Molecular characterization of a Moroccan isolate of Tomato yellow leaf curl Sardinia virus and differentiation of the Tomato yellow leaf curl virus complex by the polymerase chain reaction

The polymerase chain reaction (PCR) was used to identify an isolate of Tomato yellow leaf curl Sardinia virus (TYLCSV) from southwestern Morocco and to detect the members of the Tomato yellow leaf curl virus (TYLCV) complex. Thirty-five tomato samples with typical TYLCV symptoms were collected from infected tomato fields in the Souss-Massa region. PCR was performed with a general primer pair based on the coat protein (Cp) gene of the TYLCV complex, as well as with specific primer pairs for TYLCV and TYLCSV. Of the 35 samples tested, 29 generated a viral DNA product with the general primer pair, 29 samples gave a viral DNA product with the TYLCV-specific primers, and of these, 9 also gave a product with the TYLCSV primer pair; 6 samples did not give any PCR product with either primer pair. The full-length genome of TYLCSV was amplified with overlapping primers at the unique NcoI site in the TYLCSV genome (GenBank accession number X61153). The full-length genome of the TYLCSV isolate from Morocco is 2,777 nucleotides long (accession number AY702650) and is almost identical (97% nucleotide identity) to a TYLCSV isolate from Murcia, Spain (accession number Z25751). A PCR-based diagnostic method was developed to distinguish between TYLCV and TYLCSV in Morocco. To diagnose the TYLCV/TYLCSV complex a general primer pair was designed that anneals to a conserved region of the Cp gene. To diagnose TYLCSV exclusively, two primer pairs were designed to anneal specifically to the replication-associated protein gene (Rep) of TYLCSV from Morocco. To detect TYLCV exclusively, a primer pair previously described to amplify the intergenic region (IR) of TYLCV was used. The PCR primers were tested for their effectiveness using DNA clones of the TYLCSV from Morocco and of the TYLCV from the Dominican Republic. PCR using these primers offers a rapid means to detect the TYLCV complex and to distinguish between the two TYLCV species present in Morocco

The health system impact of false positive newborn screening results for medium-chain acyl-CoA dehydrogenase deficiency: A cohort study

Background - There is no consensus in the literature regarding the impact of false positive newborn screening results on early health care utilization patterns. We evaluated the impact of false positive newborn screening results for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) in a cohort of Ontario infants. Methods - The cohort included all children who received newborn screening in Ontario between April 1, 2006 and March 31, 2010. Newborn screening and diagnostic confirmation results were linked to province-wide health care administrative datasets covering physician visits, emergency department visits, and inpatient hospitalizations, to determine health service utilization from April 1, 2006 through March 31, 2012. Incidence rate ratios (IRRs) were used to compare those with false positive results for MCADD to those with negative newborn screening results, stratified by age at service use. Results - We identified 43 infants with a false positive newborn screening result for MCADD during the study period. These infants experienced significantly higher rates of physician visits (IRR: 1.42) and hospitalizations (IRR: 2.32) in the first year of life relative to a screen negative cohort in adjusted analyses. Differences in health services use were not observed after the first year of life. Conclusions - The higher use of some health services among false positive infants during the first year of life may be explained by a psychosocial impact of false positive results on parental perceptions of infant health, and/or by differences in underlying health status. Understanding the impact of false positive newborn screening results can help to inform newborn screening programs in designing support and education for families. This is particularly important as additional disorders are added to expanded screening panels, yielding important clinical benefits for affected children but also a higher frequency of false positive findings.This study was Funded through a Canadian Institutes of Health Research (CIHR) Emerging Team Grant (TR3-119195). Maria Karaceper received a graduate scholarship through a charitable donation to the Children’s Hospital of Eastern Ontario. This study was performed at the Institute for Clinical Evaluative Sciences (ICES), which is funded by an annual grant from the Ontario Ministry of Health and Long-Term Care (MOHLTC)

Androgen receptor signalling in Vascular Endothelial cells is dispensable for spermatogenesis and male fertility

<p>Abstract</p> <p>Background</p> <p>Androgen signalling is essential both for male development and function of the male reproductive system in adulthood. Within the adult testis, Germ cells (GC) do not express androgen receptor (AR) suggesting androgen-mediated promotion of spermatogenesis must act via AR-expressing somatic cell-types. Several recent studies have exploited the Cre/lox system of conditional gene-targeting to ablate AR function from key somatic cell-types in order to establish the cell-specific role of AR in promotion of male fertility. In this study, we have used a similar approach to specifically ablate AR-signalling from Vascular Endothelial (VE) cells, with a view to defining the significance of androgen signalling within this cell-type on spermatogenesis.</p> <p>Findings</p> <p>AR expression in VE cells of the testicular vasculature was confirmed using an antibody against AR. A Cre-inducible fluorescent reporter line was used to empirically establish the utility of a mouse line expressing Cre Recombinase driven by the Tie2-Promoter, for targeting VE cells. Immunofluorescent detection revealed expression of YFP (and therefore Cre Recombinase function) limited to VE cells and an interstitial population of cells, believed to be macrophages, that did not express AR. Mating of Tie2-Cre males to females carrying a floxed AR gene produced Vascular Endothelial Androgen Receptor Knockout (VEARKO) mice and littermate controls. Ablation of AR from all VE cells was confirmed; however, no significant differences in bodyweight or reproductive tissue weights could be detected in VEARKO animals and spermatogenesis and fertility was unaffected.</p> <p>Conclusions</p> <p>We demonstrate the successful generation and empirical validation of a cell-specific knockout of AR from VE cells, and conclude that AR expression in VE cells is not essential for spermatogenesis or male fertility.</p

Guidelines for the reliable use of high throughput sequencing technologies to detect plant pathogens and pests

High-throughput sequencing (HTS) technologies have the potential to become one of the most significant advances in molecular diagnostics. Their use by researchers to detect and characterize plant pathogens and pests has been growing steadily for more than a decade and they are now envisioned as a routine diagnostic test to be deployed by plant pest diagnostics laboratories. Nevertheless, HTS technologies and downstream bioinformatics analysis of the generated datasets represent a complex process including many steps whose reliability must be ensured. The aim of the present guidelines is to provide recommendations for researchers and diagnosticians aiming to reliably use HTS technologies to detect plant pathogens and pests. These guidelines are generic and do not depend on the sequencing technology or platform. They cover all the adoption processes of HTS technologies from test selection to test validation as well as their routine implementation. A special emphasis is given to key elements to be considered: undertaking a risk analysis, designing sample panels for validation, using proper controls, evaluating performance criteria, confirming and interpreting results. These guidelines cover any HTS test used for the detection and identification of any plant pest (viroid, virus, bacteria, phytoplasma, fungi and fungus-like protists, nematodes, arthropods, plants) from any type of matrix. Overall, their adoption by diagnosticians and researchers should greatly improve the reliability of pathogens and pest diagnostics and foster the use of HTS technologies in plant health

Huanglongbing in Texas: Report on the first detections in commercial citrus

Huanglongbing (HLB), also known as citrus greening, is a destructive citrus disease associated with 3 α-proteobacteria species of Candidatus Liberibacter. The first report of HLB in the USA was from Florida in 2005 and Ca. L. asiaticus (Las) is the only species currently confirmed in the USA. In January 2012, a Valencia sweet orange tree in a commercial orchard in San Juan, Texas, tested positive for Las by real-time and conventional PCR assays and by the sequence of its partial 16S rRNA gene. The sample tested negative for Ca. L. americanus and Ca. L. africanus. All 4 Valencia sweet orange seedlings that were graft-inoculated using budwood from the first Texas HLB-infected tree showed typical HLB symptoms 3 months post-inoculation and tested positive for the pathogen. Such HLB typical symptoms as leaf blotchy mottle, twig die-back, veinal chlorosis, lopsided and greening fruits were observed on the Las-positive tree in the orchard, which immediately triggered an intensive survey of the disease in the area. Typical HLB symptoms were found on 54 Valencia sweet orange trees in the same orchard and 18 Rio Red grapefruit trees in an adjacent orchard. All these symptomatic trees tested positive for Las by PCR and sequencing

Huanglongbing in Texas: Report on the first detections in commercial citrus

Huanglongbing (HLB), also known as citrus greening, is a destructive citrus disease associated with 3 α-proteobacteria species of Candidatus Liberibacter. The first report of HLB in the USA was from Florida in 2005 and Ca. L. asiaticus (Las) is the only species currently confirmed in the USA. In January 2012, a Valencia sweet orange tree in a commercial orchard in San Juan, Texas, tested positive for Las by real-time and conventional PCR assays and by the sequence of its partial 16S rRNA gene. The sample tested negative for Ca. L. americanus and Ca. L. africanus. All 4 Valencia sweet orange seedlings that were graft-inoculated using budwood from the first Texas HLB-infected tree showed typical HLB symptoms 3 months post-inoculation and tested positive for the pathogen. Such HLB typical symptoms as leaf blotchy mottle, twig die-back, veinal chlorosis, lopsided and greening fruits were observed on the Las-positive tree in the orchard, which immediately triggered an intensive survey of the disease in the area. Typical HLB symptoms were found on 54 Valencia sweet orange trees in the same orchard and 18 Rio Red grapefruit trees in an adjacent orchard. All these symptomatic trees tested positive for Las by PCR and sequencing