Analysis of five polymorphic DNA markers for indirect genetic diagnosis of haemophilia A in the Brazilian population

Hemophilia A is an X-linked, inherited, bleeding disorder caused by the partial or total inactivity of the coagulation factor VIII (FVIII). Due to difficulties in the direct recognition of the disease-associated mutation in the F8 gene, indirect diagnosis using polymorphic markers located inside or close to the gene is used as an alternative for determining the segregation of the mutant gene within families and thus for detecting carrier individuals and/or assisting in prenatal diagnosis. This study characterizes the allelic and haplotype frequencies, genetic diversity, population differentiation and linkage disequilibrium of five microsatellites (F8Int1, F8Int13, F8Int22, F8Int25.3 and IKBKG) in samples of healthy individuals from Sao Paulo, Rio Grande do Sul and Pernambuco and of patients from Sao Paulo with haemophilia A to determine the degree of informativeness of these microsatellites for diagnostic purposes. The interpopulational diversity parameters highlight the differences among the analyzed population samples. Regional differences in allelic frequencies must be taken into account when conducting indirect diagnosis of haemophilia A. With the exception of IKBKG, all of the microsatellites presented high heterozygosity levels. Using the markers described, diagnosis was possible in 10 of 11 families. The F8Int22, F8Int1, F8Int13, F8Int25.3 and IKBKG microsatellites were informative in seven, six, five and two of the cases, respectively, demonstrating the effectiveness of using these microsatellites in prenatal diagnosis and in carrier identification in the Brazilian population.

Effects of adding polyclonal antibody preparations on ruminal fermentation patterns and digestibility of cows fed different energy sources

Nine ruminally cannulated cows fed different energy sources were used to evaluate an avian-derived polyclonal antibody preparation (PAP-MV) against the specific ruminal bacteria Streptococcus bovis, Fusobacterium necrophorum, Clostridium aminophilum, Peptostreptococcus anaerobius, and Clostridium stick-landii and monensin (MON) on ruminal fermentation patterns and in vivo digestibility. The experimental design was three 3 x 3 Latin squares distinguished by the main energy source in the diet [dry-ground corn grain (CG), high-moisture corn silage (HMCS), or citrus pulp (CiPu)]. Inside each Latin square, animals received one of the feed additives per period [none (CON), MON, or PAP-MV]. Dry matter intake and ruminal fermentation variables such as pH, total short-chain fatty acids (tSCFA), which included acetate, propionate, and butyrate, as well as lactic acid and NH(3)-N concentration were analyzed in this trial. Total tract DM apparent digestibility and its fractions were estimated using chromic oxide as an external marker. Each experimental period lasted 21 d. Ruminal fluid sampling was carried out on the last day of the period at 0, 2, 4, 6, 8, 10, and 12 h after the morning meal. Ruminal pH was higher (P = 0.006) 4 h postfeeding in MON and PAP-MV groups when compared with CON. Acetate: propionate ratio was greater in PAP-MV compared with MON across sampling times. Polyclonal antibodies did not alter (P > 0.05) tSCFA, molar proportion of acetate and butyrate, or lactic acid and NH(3)-N concentration. Ruminal pH was higher (P = 0.01), 4 h postfeeding in CiPu diets compared with CG and HMCS. There was no interaction between feed additive and energy source (P > 0.05) for any of the digestibility coefficients analyzed. Starch digestibility was less (P = 0.008) in PAP-MV when compared with CON and MON. In relation to energy sources, NDF digestibility was greater (P = 0.007) in CG and CiPu vs. the HMCS diet. The digestibility of ADF was greater (P = 0.002) in CiPu diets followed by CG and HMCS. Feeding PAP-MV or monensin altered ruminal fermentation patterns and digestive function in cows; however, those changes were independent of the main energy source of the diet.

Surveillance of Rickettsia sp infection in capybaras (Hydrochoerus hydrochaeris) a potential model of epidemiological alert in endemic areas

Introduction. Capybaras (Hydrochoerus hydrochaeris) are considered amplifying hosts of Rickettsia sp. These rodents are usually parasitized by the tick vector, Amblyomma cajennense, the main vector of rickettsioses in humans and animals in South America. Capybaras can be used as sentinels in detection of circulation of rickettsiae. Objective. Antibodies to rickettsiae of spotted fever group were detected in capybaras in a rural area of Cordoba Province, northern Colombia. Materials and methods. Sera were analyzed from 36 capybaras in a rural area of Monteria (village of San Jeronimo) in Cordoba. For the detection of IgG antibodies, indirect immunofluorescence was performed. The antigens were derived from R. rickettsia strain Taiacu isolated in Brazil. Capybara sera were diluted 1:64 for IFA analysis. Ticks were collected from each capybara (also known as chiguiro) and identified to species. Results. The seroprevalence of spotted fever group Rickettsia was 22% (8 capybaras). Four sera had a titer of 1:64, 3 had a titer of 1:128 and one serum had a titer of 1:512. All ticks removed from the capybaras (n=933) were taxonomically identified as Amblyomma cajennense. Conclusion. Colombia has areas endemic for rickettsioses, as indicated by confirmed annual outbreaks. The current study reports the first evidence of natural rickettsial infection of the spotted fever group in capybaras from Colombia. The findings suggest that capybaras can be used as sentinels for the circulation of rickettsiae and can identify endemic areas for the transmission of rickettsial diseases.

Detection of Bartonella spp. in neotropical felids and evaluation of risk factors and hematological abnormalities associated with infection

Although antibodies to Bartonella henselae have been described in all neotropical felid species, DNA has been detected in only one species, Leopard us wiedii. The aim of this study was to determine whether DNA of Bartonella spp. could be detected in blood of other captive neotropical felids and evaluate risk factors and hematological findings associated with infection. Blood samples were collected from 57 small felids, including 1 Leopard us geoffroyi, 17 L wiedii, 22 Leopardus tigrinus, 14 Leopardus pardalis, and 3 Puma yagouaroundi; 10 blood samples from Panthera onca were retrieved from blood banks. Complete blood counts were performed on blood samples from small felids, while all samples were evaluated by PCR. DNA extraction was confirmed by amplification of the cat GAPDH gene. Bartonella spp. were assessed by amplifying a fragment of their 16S-23S rRNA intergenic spacer region; PCR products were purified and sequenced. For the small neotropical felids, risk factors [origin (wild-caught or zoo-born), gender, felid species, and flea exposure) were evaluated using exact multiple logistic regression. Hematological findings (anemia, polycythemia/hyperproteinemia, leukocytosis and leukopenia) were tested for association with infection using Fisher`s exact test. The 635 bp product amplified from 10 samples (10/67 = 14.92%) was identified as B. henselae by sequencing. Small neotropical felid males were more likely to be positive than females (95% CI = 0.00-0.451, p = 0.0028), however other analyzed variables were not considered risk factors (p > 0.05). Hematological abnormalities were not associated with infection (p > 0.05). This is the first report documenting B. henselae detection by PCR in several species of neotropical felids. (C) 2009 Elsevier B.V. All rights reserved.

Prevalence of equine Piroplasmosis and its association with tick infestation in the State of Sao Paulo, Brazil

Serum samples were collected from 582 horses from 40 stud farms in the State of Sao Paulo and tick (Acari: Ixodidae) infestations were evaluated on them. Serum samples were subjected to the complement fixation test (CFT) and a competitive inhibition ELISA (cELISA) for Babesia caballi and Theileria equi. Logistic regression analyses were performed to construct multivariate models that could explain the dependent variable (horses positive for B. caballi or T equi) as a function of the independent variables (presence or abundance of each one of the rick species found on the farms). A higher overall prevalence of B. caballi (54.1%) than of T equi (21.6%) was found by the two tests. The ticks Dermacentor nitens Neumann, 1897, Amblyomma cajennense (Fabricius, 1787) and Rbipicephalus (Boopbilus) microplus (Canestrini, 1887) were present on horses on 38 (95%), 20 (50%), and 4 (10%) farms, respectively. Infestations by D. nitens were statistically associated with B. caballi-positive horses on the farms by either the CFT or cELISA. Infestations by A. cajennense were statistically associated with T equi-positive horses on the farms by either CFT or cELISA.

Protection levels of vaccinated pigeons (Columba livia) against a highly pathogenic newcastle disease virus strain

The purposes of this study were to model a vaccination regimen for Newcastle disease virus (NDV) in pigeons, and to evaluate the susceptibility and behavior of vaccinated birds against a highly pathogenic NDV Brazilian strain. Antibody response was assessed by means of hemagglutination inhibition test (HI), and viral genome excretion by means of RT-PCR. Vaccinal strains (La Sota and Ulster) induced high antibody titers without any adverse effects, both in inoculated and in sentinel birds. A viral strain pathogenic for chickens did not produce clinical signs of the disease in experimentally infected pigeons. Only 4 out of 10 vaccinated pigeons shed NDV genome, and just for two days. Results confirmed the high infectivity of the vaccinal strains used, as all nonvaccinated pigeons showed antibody titers as high as those of vaccinated birds.

Prevention of Salmonella Typhimurium colonization and organ invasion by combination treatment in broiler chicks

The effects in broiler chicks of treatment with a competitive exclusion (CE) product, an experimental dietary probiotic, and the abiotic beta-glucan on cecal colonization, organ invasion, and serum and intestinal IgG and IgA levels to Salmonella challenge was evaluated. Four groups of 1-d-old chicks were treated by oral gavage on d 1 with an appropriate dose of a commercial CE product. Three groups received daily doses of probiotic, beta-glucan, or both, for 6 d. Three other groups were fed daily from d 1 onwards with probiotic, beta-glucan, or both. Subgroups of 30 chicks from each group were challenged on d 1, 9, 16, or 23 with 10(7) cfu/mL of Salmonella Typhimurium (1769NR) and killed 7 d later. Control groups were maintained untreated and remained unchallenged (negative control), or were challenged with Salmonella Typhimurium (1769NR; positive control), as described above. Cecum, liver, and spleen samples were examined for the presence of Salmonella, whereas serum and intestinal fluid samples were assayed for total antibody (IgG and IgA) concentrations. Data were analyzed by 1-way ANOVA, and means were compared using Duncan`s multiple range test. In comparison with other treatments, those involving CE product and beta-glucan, with or without probiotic during the first week, resulted in a superior inhibition of cecal colonization and organ invasion by Salmonella and also offered a higher level of protection (P < 0.05). During the second week, treatments containing experimental dietary probiotic and beta-glucan, with or without CE product, resulted in an inhibition of liver invasion (P < 0.05). The IgA levels were significantly higher (P < 0.05) in intestinal fluid compared with serum, whereas IgG had low levels. The results in the first and third week indicate that combination treatments involving CE product, probiotic, and beta-glucan are a more effective control of Salmonella colonization than the corresponding individual preparations.

In vitro macrophage activity: Biphasic effect of prolactin and indirect evidence of dopaminergic modulation

Objective: Prolactin (PRL), a peptide hormone produced by the pituitary gland, is involved in the interaction between the neuroendocrine and immune system. Since dopamine receptor antagonists increase serum levels of PRL, both PRL and dopamine receptors might be involved in the modulation of macrophage activity, providing means of communication between the nervous and immune systems. This study evaluated the effects of PRL and the dopamine antagonist domperidone (DOMP) on macrophage activity of female rats. Methods: Oxidative burst and phagocytosis of peritoneal macrophages were evaluated by flow cytometry. Samples of peritoneal liquid from female rats were first incubated with PRL (10 and 100 nM) for different periods. The same procedure was repeated to evaluate the effects of DOMP (10 and 100 nM). Results: In vitro incubation of macrophages with 10 nM DOMP decreased oxidative burst, after 30 min, whereas the PMA-induced burst was decreased by DOMP 10 nM after 2 and 4 h. Treatment with PRL (10 and 100 nM) for 30 min decreased oxidative burst and rate of phagocytosis (10 nM). After 2 h of incubation, 10 nM PRL decreased oxidative burst and phagocytosis intensity, but increased the rate of phagocytosis. On the other hand, after 4 h, PRL 10 and 100 nM increased oxidative burst and the rate of phagocytosis, but decreased intensity of phagocytosis. Conclusions: These observations suggest that macrophage functions are regulated by an endogenous dopaminergic tone. Our data also suggest that both PRL and dopamine exert their action by acting directly on the peritoneal macrophage. Copyright (C) 2008 S. Karger AG, Basel.

Pre-treatment of cattle sperm and/or oocyte with antibody to lipocalin type prostaglandin D synthase inhibits in vitro fertilization and increases sperm-oocyte binding

The present study was conducted to determine the affect of pre-treating of oocytes and/or sperm with a rabbit polyclonal antibody against recombinant cattle lipocalin type prostaglandin D synthase (alpha L-PGDS) on in vitro sperm-oocyte binding and fertilization. In vitro matured cattle oocytes were incubated (39 degrees C, 5% CO2 in air) for I It in the following treatments either 500 mu L of fertilization medium (FM) or FM with alpha L-PGDS (1:2000). Frozen-thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for I h either FM or FM with a L-PGDS. This study utilized five different treatments: (1) no antibody (control); (2) a rabbit IgG against a non-bovine antigen, bacterial histidase (alpha-hist); (3) a L-PGDS at fertilization time (with fertilization medium); (4) alpha L-PGDS-treated oocytes; or (5) a L-PGDS-treated sperm. Pre-treated oocytes were incubated with 10 X 10(4) washed spermatozoa per 25 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zonae pellucidae counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate-orcein and observed to determine the presence of pronuclei. More sperm bound to the zonae pellucidae when oocytes and/or sperm were pre-treated with alpha. L-PGDS: (1) 26.4 +/- 3.0; (2) 25.6 +/- 3.0; (3) 59.7 +/- 3.0; (4) 56.4 +/- 3.0; and (5) 57.1 +/- 3.0. Addition of alpha L-PGDS with sperm, oocytes, or both, decreased fertilization (P < 0.05) compared with the control: (1) 89.2 +/- 2.0%; (2) 87.5 +/- 2.0%; (3) 19.4 +/- 2.0%; (4) 27.2 +/- 3.1%; and (5) 14.1 +/- 3.4%. The alpha L-PGDS reacts with both oocytes and spermatozoa, resulting in increases of in vitro sperm-oocyte binding and inhibition of fertilization. These observations suggest that L-PGDS may have a role in cattle fertilization. (c) 2007 Elsevier B.V. All rights reserved.

Prevalence and risk factors for anti-Toxoplasma gondii antibodies in goats of the Serido Oriental microregion, Rio Grande do Norte state, Northeast region of Brazil

The prevalence and risk factors for anti-Toxoplasma gondii antibodies were investigated in goats of the Serido Oriental microregion, Rio Grande do Norte state, Northeast region of Brazil. Three hundred and sixty-six blood samples from goats collected by jugular venopuncture were used. For the serologic diagnosis of Toxoplasma gondii infection, the indirect fluorescent-anti body test (IFAT) with cut-off value 1:64 was carried out. The prevalence of anti-T. gondii antibodies was 30.6% [95% CI = 25.9-35.6%] with titers ranging from 1:64 to 1: 16,384. The multivariate logistic regression analysis showed that the risk factors associated to anti-T. gondii antibodies were presence of cats in the herd, extensive/semi-intensive management systems and lack of mineral supplementation. (c) 2008 Elsevier B.V. All rights reserved.

Direct comparison of the bond strength results of the different test methods: A critical literature review

Objective. The goal of this paper is to undertake a literature search collecting all dentin bond strength data obtained for six adhesives with four tests ( shear, microshear, tensile and microtensile) and to critically analyze the results with respect to average bond strength, coefficient of variation, mode of failure and product ranking. Method. A PubMed search was carried out for the years between 1998 and 2009 identifying publications on bond strength measurements of resin composite to dentin using four tests: shear, tensile, microshear and microtensile. The six adhesive resins were selected covering three step systems ( OptiBond FL, Scotch Bond Multi-Purpose Plus), two-step (Prime & Bond NT, Single Bond, Clear. l SE Bond) and one step (Adper Prompt L Pop). Results. Pooling results from 147 references showed an ongoing high scatter in the bond strength data regardless which adhesive and which bond test was used. Coefficients of variation remained high (20-50%) even with the microbond test. The reported modes of failure for all tests still included high number of cohesive failures. The ranking seemed to be dependant on the test used. Significance. The scatter in dentin bond strength data remains regardless which test is used confirming Finite Element Analysis predicting non-uniform stress distributions due to a number of geometrical, loading, material properties and specimens preparation variables. This reopens the question whether, an interfacial fracture mechanics approach to analyze the dentin - adhesive bond is not more appropriate for obtaining better agreement among dentin bond related papers. (C) 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Effect of Test Environment and Microstructure on the Flexural Strength of Dental Porcelains

Purpose: The objective of this study was to verify the influence of test environment on the flexural strength of dental porcelains with distinct microstructures. Material and Methods: Disk-shaped specimens from three dental porcelains with distinct leucite content (VM: zero; CE: 12; NS: 22 vol%) were manufactured and tested for biaxial flexural strength in air and immersed in artificial saliva. The results were analyzed by means of two-way ANOVA and Tukey`s test (alpha = 0.05). Results: The flexural strength (MPa) obtained for ambient air and artificial saliva environments, respectively, were: 110.0 +/- 16.0 and 81.5 +/- 10.8 for VM; 51.9 +/- 4.0 and 42.0 +/- 4.7 for CE; 72.0 +/- 11.5 and 63.6 +/- 5.8 for NS. A numerical decrease in the mean flexural strength was observed for all groups when specimens were tested under artificial saliva; however, the difference was only statistically significant for VM. Conclusions: The results indicate that the effect of water immersion on the flexural strength of dental porcelains varies according to their leucite content, as only the material without leucite in its microstructure (VM) showed significant strength degradation when tested under water.

The role of surface sealants in the roughness of composites after a simulated toothbrushing test

Objectives: To evaluate the influence of two surface sealants (BisCover/Single Bond) and three application techniques (unsealed/conventional/co-polymerization) on the roughness of two composites (Filtek Z250/Z350) after the toothbrushing test. Methods: Seventy-two rectangular specimens (5 mm x 10 mm x 3 mm) were fabricated and assigned into 12 groups (n = 6). Each sample was subjected to three random roughness readings at baseline, after 100,000 (intermediate), and 200,000 (final) toothbrushing strokes. Roughness (R) at each stage was obtained by the arithmetic mean of the reading of each specimen. Sealant removal was qualitatively examined (optical microscope) and classified into scores (0-3). Data were analyzed by Student`s paired t-test, two-way ANOVA/Tukey`s test, and by Wilcoxon, Kruskal-Wallis and Miller`s test (alpha = 0.05). Results: Z250 groups at baseline did not differ statistically from each other. Unsealed Z350 at baseline had lower R values. All the unsealed groups presented gradual decrease in R from baseline to final brushing. From baseline to the inter-mediate stage, Z250 co-polymerized groups presented a significant reduction in R (score 3). Conventionally sealed groups had no significant changes in R (scores 2-0.8). From baseline to the intermediate stage, the conventionally sealed Z350 Single Bond group had an increase in R (score 1.5). In the final stage, all the conventionally sealed groups presented a reduction in R (scores 0.7-0). Co-polymerized Single Bond groups had a significant reduction in R (scores 2.5-2.7), and co-polymerized BisCover groups an increase in R (scores 2.8-3). Conclusions: At any brushing stage, sealed composites presented superior performance when compared with unsealed composites. (C) 2009 Elsevier Ltd. All rights reserved.

Evaluation of the temperature of different refrigerant sprays used as a pulpal test

The temperature of different refrigerant sprays (Endo-Ice, Endo-Frost, Coolermatic and Sprayon Contact and Tuner Cleaner) used as pulpal tests were evaluated in vitro. A thermocouple placed inside the pulp chamber of a maxillary central incisor was used to register the temperature changes when the refrigerant sprays were applied with a cotton swab, for 10 s. Results indicate that Endo-Ice and Endo-Frost presented the lowest temperatures among the refrigerant sprays tested. Temperatures measured inside the pulp chamber, however, were statistically similar in all groups.

Effect of different surface treatments on the repair bond strength of indirect composites

Purpose: To evaluate the tensile bond strength of indirect composites repaired with different surface treatments and direct composites. Methods: 180 specimens were prepared with Targis, belleGlass HP and Sculpture indirect composites, light-activated and post-cured according to the manufacturers` recommendations. The specimens were stored in distilled water for 24 hours at 37 degrees C. The bonding surfaces were prepared with air abrasion, hydrofluoric acid or hydrofluoric acid followed by a neutralizing solution. All the treated surfaces were subject to the application of a silane and a bonding agent before the repair procedures with Tetric Ceram and Tetric Flow for the Targis specimens, Herculite XRV and Revolution for the belleGlass HP specimens and Sculp-It and Flow-It for Sculpture specimens. The tensile bond strength tests were carried out using a universal testing machine at cross-head speed of 0.5 mm/minute. The type of fracture was observed under a light microscope at x40 magnification. Data were analyzed by a two-way ANOVA and Tukey`s post-hoc tests (P<0.05). Results: Targis showed a statistically higher repair bond strength than belleGlass HP and Sculpture, which were not significantly different from each other. Air abrasion increased the repair bond strength of belleGlass HP and Sculpture. For Targis, all the surface treatments resulted in similar repair bond strength. The different viscosity of repair composites did not affect the repair of indirect composites. Fractured surfaces showed mostly adhesive failures, mainly with hydrofluoric acid treatment.