Multiclonal human origin and global expansion of an endemic bacterial pathogen of livestock.

Most new pathogens of humans and animals arise via switching events from distinct host species. However, our understanding of the evolutionary and ecological drivers of successful host adaptation, expansion, and dissemination are limited. Staphylococcus aureus is a major bacterial pathogen of humans and a leading cause of mastitis in dairy cows worldwide. Here we trace the evolutionary history of bovine S. aureus using a global dataset of 10,254 S. aureus genomes including 1,896 bovine isolates from 32 countries in 6 continents. We identified 7 major contemporary endemic clones of S. aureus causing bovine mastitis around the world and traced them back to 4 independent host-jump events from humans that occurred up to 2,500 y ago. Individual clones emerged and underwent clonal expansion from the mid-19th to late 20th century coinciding with the commercialization and industrialization of dairy farming, and older lineages have become globally distributed via established cattle trade links. Importantly, we identified lineage-dependent differences in the frequency of host transmission events between humans and cows in both directions revealing high risk clones threatening veterinary and human health. Finally, pangenome network analysis revealed that some bovine S. aureus lineages contained distinct sets of bovine-associated genes, consistent with multiple trajectories to host adaptation via gene acquisition. Taken together, we have dissected the evolutionary history of a major endemic pathogen of livestock providing a comprehensive temporal, geographic, and gene-level perspective of its remarkable success.

Identification and characterization of Staphylococcus devriesei isolates from bovine intramammary infections in KwaZulu-Natal, South Africa.

BACKGROUND: Coagulase-negative staphylococci (CoNS) are among the leading bacterial causes of bovine mastitis in many dairy-producing countries. Among the challenges associated with the specific diagnosis of CoNS infections is the biochemical heterogeneity of the species in the genus and the unavailability of accurate, cost-effective and up-to-date diagnostic tests. A previous study investigating the diversity of CoNS associated with cases of bovine mastitis in South Africa, resulted in six CoNS isolates which could not be identified despite the use of a combination of different molecular assays. The identification and characterisation of the isolates was pursued further in this study. RESULTS: The six CoNS isolates in question were identified by sequencing multiple housekeeping genes (dnaJ, hsp60, rpoB, 16S rRNA) and characterized through the use of matrix-assisted laser/desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and the Biolog GEN III Microplate™ bacterial identification system. Sequencing of housekeeping genes identified the isolates as S. devriesei. This Staphylococcus species was only described in 2010 and this is the first report documenting the isolation of S. devriesei from cases of bovine IMIs in South Africa. Analysis of mass spectra generated by the six isolates showed intra-species variation which was also observed when evaluating the metabolic profiles of the isolates using the Biolog GEN III system. Neither the MALDI-TOF MS nor the Biolog database are currently populated with data relating to S. devriesei, resulting in the isolates not being identified, in the case of MALDI-TOF MS analysis, or mis-identified as was observed with the Biolog GEN III system. CONCLUSIONS: The phenotyping data collected during this investigation provides useful information concerning Staphylococcus devriesei which could be used to populate user system databases thereby ensuring the accurate identification of isolates in future. The availability of improved diagnostics will in turn facilitate studies to elucidate the epidemiology, pathogenicity and true prevalence of this species in dairy herds.

S1PR1 Tyr143 phosphorylation downregulates endothelial cell surface S1PR1 expression and responsiveness.

Activation of sphingosine-1-phosphate receptor 1 (S1PR1) plays a key role in repairing endothelial barrier function. We addressed the role of phosphorylation of the three intracellular tyrosine residues of S1PR1 in endothelial cells in regulating the receptor responsiveness and endothelial barrier function regulated by sphingosine 1-phosphate (S1P)-mediated activation of S1PR1. We demonstrated that phosphorylation of only Y143 site was required for S1PR1 internalization in response to S1P. Maximal S1PR1 internalization was seen in 20 min but S1PR1 returned to the cell surface within 1 h accompanied by Y143-dephosphorylation. Cell surface S1PR1 loss paralleled defective endothelial barrier enhancement induced by S1P. Expression of phospho-defective (Y143F) or phospho-mimicking (Y143D) mutants, respectively, failed to internalize or showed unusually high receptor internalization, consistent with the requirement of Y143 in regulating cell surface S1PR1 expression. Phosphorylation of the five S1PR1 C-terminal serine residues did not affect the role of Y143 phosphorylation in signaling S1PR1 internalization. Thus, rapid reduction of endothelial cell surface expression of S1PR1 subsequent to Y143 phosphorylation is a crucial mechanism of modulating S1PR1 signaling, and hence the endothelial barrier repair function of S1P.

Neural Wiskott-Aldrich syndrome protein (N-WASP)-mediated p120-catenin interaction with Arp2-Actin complex stabilizes endothelial adherens junctions.

Stable adherens junctions (AJs) are required for formation of restrictive endothelial barrier. Vascular endothelial cadherin from contiguous endothelial cells forms AJs, which are stabilized intracellularly by binding of p120-catenin and cortical actin. Mechanisms inducing cortical actin formation and enabling its linkage with p120-catenin remain enigmatic. We altered the function of neural Wiskott-Aldrich syndrome protein (N-WASP), which induces actin polymerization through actin-related protein 2/3 complex (Arp2/3), to address the role of N-WASP in regulating AJ stability and thereby endothelial permeability. We show that depletion of N-WASP in endothelial cells impaired AJ adhesion and favored the organization of actin from cortical actin to stress fibers, resulting thereby in formation of leaky endothelial barrier. Exposure of the N-WASP-depleted endothelial cell monolayer to the permeability-increasing mediator, thrombin, exaggerated AJ disruption and stress fiber formation, leading to an irreversible increase in endothelial permeability. We show that N-WASP binds p120-catenin through its verprolin cofilin acid (VCA) domain, induces cortical actin formation through Arp2, and links p120-catenin with cortical actin. The interaction of N-WASP with p120-catenin, actin, and Arp2 requires phosphorylation of N-WASP at the Tyr-256 residue by focal adhesion kinase. Expression of the VCA domain of N-WASP or phosphomimicking (Y256D)-N-WASP mutant in endothelial cells stabilizes AJs and facilitates barrier recovery after thrombin stimulation. Our study demonstrates that N-WASP, by mediating p120-catenin interaction with actin-polymerizing machinery, maintains AJs and mitigates disruption of endothelial barrier function by edemagenic agents, therefore representing a novel target for preventing leaky endothelial barrier syndrome.

Conditional deletion of FAK in mice endothelium disrupts lung vascular barrier function due to destabilization of RhoA and Rac1 activities.

Loss of lung-fluid homeostasis is the hallmark of acute lung injury (ALI). Association of catenins and actin cytoskeleton with vascular endothelial (VE)-cadherin is generally considered the main mechanism for stabilizing adherens junctions (AJs), thereby preventing disruption of lung vascular barrier function. The present study identifies endothelial focal adhesion kinase (FAK), a nonreceptor tyrosine kinase that canonically regulates focal adhesion turnover, as a novel AJ-stabilizing mechanism. In wild-type mice, induction of ALI by intraperitoneal administration of lipopolysaccharide or cecal ligation and puncture markedly decreased FAK expression in lungs. Using a mouse model in which FAK was conditionally deleted only in endothelial cells (ECs), we show that loss of EC-FAK mimicked key features of ALI (diffuse lung hemorrhage, increased transvascular albumin influx, edema, and neutrophil accumulation in the lung). EC-FAK deletion disrupted AJs due to impairment of the fine balance between the activities of RhoA and Rac1 GTPases. Deletion of EC-FAK facilitated RhoA's interaction with p115-RhoA guanine exchange factor, leading to activation of RhoA. Activated RhoA antagonized Rac1 activity, destabilizing AJs. Inhibition of Rho kinase, a downstream effector of RhoA, reinstated normal endothelial barrier function in FAK-/- ECs and lung vascular integrity in EC-FAK-/- mice. Our findings demonstrate that EC-FAK plays an essential role in maintaining AJs and thereby lung vascular barrier function by establishing the normal balance between RhoA and Rac1 activities.

Impact of high-dose inotropic donor support on early myocardial necrosis and outcomes in cardiac transplantation.

BACKGROUND: Cardiac donors routinely require vasoactive agents for circulatory stability after brain death. Nevertheless, inotropes have been associated with direct cardiac toxicity. Our study evaluated whether the use of high-dose inotropic support in potential donors was associated with increased early myocardial necrosis (MN) and worse clinical outcomes after cardiac transplantation. METHODS: The UTAH Cardiac Transplant Program (UCTP) and Intermountain Donor Services databases were queried for records between 1996 and 2009. The high-dose donor inotropic support (HDIS) group was defined as patients on dopamine >10 µg/kg/min. The incidence of early MN, intensive care unit (ICU) length of stay, length of ventilator support, and mortality was evaluated. RESULTS: Two hundred and forty-four recipients undergoing transplant met study criteria. The average donor age was 27 yr. The incidence of MN in the HDIS (n=29) and non-HDIS (n=204) groups was 14.8% and 6.7%, respectively, OR 2.67. Total ischemic time, ventilator support time, ICU stay, and actuarial survival were similar between both groups. CONCLUSION: The use of high-dose inotropic support to maintain donor stability appears to have a higher trend for early post-transplant MN without an impact on clinical outcomes. With the current growing shortage of organ donors, it appears reasonable to use donors on high-dose inotropic support.

Informed consent to tissue donation: policies and practice.

Nearly 10 years ago, the tissue industry's informed consent practices with donor families in the United States were criticized. In response, the industry, along with the Inspector General of the Department of Health and Human Services, suggested elements to be included in the informed consent process. This study examines which of these elements were present in the informed consent documents of 45 (78%) of the nation's 58 Organ Procurement Organizations (OPOs). Some elements, such as involvement of for-profit companies, were present in almost all. Others, such as labeling tissue as a gift from donor families, never were. The authors conclude that the time is ripe for reexamination of the informed consent process with an eye to meaningful consent that promotes the benefits of tissue transplantation and at the same time protects the rights and interests of donor families; can be realistically implemented; and, maintains the trust of the American public.

Molecular Characterization of Staphylococcus aureus Isolated from Bovine Mastitis and Close Human Contacts in South African Dairy Herds: Genetic Diversity and Inter-Species Host Transmission.

Staphylococcus aureus is one of the most common etiological agents of contagious bovine mastitis worldwide. The purpose of this study was to genetically characterize a collection of S. aureus isolates (bovine = 146, human = 12) recovered from cases of bovine mastitis and nasal swabs of close human contacts in the dairy environment. Isolates were screened for a combination of clinically significant antimicrobial and virulence gene markers whilst the molecular epidemiology of these isolates and possible inter-species host transmission was investigated using a combination of genotyping techniques. None of the isolates under evaluation tested positive for methicillin or vancomycin resistance encoding genes. Twenty seven percent of the bovine S. aureus isolates tested positive for one or more of the pyrogenic toxin superantigen (PTSAg) genes with the sec and sell genes predominating. Comparatively, 83% of the human S. aureus isolates tested positive for one or more PTSAg genes with a greater variety of genes being detected. Genomic DNA macrorestriction followed by pulsed-field gel electrophoresis (PFGE) of the bovine isolates generated 58 electrophoretic patterns which grouped into 10 pulsotypes at an 80% similarity level. The majority of the bovine isolates, 93.2% (136/146), clustered into four major pulsotypes. Seven sequence types (ST) were identified among the representative bovine S. aureus isolates genotyped, including: ST8 (CC8), ST97 (CC97), ST351 (CC705), ST352 (CC97), ST508 (CC45), ST2992 (CC97) and a novel sequence type, ST3538 (CC97). Based on PFGE analysis, greater genetic diversity was observed among the human S. aureus isolates. Bovine and human isolates from three sampling sites clustered together and were genotypically indistinguishable. Two of the isolates, ST97 and ST352 belong to the common bovine lineage CC97, and their isolation from close human contacts suggests zoonotic transfer. In the context of this study, the third isolate, ST8 (CC8), is believed to be a human clone which has transferred to a dairy cow and has subsequently caused mastitis. The detection of indistinguishable S. aureus isolates from bovine and human hosts at three of the sampling sites is suggestive of bacterial transmission and supports the need for vigilant monitoring of staphylococcal populations at the human-animal interface.

Use of Brucella abortus species specific polymerase chain reaction assay for the diagnosis of bovine brucellosis.

Serology is primarily used in the diagnosis of bovine brucellosis. Bacterial culture and isolation is the gold standard in diagnosing brucellosis but, like serology, it does not offer complete (100%) diagnostic sensitivity and specificity. Polymerase chain reaction (PCR) has been suggested to offer better specificity and sensitivity. In this study, we evaluated the performance of Brucella abortus species specific (BaSS) PCR directly from different samples in the diagnosis of bovine brucellosis in naturally infected cattle in KwaZulu-Natal province of South Africa with known infectious status from culture. The BaSS PCR had a low diagnostic sensitivity (DSe) of 70%, but was able to identify vaccine strains using abomasal fluid from aborted foetuses and detect Brucella DNA from decomposing samples. The best sample for the BaSS PCR was abomasal fluid.

The Ohio study in light of national data and clinical experience.

The Siminoff, Burant, and Youngner study in Ohio is strikingly consistent with data from a national study. Both suggest that there might be significant public acceptance of future policies that violate the dead donor rule, or that further extend the boundary between life and death to include brain-damaged patients short of "brain death." Experience with donation suggests that many individuals would donate their loved ones' organs when they have concluded that the brain injury is not survivable, even if all the criteria for "brain death" are not met. It would be very helpful to have research on those who have gone through the real-life clinical situation. Based on the findings of this study and the increasing demand for organs, it may be appropriate for public policy to allow for ways to increase organ procurement from individuals who are not fully "brain dead" beyond the current method of procurement after cardiac death, but any change in this area should go slowly and with significant public input.

Evaluation of molecular assays for identification Campylobacter fetus species and subspecies and development of a C. fetus specific real-time PCR assay.

Phenotypic differentiation between Campylobacter fetus (C. fetus) subspecies fetus and C. fetus subspecies venerealis is hampered by poor reliability and reproducibility of biochemical assays. AFLP (amplified fragment length polymorphism) and MLST (multilocus sequence typing) are the molecular standards for C. fetus subspecies identification, but these methods are laborious and expensive. Several PCR assays for C. fetus subspecies identification have been described, but a reliable comparison of these assays is lacking. The aim of this study was to evaluate the most practical and routinely implementable published PCR assays designed for C. fetus species and subspecies identification. The sensitivity and specificity of the assays were calculated by using an extensively characterized and diverse collection of C. fetus strains. AFLP and MLST identification were used as reference. Two PCR assays were able to identify C. fetus strains correctly at species level. The C. fetus species identification target, gene nahE, of one PCR assay was used to develop a real-time PCR assay with 100% sensitivity and 100% specificity, but the development of a subspecies venerealis specific real-time PCR (ISCfe1) failed due to sequence variation of the target insertion sequence and prevalence in other Campylobacter species. None of the published PCR assays was able to identify C. fetus strains correctly at subspecies level. Molecular analysis by AFLP or MLST is still recommended to identify C. fetus isolates at subspecies level.

Prevalence of sleep disturbance and its relationship to pain in adults with chronic pain.

The study examined the prevalence and relationship between sleep disturbance and chronic pain. Research questions were: (1) What is the prevalence of sleep disturbance in adults with chronic pain, and how does this prevalence compare with healthy and insomniac adults? (2) What is the relationship between sleep disturbance and chronic pain? (3) What is the relationship of patient characteristics to sleep? This descriptive, correlational field study was done at an interdisciplinary pain clinic, sampling 99 adults, and using an 11-point pain scale and a visual analog sleep scale. For every disturbance item, more than 47% of subjects reported a score of 50 or higher, twice as high as those for healthy adults, indicating disrupted sleep. For every effectiveness item, more than 54% of subjects reported a score of 50 or less, significantly lower than for healthy adults, indicating poor sleep quality. For every supplementation item, more than 60% reported mean scores of 10 or less, indicating minimal napping, yet scores were higher than for healthy adults. For all three scales, scores were similar to the mean scores for insomniacs. Soundness of sleep showed a small but significant positive (r <.30) correlation with years of pain. Supplementation scale items were not correlated with either years of pain or pain intensity. Fragmentation was significant on the basis of gender, with men having higher scores than women. Age was a negative predictor of sleep latency. Education and age were negative predictors of the quality of sleep.

Deletion of the alternatively spliced fibronectin EIIIA domain in mice reduces atherosclerosis.

The alternatively spliced and highly conserved EIIIA domain of fibronectin (FN) is included in most FN of the extracellular matrix in embryos. In adults, both extracellular matrix and plasma FN essentially lack EIIIA. In diverse inflammatory situations however, EIIIA is specifically included by regulated RNA splicing. In atherosclerotic lesions, FN, including the EIIIA domain (EIIIA-FN), is abundant, whereas FN in the flanking vessel wall lacks EIIIA. Lesional EIIIA-FN is localized with endothelial cells and macrophage foam cells. To directly test the function of EIIIA-FN, we generated EIIIA-null (EIIIA(-/-)) mice that lack the EIIIA exon and crossed them with apolipoprotein E (ApoE)-null (ApoE(-/-)) mice that develop arterial wall lesions. Compared with ApoE(-/-) controls, EIIIA(-/-)ApoE(-/-) mice had significantly smaller lesions throughout the aortic tree. EIIIA-FN was increased in ApoE(-/-) plasma, and total plasma cholesterol was reduced in EIIIA(-/-)ApoE(-/-) mice, specifically in large lipoprotein particles, suggesting a functional role for plasma EIIIA-FN. To assess a role for macrophage EIIIA-FN in the vessel wall, we conducted in vitro foam cell assays. EIIIA(-/-)ApoE(-/-) macrophages accumulated significantly less intracellular lipid than control ApoE(-/-) cells. These results provide genetic evidence that suggests roles for EIIIA-FN in plasma lipoprotein metabolism and in foam cell formation.