Curupira-1 and Curupira-2, two novel Mutator-like DNA transposons from the genomes of human parasites Schistosoma mansoni and Schistosoma japonicum

Transposons of the Mutator superfamily have been widely described in plants, but only recently have metazoan organisms been shown to harbour them. In this work we describe novel Mutator superfamily transposons from the genomes of the human parasites Schistosoma mansoni and S. japonicum, which we name Curupira-1 and Curupira-2. Curupira elements do not have Terminal Inverted Repeats (TIRs) at their extremities and generate Target Site Duplications (TSDs) of 9 base pairs. Curupira-2 transposons code for a conserved transposase and SWIM zinc finger domains, while Curupira-1 elements comprise these same domains plus a WRKY zinc finger. Alignment of transcript sequences from both elements back to the genomes indicates that they are subject to splicing to produce mature transcripts. Phylogenetic analyses indicate that these transposons represent a new lineage of metazoan Mutator-like elements with characteristics that are distinct from the recently described Phantom elements. Description of these novel schistosome transposons provides new insights in the evolution of transposable elements in schistosomes.

Prevalence of Rickettsia species antibodies and Rickettsia species DNA in the blood of cats with and without fever

Rickettsia species antibodies have been detected in some cats but it is unknown whether infected cats develop clinical signs. The prevalence of Rickettsia species deoxyribonucleic acid (DNA) in blood from clinically ill cats has not been determined. The objective of this study was to determine if cats with fever (body temperature >= 102.5 degrees F [39.2 degrees C]) were more likely to have evidence of rickettsial infection than healthy, age-matched, control cats with a body temperature < 102.5 degrees F. Rickettsia species polymerase chain reaction (PCR) assays were performed to detect rickettsial DNA extracted from blood (71 paired samples), indirect immunofluorescence assays (IFA) were performed to detect serum antibodies against Rickettsia felis (90 paired samples) and Rickettsia rickettsii (91 paired samples), and the results between pairs were compared. All samples were negative for Rickettsia species DNA. More cats with fever were seropositive for R felis or R rickettsii than control cats, but results were not statistically significant. Results of this pilot study failed to show an association between Rickettsia species DNA or Rickettsia species antibodies and fever. (c) 2008 ESFM and AAFP. Published by Elsevier Ltd. All rights reserved.

Evaluation of DNA damage by the alkaline comet assay of the olfactory and respiratory epithelia of dogs from the city of Sao Paulo, Brazil

Animals kept as pets may be considered sentinels for environmental factors to which humans could be exposed. Olfactory and respiratory epithelia are directly subjected to airborne factors, which could cause DNA lesions, and the alkaline comet assay is considered a reliable tool for the assessment of DNA damage. The objective of this work is to evaluate the extent of DNA damage by the comet assay of the olfactory and respiratory epithelia of dogs from different regions of the city of sao Paulo, Brazil. Thirty-three clinically healthy dogs, aged 5 years or more, were used in the study, with 7 from the North region of Sao Paulo, 7 from the South region, 3 dogs from the East region, and 16 dogs from the West city region. Three dogs younger than 6 months were used as controls. DNA damage was analyzed by the alkaline comet assay. We observed no difference in histopathological analysis of olfactory and respiratory epithelia between dogs from different regions of Sao Paulo. Dogs older than 5 years presented significantly higher comet length in both olfactory and respiratory epithelia, when compared with controls, indicating DNA damage. When separated by regions, olfactory and respiratory epithelia presented similar DNA damage in dogs from different regions of Sao Paulo, corroborating with similar levels of particulate matter index (PM10) in all regions of the city. In this study, we report for the first time that the comet assay can be used to quantify the extent of DNA damage in dog olfactory and respiratory epithelia, and that comet length (DNA damage) increases with age, probably due to environmental factors. Air pollution, as measured by PM 10, can be responsible for this DNA damage. (C) 2009 Elsevier GmbH. All rights reserved.

Differentiation of field isolates and vaccine strains of infectious laryngotracheitis virus by DNA sequencing

Two different regions of the infected cell protein 4 (ICP4) gene of infectious laryngotracheitis virus (ILTV) were amplified and sequenced for characterization of field isolates and tissue culture-origin (TCO) and chicken embryo-origin (CEO) vaccine strains. Phylogenetic analysis of the two regions showed differences in nucleotide and amino acid sequences between field isolates and attenuated vaccines. The PCR-RFLP results were identical to those obtained by DNA sequencing and validated their use to differentiate ILTV strains. The approach using the sequencing of the two fragments of the ICP4 gene showed to be an efficient and practical procedure to differentiate between field isolates and vaccine strains of ILTV. (C) 2009 Elsevier Ltd. All rights reserved.

Exogenous DNA uptake by bovine spermatozoa does not induce DNA fragmentation

Sperm-mediated gene transfer (SMGT) is a fast and low-cost method used to produce transgenic animals. The objective of this study was to evaluate the effects of the concentration of exogenous DNA and the duration of incubation on DNA uptake by bovine spermatozoa and subsequently the integrity of sperm DNA and sperm apoptosis. Spermatozoa (5 X 10(6) cells/mL) were incubated with 100, 300, or 500 ng of exogenous DNA (pEYFP-Nuc plasmid) for 60 or 120 min at 39 degrees C. The amount of exogenous DNA associated with spermatozoa was quantified by real-time PCR, and the percentages of DNA fragmentation in spermatozoa were evaluated using SCSA and a TUNEL assay, coupled with flow cytometry. Uptake of exogenous DNA increased significantly as incubation increased from 60 to 120 min (0.0091 and 0.028 ng, respectively), but only when the highest exogenous DNA concentration (500 ng) was used (P < 0.05). Based on SCSA and TUNEL assays, there was no effect of exogenous DNA uptake or incubation period on sperm DNA integrity. In conclusion, exogenous DNA uptake by bovine spermatozoa was increased with the highest exogenous DNA concentration and longest incubation period, but fragmentation of endogenous DNA was apparently not induced. (C) 2010 Elsevier Inc. All rights reserved.

Effects of sperm concentration and straw volume on motion characteristics and plasma, acrosomal, and mitochondrial membranes of equine cryopreserved spermatozoa

The advantages of using cryopreserved semen in equine reproduction are well known. During cryopreservationl spermatozoa undergo many changes that lead to a decrease in fertility. There is no agreement on the ideal sperm dose and concentration to maximize fertility rates. Thus, the objectives of this experiment were to evaluate sperm motion by computer-assisted analysis (CASA), sperm membrane integrity and function with fluorescence probes of cryopreserved sperm at three concentrations: 100 (C100), 200 (C200) and 400 x 10(6) sperm/mL (C400), and two straw volumes (0.50 and 0.25 mL). There was no interaction between sperm concentration and storage volume (P > .05). Sperm motion characteristics were influenced by concentration (C100 > C200 > C400; P < .05). Curvilinear velocity (VCL) in 0.25-mL straws had higher average values (P < .05). Membrane integrity and function were not changed by straw volume (P > .05). However, sperm concentration changed the percentage of cells with intact plasma membrane (C100 > C200 > C400; P < .05) and the percentage of cells with high mitochondrial membrane potential (C100 = C200; P > .05 and C400 < C100 and C200; P < .05). According to this experiment, the best freeing method was that involving 100 x 10(6) sperm/mL, regardless of straw volume.

Circulating cell-free DNA levels in plasma increase with severity in experimental acute pulmonary thromboembolism

Background: The diagnosis of acute pulmonary thromboembolism (APT) and its severity is challenging. No previous study has examined whether there is a linear relation between plasma DNA concentrations and the severity of APT. We examined this hypothesis in anesthetized dogs. We also examined the changes in plasma DNA concentrations in microspheres lung embolization and whether the therapy of APT with nitrite could modify APT-induced changes in plasma DNA concentrations. In vitro DNA release from blood clots was also studied. Methods: APT was induced with autologous blood clots (saline, 1, 3, or 5 ml/kg) injected into the right atrium. A group of dogs received 300 pm microspheres into the inferior vena cava to produce similar pulmonary hypertension. Another group of dogs received 6.75 mu mol/kg nitrite after APT with blood clots of 5 ml/kg. Hemodynamic evaluations were carried out for 120 min. DNA was extracted from plasma samples using QIAamp DNA Blood Mini Kit and quantified using Quant-iT (TM) PicoGreen (R) dsDNA detection kit at baseline and 120 min after APT. Results: APT produced dose-dependent increases in plasma DNA concentrations. which correlated positively with pulmonary vascular resistance (P=0.002, r=0.897) and with mean pulmonary arterial pressure (P=0.006, r=0.856). Conversely, lung embolization with microspheres produced no significant changes in plasma DNA concentrations. While nitrite attenuated APT-induced pulmonary hypertension, it produced no changes in plasma DNA concentrations. Blood clots released dose-dependent amounts of DNA in vitro. Conclusions: Cell-free DNA concentrations increase in proportion to the severity of APT, probably as a result of increasing amounts of thrombi obstructing the pulmonary vessels. (C) 2009 Elsevier B.V. All rights reserved.