Development of Bioglass/PEEK Composite Coating by Cold Gas Spray for Orthopedic Implants.

Cold gas spray (CGS) technology has allowed the development of biofunctional coatings composed of 45S5 and polyetheretherketone (PEEK). The combination of a bioactive glass material embedded in a polymeric matrix makes this composite an interesting material for orthopedic applications since this composite meets the biomechanical and biological requirements of an implant. In the present study, blends of bioactive glass 45S5 and PEEK powder with different granulometry and 45S5/PEEK ratio have been prepared. These mixtures of powders have been deposited onto PEEK substrates by CGS with the goal of incorporating a bioactive additive to the biocompatible polymer, which can improve the bone-implant interaction of PEEK. The deposition efficiency (DE) of the coatings has been evaluated, and from the results obtained, it was possible to conclude that DE is significantly affected by the granulometry and by the 45S5/PEEK ratio of the blends. By scanning electron microscopy (SEM) inspection, it was observed that the use of blends with high 45S5/PEEK ratio lead to the deposition of coatings with high content of 45S5. Finally, the friction behavior of the coatings was analyzed performing ball-on-disk tests and these experiments showed that the presence of glass particles has a beneficial role in the wear resistance.

Osteoblastic cell response on high-rough titanium coatings by cold spray.

Highly rough and porous commercially pure titanium coatings have been directly produced for first time by the cold spray technology, which is a promising technology in front of the vacuum plasma spray for oxygen sensitive materials. The wettability properties as well as the biocompatibility evaluation have been compared to a simply sand blasted Ti6Al4V alloy substrate. Surface topographies were analysed using confocal microscopy. Next, osteoblast morphology (Phalloidin staining), proliferation (MTS assay), and differentiation (alkaline phosphatase activity) were examined along 1, 7 and 14 days of cell culture on the different surfaces. Finally, mineralization by alizarin red staining was quantified at 28 days of cell culture. The contact angle values showed an increased hydrophilic behaviour on the as-sprayed surface with a good correlation to the biological response. A higher cell viability, proliferation and differentiation were obtained for highly rough commercial pure titanium coatings in comparison with sand blasted substrates. Cell morphology was similar in all coatings tested; at 14 days both samples showed extended filopodia. A higher amount of calcium-rich deposits was detected on highly rough surfaces. In summary, in-vitro results showed an increase of biological properties when surface roughness increases.

Long-term results after treatment of pertrochanteric femoral fractures with percutaneous compression plate (PCCP).

OBJECTIVE: This study sought to evaluate the long-term osteosynthesis results for AO/OTA 31.A1-A2 pertrochanteric fractures treated with the percutaneous compression plate (PCCP). MATERIALS AND METHODS: This investigation was a retrospective observational descriptive study of 335 patients, with a minimum follow-up of 2 years and a maximum follow-up of 8 years (2004-2011). RESULTS: The average post-operative hospital stay was 6.2 days, and the average decrease in haemoglobin levels after the intervention was 2.7 mg/dL, with transfusion required for one-third of the patients. Complications related to the implant were observed in 4.2 % of patients; the most notable complication was cut-out (2.4 % of patients), and 3 cases involved pseudarthrosis with breakage of the implant (0.9 % of patients). CONCLUSIONS: From the results obtained in this study and an analysis of previously published work, we believe that the PCCP may be the implant of choice for AO/OTA 31.A1-A2 fractures.

Effects of lung ventilation-perfusion and muscle metabolism-perfusion heterogeneities on maximal O2 transport and utilization.

KEY POINTS: We expanded a prior model of whole-body O2 transport and utilization based on diffusive O2 exchange in the lungs and tissues to additionally allow for both lung ventilation-perfusion and tissue metabolism-perfusion heterogeneities, in order to estimate V̇O2 and mitochondrial PO2 (PmO2) during maximal exercise. Simulations were performed using data from (a) healthy fit subjects exercising at sea level and at altitudes up to the equivalent of Mount Everest and (b) patients with mild and severe chronic obstructive pulmonary disease (COPD) exercising at sea level. Heterogeneity in skeletal muscle may affect maximal O2 availability more than heterogeneity in lung, especially if mitochondrial metabolic capacity (V̇ MAX ) is only slightly higher than the potential to deliver O2 , but when V̇ MAX is substantially higher than O2 delivery, the effect of muscle heterogeneity is comparable to that of lung heterogeneity. Skeletal muscle heterogeneity may result in a wide range of potential mitochondrial PO 2 values, a range that becomes narrower as V̇ MAX increases; in regions with a low ratio of metabolic capacity to blood flow, PmO2 can exceed that of mixed muscle venous blood. The combined effects of lung and peripheral heterogeneities on the resistance to O2 flow in health decreases with altitude. ABSTRACT: Previous models of O2 transport and utilization in health considered diffusive exchange of O2 in lung and muscle, but, reasonably, neglected functional heterogeneities in these tissues. However, in disease, disregarding such heterogeneities would not be justified. Here, pulmonary ventilation-perfusion and skeletal muscle metabolism-perfusion mismatching were added to a prior model of only diffusive exchange. Previously ignored O2 exchange in non-exercising tissues was also included. We simulated maximal exercise in (a) healthy subjects at sea level and altitude, and (b) COPD patients at sea level, to assess the separate and combined effects of pulmonary and peripheral functional heterogeneities on overall muscle O2 uptake (V̇O2) and on mitochondrial PO2 (PmO2). In healthy subjects at maximal exercise, the combined effects of pulmonary and peripheral heterogeneities reduced arterial PO2 (PaO2) at sea level by 32 mmHg, but muscle V̇O2 by only 122 ml min(-1) (-3.5%). At the altitude of Mt Everest, lung and tissue heterogeneity together reduced PaO2 by less than 1 mmHg and V̇O2 by 32 ml min(-1) (-2.4%). Skeletal muscle heterogeneity led to a wide range of potential PmO2 among muscle regions, a range that becomes narrower asV̇ MAX increases, and in regions with a low ratio of metabolic capacity to blood flow, PmO2 can exceed that of mixed muscle venous blood. For patients with severe COPD, peak V̇O2 was insensitive to substantial changes in the mitochondrial characteristics for O2 consumption or the extent of muscle heterogeneity. This integrative computational model of O2 transport and utilization offers the potential for estimating profiles of PmO2 both in health and in diseases such as COPD if the extent for both lung ventilation-perfusion and tissue metabolism-perfusion heterogeneity is known.

An experimental means of transmitting pancreas disease in Atlantic salmon Salmo salar L. fry in freshwater.

A challenge model for pancreas disease in Atlantic salmon, Salmo salar L. fry, was developed comparing two salmonid alphavirus (SAV) subtypes: SAV1 and SAV5. Viral doses of 3 × 10(5) TCID50  mL(-1) for SAV1 and 3 × 10(4) for SAV5 were tested in triplicate tanks, each containing 450 salmon fry. Cumulative mortalities of 1.2% were recorded. Titres of virus recovered from the mortalities ranged from 10(2) to 10(7) TCID50  mL(-1) . Fry were sampled at 3, 5 and 7.5 weeks post-challenge. Sampling after 3 weeks revealed a high prevalence of infection in the absence of clinical signs, and infectious virus was recovered from 80% and 43% of sampled fry infected with SAV1 and SAV5, respectively. After 5 weeks pancreas, heart and red skeletal muscle lesions were generally observed, whilst degeneration in white skeletal muscle was observed only in fish infected with SAV1. In situ hybridisation confirmed the presence of viral genome in infected pancreas, heart and muscle. After 7.5 weeks, infectious virus (both isolates) was recovered from 13.3% of the fish sampled, with a viral titre of 10(2) TCID50  mL(-1) . Clearly, salmon fry are susceptible to SAV infection and pancreas disease.

Transmission of lymphocystis disease virus to cultured gilthead seabream, Sparus aurata L., larvae.

The transmission of lymphocystis disease virus (LCDV) to gilthead seabream, Sparus aurata L., larvae was investigated using fertilized eggs from a farm with previous reports of lymphocystis disease. LCDV genome was detected by PCR-hybridization in blood samples from 17.5% of the asymptomatic gilthead seabream broodstock analysed. Using the same methodology, eggs spawned from these animals were LCDV positive, as well as larvae hatched from them. The presence of infective viral particles was confirmed by cytopathic effects development on SAF-1 cells. Whole-mount in situ hybridization (ISH) and immunohistochemistry (IHC) showed the presence of LCDV in the epidermis of larvae hatched from LCDV-positive eggs. When fertilized eggs were disinfected with iodine, no viral DNA was detected either in eggs (analysed by PCR-hybridization) or in larvae (PCR-hybridization and ISH). These results suggest the vertical transmission of LCDV, the virus being transmitted on the egg surface. Larvae hatched from disinfected eggs remain LCDV negative during the endotrophic phase, as showed by PCR-hybridization, ISH and IHC. After feeding on LCDV-positive rotifers, viral antigens were observed in the digestive tract, which suggests that viral entry could be achieved via the alimentary canal, and that rotifers can act as a vector in LCDV transmission to gilthead seabream larvae.

[Current problems in training of minimally invasive surgery: vision of residents in pediatric surgery]. / Problemas actuales en la formación en cirugía mínimamente invasiva: visión de los residentes de Cirugía pediátrica.

INTRODUCTION: Minimally Invasive Surgery (MIS) is getting more and more important in our specialty. However, the formation of the residents on MIS is, in many cases, irregular. The purpose of this study is to assess the state of training in MIS among the residents of Pediatric Surgery and their potential weaknesses. MATERIAL AND METHODS: An electronic survey was distributed among 71 residents of Pediatric Surgery from 17 national hospitals. RESULTS: The response rate was 70.2%.100% of the residents are interested in a broadening of training activities in MIS. The main areas of interest are gastrointestinal (92%) and thoracic (47%) surgery. Only 57% have access to training facilities and less than half of them attend to courses and conferences. 80% believe that they are not given adequate attention from specialized associations. 52% think they should do rotations at referral centers, 86% that courses and seminars should be enhanced, and 44% that the responsibility of the resident in surgery should be increased. The main defects encountered in their training are scarce volume of patients, lack of financial support and overcoming the learning curve of surgeons in their services. CONCLUSIONS: Despite the advance of the MIS, resident's training in this discipline still has shortcomings, as expressed in their views. Knowledge of the current state of training should be the starting point for designing a training strategy that ensures adequate skills.

Fluoxetine increased adult neurogenesis is mediated by 5-HT3 receptor.

Adult neurogenesis is an aspect of structural plasticity that remains active during adulthood in some brain regions. One of them is the subgranular zone (SGZ) of the dentate gyrus of the hippocampus. Adult neurogenesis is reduced by different factors and in disorders of the CNS, including major depression. Antidepressant treatments, such as chronic fluoxetine administration, recover the normal level of adult neurogenesis. Fluoxetine treatment increases the free concentration of the neurotransmitter serotonin and this monoamine is implicated in the regulation of the neurogenic process; however, the target of the action of this neurotransmitter has not been fully elucidated. In this study, we have tried to determine the relevance of the serotonin receptor 3 (5-HT3) in the hippocampal neurogenesis of adult rats. We have used fluorescent immunohistochemistry to study the expression of the 5-HT3 receptor in different neurogenesis stages in the SGZ, identifying its expression in stem cells, amplifying neural progenitors and immature neurons. Moreover, we have studied the impact of a 5-HT3 antagonist (ondansetron) in the fluoxetine-induced adult neurogenesis. We observed that fluoxetine alone increases the number of both proliferating cells (ki67 positive) and immature neurons (DCX positive) in the SGZ. By contrast, co-treatment with ondansetron blocked the increase in proliferation and neurogenesis. This study demonstrates that the activation of 5-HT3 receptors is necessary for the increase of adult neurogenesis induced by fluoxetine.

Dimensioning of OFDMA PON with non-preselected independent ONUs sources and wavelength-control.

A simple and low cost method for wavelength control of economical random non-preselected independent ONU sources is shown to increase the number of users in an OFDMA-PON. The method is based on OLT monitoring and thermal tuning control; it has been validated through Monte-Carlo simulations and a probabilistic model. The minimum optical spectral gap between the ONUs wavelengths that guarantees a tolerable amount of optical beat interference has been determined through an experiment.

[Cardiovascular and respiratory changes during laparoscopy in neonates and infants]. / Cambios cardiovasculares y ventilatorios durante la laparoscopia en neonatos y lactantes pequeños.

The pneumoperitoneum induced during laparoscopy has cardiovascular and respiratory effects that have to be taken into account to perform laparoscopic procedures in neonates and small infants. The aim of this paper is to describe the hemodynamic and ventilatory changes that occur in a group of these patients undergoing standard laparoscopic procedure. We performed a retrospective review of patients undergoing laparoscopic pyloromyotomy in our institution. We collected demographic data, data from the intervention, outcome and complications and hemodynamic parameters (heart rate--HR--and blood pressure--BP--) and respiratory (O2 saturation--SO2--and exhaled CO2--ECO2--) at different times during surgery. We performed statistical analysis of these data. We reviewed a total of 55 charts. Mean age was 34 days and average weight 3,785 g. All patients were ventilated with pressure control mode and received maintenance fluid therapy. BP, HR and SO2 were stable throughout the surgery not showing significant differences. The ECO2 increased significantly after inflation, however it was controlled by increasig respiratory rate. In conclusion, the hemodynamic and ventilatory response in these patients is controllable with anesthetic and surgical measures but it is important to consider the possible consequences of pneumoperitoneum in this group of patients.

Changes in complement responses in Gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) under crowding stress, plus viral and bacterial challenges.

Gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) were subjected to either experimental infection with Photobacterium damselae subsp. piscicida or Nodavirus after a period of 2 weeks of crowding in which fish were subjected to a 5-fold increase in density (10-50 kg/m(3)). Samples were obtained before the crowding period (0 h or control) and at 24h and 72 h after crowding from both groups of infected fish. The Complement haemolytic activity and the expression of the C3 gene were evaluated in blood and liver samples respectively. The bacteriolytic and lysozyme activities were also assessed. The results showed that Complement haemolytic activity was reduced at 72 h with both bacteria and virus in high density Gilthead seabream, and a similar increase was observed at low density. Bacteriolytic activity under both bacterial and viral challenges for both species was increased at 24h, under low density. At high density, the bacterial challenge did not induce significant changes. C3 mRNA abundance was substantially increased after pathogen treatments in low density groups at 24h but no significant changes were detected at high densities. These results support the idea of the suppressor effect of stressors on the immune system since a reduction of Complement activity under virus and high density, or lack of response in C3 expression under high density were observed.

Detection, cellular localization and antibacterial activity of two lytic enzymes of Pediococcus acidilactici ATCC 8042.

AIM: In Pediococcus acidilactici ATCC 8042, two activities of peptidoglycan hydrolase (PGH) with lytic effect against Micrococcus lysodeikticus and Staphylococcus aureus have been detected. This work intends to elucidate the growth phase of maximum lytic activity, the localization and the effectiveness of the activity against pathogenic Gram-negative and Gram-positive bacteria. METHODS AND RESULTS: Cells were grown in MRS medium and collected at different growth stages, and the proteins were extracted. The highest PGH activity was found during the logarithmic growth phase in the protein fraction bound to the cell membrane. From this fraction, two distinct proteins bands (110- and 99-kDa) in SDS-PAGE were partially purified with a three-step procedure. Both bands showed lytic activity against M. lysodeikticus. Mass spectrometry analysis (LC/ESI-MS/MS) indicated that the 110-kDa band corresponded to a protein of unknown function. The 99-kDa band corresponded to a N-acetylmuramidase that harboured catalytic sites with N-acetylmuramoyl-L-alanine amidase and N-acetylglucosaminidase activities. Both proteins are reported in the Ped. acidilactici 7_4 genome. The fraction containing the concentrated proteins (110 and 99 kDa) inhibited the growth of several pathogenic strains as: Bacillus cereus, Listeria monocytogenes and Salmonella typhimurium. The growth of S. aureus was diminished by 3 logarithmic units as early as 0.5 h of growth, while inhibition of Escherichia coli and Ped. acidilactici was observed after 18 and 8 h, respectively (both in one logarithmic unit). The minimum inhibitory concentration against S. aureus was 10 µg ml(-1). CONCLUSION: Pediococcus acidilactici harbours at least two lytic enzymes, one of them recognized as PGH for the first time, which exert antibacterial activity against several bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Both PGH activities have a broad growth inhibition spectrum and could be used to control pathogenic bacteria. Because this activity comes from a lactic acid bacterium, it could be safely used in manufacturing processes of fermented foods.

[Conservative treatment of the pectus carinatum]. / Tratamiento conservador del pectus carinatum.

INTRODUCTION: Pectus carinatum (PC) is a deformity that involves the protrusion of the anterior chest wall. It is 10 times less frequent than pectus excavatum. It has a progressive growth and is more common with men. There are two different types, the lower or condrocorporal which is the most common one, and the upper or condromanubrial. Most of the time there are no cardio-respiratory symptoms. OBJECTIVE: We present our experience in the orthopedic treatment of the pectus carinatum. METHOD: Retrospective review of patients treated in our hospital from 2002 until 2009. Patients were treated with observation, aerobic exercises, postural change and/or compression braces. Literature review was performed of the treatment for this pathology. RESULTS: 18 patients have been diagnosed with PC, 16 were men and 2 women. All were treated in a nonoperative way. Only 11 of them used a compression brace. We missed two follow-ups and another has just yet begun to achieve proper results. All the rest have had excellent results with nonoperative treatment. None of them have had a surgical treatment. CONCLUSION: The PC is a disease that most often is a cosmetic problem, with no impact on a cardio-respiratory level. Classically it has been a surgical entity. In our experience we have found that the orthopedic method is an effective alternative, safe and with a significant reduction in morbidity. But we need the collaboration of the patient to accept and maintain continuity in the use of the prostheses.

[Laparoscopy as diagnostic-therapeutic method in abdominal traumatism in the pediatric age]. / Laparoscopia como método diagnóstico-terapéutico en el traumatismo abdominal en la edad pediátrica.

The management of blunt abdominal traumatism with a moderate amount of free peritoneal fluid and without solid organ injury as well as the one of minimal penetrating trauma is controversial. We present three cases of blunt abdominal trauma and two of penetrating trauma that underwent diagnostic laparoscopy in our department. We found a small bowel perforation in one of the cases of blunt trauma that was repaired by externalization of the jejuna loop by one of the ports. In the other two cases we found intestinal and mesenteric contusions and free fluid that were treated by peritoneal drainage. One of the cases of penetrating trauma presented omentum evisceration with no other injuries and the second presented a gastric perforation that needed reconversion to laparotomy. In our experience and according to literature, laparoscopy should be taken into account as a diagnostic procedure and sometimes also therapeutic in selected cases of both blunt and penetrating abdominal trauma in pediatric population.

[Minimally invasive surgery in pediatric patients with ventriculo-peritoneal shunts]. / Cirugía laparoscópica en pacientes con válvula de derivación ventrículo-peritoneal.

The use of laparoscopy in patients with ventriculo-peritoneal shunts has been controversial. There is concern about the risk malfunction or infection of the shunt as well as about the possibility of adhesions that could make the surgical procedures too difficult to be undertaken by minimally invasive surgery. We present our results in pediatric patients with ventriculoperitoneal shunts that have undergone laparoscopic procedures in our department. We have performed 16 surgical procedures. The most frequent indication was fundoplication. During surgery there were no complications or intracranial hypertension episodes. Two of the procedures were considered by the surgeons more difficult and longer than usual. None of them required conversion to open technique. One shunt had to be replaced after surgery because of infection. According to our experience minimally invasive surgery is feasible and safe in pediatric patients with ventriculo-peritoneal shunts.

Role of the orthopaedic surgeon in 3D printing: current applications and legal issues for a personalized medicine. / Papel del cirujano ortopédico y traumatólogo en la impresión 3D: aplicaciones actuales y aspectos legales para una medicina personalizada.

3D printing (I3D) is an additive manufacturing technology with a growing interest in medicine and especially in the specialty of orthopaedic surgery and traumatology. There are numerous applications that add value to the personalised treatment of patients: advanced preoperative planning, surgeries with specific tools for each patient, customised orthotic treatments, personalised implants or prostheses and innovative development in the field of bone and cartilage tissue engineering. This paper provides an update on the role that the orthopaedic surgeon and traumatologist plays as a user and prescriber of this technology and a review of the stages required for the correct integration of I3D into the hospital care flow, from the necessary resources to the current legal recommendations.

Circulating miR-30b-5p levels in plasma as a novel potential biomarker for early detection of breast cancer.

BACKGROUND: Recently, microRNAs have been demonstrated to be potential non-invasive biomarkers for diagnosis, prognosis assessment or prediction of response to treatment in cancer. In this study, we evaluate the potential of miR-30b-5p as a biomarker for early diagnosis of breast cancer (BC) in tissue and plasma. METHODS: Expression of miR-30b-5p was determined in a series of 112 BC and 40 normal breast tissues. Circulating miR-30b-5p levels in plasma samples were determined in a discovery cohort of 38 BC patients and 40 healthy donors and in a validation cohort of 83 BC patients and 83 healthy volunteers. miR-30b-5p expression was measured by quantitative real-time PCR and receiver operating characteristics curve analysis was carried out. RESULTS: The miR-30b-5p expression was significantly lower in BC tissue than in healthy breast samples. In contrast, circulating miR-30b-5p levels were significantly higher in BC patients compared with healthy donors. Furthermore, circulating miR-30b-5p levels were significantly higher in patients with positive axillary lymph node and de novo metastatic patients. Receiver operating characteristics curve analysis demonstrated a good diagnostic potential of miR-30b-5p to detect BC even at an early stage of the disease. CONCLUSION: Thus, we highlight the potential of miR-30b-5p as a non-invasive, fast, reproducible and cost-effective diagnostic biomarker of BC.

Cellular and molecular immune responses of the sea bass (Dicentrarchus labrax) experimentally infected with betanodavirus.

Naïve sea bass juveniles (38.4 + or - 4.5 g) were intramuscularly infected with a sublethal dose of betanodavirus isolate 378/I03, followed after 43 days by a similar boosting. This infection resulted in an overall mortality of 7.6%. At various intervals, sampling of fish tissues was performed to investigate: i) B and T lymphocyte content in organs and tissues; ii), proliferation of leucocytes re-stimulated in vitro with inactivated virus; iii) presence of serum antibody specific for betanodavirus; iv) expression of genes coding for the following immunoregulatory molecules involved in innate and acquired responses: type I IFN, Mx, IL-1, Cox-2; IL-10, TGF-beta, TCRbeta, CD4, CD8alpha, IgM, by using a quantitative PCR array system developed for sea bass. The obtained results showed a detectable increase of T cells and B cells in PBL during betanodavirus infection. Furthermore, leucocytes obtained from blood, head kidney, and gills showed a detectable "in vitro" increase in viability upon addition of inactivated viral particles, as determined by measuring intracellular ATP concentration. ELISA analysis of sera showed that exposure to nodavirus induced a low, but specific antibody titer measured 43 days after infection, despite the presence of measurable levels of natural antibody. Finally, a strong upregulation of genes coding for type I IFN, Mx, and IgM was identified after both infection and boosting. Interestingly, an upregulation of Cox-2 until boosting, and of TGF-beta and IL-10 after boosting was also observed, while the other tested genes did not show any significant variations with respect to mock-treated fish. Overall, our work represents a first comprehensive analysis of cellular and molecular immune parameters in a fish species exposed to a pathogenic virus.

A combined RT-PCR and dot-blot hybridization method reveals the coexistence of SJNNV and RGNNV betanodavirus genotypes in wild meagre (Argyrosomus regius).

AIMS: To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study. METHODS AND RESULTS: The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin-labelled probes resulted in the identification of both genotypes separately. The application of the RT-PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46.87% of viral nervous necrosis virus carriers. The combination of RT-PCR and blot hybridization increases the detection rate up to 90.62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56.25%). CONCLUSIONS: This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time.

Detection of infectious pancreatic necrosis virus (IPNV) from asymptomatic redbanded seabream, Pagrus auriga Valenciennes, and common seabream, Pagrus pagrus (L.), using a non-destructive procedure.

A non-destructive procedure based on nested RT-PCR and dot-blot hybridization has been developed for the detection of asymptomatic IPNV-carrier fish. The pair of primers designed for RT-PCR amplified a 599-bp fragment of the pVP2 region within the polyprotein gene, resulting in the detection of IPNV genotype III.1. The use of a nested RT-PCR allowed the amplification of IPNV genotypes III.1 and I.2. In addition, a 191-bp probe was designed for hybridization studies used in combination with the nested RT-PCR. The application of the nested RT-PCR to analyse blood samples from asymptomatic redbanded seabream, Pagrus auriga, and common seabream, P. pagrus, specimens showed a 53.1% and 77.8% prevalence of IPNV-carriers, respectively. The combination of nested RT-PCR and dot-blot hybridization increased the detection rates up to 100% for redbanded seabream and 94.4% for common seabream. Therefore, the protocol described in this study is highly sensitive and specific for the detection of IPNV in asymptomatic carrier fish, and, in addition, the results demonstrate the carrier state in two newly cultured sparid species in southern Spain.