Functional Validation of Apoptosis Genes IAP1 and DRONC in Midgut Tissue of the Biting Midge Culicoides sonorensis (Diptera: Ceratopogonidae) by RNAi.

Culicoides biting midges transmit multiple ruminant viruses, including bluetongue virus and epizootic hemorrhagic disease virus, causing significant economic burden worldwide. To further enhance current control techniques, understanding vector-virus interactions within the midge is critical. We developed previously a double-stranded RNA (dsRNA) delivery method to induce RNA interference (RNAi) for targeted gene knockdown in adult Culicoides sonorensis Wirth & Jones. Here, we confirm the C. sonorensis inhibitor of apoptosis 1 (CsIAP1) as an anti-apoptotic functional ortholog of IAP1 in Drosophila, identify the ortholog of the Drosophila initiator caspase DRONC (CsDRONC), and demonstrate that injection of dsRNA into the hemocoel can be used for targeted knockdown in the midgut in C. sonorensis. We observed CsIAP1 transcript reduction in whole midges, with highest transcript reduction in midgut tissues. IAP1knockdown (kd) resulted in pro-apoptotic caspase activation in midgut tissues. In IAP1kd midges, midgut tissue integrity and size were severely compromised. This phenotype, as well as reduced longevity, was partially reverted by co-RNAi suppression of CsDRONC and CsIAP1. Therefore, RNAi can be directed to the midgut of C. sonorensis, the initial site of virus infection, using dsRNA injection into the hemocoel. In addition, we provide evidence that the core apoptosis pathway is conserved in C. sonorensis and can be experimentally activated in the midgut to reduce longevity in C. sonorensis. This study thus paves the way for future reverse genetic analyses of midgut-virus interactions in C. sonorensis, including the putative antiviral properties of RNAi and apoptosis pathways.

Dose-dependent fate of GFP-expressing Escherichia coli in the alimentary canal of adult house flies.

The adult house fly Musca domestica (L.) (Diptera: Muscidae) can disseminate bacteria from microbe-rich substrates to areas in which humans and domesticated animals reside. Because bacterial abundance fluctuates widely across substrates, flies encounter and ingest varying amounts of bacteria. This study investigated the dose-dependent survival of bacteria in house flies. Flies were fed four different 'doses' of green fluorescent protein (GFP)-expressing Escherichia coli (GFP E. coli) (very low, low, medium, high) and survival was determined at 1, 4, 10 and 22 h post-ingestion by culture and epifluorescent microscopy. Over 22 h, the decline in GFP E. coli was significant in all treatments (P < 0.04) except the very low dose treatment (P = 0.235). Change in survival (ΔS) did not differ between flies fed low and very low doses of bacteria across all time-points, although ΔS in both treatments differed from that in flies fed high and medium doses of bacteria at several time-points. At 4, 10 and 22 h, GFP E. coli ΔS significantly differed between medium and high dose-fed flies. A threshold dose, above which bacteria are detected and destroyed by house flies, may exist and is likely to be immune-mediated. Understanding dose-dependent bacterial survival in flies can help in predicting bacteria transmission potential.

Inducing RNA interference in the arbovirus vector, Culicoides sonorensis.

Biting midges in the genus Culicoides are important vectors of arboviral diseases, including epizootic haemorrhagic disease, bluetongue and most likely Schmallenberg, which cause significant economic burdens worldwide. Research on these vectors has been hindered by the lack of a sequenced genome, the difficulty of consistent culturing of certain species and the absence of molecular techniques such as RNA interference (RNAi). Here, we report the establishment of RNAi as a research tool for the adult midge, Culicoides sonorensis. Based on previous research and transcriptome analysis, which revealed putative small interfering RNA pathway member orthologues, we hypothesized that adult C. sonorensis midges have the molecular machinery needed to perform RNA silencing. Injection of control double-stranded RNA targeting green fluorescent protein (dsGFP), into the haemocoel of 2-3-day-old adult female midges resulted in survival curves that support virus transmission. dsRNA injection targeting the newly identified C. sonorensis inhibitor of apoptosis protein 1 (CsIAP1) orthologue resulted in a 40% decrease of transcript levels and 73% shorter median survivals as compared with dsGFP-injected controls. These results reveal the conserved function of IAP1. Importantly, they also demonstrate the feasibility of RNAi by dsRNA injection in adult midges, which will greatly facilitate studies of the underlying mechanisms of vector competence in C. sonorensis.

Molecular phylogenetic profiling of gut-associated bacteria in larvae and adults of flesh flies.

Flesh flies of the genus Sarcophaga (Diptera: Sarcophagidae) are carrion-breeding, necrophagous insects important in medical and veterinary entomology as potential transmitters of pathogens to humans and animals. Our aim was to analyse the diversity of gut-associated bacteria in wild-caught larvae and adult flesh flies using culture-dependent and culture-independent methods. Analysis of 16S rRNA gene sequences from cultured isolates and clone libraries revealed bacteria affiliated to Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes in the guts of larval and adult flesh flies. Bacteria cultured from larval and adult flesh fly guts belonged to the genera Acinetobacter, Bacillus, Budvicia, Citrobacter, Dermacoccus, Enterococcus, Ignatzschineria, Lysinibacillus, Myroides, Pasteurella, Proteus, Providencia and Staphylococcus. Phylogenetic analysis showed clone sequences of the genera Aeromonas, Bacillus, Bradyrhizobium, Citrobacter, Clostridium, Corynebacterium, Ignatzschineria, Klebsiella, Pantoea, Propionibacterium, Proteus, Providencia, Serratia, Sporosarcina, Weissella and Wohlfahrtiimonas. Species of clinically significant genera such as Ignatzschineria and Wohlfahrtiimonas spp. were detected in both larvae and adult flesh flies. Sequence analysis of 16S rRNA gene libraries supported culture-based results and revealed the presence of additional bacterial taxa. This study determined the diversity of gut microbiota in flesh flies, which will bolster the ability to assess microbiological risk associated with the presence of these flies. The present data thereby establish a platform for a much larger study.

Temporospatial fate of bacteria and immune effector expression in house flies fed GFP-Escherichia coli O157:H7.

The house fly Musca domestica L. (Diptera: Muscidae) harbours and transmits a variety of human enteropathogens including Escherichia coli (Enterobacteriales: Enterobacteriaceae) O157:H7. Interactions between ingested bacteria and the fly gut directly impact bacterial persistence, survival and ultimately fly vector competence. We assessed the temporospatial fate of green fluorescent protein (GFP)-expressing E. coli O157:H7 (GFP-ECO157) in house flies along with fly antimicrobial responses up to 12 h post-ingestion. In flies fed GFP-ECO157, culture and microscopy revealed a steady decrease in bacterial load over 12 h, which is likely to be attributable to the combined effects of immobilization within the peritrophic matrix, lysis and peristaltic excretion. However, flies can putatively transmit this pathogen in excreta because intact bacteria were observed in the crop and rectum. Quantitative reverse-transcriptase polymerase chain reaction analysis of antimicrobial peptides (AMPs) and lysozyme gene expression showed minimal upregulation in both the gut and carcass of house flies fed GFP-ECO157. However, these genes were upregulated in fly heads and salivary glands, and effector proteins were detected in the gut in some flies. Collectively, these data indicate that house flies can serve as reservoirs of E. coli O157:H7 for up to 12 h, and factors in addition to AMPs and lysozyme may contribute to bacteria destruction in the gut.

Vector potential of houseflies for the bacterium Aeromonas caviae.

Houseflies, Musca domestica Linnaeus (Diptera: Muscidae), have been implicated as vectors or transporters of numerous gastrointestinal pathogens encountered during feeding and ovipositing on faeces. The putative enteropathogen Aeromonas caviae (Proteobacteria: Aeromonadaceae) may be present in faeces of humans and livestock. Recently A. caviae was detected in houseflies by PCR and isolated by culture methods. In this study, we assessed the vector potential of houseflies for A. caviae relative to multiplication and persistence of the bacterium in the fly and to contamination of other flies and food materials. In experimentally fed houseflies, the number of bacteria increased up to 2 days post-ingestion (d PI) and then decreased significantly 3 d PI. A large number of bacteria was detected in the vomitus and faeces of infected flies at 2-3 d PI. The bacteria persisted in flies for up to 8 d PI, but numbers were low. Experimentally infected flies transmitted A. caviae to chicken meat, and transmissibility was directly correlated with exposure time. Flies contaminated the meat for up to 7 d PI; however, a significant decrease in contamination was observed 2-3 d PI. In the fly-to-fly transmission experiments, the transmission of A. caviae was observed and was apparently mediated by flies sharing food. These results support houseflies as potential vectors for A. caviae because the bacterium multiplied, persisted in flies for up to 8 d PI, and could be transmitted to human food items.

Detection of Aeromonas caviae in the common housefly Musca domestica by culture and polymerase chain reaction.

Aeromonas caviae has been implicated in diarrhoeal disease of livestock and humans. The potential role of houseflies in the epidemiology of this pathogen was investigated by examining the prevalence of A. caviae in houseflies collected from two South Carolina farms and one restaurant. Isolation was accomplished by culture of flies in alkaline peptone water followed by identification with Aeromonas-specific PCR using novel primers (APW-PCR). All isolates cultured from houseflies were identified as A. caviae by biochemical characteristics and direct sequencing approximately 800 bp of the 16S rRNA gene. Aeromonas caviae was detected in 78% (272/349) dairy farm flies, 55% (54/99) pig farm flies and 39% (77/200) restaurant flies. Faeces from cows and pigs at the farms also were positive for A. caviae (58% and 100%, respectively). The APW PCR method provided a rapid, convenient way to identify A. caviae from faeces and houseflies that contained hundreds of bacterial species.

Trauma wound management.

This article provides a review of wound healing and management after injury. Wound management includes cleansing, primary and delayed closure, and a discussion of gunshot wounds. Forensic issues for evidence collection and the accurate documentation of wounds are reviewed.

Abdominal compartment syndrome.

TOPIC: Abdominal compartment syndrome (ACS) in blunt and penetrating abdominal trauma. PURPOSE: To review the clinical manifestations, pathophysiology, and nursing management of patients with ACS. SOURCES: Published literature, case study. CONCLUSIONS: Trauma nurses play a key role in early identification and management of ACS. Priorities include knowledge of signs and symptoms, methods of measurement of ACS; specifics of wound care, psychosocial interventions, and key factors in discharge planning.

Thoracic aorta injuries: management and outcome of 144 patients.

Rupture of the thoracic aorta from blunt injury is often lethal. Methods of operative repair vary, based on the surgeon's preference and circumstances. The primary hypothesis of this study was that operative management choices would correlate with outcome. Data on demographics, injury mechanism, initial evaluation, diagnostic procedures, operative treatment, and outcome were obtained from chart review at the state's eight trauma centers. Rates of paraplegia and survival were compared for different methods of operative repair. Of 63,507 hospitalized trauma patients, 144 patients sustained thoracic aortic injury (incidence = 0.23%). Sixty-four died (44.1%), most of whom died in the emergency department (26) or the operating room (12). Eighty-six patients had complete operative data for analysis, including cross-clamp time and methods of repair. No patient in the group with a cross-clamp time of less than 35 minutes developed paraplegia (p = 0.02). For the patients with longer cross-clamp times, 6 of 14 patients (42.9%) undergoing clamp and sew repair developed paraplegia, as compared to 2 of 37 patients (5.4%) repaired on bypass (p = 0.005). This study suggests that the rate of paraplegia after repair of thoracic aortic injury can be minimized with short cross-clamp times or the use of bypass when long cross-clamp times can be anticipated.

American College of Surgeons trauma quality indicators: an analysis of outcome in a statewide trauma registry.

Quality assurance/quality improvement (QA-QI) is a priority for maintaining the highest standards of care in trauma systems. To be an effective tool for system review, the QA-QI indicators should identify patients with higher rates of morbidity and mortality from injury. While the American College of Surgeons (ACS) and the Joint Commission on Accreditation of Health Care Operations have identified certain audit filters within the trauma system, there are few data to substantiate the value of these audit filters for trauma care. The purpose of this study was to analyze the ability of the ACS trauma indicators to predict adverse patient outcome following injury requiring review. The study population consisted of 44,019 patients from the North Carolina State Trauma Registry from 1987 to 1992. Of the 22 audit filters nine were available for analysis. Mortality rate, length of stay, and total charges were used as measures of outcome. The hypotheses tested were that patients who met the indicator criteria would have higher mortality rates and worse outcomes than the non-indicator group. Student's t test and Chi-square analysis were used to test the differences between the group which met the criteria for the indicator and those without. Of the nine audit filters tested, only three were found to have significantly worse outcomes than their non-indicator comparison group: gunshot wound to the abdomen with non-surgical management, femur fracture without fixation, and complications from pulmonary embolism-deep vein thrombosis-decubitus ulcer (p < 0.05). Contrary to expectations, four of the audit filters, coma without intubation, laparotomy > 2 hours, transfer > 6 hours, and admission to non-surgical service, actually had significantly better outcomes than their non-indicator counterpart. Scene time > 20 minutes, laparotomy > 2 hours after arrival, and craniotomy > 4 hours after arrival may be indicators of patients at risk for morbidity. This study demonstrates that several ACS clinical indicators, as currently written, are not useful in identifying patients at higher risk for poor outcome. The indicators need further definition to be of value in the quality review process. Specifically, the study suggests that audit filters should be data driven and based upon analyses of large populations of injured patients and their outcomes to be valid QA-QI tools.

High-dose methylprednisolone after acute spinal cord injury.

The spinal cord-injured patient has presented a dilemma for nursing care since the earliest survivor of such an injury. Recent research has identified a means of improving neurologic recovery for these patients. Proper administration of high-dose MP within 8 hours after injury provides the patient with the best potential for improvement. Ensuring immobilization and prevention of the complications of immobility are the next most significant actions the nurse can undertake to assist such a patient through a devastating injury toward optimal functional outcome. With continued research and education of caregivers, the residual effects of spinal cord injury may be further abated.

The problems of intrahospital transfer of patients with trauma and one solution: the "Trauma Transfer Backpack".

With long stays for computed tomographic scans and x-ray exams and long trips to ICUs, patients with trauma may need extra fluids, drugs, or equipment outside the emergency department. Putting everything in a backpack saves time, leaves the nurse's hands free, and does not take up room on the stretcher.

Comparison of the ability of adult and pediatric trauma scores to predict pediatric outcome following major trauma.

The Pediatric Trauma Score (PTS) has been identified as the only accurate and adequate means of predicting outcome in pediatric trauma. In answer to the increasing number of trauma patients arriving at local hospitals, the ability of the adult Trauma Score (TS) to predict pediatric trauma outcome was tested. Of the total 2,604 pediatric trauma cases in the North Carolina State Trauma Registry, 441 had both a PTS and TS available for analysis. The primary measures of outcome were emergency department and hospital dispositions. Logistic regression demonstrated that TS (R2 = 0.50) was a stronger predictor of pediatric outcome and PTS (R2 = 0.35) for emergency department disposition and TS (R2 = 0.63) with PTS (R2 = 0.51) for hospital disposition. The correlation between TS and PTS was high (R = 0.8). Stepwise discriminant analysis demonstrated that TS was the stronger predictor of outcome and the PTS added only 9% (partial R2 = 0.09) more accuracy to TS for emergency department disposition and only 6% (partial R2 = 0.06) for hospital disposition. The results of this research demonstrate that TS is a useful method of predicting outcome in pediatric trauma. The use of both scores for each patient does not increase the predictive value of the scores.

Thoracic aorta rupture as the cause of paraplegia: a diagnostic dilemma (case report).

An unusual case of traumatic aortic transection in an 8-year-old boy: the child presented with paraplegia after being struck by an automobile and thrown into a telephone pole. No bony abnormalities were noted on radiologic examination. The case emphasizes the importance of a high risk index of suspicion and the mechanism of injury.

Medicinal leech therapy: a case study.

Essential to the outcome of the replantation of digits is adequate arterial inflow and venous outflow. A nonoperative solution to the problem of venous insufficiency is leeching. The leech relieves venous congestion while attached, as the hirudin released by the leech continues to decongest the digit for 1-2 more hours. The emphasis of this article is a case study using leeches for venous congestion as an alternative to surgery.