Sero-prevalence and epidemiology of peste des petits ruminants in Libya.

We conducted a cross-sectional study during 2013 to quantify the serological prevalence of peste des petits ruminants (PPR) infection and to investigate host factors associated with PPR infection in small ruminants in Libya. A two-stage sampling design was carried out. A total number of 148 flocks owning at least 100 heads each were randomly selected. Sixteen to forty-eight samples were collected from each selected flock. A total number of 3,508 serum samples from unvaccinated animals were collected and analysed at IZSLER Brescia, Italy, by using competitive ELISA, IDvet innovative diagnostics (IDvet 310, France). The overall serological prevalence among SR was 33% (95% CI: 31.4-34.5). Significant differences between the prevalence in the geographical branches were observed. The lowest prevalence level was observed in Zawiyah branch (16.1%), whereas the highest value was obtained for the Sabha branch (56.8%). Considering the age, a serological prevalence of 24.7%, 31.5% and 42.1% was observed in SR <1 year, between 1 and 2 years and more than 2 years, respectively. Statistically significant differences (p < .001) in the sero-prevalence levels were also observed between the age groups. Our findings suggest that the southern part of Libya could be more exposed to the infections coming from the neighbouring countries and this should be better investigated to correctly identify wherever specific entry points can be considered at higher risk than others. The results also confirmed the endemic status of PPR in Libya, with a constant exposure to the infection of the animals during their life. In the framework of the global strategy for control and eradication of PPR, our results, even if obtained by a preliminary study, can contribute to the assessment of the epidemiological situation of PPR in Libya as required by the Stage 1 of the plan.

Molecular detection and phylogenetic analysis of hepatitis E virus strains circulating in wild boars in south-central Italy.

Hepatitis E virus (HEV) is a zoonotic pathogen with a worldwide distribution, and infects several mammalian species, including pigs and wild boars, which are recognized as its natural reservoirs. The virus causes a usually self-limiting liver disease with a mortality rate generally below 1%, although mortality rates of 15%-25% have been recorded in pregnant woman. Chronic infections can also occur. The prevalence of HEV has been extensively studied in wild boars and pigs in northern Italy, where intensive pig herds are predominantly located. In contrast, few data have been collected in south-central Italy, where small pig herds are surrounded by large regional parks populated with heterogeneous wild fauna. In this study, 291 liver samples from wild boars caught in south-central Italy were analysed with the molecular detection of viral RNA. Our results confirm the circulation of HEV in these animals, with a mean prevalence of 13.7% (40 of 291). A nucleotide sequence analysis showed that the HEV strains were highly conserved within the same geographic areas. The wild boar HEV strains belonged to the HEV-3c subtype, which is frequently described in wild boars, and to an uncommon undefined subtype (HEV-3j-like).The viral prevalence detected is concerning because it could represent a potential risk to hunters, meat workers and consumers of wild boar liver and derivative products. The hypothesized inter-species transmission of HEV to pigs and the possibility that the virus maintains its virulence in the environment and the meat chain also present potential risks to human health, and warrant further investigations in the near future.

Brucella suis biovar 2 multi locus sequence type ST16 in wild boars (Sus scrofa) from Abruzzi region, Italy. Introduction from Central-Eastern Europe?

Porcine brucellosis occurs in many countries where pigs are farmed, often representing an underrated problem. B. suis biovar 2 is the most common isolate in Europe, with high prevalence reported in wild boars in which it is generally isolated in the absence of gross lesions. In the last five years, we tested for Brucella spp. 389 lymph nodes of wild boars collected during hunting seasons or during necropsy procedures. In this paper, we describe the first case of isolation of B. suis biovar 2 from a wild boar aborted foetus, and we analyse the genomic relationships with B.suis biovar 2 strains isolated in the past five years in Abruzzi Region, Central Italy. The genetic fingerprint revealed that the isolates under study belong to the MLST ST16 and to the MLVA11 Gt 57, similar to the Central-Eastern European strains. Massive restocking (for hunting purpose) of wild boars from Eastern Europe have been done since 1950 in Italy contributing to the increasing of population size and distribution, as well as to the interbreeding between these foreign breeds and the local population. The contamination of pastures with infected material such as aborted wild boars foetuses can increase the risk of transmission of Brucella among wild and domestic animals. The contact of B. suis with domestic ruminants may also cause serological reactions to brucellosis serological testing, and even unapparent infection, thus hampering the efforts made in the brucellosis eradication campaign.

Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A.

Essentials Factor (F) VIII is an inefficiently expressed protein. Furin deletion FVIII variants were purified and characterized using in vitro and in vivo assays. These minimally modified novel FVIII variants have enhanced function. These variants provide a strategy for increasing FVIII expression in hemophilia A gene therapy. SUMMARY: Background The major challenge for developing gene-based therapies for hemophilia A is that human factor VIII (hFVIII) has intrinsic properties that result in inefficient biosynthesis. During intracellular processing, hFVIII is predominantly cleaved at a paired basic amino acid cleaving enzyme (PACE) or furin cleavage site to yield a heterodimer that is the major form of secreted protein. Previous studies with B-domain-deleted (BDD) canine FVIII and hFVIII-R1645H, both differing from hFVIII by a single amino acid at this site, suggested that these proteins are secreted mainly in a single polypeptide chain (SC) form and exhibit enhanced function. Objective We hypothesized that deletion(s) of the furin site modulates FVIII biology and may enhance its function. Methods A series of recombinant hFVIII-furin deletion variants were introduced into hFVIII-BDD [Δ1645, 1645-46(Δ2), 1645-47(Δ3), 1645-48(Δ4), or Δ1648] and characterized. Results In vitro, recombinant purified Δ3 and Δ4 were primarily SC and, interestingly, had 2-fold higher procoagulant activity compared with FVIII-BDD. In vivo, the variants also have improved hemostatic function. After adeno-associated viral (AAV) vector delivery, the expression of these variants is 2-4-fold higher than hFVIII-BDD. Protein challenges of each variant in mice tolerant to hFVIII-BDD showed no anti-FVIII immune response. Conclusions These data suggest that the furin deletion hFVIII variants are superior to hFVIII-BDD without increased immunogenicity. In the setting of gene-based therapeutics, these novel variants provide a unique strategy to increase FVIII expression, thus lowering the vector dose, a critical factor for hemophilia A gene therapy.

A Transitional Model for the Evaluation of West Nile Virus Transmission in Italy.

In August 2008, after 10 years of apparent silence, West Nile virus (WNV) infection re-emerged in northern Italy, spreading through the territories of three regions. In the following years, new cases occurred in the same area and additional foci of infection were observed in central and southern Italy, involving also Sicily and Sardinia islands. The Italian Ministry of Health ordered to test by RT-PCR all blood and organ donors from 15th June to 15th November of each year in the infected areas. The period at risk of WNV transmission was defined on the basis of literature data, but a more scientific estimation of the transmission season, under Italian circumstances, needs to be performed. A transitional model previously developed by other Authors was applied and adapted to Italian circumstances, to describe and quantify the WNV transmission cycle between birds and mosquitoes. Culex spp. was considered the main vector, and mosquito parameters were adapted to this genus. Magpies (Pica pica) were considered the main bird host. The model was partially validated through the results of the entomological surveys carried out in central Italy and in Po Valley. The results of the transitional model permitted to calculate the basic reproduction number (R0 ) during 2010 for the whole Italian territory at 1 km of spatial resolution, estimating the risk of WNV transmission during the year and creating detailed risk maps for Italy. The mean values of R0 for the whole Italy varied between 0.4 and 4.8, with values >1 from the end of May to the middle of September. The coastal and flat zones of Italy showed the highest R0 values. Although partially validated, the model showed a substantial acceptable capacity of defining the period at major risk of WNV transmission in Italy, helping Public health authorities in the application of appropriate and timely control and preventive measures.

Platelet-delivered therapeutics.

We have proposed that modified platelets could potentially be used to correct intrinsic platelet defects as well as for targeted delivery of therapeutic molecules to sights of vascular injury. Ectopic expression of proteins within α-granules prior to platelet activation has been achieved for several proteins, including urokinase, factor (F) VIII, and partially for FIX. Potential uses of platelet-directed therapeutics will be discussed, focusing on targeted delivery of urokinase as a thromboprophylactic agent and FVIII for the treatment of hemophilia A patients with intractable inhibitors. This presentation will discuss new strategies that may be useful in the care of patients with vascular injury as well as remaining challenges and limitations of these approaches.

A rare case of myocardial infarction by myocardial bridging of circumflex artery.

Myocardial Bridging (MB) is a benign condition that may be considered as a possible cause of ischaemic cardiopathy up to myocardial infarction. The authors describe the autopsy case of an MB of circumflex artery in a person with a myocardial infarction of the posterior wall of the left ventricle. In this case, the chronic ischemic heart disease can be explained by the congenital anomaly and in the literature there is scientific evidence to support the thesis. The MB is a benign condition that in combination with other risk factors can cause ischemic heart disease.

Apoptotic effects of platelet factor VIII on megakaryopoiesis: implications for a modified human FVIII for platelet-based gene therapy.

BACKGROUND: Ectopically expressed B-domainless factor VIII in megakaryocytes is stored in α-granules, is effective in a number of murine hemostatic models, and is protected from circulating inhibitors. However, this platelet (p) FVIII has different temporal-spatial availability from plasma FVIII, with limited efficacy in other murine hemostatic models. OBJECTIVES AND METHODS: We sought to improve pFVIII hemostatic efficacy by expressing canine (c) FVIII, which has higher stability and activity than human (h) FVIII in FVIII(null) mice. RESULTS AND CONCLUSIONS: We found that pcFVIII was more effective than phFVIII at restoring hemostasis, but peak pcFVIII antigen levels were lower and were associated with greater megakaryocyte apoptosis than phFVIII. These new insights suggest that pFVIII gene therapy strategies should focus on enhancing activity rather than levels. We previously showed that modification of the PACE/furin cleavage site in hFVIII resulted in secretion of hFVIII primarily as a single-chain molecule with increased biological activity. In megakaryocytes, this variant was expressed at the same level as phFVIII with a lentiviral bone marrow transplant approach to reconstitute FVIII(null) mice, but was more effective, resulting in near-normal hemostasis in the cremaster laser injury model. These studies may have implications for pFVIII gene therapy in hemophilia A.

The components of 'One World - One Health' approach.

The interaction between living beings, including men, animals and pathogens, sharing the same environment, should be considered as a unique dynamic system, in which the health of each component is inextricably interconnected and dependent with the others. Nowadays, a new integrated One Health approach is reflecting this interdependence with a holistic view to the ecological system. The One Health approach can be defined as a collaborative and a multidisciplinary effort at local, national and global level to guarantee an optimal healthy status for humans, animals and environment. Strictly related to the One Health concept is to be considered the control of infectious diseases, which have influenced the course of human history. Four different components might be identified as key elements within the 'One World - One Health' (OWOH) approach: the geographical component, the ecological one, the human activities and the food-agricultural ones.

Analysis of surveillance systems in place in European Mediterranean countries for West Nile virus (WNV) and Rift Valley fever (RVF).

West Nile virus (WNV) and Rift Valley fever virus (RVFV) represent an important group of viral agents responsible for vector-borne zoonotic diseases constituting an emerging sanitary threat for the Mediterranean Basin and the neighbouring countries. WNV infection is present in several Mediterranean countries, whereas RVF has never been introduced into Europe, but it is considered a major threat for North African countries. Being vector-borne diseases, they cannot be prevented only through an animal trade control policy. Several approaches are used for the surveillance of WNV and RVFV. With the aim of assessing the surveillance systems in place in Mediterranean countries, two disease-specific questionnaires (WNV, RVFV) have been prepared and submitted to Public Health and Veterinary Authorities of six EU countries. This study presents the information gathered through the questionnaires and describes some critical points in the prevention and surveillance of these diseases as emerged by the answers received.

West Nile transmission in resident birds in Italy.

Migratory birds are considered one of the main sources for West Nile virus (WNV) introduction into European countries. Following the WNV epidemic in the late summer of 1998 in a marshy area of Tuscany (Padule of Fucecchio), an extensive ornithological surveillance programme was carried out in the infected areas from 2006 to 2008. Several species of migratory and resident birds were trapped, sampled and serologically tested. The results of this surveillance programme gave a useful indication of potential sources of WNV re-introduction and spread into Italy. The area under study was also investigated and classified into ecological areas through satellite image processing. In August 2008, the WNV infection re-emerged in Italy in the area surrounding the Po river delta, involving three regions: Lombardy, Emilia Romagna and Veneto. Several surveillance activities were immediately put in place, including the extensive monitoring of wild birds found dead or trapped in the framework of other surveillance programmes. These activities were also prolonged in the 2009, when the virus circulation re-occurred at the border of the area already infected in 2008. The possible epidemiological role of the different species of migratory and resident birds is discussed, in relation to the different ecological patterns identified in the area and their potential ability to introduce, spread and support the endemization of WNV infection.

Volumetric modulated arc therapy with simultaneous integrated boost for locally advanced rectal cancer.

AIMS: To report the feasibility of volumetric modulated arc therapy (VMAT) for neoadjuvant radiotherapy in locally advanced rectal cancer in a dose-escalation protocol and simultaneous integrated boost (SIB) approach. Moreover, the VMAT technique was compared with three-dimensional conformal radiotherapy (3D-CRT) and fixed-field intensity modulated radiotherapy (IMRT), in terms of target coverage and irradiation of organs at risk. MATERIALS AND METHODS: Eight patients with locally advanced rectal cancer were treated with the SIB-VMAT technique. The VMAT plans were compared with 3D-CRT and IMRT techniques in terms of several clinically dosimetric parameters. The number of monitor units and the delivery time were analysed to score the treatment efficiency. All plans were verified in a dedicated solid water phantom using a two-dimensional array of ionisation chambers. RESULTS: All techniques meet the prescription goal for planning target volume coverage, with VMAT showing the highest level of conformality. VMAT is associated with 40, 53 and 58% reduction in the percentage of volume of small bowel irradiated to 30, 40 and 50Gy, compared with 3D-CRT. No significant differences were found with respect to SIB-IMRT. VMAT plans showed a significant reduction of monitor units by nearly 20% with respect to IMRT and reduced treatment time from 14 to 5min for a single fraction. CONCLUSIONS: SIB-VMAT plans can be planned and carried out with high quality and efficiency for rectal cancer, providing similar sparing of organs at risk to SIB-IMRT and resulting in the most efficient treatment option. SIB-VMAT is currently our standard approach for radiotherapy of locally advanced rectal cancer.

Real-time dose reconstruction for wedged photon beams: a generalized procedure.

A practical and accurate generalized procedure to reconstruct the isocenter dose D(iso) for 3D conformal radiotherapy (3DCRT) has been developed for X-ray open beams supplied by linacs of different manufacturers and equipped with aSi electronic portal imaging devices (aSi EPIDs). This paper reports an extension of the method, to be applied at the wedged X-ray beams characterized by the wedge attenuation factor W(AF). Using water-equivalent solid phantoms (SPs) of different thicknesses, w, and photon square fields of sizes, L, the generalized midplane doses D(0)(W(AF), w/2,L) and generalized transit signals s(t)(0)(W(AF),w,L) by 38 beams of six different linacs were determined. The generalized data were fitted by surface equations and used together with the information of the 'record & verify' network of the centers. In this manner, for every beam, the D(iso) reconstruction was obtained in about 25 seconds after the treatment. To test the in vivo dosimetric procedure, six pelvic treatments that used conformed wedged beams were carried out with three linacs of different manufacturers. For every beam, the comparison between the reconstructed D(iso) and the D(iso,TPS) computed by the TPS, resulted in an acceptable tolerance level of ±5%, estimated for this kind of treatment. Generally the in vivo dosimetry methods that use EPIDs require: (i) a special effort for the dosimetric commissioning with SPs of different thicknesses, and (ii) extra time for the analysis of the EPID signals. The proposed procedure simplifies the commissioning step and supplies for Varian, Elekta, and Siemens linacs equipped with the aSi EPIDs a quasi-real time in vivo dosimetry for open and wedged 3DCRT fields.

Calibration of Elekta aSi EPIDs used as transit dosimeter.

The transit in vivo dosimetry performed by the Electronic Portal Imaging Device (EPID), avoids the problem of solid-state detector positioning on the patient. Moreover, the dosimetric characterization of the recent Elekta aSi EPIDs in terms of signal stability and linearity enables these detectors adaptable for the transit in vivo dosimetry with 6, 10 and 15 MV photon beams. However, the implementation of the EPID transit dosimetry requires several measurements. Recently, the present authors have developed an in vivo dosimetry method for the 3D CRT based on correlation functions defined by the ratios between the transit signal, s(t) (w,L), by the EPID and the phantom mid-plane dose, D(m)(w,L), at the Source to Axis Distance (SAD) as a function of the phantom thickness, w, and the square field dimensions, L. When the phantom mid-plane was positioned at distance d from the SAD, the ratios st(w,L)/s't(d,w,L), were used to take into account the variation of the scattered photon contributions on the EPID as a function of, d and L. The aim of this paper was the implementation of a procedure that uses generalized correlation functions obtained by nine Elekta Precise linac beams. The procedure can be used by other Elekta Precise linacs equipped with the same aSi EPIDs assuring the stabilities of the beam output factors and the EPID signals. The calibration procedure of the aSi EPID here reported avoids measurements in solid water equivalent phantoms needed to implement the in vivo dosimetry method in the radiotherapy center. A tolerance level ranging between ±5% and ±6% (depending on the type of tumor) was estimated for the comparison between the reconstructed isocenter dose, D(iso) and the computed dose D(iso,TPS) by the treatment planning system (TPS).

gamma-ray spectrometry of soil samples from the Provincia dell'Aquila (Central Italy).

In the Abruzzo Region (Central Italy) there is a lack of measurements of gamma-ray activity in soils and waters. For this reason, we have planned to carry out a systematic measurement of soils in the area of the Provincia dell'Aquila, which covers about one-half of the entire region. In this paper we report the results obtained from 56 soil samples, collected in the northern part of the area of interest (about one-fourth of the total area under study). The results, in terms of content of uranium, thorium and potassium and the activity of caesium are reported, as well as the details on the experimental procedure. The results show a limited content of K and U, with no large variations from site to site, in agreement with the expectations based on the knowledge of the geo-lithological nature of the soil. The amount of Th is also quite limited, with a few exceptions where the Th content is up to five times the average value. Caesium, originated from the fall-out following the Chernobyl accident, is very irregularly distributed owing to the complicated orography of the land. Future plans are also shortly discussed.

Solid and papillary epithelial neoplasm of the pancreas in an 11-year-old girl: case report and literature review.

An 11-year-old girl presented with episodic abdominal pain of 2 years' duration. CT scan of the abdomen showed a mass in the tail of the pancreas. A distal pancreatectomy was done and the tumor was excised. Macroscopic and immunohistochemical studies were compatible with a solid and papillary epithelial neoplasm. This is a rare neoplasm with a decidedly female predominance. It has a very low malignant potential with a good prognosis. Surgical removal of the tumor is usually curative.

Optimized production of high-titer recombinant adeno-associated virus in roller bottles.

Adeno-associated viral (AAV) vectors are used for in vivo gene transfer in a number of preclinical models of genetic diseases (including large-animal models) and are currently being tested in clinical trials for treatment of hemophilia B and cystic fibrosis. Protocols for production of AAV vectors in a helper virus-free system are available and are based on transient transfection of HEK-293 cells with multiple plasmids. Scale-up of vector production has been labor intensive and inefficient because of a lack of larger culture vessels suitable for growth of adherent cells, large-scale transfection, and vector production. Here we report efficient production of AAV vector in roller bottles, which represents a 10-fold scale-up from the conventional flask or plate method. Optimized production yielded greater than 10(13) vector genomes per bottle and was as cost effective as published protocols using plates. Successful vector production by this method was dependent on optimization of transfection by calcium phosphate precipitation, of monitoring of cell growth (by measurement of glucose consumption), of cell culture conditions, and CO2/air exchange with the culture vessel.

Erythrocyte ankyrin promoter mutations associated with recessive hereditary spherocytosis cause significant abnormalities in ankyrin expression.

Ankyrin defects are the most common cause of hereditary spherocytosis (HS). In several kindreds with recessive, ankyrin-deficient HS, mutations have been identified in the ankyrin promoter that have been proposed to decrease ankyrin synthesis. We analyzed the effects of two mutations, -108T to C and -108T to C in cis with -153G to A, on ankyrin expression. No difference between wild type and mutant promoters was demonstrated in transfection or gel shift assays in vitro. Transgenic mice with a wild type ankyrin promoter linked to a human (A)gamma-globin gene expressed gamma-globin in 100% of erythrocytes in a copy number-dependent, position-independent manner. Transgenic mice with the mutant -108 promoter demonstrated variegated gamma-globin expression, but showed copy number-dependent and position-independent expression similar to wild type. Severe effects in ankyrin expression were seen in mice with the linked -108/-153 mutations. Three transgenic lines had undetectable levels of (A)gamma-globin mRNA, indicating position-dependent expression, and four lines expressed significantly lower levels of (A)gamma-globin mRNA than wild type. Two of four expressing lines showed variegated gamma-globin expression, and there was no correlation between transgene copy number and RNA level, indicating copy number-independent expression. These data are the first demonstration of functional defects caused by HS-related, ankyrin gene promoter mutations.

Development of a stable retrovirus vector capable of long-term expression of gamma-globin mRNA in mouse erythrocytes.

Gene therapy for patients with hemoglobin disorders such has been hampered by the inability of retrovirus vectors to transfer globin genes and the locus control region (LCR) into hematopoietic stem cells without rearrangement. In addition, the expression from intact globin gene vectors has been variable in red blood cells as a result of position effects and retrovirus silencing. We hypothesized that by substituting the globin gene promoter for the promoter of another gene expressed in red blood cells, we could generate stable retrovirus vectors that would express globin at sufficient levels to treat hemoglobinopathies. Transgenic mice containing the human ankyrin (Ank) gene promoter fused to the human gamma-globin gene showed position-independent, copy number-dependent expression of a linked gamma-globin mRNA. We generated a "double-copy" Ank/A gamma-globin retrovirus vector that transferred two copies of the Ank/A gamma-globin gene into target cells. Stable gene transfer was observed in primary primary mouse progenitor cells and long-term repopulating hematopoietic stem cells. Expression of Ank/A gamma-globin mRNA in mature red blood cells was approximately 8% of the level of mouse alpha-globin mRNA. We conclude that this novel retrovirus vector may be valuable for treating a variety of hemoglobinopathies by gene therapy if the level of expression can be further increased.

Functional requirements for phenotypic correction of murine beta-thalassemia: implications for human gene therapy.

As initial human gene therapy trials for beta-thalassemia are contemplated, 2 critical questions important to trial design and planning have emerged. First, what proportion of genetically corrected hematopoietic stem cells (HSCs) will be needed to achieve a therapeutic benefit? Second, what level of expression of a transferred globin gene will be required to improve beta-thalassemic erythropoiesis? These questions were directly addressed by means of a murine model of severe beta-thalassemia. Generation of beta-thalassemic mice chimeric for a minority proportion of genetically normal HSCs demonstrated that normal HSC chimerism levels as low as 10% to 20% resulted in significant increases in hemoglobin (Hb) level and diminished extramedullary erythropoiesis. A large majority of the peripheral red cells in these mice were derived from the small minority of normal HSCs. In a separate set of independent experiments, beta-thalassemic mice were bred with transgenic mice that expressed different levels of human globins. Human gamma-globin messenger RNA (mRNA) expression at 7% of the level of total endogenous alpha-globin mRNA in thalassemic erythroid cells resulted in improved red cell morphology, a greater than 2-g/dL increase in Hb, and diminished reticulocytosis and extramedullary erythropoiesis. Furthermore, gamma-globin mRNA expression at 13% resulted in a 3-g/dL increase in Hb and nearly complete correction of red cell morphology and other indices of inefficient erythropoiesis. These data indicate that a significant therapeutic benefit could be achieved with expression of a transferred globin gene at about 15% of the level of total alpha-globin mRNA in patients with severe beta-thalassemia in whom 20% of erythroid precursors express the vector genome.