Deep sequencing and variant analysis of an Italian pathogenic field strain of equine infectious anaemia virus.

Equine infectious anaemia virus (EIAV) is a lentivirus with an almost worldwide distribution that causes persistent infections in equids. Technical limitations have restricted genetic analysis of EIAV field isolates predominantly to gag sequences resulting in very little published information concerning the extent of inter-strain variation in pol, env and the three ancillary open reading frames (ORFs). Here, we describe the use of long-range PCR in conjunction with next-generation sequencing (NGS) for rapid molecular characterization of all viral ORFs and known transcription factor binding motifs within the long terminal repeat of two EIAV isolates from the 2006 Italian outbreak. These isolates were from foals believed to have been exposed to the same source material but with different clinical histories: one died 53 days post-infection (SA) while the other (DE) survived 5 months despite experiencing multiple febrile episodes. Nucleotide sequence identity between the isolates was 99.358% confirming infection with the same EIAV strain with most differences comprising single nucleotide polymorphisms in env and the second exon of rev. Although the synonymous:non-synonymous nucleotide substitution ratio was approximately 2:1 in gag and pol, the situation is reversed in env and ORF3 suggesting these sequences are subjected to host-mediated selective pressure. EIAV proviral quasispecies complexity in vivo has not been extensively investigated; however, analysis suggests it was relatively low in SA at the time of death. These results highlight advantages of NGS for molecular characterization of EIAV namely it avoids potential artefacts generated by traditional composite sequencing strategies and can provide information about viral quasispecies complexity.

Umbilical infections in foals: microbiological investigation and management.

This study aims to investigate the bacteria involved in equine omphalitis and their susceptibility to antimicrobial drugs, and consequently to provide guidelines concerning the most suitable treatment protocol in accordance with the clinical, ultrasound and laboratory findings. Forty foals aged between one and 30 days were evaluated in the course of this investigation. An ultrasound examination of all umbilical remnants was carried out carefully in all foals; umbilical swabs were collected for bacteriological examination, and blood samples were collected for blood culture from 19 foals with fever and abnormal blood values. Bacterial omphalitis was observed in 95 per cent of foals and bacterial septicaemia was diagnosed in 11 cases. Enterobacteria and coccoid Gram-positive bacteria were isolated more frequently than Serratia marcescens, Pantoea agglomerans and Trueperella pyogenes Omphalectomy was performed in 77.5 per cent of the foals examined; the remainder were treated only medically with antimicrobial drugs as recommended by antibiotic susceptibility testing performed for all bacteria isolated. Antibiotic therapy was successful in all foals that only received medical treatment; nevertheless, omphalectomy was performed in most cases particularly in situations of clinical decline despite antibiotic therapy and when involvement of umbilical vein, fever and joint disorders were observed.

Stored Canine Whole Blood Units: What is the Real Risk of Bacterial Contamination?

BACKGROUND: Bacterial contamination of whole blood (WB) units can result in transfusion-transmitted infection, but the extent of the risk has not been established and may be underestimated in veterinary medicine. OBJECTIVES: To detect, quantify, and identify bacterial microorganisms in 49 canine WB units during their shelf life. ANIMALS: Forty-nine healthy adult dogs. METHODS: Forty-nine WB units were included in the study. Immediately after collection, 8 sterile samples from the tube segment line of each unit were aseptically collected and tested for bacterial contamination on days 0, 1, 7, 14, 21, 28, 35, and 42 of storage. A qPCR assay was performed on days 0, 21, and 35 to identify and quantify any bacterial DNA. RESULTS: On bacterial culture, 47/49 blood units were negative at all time points tested, 1 unit was positive for Enterococcus spp. on days 0 and 1, and 1 was positive for Escherichia coli on day 35. On qPCR assay, 26 of 49 blood units were positive on at least 1 time point and the bacterial loads of the sequences detected (Propionobacterium spp., Corynebacterium spp., Caulobacter spp., Pseudomonas spp., Enterococcus spp., Serratia spp., and Leucobacter spp.) were <80 genome equivalents (GE)/µL. CONCLUSIONS AND CLINICAL IMPORTANCE: Most of the organisms detected were common bacteria, not usually implicated in septic transfusion reactions. The very low number of GE detected constitutes an acceptable risk of bacterial contamination, indicating that WB units have a good sanitary shelf life during commercial storage.

Aortic rupture of acute aortic dissection type treated with thoracic endovascular aortic repair (TEVAR).

OBJECTIVE: Acute aortic dissection (AAD) is one of the most frequent aortic emergencies, which occurs to the vascular specialist. Endovascular reconstruction of the true lumen using minimally invasive stent grafting or stenting has become increasingly popular and widespread among institutions. The aim of this paper is to report a case series composed by twenty-eight patients, who underwent endovascular intervention for acute type B aortic dissections complicated by rupture using thoracic endovascular aortic repair (TEVAR). PATIENTS AND METHODS: All patients with type B-AAD were admitted to the surgical intensive care unit and initially managed with a standing protocol for medical management of AD and observed for evidence of visceral or extremity malperfusion. RESULTS: No major complications or adverse reactions occurred during the immediate postoperative period. Two patients died in the first three months of the study; both developed a cerebral ischemia. Three patients were lost at follow-up, the remaining twenty-three had a mean follow-up of 41.12±3.55 months (range: 36-58). CT scans were routinely performed at 3 months, 6 months, and yearly after the intervention for all patients. CONCLUSIONS: Endovascular repair is developing as a strong alternative to surgery and may eventually evolve as a superior method for definitive treatment for patients with appropriate indications, such as complicated dissections. AD rupture may be more common in arch stent-graft patients with an ascending aortic diameter >4 cm and with a multi-stents placement.

Rhodococcus equi pneumonia in foals: an assessment of the early diagnostic value of serum amyloid A and plasma fibrinogen concentrations in equine clinical practice.

Early diagnosis and prevention of Rhodococcus equi pneumonia in foals represent important goals for equine clinicians. Recent protocols for diagnosis and treatment of Rhodococcosis in foals typically rely on a multimodal approach based on sonographic evidence suggestive of pyogranulomas, sonographic abscess scores and laboratory findings including plasma fibrinogen concentrations, blood biochemistry testing and platelet and leukocyte counts. The aim of this study was to assess the utility of weekly testing of serum amyloid A (SAA) and plasma fibrinogen concentrations in foals to achieve early diagnosis of R. equi pneumonia prior to the onset of clinical signs. This testing was used to simulate a clinically practical screening procedure and compared with thoracic ultrasonography performed in parallel. The present study suggests that SAA does not represent a reliable early marker of Rhodococcosis when plasma concentrations are tested weekly. However, when clinical signs of R. equi pneumonia are present, SAA concentrations may allow clinicians to obtain 'real-time' indications concerning both the progress of infection and the effectiveness of therapy. This study raises the possibility that plasma fibrinogen monitoring starting at 1 week of age and repeated on a weekly basis, could serve as a screening test allowing clinicians to identify foals as suspected of R. equi infection. Future investigations regarding both physiological plasma fibrinogen concentrations in foals as well as fibrinogen kinetics in foals affected with R. equi pneumonia, including the establishment of appropriate reference intervals for the test method employed in this study, will be necessary in order to clarify this possibility.

Insertion sequence IS256 in canine pyoderma isolates of Staphylococcus pseudintermedius associated with antibiotic resistance.

Staphylococcus pseudintermedius is the most frequent staphylococcal species isolated from canine pyoderma. The control of S. pseudintermedius infection is often difficult due to the expanded antimicrobial resistance phenotypes. Antibiotic resistance in staphylococcal pathogens is often associated to mobile genetic elements such as the insertion sequence IS256 that was first described as a part of the transposon Tn4001, which confers aminoglycoside resistance in Staphylococcus aureus and in Staphylococcus epidermidis. In this study a collection of 70 S. pseudintermedius isolates from canine pyoderma was used to investigate antimicrobial susceptibility to 15 antibiotics and the presence of IS256, not revealed in S. pseudintermedius yet. Antibiotic resistance profiling demonstrated that all S. pseudintermedius isolates had a multi-drug resistance phenotype, exhibiting simultaneous resistance to at least five antibiotics; indeed methicillin resistant S. pseudintermedius isolates were simultaneously resistant to at least nine antibiotics and all were also gentamicin resistant. PCR analyses revealed the presence of IS256 in 43/70 S. pseudintemedius isolates. The association between the presence of IS256 and the resistance was particularly significant for certain antibiotics: cefovecin, amikacin, gentamicin and oxacillin (χ(2)p-value<0.05). However, there was a striking result in frequency of strains resistant to gentamicin and oxacillin, suggesting a specific association between the presence of the IS256 element and the determinants for the resistance to these antibiotics. To the best of our knowledge, this is the first report showing the detection of IS256 in S. pseudintermedius isolates and its association with antibiotic resistance. Our findings suggest that S. pseudintermedius may acquire antibiotic resistance genes through mobile genetic elements which may play a predominant role in the dissemination of multi-drug resistance.

Isolation and characterization of ß-haemolytic-Streptococci from endometritis in mares.

The objective of this manuscript was to validate published PCR-based methods for detection of ß-haemolytic Streptococci by comparison with established bacteriological techniques using 85 clinical isolates recovered from uterine swabs of mares with clinical signs of endometritis and to determine the distribution of SeeL/SeeM and SzeL/SzeM superantigens in isolates of Streptococcus equi subsp. equi (S. equi) and S. equi subsp. zooepidemicus (S. zooepidemicus). The conventional bacteriological techniques showed the vast majority of these isolates (78) were S. zooepidemicus with just 5 Streptococcus dysgalactiae subsp. equisimilis (S. equisimilis) and 2 S. equi strains detected. The PCR analyses confirmed the bacteriological results demonstrating the reliability of the 16S rRNA PCR assay for detecting Streptococci, the multiplex PCR for differentiating between S. zooepidemicus, and S. equi, and PCR assays based on streptokinase genes for identification of S. equisimilis. PCRs for genes encoding superantigens revealed seeL and seeM specific amplicons with size of approximately 800 and 810 bp respectively for the S. equi strains and for 2 S. zooepidemicus strains. To our knowledge, this is the first report of szeL and szeM possession by S. zooepidemicus isolates derived from endometritis in mares.

Clinical, serological and molecular investigations of EHV-1 and EHV-4 in 15 unweaned thoroughbred foals.

Fifteen unweaned thoroughbred foals, born on a stud farm to vaccinated mares, were clinically monitored during their first six months of life and repeatedly tested for equine herpesvirus type 1 (EHV-1) and equine herpesvirus type 4 (EHV-4). Nasopharyngeal swabs and blood samples were collected and screened respectively by PCR and seroneutralisation to detect the presence of the virus, explore its role as a possible cause of respiratory disease, and to assess the efficiency of the pcr for the diagnosis of this disease. The foals were divided into three groups on the basis of their clinical signs and whether they had seroconverted to EHV-1 and/or EHV-4: first, foals with no clinical signs of disease that had not seroconverted; secondly, foals with clinical signs that had seroconverted, and thirdly, foals with clinical signs that had not seroconverted. The results indicated that the viruses circulated on the stud farm despite stringent vaccination regimens against them, and confirmed their association with respiratory disease. The absence of significantly different pcr results among the three groups of foals showed that the pcr was effective in confirming the circulation of the viruses on the premises without being particularly helpful as a diagnostic tool.

Assessment in the context of uncertainty: how many members are needed on the panel of reference of a script concordance test?

PURPOSE: The script concordance test (SCT) assesses clinical reasoning in the context of uncertainty. Because there is no single correct answer, scoring is based on a comparison of answers provided by examinees with those provided by members of a panel of reference made up of experienced practitioners. This study aims to determine how many members are needed on the panel to obtain reliable scores to compare against the scores of examinees. METHODS: A group of 80 residents were tested on 73 items (Cronbach's alpha: 0.76). A total of 38 family doctors made up the pool of experienced practitioners, from which 1000 random panels of reference of increasing sizes (5, 10, 15, 20, 25 and 30) were generated with a resampling procedure. Residents' scores were computed for each panel sample. Units of analysis were means of residents' score, test reliability coefficient and correlation coefficient between scores obtained with a given panel of reference versus the scores obtained with the full panel of 38. Statistics were averaged across the 1000 samples for each panel size for the mean and test reliability computations, and across 100 samples for the correlation computation. RESULTS: For sample variability, there was a 3-fold increase in standard deviation of means between a sample panel size of 5 (SD=1.57) and a panel size of 30 (SD=0.50). For reliability, there was a large difference in precision between a panel size of 5 (0.62) and a panel size of 10 (0.70). When the panel size was over 20, the gain became negligible (0.74 for 20 and 0.76 for 38). For correlation, the mean correlation coefficient values were 0.90 with 5 panel members, 0.95 with 10 members and 0.98 with 20 members. CONCLUSION: Any number over 10 is associated with acceptable reliability and good correlation between the samples versus the full panel of 38. For high stake examinations, using a panel of 20 members is recommended. Recruiting more than 20 panel members shows only a marginal benefit in terms of psychometric properties.

[Small intestine tumors: our experience in emergencies]. / Tumori dell'intestino tenue: nostra esperienza in urgenza.

Small bowel tumours are relatively rare neoplasms; unusual occurrence associated with nonspecific symptoms, and low-sensitivity tests availability, are responsible for diagnostic delay. A retrospective study was performed on 42 cases with acute presentation, from 1972 to 2001; median age was 52 years (range 14-79) and there was a slight female prevalence (57.1% vs 42.9%). The most common acute presentation was occlusion (57.1%), followed by gastrointestinal (GI) bleeding (23.8%), perforation (14.3%) and occlusion/perforation (4.8%). Benign neoplasia were 38.1% (16 cases) and adenoma is was the most common type; malignant forms were 61.9 (26 cases) and adenocarcinoma and lymphomas were the most common histotype. Radical surgical procedures were possible only in 57% of malignant forms (24 patients); morbility was 4.8% (2 cases: 1 anastomotic dehiscence and 1 subphrenic abscess); mortality was 14.3%. From our retrospective study, we can state that survival for malignant lesions is strictly dependent of early TNM staging and possibility of radical surgical procedure. An extremely high index of suspicion in evaluating mild and often misleading symptoms, integrated with specific diagnostic studies, should be the proper approach. Prognosis for benign from is excellent in all cases.

Thoracic outlet syndrome in paediatrics: clinical presentation, surgical treatment, and outcome in a series of eight children.

BACKGROUND/PURPOSE: This report reviews the clinical presentation, surgical treatment, and outcome of 8 children treated for the thoracic outlet syndrome (TOS) during the last 3 years. METHODS: From 1998 through 2001 31 patients were admitted to our Vascular Surgery Unit with TOS. Eight of them (25.8%) were in the paediatric age group, 8 to 16 years (mean, 13 years). No sex prevalence was found. The presenting symptoms were neurologic in 2 patients (25%) and secondary to venous flow impairment in 6 (75%). At phlebography, venous thrombosis was seen in 2 cases, and functional intermittent obstruction was seen in 4. Seven patients underwent decompressive surgical partial resection of the first rib with transaxillary or supraclavicular access. One patient was treated conservatively. RESULTS: There were no major postoperative complications. Mean hospital stay was 2.7 days. In no patient were there signs of recurrence after a mean follow-up of 18 months (range, 3 to 36 months). CONCLUSIONS: In the authors' experience TOS in paediatric patients occurs with the same symptoms and thrombotic complications as in adults. The same surgical strategy adopted in adult patients is advisable for affected children.

[Abdominal "packing": indications and method]. / Il packing addominale: indicazioni e metodo.

Abdominal packing is a lifesaving technique for temporary control of severe injury and it is used in damage control surgery schedule. Technically bleeding from abdominal cavity can generally be achieved by applying pressure with several large abdominal packs. Its possible too applying packs in organ-specific techniques (early abdominal packing). A wide review of the literature has allowed to emphasize the most common problem of this technique, the adequacy of the particular indications, their evolution, timing, the results in general and particular which multiple critical situations and not always predictable when an intensive diagnostic and methodological approach is necessary in. The principal indications are when complex anatomic lesions are diagnosed with not ruleable hemorrhages, in presence of metabolic failure (hypothermia < 35 degrees C, acidosis > 7.2, coagulopathy PTT > 16 seconds: These three derangements become established quickly in the exsanguinating trauma patient and, once established, form a vicious circle which may be impossible to overcome. The results are encouraging and we can evaluate a median survival of the 70%, certainly superior to the obtainable survival with immediate surgical repair. Immediate failures are substantially due to bleeding, especially in "underpacking" case, and remote: these last can be premises, fundamentally septic and bound at the time of stay (above the 72 hours) and associated by the coexistence of lesions: in these situations is possible a MOF syndrome due to excessive intra-abdominal pressure (overpacking) or to an Abdominal Compartment Syndrome.

[Surgical management of trauma: from the "golden hour" to the "Golden Day"]. / La gestione chirurgica del trauma: dalla "golden hour" al "Golden Day".

The treatment of the "trauma" has individualized objective therapeutic to reach in the first phase of clinical management defined by Mattox in the concept of the Golden Hour, that consist to transport the patient to the fittest hospital and to recognize and to treat the priorities "ABCDE", identifies in ATLS Guideline. The evolution of the organization for the treatment of the trauma has developed the concept of Trauma System to whose apex there are of the structures devoted define Trauma Center, with specificity of structures and functions, personal devoted fully grown with a specific run. In the Trauma Center is possible to get a therapeutic planning according to the priorities of the case in few times so that to not only achieve the objective to treat in emergency the vital lesions, but to do the necessary treatments precociously to prevent the compliances of the patient, that is checked in environment then multidisciplinary intensive care. This systematic treatment by objective it allows to identify some Guideline of surgical treatment for priority in once defined Golden Day.

[Damage control surgery: the technique]. / Damage control surgery: le tecniche.

Abdominal packing and planned reoperation is a lifesaving technique for temporary control of haemorrhage in severely injured patients. Morbidity and mortality, however, remain significant. The purpose of this study is to evaluate all surgical technique and our results during 31 years of trauma surgery. In the last 12 years the Authors have performed 11 packing. They stressed fully "damage control technique" in trauma surgery in the last four years. Overall mortality was 45.5%.

Early surgical management of Klippel-Trenaunay syndrome in childhood can prevent long-term haemodynamic effects of distal venous hypertension.

PURPOSE: The aim of this study was to evaluate the authors' preliminary experience in early surgical treatment of distal venous hypertension (DVH) in children affected by Klippel-Trenaunay syndrome (KTS). METHODS: Clinical assessment, surgical management, and outcome of 29 children (18 girls, 11 boys) affected by KTS observed from October 1998 to October 2000 were reviewed retrospectively. RESULTS: Patients ranged in age from 8 months to 17 years (median age at surgery, 10.3 years). The clinical findings are presented. Surgical treatments included stripping of persistent marginal vein (n = 16), multiple legation of bulky varicosities (n = 10), complementary sclerotherapy (n = 14) and laser photocoagulation (n = 13), and excision of associated lymphatic malformations (n = 5). No mortality or major postoperative morbidity occurred. Follow-up period ranged from 6 months to 2 years. CONCLUSION: These preliminary results suggest that early surgical management of DVH in KTS is safe and could be effective in preventing or minimising the long-term haemodynamic effects of DVH in absence of associated deep venous system anomalies.

[Superior vena cava syndrome]. / Sindrome della vena cava superiore.

Superior vena cava syndrome is due to an intrinsic or extrinsic caval obstruction that evolves in acute or subacute way with distinctive clinical feature such as respiratory symptoms and venous stasis. Since 1998 we have treated three cases of spontaneous superior vena cava thrombosis in neoplastic patients who underwent several infusion of chemotherapy, respectively for a breast, uterine and rectum cancer. All patients was female, 52, 58 and 70 years old. The first two cases was treated with locoregional thrombolysis by infusing Urokinase 50,000 UI/h during 24 hours and Urokinase 50,000 UI/h during 12 h the third one. After that, we have positioned a 16/9 wallstent: in the first two cases directly into the superior vena cava, in the third case in the subclavian-anonyma truncus. We had in all cases the complete opening of the stent within the first 48 hours without complications, enabling us to reach a free caval diameter of about 2 cm with resolution of the clinical signs. In patients with high surgical risk, the caval wall-stent is the first choice to solve the vein recanalization.

Retrospective ultramicroscopic investigation on naturally cryptosporidial-infected commercial turkey poults.

The morphometric characteristics and the ultramicroscopic findings of Cryptosporidium spp. at various stages of their life cycle in the intestinal and bursal epithelial cells of naturally infected 30-day-old commercial turkeys are reported. Small, sporulated oocysts, observed in the small intestinal content after flotation, were identified as Cryptosporidium meleagridis on the basis of morphometric characteristics (round in shape and 4.5-5.0 microm in size) and the small intestinal localization. Light section examinations revealed the presence of the protozoon in multiple organs, but its prevalence was highest in the intestinal and bursal epithelial cells. Ultramicroscopic studies on ileum and bursal samples showed the presence of all the life cycle stages in the microvillar brush epithelial cells in both the organs examined. On the basis of the comparison of the morphology and the sizes of the microorganisms parasitizing the ileum and the bursa, hypotheses are considered on the possible species involved.

Medical subject headings used to search the biomedical literature.

The National Library of Medicine's MEDLINE (MEDLARS Online) database was the first database to be searched nationwide via value-added telecommunication networks. Now available on the World Wide Web free of charge from the National Library of Medicine and from many other sources, it is the world's most heavily used medical database. MEDLINE is unique in that each reference to the medical literature is indexed under a controlled vocabulary called Medical Subject Headings (MeSH). These headings are the keys that unlock the medical literature. MeSH multiplies the usefulness of the MEDLINE database and makes it possible to search the medical literature as we do today. This paper commemorates the 40th anniversary of the introduction of MeSH and salutes some of the farsighted persons who conceived and developed the MEDLINE database.