Turning the "Page" Again: Decreasing Inappropriate After-Hours Pages.

OBJECTIVE: To decrease the number of inappropriate pages after-hours by 50%. METHODS: Over 9 weeks, after-hours pages to the on-call resident at University of Minnesota Department of Urology were documented. Pages were deemed "appropriate" or "inappropriate" by 3 independent reviewers. The source of each page was reviewed and after several plan-do-study-act (PDSA) cycles, 3 separate interventions were created, directed at floor nursing, patient phone calls, and the call triage center. After a 6-week implementation period, data were re-collected for 9 weeks. Descriptive statistics were generated, and a Mann-Whitney U test was used to compare inappropriate pages pre- and post- interventions. RESULTS: In phase I, 48 shifts were recorded. The mean number of pages per shift was 8.9 (range 1-27), with 52.7% of pages considered "inappropriate." Most pages came from the wards (48.9%) followed by the emergency department (20.4%), patient phone calls (19.4%), and the call center (2.6%). From these domains, the call center had the highest proportion of inappropriate pages (90.9%). In phase II, 39 shifts were recorded. The mean number of pages per shift decreased to 6.0 (range 1-20), and inappropriate pages per shift decreased by 68.4%, (P <.0001). CONCLUSION: By improving communication to nurses, patients, and call center personnel, inappropriate after-hours pages decreased by 68.4%-exceeding our aim. Future studies are needed to assess if the impact was sustainable over time.

'Candidatus Liberibacter brunswickensis' colonization has no effect to the early development of Solanum melongena.

This study is the first to investigate the presence and movement of the novel Liberibacter species 'Candidatus Liberibacter brunswickensis' (CLbr) in eggplant, Solanum melongena. The psyllid, Acizzia solanicola can transmit CLbr to eggplant and CLbr can be acquired by CLbr-negative A. solanicola individuals from CLbr-positive eggplants. In planta, CLbr can replicate, move and persist. Investigation into the early development of eggplants showed that CLbr titres had increased at the inoculation site at 14 days post inoculation access period (DPIAP). CLbr had become systemic in the majority of plants tested by 28 DPIAP. The highest bacterial titres were recorded at 35 DPIAP in all samples of the inoculated leaf, the roots, stems and the midrib and petiole samples of the newest leaf (the top leaf). This finding strongly suggests that CLbr movement in planta follows the source to sink relationship as previously described for 'Ca. Liberibacter asiaticus' (CLas) and 'Ca. Liberibacter solanacearum' (CLso). No symptoms consistent with Liberibacter-associated diseases were noted for plants colonised by CLbr during this study, consistent with the hypothesis that CLbr does not cause disease of eggplant during the early stages of host colonisation. In addition, no significant differences in biomass were found between eggplant colonised with CLbr, compared to those that were exposed to CLbr-negative A. solanicola, and to control plants.

In vivo editing of lung stem cells for durable gene correction in mice.

In vivo genome correction holds promise for generating durable disease cures; yet, effective stem cell editing remains challenging. In this work, we demonstrate that optimized lung-targeting lipid nanoparticles (LNPs) enable high levels of genome editing in stem cells, yielding durable responses. Intravenously administered gene-editing LNPs in activatable tdTomato mice achieved >70% lung stem cell editing, sustaining tdTomato expression in >80% of lung epithelial cells for 660 days. Addressing cystic fibrosis (CF), NG-ABE8e messenger RNA (mRNA)-sgR553X LNPs mediated >95% cystic fibrosis transmembrane conductance regulator (CFTR) DNA correction, restored CFTR function in primary patient-derived bronchial epithelial cells equivalent to Trikafta for F508del, corrected intestinal organoids and corrected R553X nonsense mutations in 50% of lung stem cells in CF mice. These findings introduce LNP-enabled tissue stem cell editing for disease-modifying genome correction.

Rhodopsin mislocalization drives ciliary dysregulation in a novel autosomal dominant retinitis pigmentosa knock-in mouse model.

Rhodopsin mislocalization encompasses various blind conditions. Rhodopsin mislocalization is the primary factor leading to rod photoreceptor dysfunction and degeneration in autosomal dominant retinitis pigmentosa (adRP) caused by class I mutations. In this study, we report a new knock-in mouse model that harbors a class I Q344X mutation in the endogenous rhodopsin gene, which causes rod photoreceptor degeneration in an autosomal dominant pattern. In RhoQ344X/+ mice, mRNA transcripts from the wild-type (Rho) and RhoQ344X mutant rhodopsin alleles are expressed at equal levels. However, the amount of RHOQ344X mutant protein is 2.7 times lower than that of wild-type rhodopsin, a finding consistent with the rapid degradation of the mutant protein. Immunofluorescence microscopy indicates that RHOQ344X is mislocalized to the inner segment and outer nuclear layers of rod photoreceptors in both RhoQ344X/+ and RhoQ344X/Q344X mice, confirming the essential role of the C-terminal VxPx motif in promoting OS delivery of rhodopsin. The mislocalization of RHOQ344X is associated with the concurrent mislocalization of wild-type rhodopsin in RhoQ344X/+ mice. To understand the global changes in proteostasis, we conducted quantitative proteomics analysis and found attenuated expression of rod-specific OS membrane proteins accompanying reduced expression of ciliopathy causative gene products, including constituents of BBSome and axonemal dynein subunit. Those studies unveil a novel negative feedback regulation involving ciliopathy-associated proteins. In this process, a defect in the trafficking signal leads to a reduced quantity of the trafficking apparatus, culminating in a widespread reduction in the transport of ciliary proteins.

Zoo professionals and volunteers in the U.S: experiences and prevalence of burnout, mental health, and animal loss.

Introduction: Burnout and mental health among animal care and health professionals (ACHPs) has received increasing attention in recent years. Despite rapid growth of research in this area, the wellbeing of individuals who work and/or volunteer in zoo settings has received minimal attention. Method: An anonymous online survey was created to evaluate zoo staff and volunteers' experiences of animal-related loss, rates of professional fulfillment and burnout, mental health, perceived organizational support, and resilience. Participants included 1695 zoo professionals (72% ACHPs, 20% other staff) and volunteers (7%) who were recruited through relevant professional listservs and online platforms, and flyers on zoo grounds. Results: ACHPs reported higher levels of anxiety, depression, and burnout and lower levels of professional fulfillment than other zoo staff and volunteers. The most common animal-related losses experienced by ACHPs in the past year were unexpected death (80%) and anticipated loss (74%), with more than half of these losses occurring within the past 3 months. ACHPs' reported bond with animals under their care was positively associated with depression and anxiety. Having a formal ritual or process following the death of an animal was positively associated with job fulfillment and perceived organizational support and negatively associated with depression and burnout-yet only 17% of participants in our sample indicated that their zoo had such a process or ritual. Discussion: Our findings suggest that many ACHPs are struggling with burnout, anxiety, depression, and low rates of professional fulfilment and perceived organizational support. We recommend that zoos develop organizational plans that foster a culture which normalizes and validates grief/loss experiences and is proactive in responding to animal loss, related trauma, and other occupational stressors. The results of this research demonstrate the need for systemic changes within the zoo industry, for the betterment and welfare of both humans and the animals under their care.

Disparities in Access to Robotic Knee Arthroplasty: A Geospatial Analysis.

BACKGROUND: The utilization of robotic knee arthroplasty (RKA) continues to increase across the United States. The aim of this geospatial analysis was to elucidate if RKA is distributed uniformly across the United States or if disparities exist in patient access. METHODS: Publicly available provider-finding functions for 5 major manufacturers of RKA systems were used to obtain the practice locations of surgeons performing RKA along with their associated RKA system manufacturer. The average travel distance for each county to the nearest RKA surgeon was calculated and Moran's index clustering analysis was used to find hotspots and coldspots of RKA access. A logistic regression model was used to identify the predictive odds ratios between robotic hotspots and coldspots with county-level sociodemographic variables. Of the 34,216 currently practicing orthopedic surgeons in 2022, 2,571 have access to robotic assistance for knee arthroplasty. RESULTS: Hotspots of increased travel time were predominantly in West South Central and West North Central census regions. Hotspots were significantly more rural and consisted of predominantly White populations, with lower median income and health insurance coverage. CONCLUSIONS: The results of the current study align with existing literature, demonstrating absolute geographic access disparities for rural and economically disadvantaged populations. Additionally, relative access disparities persist for minority populations and individuals with high comorbidity burdens residing in urban areas.

The effectiveness of community friendship groups on participant social and mental health: a meta-analysis.

Introduction: Social disconnectedness and isolation are risk factors for poor mental health. Community-based friendship group interventions have been designed to increase an individual's social capital and consequently their mental wellbeing. Structured and unstructured friendship groups reflect two distinct approaches to friendship group interventions. Methods: This meta-analysis investigated whether structured or unstructured community friendship groups are more effective for mental health and social capital outcomes. A systematic search of quantitative studies was conducted across seven databases and study quality was assessed using the Effective Public Health Practice Project (EPHPP) tool. Eight studies (2 unstructured and 6 structured friendship groups) were included in the review, published between 2005 and 2020. Results: Structured friendship groups had a small significant effect on reducing participant depression symptoms. There was not enough available data to compare the effectiveness of structured and unstructured groups for mental health outcomes. There was substantial heterogeneity between studies to calculate pooled effect sizes for any social capital outcomes. Data synthesis indicated mixed reviews for social capital outcomes, likely due to the large heterogeneity and limited studies. Discussion: This meta-analysis provides limited support for positive mental health outcomes following structured community-based friendship group interventions. There is a need for additional research as a large research gap remains, particularly for unstructured friendship groups. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=260639, CRD42021260639.

The pan-genome of Mycobacterium avium subsp. paratuberculosis (Map) confirms ancestral lineage and reveals gene rearrangements within Map Type S.

BACKGROUND: To date genomic studies on Map have concentrated on Type C strains with only a few Type S strains included for comparison. In this study the entire pan-genome of 261 Map genomes (205 Type C, 52 Type S and 4 Type B) and 7 Mycobacterium avium complex (Mac) genomes were analysed to identify genomic similarities and differences between the strains and provide more insight into the evolutionary relationship within this Mycobacterial species. RESULTS: Our analysis of the core genome of all the Map isolates identified two distinct lineages, Type S and Type C Map that is consistent with previous phylogenetic studies of Map. Pan-genome analysis revealed that Map has a larger accessory genome than Mycobacterium avium subsp. avium (Maa) and Type C Map has a larger accessory genome than Type S Map. In addition, we found large rearrangements within Type S strains of Map and little to none in Type C and Type B strains. There were 50 core genes identified that were unique to Type S Map and there were no unique core genes identified between Type B and Type C Map strains. In Type C Map we identified an additional CE10 CAZyme class which was identified as an alpha/beta hydrolase and an additional polyketide and non-ribosomal peptide synthetase cluster. Consistent with previous analysis no plasmids and only incomplete prophages were identified in the genomes of Map. There were 45 hypothetical CRISPR elements identified with no associated cas genes. CONCLUSION: This is the most comprehensive comparison of the genomic content of Map isolates to date and included the closing of eight Map genomes. The analysis revealed that there is greater variation in gene synteny within Type S strains when compared to Type C indicating that the Type C Map strain emerged after Type S. Further analysis of Type C and Type B genomes revealed that they are structurally similar with little to no genetic variation and that Type B Map may be a distinct clade within Type C Map and not a different strain type of Map. The evolutionary lineage of Maa and Map was confirmed as emerging after M. hominissuis.

High-quality full genome assembly of historic Xylella fastidiosa strains from ICMP collection using a hybrid sequencing approach.

High-quality complete genomes of five Xylella fastidiosa strains were assembled by combining Nanopore and Illumina sequencing data. Among these, International Collection of Micro-organisms from Plants (ICMP) 8731, ICMP 8742 and ICMP 8745 belong to subspecies fastidiosa while ICMP 8739 and ICMP 8740 were determined as subspecies multiplex. The strains were further classified into sequence types.

Complex ureteral stricture from chronic schistosomiasis requiring ileal ureter interposition: a case report.

Background: Schistosomiasis is most notably associated with squamous cell carcinoma of the bladder, and it is estimated that approximately 10% of people infected will develop a urologic complication. Ureteral pathology is rare and has only been described in a handful of case reports. Increasing awareness of this condition is needed given a recent increase in sub-Saharan immigrant population in the United States (US), as prompt recognition is key to providing optimal care. Case Description: A 40-year-old Kenyan immigrant presented to the emergency department with left-sided flank pain and was found to have left hydronephrosis and three mid-ureteral calcifications. He underwent ureteroscopy where the left ureter appeared blind-ending just proximal to the iliac vessels. A percutaneous nephrostomy tube was placed, and renal pelvis urine was analyzed for mycobacterium tuberculosis and acid fast bacilli which were negative. Antegrade ureteroscopy demonstrated a second, proximal ureteral stricture with a pinpoint lumen. Antegrade and retrograde pyelography revealed a 3 cm mid-ureteral stricture with no contrast passage. Given the stricture length, multifocality, and unclear pathology, we opted to perform ureterectomy with ileal interposition. Final pathology revealed schistosomiasis with calcifications. The patient received two doses of Praizquantel and his stent was removed 6 weeks postoperatively. He is doing well without complications. Conclusions: There is a wide range of urologic complications caused by schistosomiasis infection, and this case highlights an extreme case. Although many patients will present with a fixed urologic complaint, they remain at risk for additional urologic pathology in the future without antihelminthic therapy. This highlights the need for an accurate diagnosis and a high index of suspicion for at-risk populations.

Comparative genomics and genomic diversity of Pseudomonas syringae clade 2b-a in Australia.

BACKGROUND: A zucchini disease outbreak with unusual symptoms associated with Pseudomonas syringae clade 2b was identified in Bundaberg, Australia during autumn 2016. To investigate the genetic diversity of the 11 Australian isolates obtained from the outbreak, the genomes were compared to the publicly available P. syringae strains in phylogroup 2. RESULTS: Average nucleotide identity refined the P. syringae clade 2b-a into four clusters (Cluster A, B, C1 and C2), an expansion from the previously identified A, B and C. Australian isolates were in Cluster A, C1 and C2. Genomic analyses highlighted several key factors that may contribute to the virulence of these isolates. Six orthologous groups, including three virulence factors, were associated with P. syringae phylogroup 2 cucurbit-infecting strains. A region of genome plasticity analysis identified a type VI secretion system pathway in clade 2b-a strains which could also contribute to virulence. Pathogenicity assays on isolates KL004-k1, KFR003-1 and 77-4C, as representative isolates of Cluster A, C1 and C2, respectively, determined that all three isolates can infect pumpkin, squash, watermelon and zucchini var. Eva with different levels of disease severity. Subsequently, type III effectors were investigated and four type III effectors (avrRpt2, hopZ5, hopC1 and hopH1) were associated with host range. The hopZ effector family was also predicted to be associated with disease severity. CONCLUSIONS: This study refined the taxonomy of the P. syringae clade 2b-a, supported the association between effector profile and pathogenicity in cucurbits established in a previous study and provides new insight into important genomic features of these strains. This study also provided a detailed and comprehensive resource for future genomic and functional studies of these strains.

Gender Representation in Orthopaedic Surgery: A Geospatial Analysis From 2015 to 2022.

Introduction The gender disparity in orthopaedic surgery is well-established. According to our analysis, only 7.4% of practicing orthopaedic surgeons in the US are female in 2022. While there are several theories attempting to explain this gender gap, our eight years of data show that limited female representation is a self-perpetuating cycle as areas without female representation almost never improve in that regard. It appears that existing female mentorship is critical to the growth of a female orthopaedic presence in an area. In the present work, we aim to describe how gender diversity in orthopaedic surgery differs across the country, how this diversity is changing over time, and how surgeon gender diversity may be affected by the sociodemographic characteristics making up the counties where orthopaedic surgery is practiced. Methods A retrospective study was conducted using publicly available National Provider Identifier (NPI) data from 2015 to 2022. Orthopaedic surgeons and their genders were identified using the Provider Type and Gender data elements associated with an individual NPI. Rural-urban and metro characters were defined using the USDA Economic Research Reserve's rural-urban continuum codes. Python was used for database building and data cleaning. GeoDa, a statistical map-based graphing software, was used to plot and assess demographic, geographic, and socioeconomic trends. Trends in gender diversity from 2015 to 2019 were analyzed for each individual year as well as the time period as an aggregate. Cluster analysis was performed to assess complex spatial patterns of variables that could not be condensed linearly or logarithmically. Moran's I was used to measure the similarity of a Federal Information Processing System (FIPS) area code to its neighbors. Within the clustering analysis, spatial clusters were broken down into four groups of spatial outliers (High-High, High-Low, Low-High, and Low-Low) referencing a given area's relationship with its neighbors. Factorial ANOVA between each of the four cluster types was performed using the variables provided in the article to identify significant demographic variables within the cluster analysis. Results There are relative hotspots of gender diversity in the Northwest, Northeast, and Southwest with relative coldspots in the Midwest and Southern US. In counties that are considered gender diversity hotspots, the total population of orthopaedic surgeons increases by 0.94 each year while the population of female orthopaedic surgeons increases by 0.2, suggesting that in areas with high gender diversity, 4.7 male orthopaedic surgeons are joining practices for every 1.0 female. In areas with low gender diversity, the population of orthopaedic surgeons increases by 0.11 surgeons each year while the slope for an increase in female orthopaedic surgeons is 0. Conclusions  Orthopaedic surgery lags behind other male-dominated surgical specialties in gender parity. Our analysis demonstrates that certain areas of the country including the Northwest, Northeast, and Arizona have improved gender diversity compared to the rest of the country. We also see that the rate of increase of female orthopaedic surgeons in the past seven years is highest in areas with more preexisting female orthopaedic surgeons, suggesting the importance of a "trailblazer" phenomenon in recruiting female surgeons.

The "Fragile" Urethra as a Predictor of Early Artificial Urinary Sphincter Erosion.

OBJECTIVES: To identify predictors of early artificial sphincter (AUS) erosion among a cohort of men with erosion, who underwent AUS placement by either university or community-based surgeons. METHODS: The records of all patients with AUS erosions, including men who underwent AUS placement at outside facilities, were retrospectively reviewed. A Cox proportional-hazards model for time to erosion was performed with the predictors being the components of a fragile urethra (history of radiation, prior AUS, prior urethroplasty), androgen deprivation therapy (ADT), trans-corporal (TC), and 3.5 cm cuff, controlling for other risk factors. Kaplan-Meier survival curves and log-rank test compared "fragile" urethras with "not fragile" urethras. All statistical analysis was done using R version 3.5.2. RESULTS: Of the 156 men included, 36% had undergone AUS placement in the community. Median time to erosion was 16.0 months (1.0-240.0 months), and 122 (78%) met at least one fragility criteria. Radiation (HR 2.36, 95% CI 1.52-3.64) and prior urethroplasty (HR 2.12, 95% CI 1.18-3.80) were independently associated with earlier time to erosion. The Kaplan-Meier estimates demonstrate 1- and 5-year survival rates of 76.5% and 50.0%, respectively, for "non-fragile" and 44.1% and 14.8% for "fragile" urethras (P < .0001). CONCLUSION: In a diverse cohort of men with AUS erosion, men with "fragile" urethras eroded sooner. Radiation and prior urethroplasty were independent risk factors for earlier time to erosion, but prior AUS, ADT, TC and 3.5 cm cuff were not.

Adolescents and social media: The effects of frequency of use, self-presentation, social comparison, and self esteem on possible self imagery.

INTRODUCTION: American adolescents continue to show a strong social media presence. This presence entails consistent monitoring of how they present themselves, and how that presentation compares to others, which in turn impacts the versions of the self they share with others. These versions include their hoped for (e.g. high academic achiever) or feared selves (e.g. inconsistent academic achiever) which have been referred to as possible selves. Here, we explored the contributions of social media use, self-presentation, social comparison, and self-esteem on adolescents' reflection of their possible selves. We also examined age as a factor to determine whether early and late adolescents reflect on and craft their social media presence in similar fashions. Ideally, all adolescents would seek to share a balance of possible selves whereby their social media presence includes both a hoped for self and a feared self in the same aspect of their lives (e.g. academic achievement and failure). These balances have been linked to positive outcomes in offline contexts. METHODS: We explored the relative contributions of age, self-esteem, social media use, online self-presentation behaviors, and online social comparison tendencies to the ideation of balanced possible selves among 152 adolescents aged 11 to 18 years (55.9% female). We also included an exploratory analysis of gender differences within the content of possible selves reported. RESULTS: The results of a sequential regression analysis, regressing the balance of possible selves on each of the independent variables, yielded no significant differences. A chi square analysis, however, revealed differences among males and females in the content of feared selves as female participants reported more feared selves involving interpersonal relationships than males. CONCLUSION: Further exploration of possible selves in the digital context is warranted given our findings that adolescent females, in particular, focus on interpersonal relationships as they develop their self-esteem and self-concept.

Characterization of Pseudomonas syringae Isolated from Systemic Infection of Zucchini in Australia.

Zucchini plants with symptoms including twisted petioles, necrotic leaves, crown rot, and internal fruit rot were found in Bundaberg, Australia, at a commercial field for the first time during late autumn 2016, resulting in direct yield losses of 70 to 80%. Three Pseudomonas syringae strains isolated from symptomatic leaf (KL004-k1), crown (77-4C), and fruit (KFR003-1) were characterized and their pathogenicity evaluated on pumpkin, rockmelon, squash, and zucchini. Biochemical assays showed typical results for P. syringae. The three isolates differed, however, in that two produced fluorescent pigment (KFR003-1 and 77-4C) whereas the third, KL004-k1, was nonfluorescent. Multilocus sequence analysis classified the isolates to phylogroup 2b. The single-nucleotide polymorphism analysis of core genome from the Australian and closely related international isolates of P. syringae showed two separate clusters. The Australian isolates were clustered based on fluorescent phenotype. Pathogenicity tests demonstrated that all three isolates moved systemically within the inoculated plants and induced necrotic leaf symptoms in zucchini plants. Their identities were confirmed with specific PCR assays for P. syringae and phylogroup 2. Pathogenicity experiments also showed that the Eva variety of zucchini was more susceptible than the Rosa variety for all three isolates. Isolate KL004-k1 was more virulent than 77-4C on pumpkin, rockmelon, squash, and zucchini. This study expands the knowledge of P. syringae isolates that infect cucurbits and provides useful information for growers about the relative susceptibility of a range of cucurbit species.

Field-Deployable Recombinase Polymerase Amplification Assay for Specific, Sensitive and Rapid Detection of the US Select Agent and Toxigenic Bacterium, Rathayibacter toxicus.

Rathayibacter toxicus is a toxigenic bacterial pathogen of several grass species and is responsible for massive livestock deaths in Australia and South Africa. Due to concern for animal health and livestock industries, it was designated a U.S. Select Agent. A rapid, accurate, and sensitive in-field detection method was designed to assist biosecurity surveillance surveys and to support export certification of annual ryegrass hay and seed. Complete genomes from all known R. toxicus populations were explored, unique diagnostic sequences identified, and target-specific primers and a probe for recombinase polymerase amplification (RPA) and endpoint PCR were designed. The RPA reaction ran at 37 °C and a lateral flow device (LFD) was used to visualize the amplified products. To enhance reliability and accuracy, primers and probes were also designed to detect portions of host ITS regions. RPA assay specificity and sensitivity were compared to endpoint PCR using appropriate inclusivity and exclusivity panels. The RPA assay sensitivity (10 fg) was 10 times more sensitive than endpoint PCR with and without a host DNA background. In comparative tests, the RPA assay was unaffected by plant-derived amplification inhibitors, unlike the LAMP and end-point PCR assays. In-field validation of the RPA assay at multiple sites in South Australia confirmed the efficiency, specificity, and applicability of the RPA assay. The RPA assay will support disease management and evidence-based in-field biosecurity decisions.

The experience of loneliness and living with sight loss in English care homes.

Loneliness is a serious societal concern, especially the loss of social connections in later life. Those with sight loss living in care homes are thought to face particular challenges in social engagement, but research is scarce. This study aimed to examine the experience of loneliness amongst residents with sight loss; the different dimensions of loneliness; and their attitudes about the role of care homes in addressing these needs. Through in-depth interviews with 18 residents, supplemented by interviews with family members and care home managers, the research identified that residents were generally satisfied with their social situation. However, there was evidence that sight loss was not appropriately accommodated within the care home. In their efforts to enable all residents to engage in social activities, attempts to include those with sight loss could feel contrived, and residents with sight loss did not feel meaningfully involved. Residents described barriers to forming close associations, including the challenges of background noise in communal spaces and a sense that conversation in the care home lacked stimulation. Residents felt that opportunities to forge and maintain bonds with people outside the care home were limited. The research concludes that care homes should avoid inclusion-for-inclusion's sake, and future research could study the effectiveness of interventions to improve care home links to the wider community.

Multiple internal controls enhance reliability for PCR and real time PCR detection of Rathayibacter toxicus.

Rathayibacter toxicus is a toxigenic bacterial plant pathogen indigenous to Australia and South Africa. A threat to livestock industries globally, the bacterium was designated a U.S. Select Agent. Biosecurity and phytosanitary concerns arise due to the international trade of seed and hay that harbor the bacterium. Accurate diagnostic protocols to support phytosanitary decisions, delineate areas of freedom, and to support research are required to address those concerns. Whole genomes of three genetic populations of R. toxicus were sequenced (Illumina MiSeq platforms), assembled and genomic regions unique to each population identified. Highly sensitive and specific TaqMan qPCR and multiplex endpoint PCR assays were developed for the detection and identification of R. toxicus to the population level of discrimination. Specificity was confirmed with appropriate inclusivity and exclusivity panels; no cross reactivity was observed. The endpoint multiplex PCR and TaqMan qPCR assays detected 10 fg and 1 fg of genomic DNA, respectively. To enhance reliability and increase confidence in results, three types of internal controls with no or one extra primer were developed and incorporated into each assay to detect both plant and artificial internal controls. Assays were validated by blind ring tests with multiple operators in three international laboratories.

Molecular characterisation of Mycobacterium avium subsp. paratuberculosis in Australia.

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (Map) causes Johne's disease (JD), a chronic enteritis widespread in ruminants, resulting in substantial economic losses, especially to the dairy industry. Understanding the genetic diversity of Map in Australia will assist epidemiological studies for tracking disease transmission and identify subtype characteristics for use in development of improved diagnostic typing methods. Here we investigated the phylogenetic relationships of 351 Map isolates and compared different subtyping methods to assess their suitability for use in diagnostics and accuracy. RESULTS: SNP-based phylogenetic analysis of 228 Australian isolates and 123 publicly available international isolates grouped Type S and Type C strains into two distinct lineages. Type C strains were highly monomorphic with only 20 SNP differences separating them. Type S strains, when aligned separately to the Telford strain, fell into two distinct clades: The first clade contained seven international isolates while the second clade contained one international isolate from Scotland and all 59 Australian isolates. The Australian Type B strain clustered with US bison strains. IS1311 PCR and Restriction Enzyme Analysis (REA) intermittently generated incorrect results when compared to Long Sequence Polymorphism (LSP) analysis, whole genome SNP-based phylogenetic analysis, IS1311 sequence alignment and average nucleotide identity (ANI). These alternative methods generated consistent Map typing results. A published SNP based assay for genotyping Map was found to be unsuitable for differentiating between Australian and international strain types of Map. CONCLUSION: This is the first phylogenetic analysis of Australian Map isolates. The Type C lineage was highly monomorphic, and the Type S lineage clustered all Australian isolates into one clade with a single Scottish sheep strain. The Australian isolate classified as Type B by IS1311 PCR and REA is likely to be descended from bison and most closely related to US bison strains. Limitations of the current typing methods were identified in this study.