device was replaced into the mouth. After 60 min, the other 4 specimens were brushed. In the second phase, the procedures were repeated but, after the immersions, the volunteers stimulated the salivary flow rate by chewing a sugar-free gum for 30 min. Enamel superficial alterations of all specimens were then evaluated using a scanning electron microscope. Enamel prism core dissolution was seen on the surfaces submitted to erosion, while on those submitted to erosion and to abrasion (both at 0 and 60 min) a more homogeneous enamel surface was observed, probably due to the removal of the altered superficial prism layer. For all the other variables - enamel substrate and salivary stimulation the microscopic pattern of the enamel specimens was similar.

β-casein gene polymorphism permits identification of bovine milk mixed with bubaline milk in mozzarella cheese

Mozzarella cheese is traditionally prepared from bubaline (Bubalus bubalis) milk, but product adulteration occurs mainly by addition of or full substitution by bovine milk. The aim of this study was to show the usefulnes of molecular markers to identify the admixture of bovine milk to bubaline milk during the manufacturing process of mozzarella cheese. Samples of mozzarella cheese were produced by adding seven different concentrations of bovine milk: 0%, 1%, 2%, 5%, 8%, 12% and 100%. DNA extracted from somatic cells found in cheese were submitted to PCR-RFLP analysis of casein genes: α-s1-CN - CSN1S1 that encompasses 954 bp from exon VII to intron IX (AluI and HinfI), β-CN - CSN2 including 495 bp of exon VII (Hae III and HinfI), and κ-CN - CSN3, encompassing 373 bp of exon IV (AluI and HindIII). Our results indicate that Hae III-RFLP of CSN2exon VII can be used as a molecular marker to detect the presence of bovine milk in mozzarella cheese. Copyright © 2008, Sociedade Brasileira de Genética.

Comparative analysis of human and bovine teeth: Radiographic density

Since bovine teeth have been used as substitutes for human teeth in in vitro dental studies, the aim of this study was to compare the radiographic density of bovine teeth with that of human teeth to evaluate their usability for radiographic studies. Thirty bovine and twenty human teeth were cut transversally in 1 millimeter-thick slices. The slices were X-rayed using a digital radiographic system and an intraoral X-ray machine at 65 kVp and 7 mA. The exposure time (0.08 s) and the target-sensor distance (40 cm) were standardized for all the radiographs. The radiographic densities of the enamel, coronal dentin and radicular dentin of each slice were obtained separately using the histogram tool of Adobe Photoshop 7.0 software. The mean radiographic densities of the enamel, coronal dentin and radicular dentin were calculated by the arithmetic mean of the slices of each tooth. One-way ANOVA demonstrated statistically significant differences for the densities of bovine and human enamel (p < 0.05) and for bovine and human coronal dentin (p < 0.05). No statistically significant differences were found for the bovine and human radicular dentin (p > 0.05). Based on the results, the authors concluded that: a) the radiographic density of bovine enamel is significantly higher than that of human enamel; b) the radiodensity of bovine coronal dentin is statistically lower than the radiodensity of human coronal dentin; bovine radicular dentin is also less radiodense than human radicular dentin, although this difference was not statistically significant; c) bovine teeth should be used with care in radiographic in vitro studies.

Semen variables of sheep (Ovis aries) experimentally infected with Toxoplasma gondii

The influence of Toxoplasma gondii on semen variables and sperm morphology of sheep was evaluated in eight reproductive males distributed into three experimental groups: GI, three sheep inoculated with 2.0 × 105 of P strain oocytes; GII, three sheep infected with 1.0 × 106 of RH strain tachyzoites and; GIII two control sheep. Clinical (rectal temperature, cardiac and respiratory frequencies), parasite and serology exams (IIF) were realized. Sperm variables (volume, motility, vigor and concentration) and semen morphology for each sheep were also evaluated. Thus, semen and blood collections were assessed on post-inoculation days (PIDs)-1,3,5,7,11,14 and weekly thereafter up to PID 70. Clinical alterations were observed (hypothermia and anorexia) in infected sheep from groups GI and GII. Parasitic outbreaks were detected in five sheep. All the infected sheep produced antibodies against T. gondii from PID 5 onwards, reaching a peak of 4096 and 8192 for group GI and GII sheep, respectively. Differences (P < 0.05) were observed regarding the ejaculate volume between the inoculated groups (oocytes and tachyzoites) and control. Even though experimental toxoplasmic infection resulted in clinical symptomology in the inoculated sheep, the minimal alterations in sperm pathologies could not be directly attributed to T. gondii. © 2008 Elsevier B.V. All rights reserved.