Identification and characterization of two novel genomic RNA segments RNA5 and RNA6 in rose rosette virus infecting roses.

Rose rosette virus (RRV), a negative-strand RNA virus belonging to the genus Emaravirus, has recently been characterized to be the causal agent of rose rosette disease. Roses showing typical symptoms of RRV collected from a rose nursery in Florida were subjected to reverse transcription-PCR (RT-PCR) assay using primers corresponding to the conserved inverted 13 nucleotide long stretches found at the termini of the RRV genomic RNA segments. RT-PCR analysis yielded two novel genomic RNA segments, RNA5 and RNA6, in addition to the previously identified four RNA segments. The RNA5 is 1650 bp long and encodes for a polypeptide of 465 amino acids (54.3 K), while RNA6 is 1400 bp long and encodes for a polypeptide of 233 amino acids (27.05 K). RACE analysis showed that, both the RNA segments posses at their 5' and 3' termini, stretches of conserved inverted complementary13 nucleotides long sequence with two nucleotide mismatches as previously identified in other genomic RNA segments. Northern blot analysis as well as RT-PCR using specific primers showed the presence of the novel genomic RNA segments in infected plants, but absent in the non-infected plants. The GenBank Acc. Nos. for the sequences reported in this paper are KT007556 and KT007557.

First Report of Rose rosette virus Associated with Rose Rosette Disease Infecting Knockout Roses in Florida.

Roses are one of the most popular flowering shrubs in the United States, with a total wholesale value of US$194 million. Among the major states, Florida is the fourth largest producer of roses with a total value exceeding US$20 million (4). In Florida, the roses have become especially popular in recent years with the introduction of Knock Out and other shrub roses. Virus-like symptoms including witches'-broom, excessive thorns, abnormal red discoloration of shoots and foliages, distorted leaves, and deformed buds and flowers were initially observed on Knock Out roses in a commercial nursery in Quincy, FL, in November 2013. Fifteen plants out of ~250,000 plants showed these characteristic symptoms. Total RNA extracts (RNeasy Plant Mini Kit, Qiagen, Valencia, CA) from eight symptomatic and two non-symptomatic rose samples were subjected to reverse-transcription (RT) assays using SuperScript III Reverse transcriptase (Invitrogen, Life Technologies, NY) and random hexamer primers. The cDNA synthesized was then subjected to PCR assay using Platinum Taq DNA polymerase (Invitrogen, Life Technologies) and using Rose rosette virus (RRV) specific primers RRV-F and RRV-R (1), targeting the core region of the RNA1 genome of the virus. The RT-PCR assays using the specific primers produced amplicons of 375 bp, only in the symptomatic leaf samples. The obtained amplicons were PCR purified and sequenced directly (GenBank Accession Nos. KF990370 to KF990377). BLAST analysis of these sequences revealed a higher identity of 99% with the RRV (HQ871942) in the NCBI database. Pairwise comparison of the eight RRV sequences exhibited 99 to 100% identity among themselves. These results revealed the association of RRV with the symptomatic rose plants. Eight symptomatic and two non-symptomatic rose plant samples were tested for RRV using blot hybridization assay, utilizing a digoxigenin-labeled DNA probe of 511 bp, targeting the RNA1 genome of the RRV. All eight symptomatic rose plants showed a positive reaction to the RRV-specific probes, confirming the presence of RRV in the samples, while the non-symptomatic and the buffer control did not produce any reactions. Even though the virus is reported to spread by an eriophyid mite Phyllocoptes fructiphilus, thorough examination of the infected samples showed absence of the vector. The samples were also tested using RT-PCR for the presence of Rose cryptic virus (RCV) and Blackberry chlorotic ringspot virus (BCRV) using specific primers (2,3). The samples tested negative for the RCV and BCRV. This is the first report of occurrence of RRV on rose in Florida. Considering the economic importance of the rose plants and the highly destructive nature of RRV, this report underscores the need for immediate effective quarantine and management of the virus for protecting the economically important rose industry in Florida. References: (1) A. G. Laney et al. J. Gen. Virol. 92:1727, 2011. (2) S. Sabanadzovic and N. Abou Ghanem-Sabanadzovic. J. Plant Pathol. 90:287, 2008. (3) I. E. Tzanetakis et al. Plant Pathol. 55:568, 2006. (4) USDA. 2007 Census of Agriculture 3:25, Washington, DC, 2010.

First Report of Xanthomonas axonopodis Causing Bacterial Leaf Spot on Crape Myrtle.

Crape myrtle (Lagerstroemia sp.) is a popular ornamental tree in the United States and the industry produced 2,781,089 trees in 2010 with a value exceeding US $42.8 million (1,4). A new disorder of crape myrtle has been observed since 2011 in numerous nurseries in Florida, which was characterized by dark brown, angular to irregularly shaped, oily-looking spots surrounded by yellow halos. The disease primarily affects lower leaves that eventually turn yellow and can lead to rapid defoliation of susceptible cultivars. Plants examined in field surveys at the University of Florida, North Florida Research and Education Center, Quincy, FL in 2012 and 2013 also had similar symptoms on cvs. Arapaho, Carolina Beauty, Tuscarora, Whit IV Red Rocket, Whit VIII Rhapsody in Pink, and White Chocolate. The disease severity ranged from 20 to 70% and all the plants were infected. A yellow-pigmented, gram-negative, oxidase negative bacterium was consistently isolated from symptomatic leaves (two leaves from each of five plants). Pathogenicity tests were performed using five isolated bacterial strains on potted crape myrtle cv. Arapaho. Three plants were inoculated with a 108 CFU/ml suspension of bacterial strains in sterile deionized water, and covered with transparent plastic bag for 48 h. Two control plants were inoculated with sterile distilled water. The inoculated plants were then incubated in a greenhouse at 30 to 34°C for 14 days. Symptoms of dark brown, angular to irregularly shaped lesions were observed only on the inoculated plants after 7 days. The bacterium was re-isolated from the inoculated symptomatic plants as described above, thus fulfilling Koch's postulates. Fatty acid methyl ester profiling of the five isolated bacteria using GC-MIDI (Microbial IDentification Inc, Newark, DE) revealed the identity of the bacterium as Xanthomonas axonopodis with an identity index of ~0.80, but matched multiple pathovars. Total genomic DNA was extracted from the pure bacterial culture using UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA). The genomic DNA was subjected to PCR assay using universal primers 27f/1492R (3) targeting the complete 16S rRNA gene and primers 16F945/23R458 (2), which target the partial 16S-23S internal transcribed spacer region. PCR amplification using primer pairs 27f/1492R and 16F945/23R458 resulted in amplicons of 1,450 and 1,500 bp, respectively. The amplicons were gel purified and sequenced directly at Florida State University. BLAST analysis of the sequences (Accession Nos. KF926678, KF926679, KF926680, KF926681, and KF926682) revealed the identity of the bacterium as X. axonopodis, ranging from 98 to 99%, with several strains in the NCBI database. Phylogenetic analysis using the neighbor-joining method showed that our strains were distantly clustered with X. axonopodis pv. dieffenbachiae when compared to other available strains in the database. To our knowledge, this is the first worldwide report of a bacterial leaf spot on crape myrtle caused by X. axonopodis. This information should aid in the development of breeding lines with resistance to bacterial leaf spot and effective disease management practices. References: (1) C. S. Furtado. Garden Bull. 24:185, 1969. (2) C. Guasp. Int. J. Syst. Evol. Microbiol. 50:1629, 2000. (3) D. J. Lane. Page 115 in: Nucleic Acid Techniques in Bacterial Systematics, 1991. (4) USDA. 2007 Census of Agriculture, Washington, DC. 3:25, 2010.

Incidence of inferior vena cava thrombosis detected by transthoracic echocardiography in the immediate postoperative period after adult cardiac and general surgery.

Venous thromboembolism is an important complication after general and cardiac surgery. Using transthoracic echocardiography, this study assessed the incidence of inferior vena cava (IVC) thrombosis among a total of 395 and 289 cardiac surgical and major surgical patients in the immediate postoperative period after cardiac and major surgery, respectively. All transthoracic echocardiography was performed by a specialist intensivist within 24 hours after surgery with special emphasis on using the subcostal view in the supine position to visualise the IVC. Of the 395 cardiac surgical patients studied, the IVC was successfully visualised using the subcostal view in 315 patients (79.8%) and eight of these patients (2.5%) had a partially obstructive thrombosis in the IVC. In 250 out of 289 (85%) general surgical patients, the IVC was also clearly visualised, but only one patient (0.4%) had an IVC thrombosis (2.5 vs 0.4%, P <0.05). In summary, visualisation of the IVC was feasible in most patients in the immediate postoperative period after both adult cardiac and major surgery. IVC thrombosis appeared to be more common after adult cardiac surgery than general surgery. A large prospective cohort study is needed to define the risk factors for IVC thrombus and whether early thromboprophylaxis can reduce the incidence of IVC thrombus after adult cardiac surgery.

Perinatal high reliability.

Perinatal high reliability is achievable with principles of high reliability organizations. Key organizational, leadership, and clinical characteristics that are essential for developing and sustaining a highly reliable perinatal unit are presented. Interdisciplinary collaboration and commitment to safe care that are founded on standardization are the hallmarks of perinatal high reliability.

Michigan Health & Hospital Association Keystone Obstetrics: a statewide collaborative for perinatal patient safety in Michigan.

BACKGROUND: Preventable harm to mothers and infants during labor and birth is a significant patient safety and professional liability issue. A Michigan Health & Hospital Association Keystone Center for Patient Safety & Quality Obstetric Collaborative Project involved perinatal teams from 15 Michigan hospitals during an 11-month period in 2009. The purpose of the project was to promote safe care practices during labor and birth using the Comprehensive Unit-based Safety Program (CUSP). Consistent with the CUSP model, this project's components included assessing and promoting a culture of safety; interdisciplinary team building; case review; learning from defects through multiple methods of education; team and individual coaching and peer encouragement; administrative support for the establishment of a fundamental safety infrastructure; and ongoing evaluation of care processes and outcomes. METHODS: Study measures included 32 components of a perinatal patient infrastructure, 6 care processes during labor and birth, and 4 neonatal outcomes. RESULTS: Significant improvements were found in the safety culture (Safety Attitudes Questionnaire), the perinatal patient safety infrastructure components, and all care processes. CONCLUSIONS: Although the project was successful, getting buy-in from all members of the clinical team in each hospital for all of the measures was challenging at times. There was initial resistance to some of the measures and their various expected aspects of care. For example, some of the clinicians were initially reluctant to adopt the recommended standardized oxytocin protocol. Peer encouragement and unit-based feedback on progress in minimizing early elective births proved useful in many hospitals. A CUSP in obstetrics can be beneficial in improving the care of mothers and infants during labor and birth.

A comprehensive perinatal patient safety program to reduce preventable adverse outcomes and costs of liability claims.

BACKGROUND: To achieve the goal of safe care for mothers and infants during labor and birth, Catholic Healthcare Partners (CHP; Cincinnati) conducted on-site risk assessments at the 16 hospitals with perinatal units in 2004-2005, with follow-up visits in 2006 through 2008. ON-SITE RISK ASSESSMENTS: In addition to assessing overall organizational risk, the assessments provided each hospital a gap analysis demonstrating up-to-date and outdated practices and strategies and resources necessary to make all practices consistent with current evidence and national guidelines and standards. CRITICAL ASPECTS OF CLINICAL CARE: Review of claims and near-miss data indicate that fetal assessment, labor induction, and second-stage labor care comprise the majority of risk of perinatal harm. Therefore, these clinical areas were the focus of strategies to promote safety. To promote consistency in knowledge and practice, in 2004 a variety of strategies were recommended, including interdisciplinary fetal monitoring education and routine medical record reviews to monitor ongoing adherence to appropriate practice and documentation. OUTCOMES: Success in implementing essential structural and process components of the perinatal patient safety program have resulted in improvement from 2003 to 2008 in specific outcomes for the 16 perinatal units surveyed, including reduction of perinatal harm, number of claims, and costs of claims. FUTURE DIRECTIONS: The program continues to evolve with modifications as needed as more evidence becomes available to guide best perinatal practices and new guidelines/standards are published. A patient safety program guided and supported by a health care system can result in safer clinical environments in individual hospitals and in decreased risk of preventable perinatal harm and liability costs.

Oxytocin as a high-alert medication: implications for perinatal patient safety.

Patient injury from drug therapy is the single most common type of adverse event that occurs in the in-patient setting. When medication errors result in patient injury, there are significant costs to the patient, healthcare providers, and institution. Some medications that have a heightened risk of causing significant patient harm when they are used in error are called "high-alert medications."In 2007, the Institute for Safe Medication Practices added intravenous (IV) oxytocin to their list of high-alert medications. This is significant for perinatal care providers because oxytocin is a drug that they use quite freguently. Errors that involve IV oxytocin administration for labor induction or augmentation are most commonly dose related and often involve lack of timely recognition and appropriate treatment of excessive uterine activity (tachysystole). Other types of oxytocin errors involve mistaken administration of IV fluids with oxytocin for IV fluid resuscitation during nonreassuring (abnormal or indeterminate) fetal heart rate patterns and/or maternal hypotension and inappropriate elective administration of oxytocin to women who are less than 39 completed weeks' gestation. Oxytocin medication errors and subsequent patient harm are generally preventable. The perinatal team can develop strategies to minimize risk of maternal-fetal injuries related to oxytocin administration consistent with safe care practices used with other high-alert medications.

Oxytocin: new perspectives on an old drug.

Oxytocin is the drug most commonly associated with preventable adverse perinatal outcomes and was recently added by the Institute for Safe Medication Practices to a small list of medications "bearing a heightened risk of harm," which may "require special safeguards to reduce the risk of error." Current recommendations for the administration of this drug are vague with respect to indications, timing, dosage, and monitoring of maternal and fetal effects. A review of available clinical and pharmacologic data suggests that specific, evidence-based guidelines for the intrapartum administration of oxytocin may be derived from available data. If implemented, such practices may reduce the likelihood of patient harm. These suggested guidelines focus on limited elective administration of oxytocin, consideration of strategies that have been shown to decrease the need for indicated oxytocin use, reliance on low-dose oxytocin regimens, adherence to specific semiquantitative definitions of adequate and inadequate labor, and an acceptance that once adequate uterine activity has been achieved, more time rather than more oxytocin is generally preferable. The use of conservative, specific protocols for monitoring the effects of oxytocin on mother and fetus is likely not only to improve outcomes but also reduce conflict between members of the obstetric team. Implementation of these guidelines would seem appropriate in a culture increasingly focused on patient safety.