Development and validation of nested-PCR for the diagnosis of clinical and subclinical infectious laryngotracheitis

A standardised nested-PCR method that amplifies a region of the glycoprotein E gene of avian infectious laryngotracheitis virus (ILTV) has been developed for the diagnosis of infection by Gallid herpesvirus 1. The two sets of primers employed produced the expected ampIification products of 524bp(externa I primers) and 219bp (internal primers) in the presence of ILTV DNA, whereas no Such amplicons were obtained with other avian respiratory pathogens or with DNA extracted from the cells of uninfected chickens. The identity of the 219bp amplified product was con firmed by DNA sequencing. The standardised nested-PCR method detected ILTV DNA from trachea, lung, conjunctiva and trigeminal ganglia samples from flocks of birds with and without clinical signs. and showed hi.-h sensitivity (95.4%) and specificity (93.1%) when compared with the reference test involving virus isolation in specific-pathogen-free chicken embryos. The standardised nested-PCR method described may be used to detect clinical and latent ILTV infections, and will be of significant value for both diagnostic and epidemiological Studies. (c) 2008 Elsevier B.V. All rights reserved.

NONINVASIVE MONITORING OF ANDROGENS IN MALE AMAZONIAN MANATEE (TRICHECHUS INUNGUIS): BIOLOGIC VALIDATION

The Amazonian manatee (Trichechus inunguis) is endemic in the Amazonian basin and is the only exclusively fresh water sirenian. Historically hunted on a large scale, this species is now considered endangered, and Studies on the reproductive physiology are critical for the improvement of reproductive management of captive and wild Populations of manatees. The aim of this Study was to verify the viability of androgen measurement in saliva, lacrimal, urine, and fecal samples of the Amazonian manatee by conducting a hormone challenge. Two adult male manatees (A-1 and A-2) were Submitted to an experimentation protocol of 12 day (D1 to D10). On D0, the animals received an intramuscular injection of gonadotropin-releasing hormone (GnRH)-analogue. Salivary, lacrimal, urinary, and fecal samples were collected daily (between 0800 hours and 0900 hours) and frozen at -20 degrees C until assayed. Fecal samples were lyophilized, extracted with 80% methanol, and diluted in buffer before the radioimmunoassay (RIA). Urine samples underwent acid hydrolysis and were diluted in depleted bovine serum. Salivary and lacrimal samples were assayed without the extraction step. Hormonal assays were conducted with a commercial testosterone RIA kit. An androgen peak (>median + 2 interquartile range [IQR]) was observed in all matrices of both animals, although it was less prominent in the lacrimal samples of A-2. However, the fecal androgen peak (A-1 peak = 293.78 ng/g dry feces, median [IQR] = 143.58 [32.38] ng/g dry feces; A-2 peak = 686.72 ng/g dry feces, median [IQR] = 243.82 [193.16] ng/g dry feces) occurred later than urinary (A-1 peak = 648.16 ng/mg creatinine [Cr], median [IQR] = 23.88 [30.44] ng/mg Cr; A-2 peak = 370.44 ng/mg Cr, median [IQR] = 113.87 [117.73] ng/mg Cr) and salivary (A-1 peak = 678.89 pg/ml, median [IQR] = 103.69 [119.86] pg/ml; A-2 peak = 733.71 pg/ml, median [IQR] = 262.92 [211.44] pg/ml) androgen peaks. These intervals appear to be correlated with the long digesta passage time in this species. The salivary and urinary peaks were closely associated. These results demonstrate that androgen concentrations in saliva, urine, or feces samples reflect reliably physiologic events and are a powerful tool for noninvasive reproductive monitoring of Amazonian manatees.

Validation of body condition score as a predictor of subcutaneous fat in Nelore (Bos indicus) cows

The aim of the present study was to determine the relationship among body weight (BW), body condition score (BCS) and rump fat thickness (RFAT) measured by ultrasonography, and validate the relationship between BCS and RFAT over the time. Two hundred sixty and six Nelore cows had their BW, BCS and RFAT evaluated at five different moments during the production cycle: M1) weaning: M2) parturition, M3) 42 days post-partum; M4) 82 days postpartum and M5) 112 days post-partum. A BCS value was attributed for each cow following a I to 5 points scale. Ultrasonographic images for RFAT measurement were obtained using a 3.5 MHz linear transducer. Images were immediately analyzed as soon as they were formed and frozen. Body condition scores and ultrasound measurements were collected on the same day by a single trained technician. The relationship between BCS and RFAT values was investigated by regression models. The analysis of similarity among the five obtained models was performed using the proc MIXED from SAS and the correlations among variables were analyzed with proc CORR from SAS. The BCS was able to predict RFAT in Nelore cows in all different moments evaluated. Also, it was shown that BCS presented high correlation (r=0.82 to 0.93) and relationship (R(2) = 0.73 to 0.92) with RFAT. However, both BCS and RFAT showed low correlation (r=0.37 to 0.50) and relationship (R(2) = 0.13 to 0.25) with BW. The BCS classification by visual method using a 1 to 5 point scale, was able to predict the RFAT in Nelore cows over the time. (C) 2008 Elsevier B.V. All rights reserved.

Validation of non-fluorescent methods to reliably detect acrosomal and plasma membrane integrity of common marmoset (Callithrix jacchus) sperm

Simple, rapid and stable sperm evaluation methods which have been optimized for common marmoset (Callithrix jacchus) are critical for studies involving collection and evaluation of sperm in the field. This is particularly important for new species groups such as Callitrichidae where the sperm have been little studied. Of this family, C jacchus is the best known, and has been chosen as a model species for other members of the genus Callithrix. The fundamental evaluation parameters for sperm of any species are viability and acrosomal status. Semen samples were collected by penile vibratory stimulation. To evaluate sperm plasma membrane integrity, Eosin-Nigrosin was tested here for the common marmoset sperm to be used under field conditions. Further, a non-fluorescent stain for acrosome, the ""Simple"" stain, developed for domestic and wild cats, was tested on common marmoset sperm. This was compared with a fluorescent staining, Fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA), routinely used and validated for common marmoset at the German Primate Centre to evaluate acrosomal integrity. Results obtained with the ""Simple"" stain showed a marked differentiation between sperm with intact and non-intact acrosome both with and without ionophore treatment and closely correlated with results obtained with FITC-PSA. Temperature had no effect on the results with the ""Simple"" stain and the complete processing is simple enough to be carried out under field conditions. These findings indicated that the ""Simple"" stain and Eosin-Nigrosin provide rapid and accurate results for C. jacchus sperm and that those methods can be reliably used as field tools for sperm evaluation for this species. (c) 2008 Elsevier Inc. All rights reserved.

Long-term stability of dentin matrix following treatment with various natural collagen cross-linkers

Objectives: Collagen disorganization is one of the main degradation patterns found in unsuccessful adhesive restorations. The hypothesis of this study was that pretreatment using natural collagen cross-linking agents rich in proanthocyanidin (PA) would improve mechanical properties and stability over time of the dentin collagen and, thus, confer a more resistant and lasting substrate for adhesive restorations. Methods: PA-based extracts, from grape seed (GSE), cocoa seed (CSE), cranberry (CRE), cinnamon (CNE) and acai berry (ACE) were applied over the demineralized dentin. The apparent elastic modulus (E) of the treated dentin collagen was analyzed over a 12 month period. Specimens were immersed in the respective solution and E values were obtained by a micro-flexural test at baseline, 10, 30, 60, 120 and 240 min. Samples were stored in artificial saliva and re-tested after 3, 6 and 12 months. Data was analyzed using ANOVA and Tukey test. Results: GSE and CSE extracts showed a time-dependent effect and were able to improve [240 min (MPa): GSE = 108.96 +/- 56.08: CSE = 59.21 +/- 24.87] and stabilize the E of the organic matrix [12 months (MPa): GSE = 40.91 +/- 19.69; CSE = 42.11 +/- 13.46]. CRE and CNE extracts were able to maintain the E of collagen matrices constant over 12 months [CRE = 11.17 +/- 7.22; CNE = 9.96 +/- 6.11; MPa]. ACE (2.64 +/- 1.22 MPa) and control groups immersed in neat distilled water (1.37 +/- 0.69 MPa) and ethanol-water (0.95 +/- 0.33 MPa) showed no effect over dentin organic matrix and enable their degradation and reduction of mechanical properties. Significance: Some PA-based extracts were capable of improving and stabilizing collagen matrices through exogenous cross-links induction. (C) 2011 Elsevier Ltd. All rights reserved.

Solubility study of phytochemical cross-linking agents on dentin stiffness

Objectives: The effects of interactions between cross-linking proanthocyanidins (PA) in polar solvents and type-I collagen of demineralized dentin were investigated. Methods: Three PA-rich extracts, two from grape seed (GSEP and GSES) and one from cocoa (COE), were dissolved (water, ethanol:water and acetone:water) and analyzed for their ability to increase the modulus of elasticity of demineralized dentin. Sound dentin beams (0.5 mm x 1.7 mm x 7 mm) were fully demineralized and divided into 12 groups according to the type of cross-linking agent and solvents used. Specimens were immersed in the respective solutions and tested at baseline, 10, 30, 60, 120 and 240 min. Results: The elastic modulus (EM) of dentin was significantly increased by the PA treatment regardless of time (p < 0.05 for all times). The extracts showed different solubility in different solvents. GSEP showed the highest increase in EM when diluted in distilled water and acetone at all exposure times. Both GSEs showed superior results when diluted in distilled water and after 4 h of treatment, while COE produced strongest enhancement when dissolved in ethanol:water. Conclusions: The results indicates that herbal extraction process and other pharmacognostic parameters have an important influence on extract solubility as well as constitution and, consequently, on the PA-dentin matrix interaction. (C) 2010 Elsevier Ltd. All rights reserved.

Cross-Sectional Microhardness of Human Enamel Subjected to Erosive, Cariogenic or Combined Erosive/Cariogenic Challenges

This in situ study evaluated the interaction between caries and erosion processes. In the first phase, enamel specimens were subjected to erosion without dental plaque (EO) or to erosion with plaque (EP); in the second phase, they were subjected to erosion plus cariogenic challenge (EC) or cariogenic challenge (CO), both with plaque accumulation. Cross-sectional hardness data (10-330 mu m depth) were tested using ANOVA (alpha = 0.05). EO and EP showed surface softening to 10 mu m depth. CO and EC produced subsurface lesions, of similar depth (up to 220 mu m), with CO showing higher integrated loss of hardness than EC, indicating that cariogenic and erosive challenges did not have an additive effect. Copyright (C) 2010 S. Karger AG, Basel

Comparison of Cross-Sectional Hardness and Transverse Microradiography of Artificial Carious Enamel Lesions Induced by Different Demineralising Solutions and Gels

The aims of this study were: (1) to correlate surface (SH) and cross-sectional hardness (CSH) with microradiographic parameters of artificial enamel lesions; (2) to compare lesions prepared by different protocols. Fifty bovine enamel specimens were allocated by stratified randomisation according to their initial SH values to five groups and lesions produced by different methods: MC gel (methylcellulose gel/lactic acid, pH 4.6, 14 days); PA gel (polyacrylic acid/lactic acid/hydroxyapatite, pH 4.8, 16 h); MHDP (undersaturated lactate buffer/methyl diphosphonate, pH 5.0, 6 days); buffer (undersaturated acetate buffer/fluoride, pH 5.0, 16 h), and pH cycling (7 days). SH of the lesions (SH(1)) was measured. The specimens were longitudinally sectioned and transverse microradiography (TMR) and CSH measured at 10- to 220-mu m depth from the surface. Overall, there was a medium correlation but non-linear and variable relationship between mineral content and root CSH. root SH(1) was weakly to moderately correlated with surface layer properties, weakly correlated with lesion depth but uncorrelated with integrated mineral loss. MHDP lesions showed the highest subsurface mineral loss, followed by pH cycling, buffer, PA gel and MC gel lesions. The conclusions were: (1) CSH, as an alternative to TMR, does not estimate mineral content very accurately, but gives information about mechanical properties of lesions; (2) SH should not be used to analyse lesions; (3) artificial caries lesions produced by the protocols differ, especially considering the method of analysis. Copyright (C) 2009 S. Karger AG, Basel

Cross-Sectional Hardness of Enamel from Human Teeth at Different Posteruptive Ages

This study evaluated, by cross-sectional hardness (CSH), enamel maturation at different depths in sound human teeth at different posteruptive ages (12 per age group): unerupted (molars; control), 2-3 years (premolars), 4-10 years (premolars) and >10 years (molars). Cross-sectional Knoop hardness was measured at 10, 30, 50, 70, 90, 110, 220 and 330 mu m depth from the surface. The data were subjected to two-way ANOVA followed by Bonferroni`s pairwise tests (alpha = 0.05). The CSH significantly increased with posteruptive age, indicating that the time of exposure in the oral cavity might result in deep enamel maturation. Copyright (C) 2009 S. Karger AG, Basel

Testing the retention of attachments for implant overdentures - validation of an original force measurement system

P>The aim of this study was to validate an original portable device to measure attachment retention of implant overdentures both in the lab and in clinical settings. The device was built with a digital force measurement gauge (Imada) secured to a vertical wheel stand associated with a customized support to hold and position the denture in adjustable angulations. Sixteen matrix and patrix cylindrical stud attachments (Locator (R)) were randomly assigned as in vitro test specimens. Attachment abutments were secured in an implant analogue hung to the digital force gauge or to the load cell of a traction machine used as the gold standard (Instron Universal Testing Machine). Matrices were secured in a denture duplicate attached to the customized support, permitting reproducibility of their position on both pulling devices. Attachment retention in the axial direction was evaluated by measuring maximum dislodging force or peak load during five consecutive linear dislodgments of each attachment on both devices. After a wear simulation, retention was measured again at several time periods. The peak load measurements with the customized Imada device were similar to those obtained with the gold standard Instron machine. These findings suggest that the proposed portable device can provide accurate information on the retentive properties of attachment systems for removable dental prostheses.