Transition of Adolescents With Severe Asthma From Pediatric to Adult Care in Spain: The STAR Consensus.

OBJECTIVES: To assess the degree of consensus among a multidisciplinary expert panel on the transition of adolescents with severe asthma from pediatric to adult care. METHODS: A 61-item survey was developed based on guidelines for other chronic diseases, covering transition planning, preparation, effective transfer, and follow-up. A 2-round Delphi process assessed the degree of consensus among 98 experts (49 pediatricians, 24 allergists, and 25 pulmonologists). Consensus was established with ≥70% agreement. RESULTS: Consensus was reached for 42 items (70%). Panelists were unable to agree on an age range for initiation of transition. The main goal during the transition identified by the experts is for adolescents to gain autonomy in managing severe asthma and prescribed treatments. The panelists agreed on the importance of developing an individualized plan, promoting patient autonomy, and identifying factors associated with the home environment. They agreed that the adult health care team should have expertise in severe asthma, biologics, and management of adolescent patients. Pediatric and adult health care teams should share clinical information, agree on the criteria for maintaining biological therapy, and have an on-site joint visit with the patient before the effective transfer. Adult health care professionals should closely follow the patient after the effective transfer to ensure correct inhaler technique, adherence, and attendance at health care appointments. CONCLUSION: This consensus document provides the first roadmap for Spanish pediatric and adult teams to ensure that key aspects of the transition process in severe asthma are covered. The implementation of these recommendations will improve the quality of care offered to the patient.

Outcomes of an enhanced recovery after radical cystectomy program in a prospective multicenter study: compliance and key components for success.

OBJECTIVE: To investigate the effect of an Enhanced Recovery After Surgery (ERAS) program on complications and length of stay (LOS) after radical cystectomy (RC) and to assess if the number and type of components of ERAS play a key role on the decrease of surgical morbidity. MATERIALS AND METHODS: We analyzed the data of 277 patients prospectively recruited in 11 hospitals undergoing RC initially managed according to local practice (Group I) and later within an ERAS program (Group II). Two main outcomes were defined: 90-day complications rate and LOS. As secondary variables we studied 90-day mortality, 30-day readmission and transfusion rate. RESULTS: Patients in Group II had a higher use of ERAS measures (98.6%) than those in Group I (78.2%) (p < 0.05). Patients in Groups I and II experienced similar complications (70.5% vs. 66%, p = 0.42). LOS was not different between Groups I and II (12.5 and 14 days, respectively, p = 0.59). The risk of having any complication decreases for patients having more than 15 ERAS measures adopted [RR = 0.815; 95% confidence interval (CI) 0.667-0.996; p = 0.045]. Avoidance of transfusion and nasogastric tube, prevention of ileus, early ambulation and a fast uptake of a regular diet are independently associated with the absence of complications. CONCLUSIONS: Complications and LOS after RC were not modified by the introduction of an ERAS program. We hypothesize that at least 15 measures should be applied to maximize the benefit of ERAS.

Immune gene expression in gilthead seabream (Sparus aurata) after Lymphocystis disease virus (LCDV-Sa) challenge resulting in asymptomatic infection.

AIMS: To determine the immune gene expression response of gilthead seabream (Sparus aurata) that is experimentally infected with the lymphocystivirus LCDV-Sa. METHODS AND RESULTS: Viral DNA and transcripts were detected by qPCR in all samples from fish injected with LCDV-Sa, demonstrating that the virus establish a systemic and asymptomatic infection. The expression of 23 immune-related genes was also analysed by RT-qPCR in the head kidney (HK) and intestine at several times post-infection (dpi). In HK, the expression of five type I interferon (IFN)-related genes (ifn, irf3, mx2, mx3 and isg15), il10 and ck10 was upregulated at 1-3 dpi, while genes related to the inflammation process (tnfα, il1ß, il6, casp1) were not differentially expressed or even downregulated. The expression profile in the intestine was different regarding type I INF-related genes. An upregulated c3 and ighm expression was observed in both HK and intestine at 3-8 dpi. Finally, the transcription of nccrp1 and mhcIIα was induced in HK, whereas tcrß expression was downregulated in both organs. CONCLUSIONS: LCDV-Sa seems to trigger an immune response in gilthead seabream characterized by a partial activation of type I IFN system and a lack of systemic inflammatory response which may be related to viral persistence. SIGNIFICANCE AND IMPACT OF THE STUDY: The immune response observed in gilthead seabream infected by LCDV-Sa could be implicated in the establishment of an asymptomatic persistent infection.

Virulence properties of three new Photobacterium species affecting cultured fish.

AIMS: Several virulence factors of three new Photobacterium species: Photobacterium toruni, Photobacterium malacitanum and Photobacterium andalusiense associated with diseases of cultured redbanded seabream (Pagrus auriga) were studied. The exoenzymatic activities, adherence and cytotoxic capabilities, and iron-uptake mechanisms were determined both in bacterial extracellular products (ECP) and whole bacterial cells. The histopathology damages provoked on redbanded seabream by the ECP was also studied. METHODS AND RESULTS: The highest exoenzymatic activities of the ECP were alkaline- and acid-phosphatase, phosphohydrolase and lipase. The ECP were strongly lethal for fish at 4-96 h post-inoculation (p.i). Histological changes were evident at 96 hpi of ECP, affecting head kidney, splenic parenchyma and heart. Cytotoxicity assays, on three fish lines and one human cell line, were conducted using whole bacterial cells and their ECP. The new species tested were cytotoxic only for fish cell lines using whole bacterial cells. Bacterial adherence showed an adherence index moderate on CHSE-214 cell line. All strains showed variable haemolytic activity, and were able to grow under iron-limiting conditions, although the CAS reactivitiy was very low. However, all strains produced high amounts of extracelullar citrate that could be used as iron carrier, and use haem as iron source, except the P. toruni strains because a deletion in the genomic region encoding this ability in all Vibrionaceae members. CONCLUSIONS: The toxic activity of the bacterial ECPs was thermolabile, and not associated with their thermoresistant lipopolysaccharide content. The virulence of the strains tested could not be related to the haemolytic activity. Iron uptake could be based on the use of endogenous citrate as iron carrier and P. toruni lacks the ability to use haem as iron source. SIGNIFICANCE AND IMPACT OF THE STUDY: The study analyses for the first time the virulence properties of three new species of Photobacterium pathogenic for fish.

Asthma diagnosis in infants and preschool children: a systematic review of clinical guidelines.

BACKGROUND AND AIM: The definition and diagnosis of asthma are the subject of controversy that is particularly intense in the case of individuals in the first years of life, due to reasons such as the difficulty of performing objective pulmonary function tests or the high frequency with which the symptoms subside in the course of childhood. Since there is no consensus regarding the diagnosis of asthma in preschool children, a systematic review has been carried out. MATERIALS AND METHODS: A systematic search was made of the clinical guidelines published in the last 10 years and containing information referred to the concept or diagnosis of asthma in childhood - including the first years of life (infants and preschool children). A series of key questions were established, and each selected guide was analyzed in search of answers to those questions. The review protocol was registered in the international prospective register of systematic reviews (PROSPERO), with registration number CRD42017074872. RESULTS: Twenty-one clinical guidelines were selected: 10 general guides (children and adults), eight pediatric guides and three guides focusing on preschool children. The immense majority accepted that asthma can be diagnosed from the first years of life, without requiring pulmonary function tests or other complementary techniques. The response to treatment and the exclusion of other alternative diagnoses are key elements for establishing the diagnosis. Only one of the guides denied the possibility of diagnosing asthma in preschool children. CONCLUSIONS: There is generalized although not unanimous agreement that asthma can be diagnosed in preschool children.

The theoretical reliability of PCR-based fish viral diagnostic methods is critically affected when they are applied to fish populations with low prevalence and virus loads.

AIMS: The reliability of polymerase chain reaction (PCR) techniques is an important issue in viral diagnosis, and it is even crucial when they must be applied for detection of viruses in asymptomatic carriers. The problems will arise when the aim is to study wild fish populations, where the viral loads and prevalence values are extremely low. We have evaluated several PCR procedures employed by two laboratories for monitoring fish captured in several oceanographic campaigns performed in the Gulf of Cádiz. METHODS AND RESULTS: To evaluate the reliability of different diagnostics test used, we have re-analysed fish samples that had been previously subjected to diagnosis for a surveillance of viruses performed in 2010-2011 in wild fish populations. The following parameters were employed: the clinical sensitivity (Ss), the clinical specificity (Sp), the predictive positive value, the predictive negative value, and the positive and negative likelihood ratio (LR+ and LR- ). For viral nervous necrosis virus, a RT-PCR procedure supplemented by nested PCR showed the highest values (100%) for all the parameters. For viral haemorrhagic septicaemia virus, the highest values were provided by RT-PCR supplemented by dot-blot hybridization. In the case of infectious pancreatic necrosis virus, none of the procedures yielded 100% for any parameter. CONCLUSIONS: The results obtained for viral prevalence indicate: (i) that the conservation of the samples at -80°C did not affect to the capacity of detection of the virus in the tissues, and (ii) that the reproducibility of the diagnosis can be affected by factors including the staff experience and/or the materials employed. Finally, the use of a combination of procedures in advised to ensure the maximum reliability of the diagnosis when it is applied to asymptomatic fish populations. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper describes a strategy of combining diagnostic tests for the surveillance and monitoring of wild fish populations to reduce underestimation of the prevalence of viruses this type of populations.

Analysis of the local and systemic inflammatory response in hospitalized infants with respiratory syncitial virus bronchiolitis.

BACKGROUND: Respiratory syncytial virus acute bronchiolitis (RSV-AB) is a major cause of hospital admission among our infants. The immune and inflammatory mechanisms involved in the RSV-AB and factors influencing severity have not been clearly established, although an imbalanced Th1 and Th2 response seems to be crucial. OBJECTIVES: To assess the local and systemic inflammatory response in RSV-AB. To find a possible marker of clinical severity and/or oxygen requirements. PATIENTS AND METHODS: Levels of nine cytokines were measured in nasopharyngeal aspirate (NPA) and peripheral blood (PB) of 45 infants with RSV-AB and 27 peer controls, including IFNγ, TNFα, VEGF, interleukins 4, 6 and 10, and chemokines (IL-8 and macrophage inflammatory proteins 1-α and 1-ß). RESULTS: The levels of the analyzed cytokines and chemokines were significantly higher in the NPA of RSV-AB group, with a decrease in IL-4/IFNγ ratio. IL-6 and MIP-1ß levels in NPA were directly correlated to oxygen therapy. PB showed an increase in IL-8 and a decrease in MIP-1α and MIP-1ß in the RSV-AB group (only MIP-1ß associated to the need for oxygen therapy). No correlation was found between cytokines and chemokines levels in NPA and PB. CONCLUSIONS: This study shows that RSV triggers an inflammatory response fundamentally at the respiratory level, with scant systemic repercussion. This local response is characterized by an increase in Th1 and Th2 cytokines, although with a relative predominance of Th1. The determination upon patient admission of IL-6 and MIP-1ß levels in NPA, and of MIP-1ß in PB could help predict severe forms and the need for oxygenotherapy.

Stratification of Metal and Sulphate Loads in Acid Mine Drainage Receiving Water Dams - Variables Regionalization by Cluster Analysis.

The Sancho Reservoir (Iberian Pyrite Belt, SW Spain) is nourished by the waters of the river Meca, which is affected by acid mine drainage (AMD) processes from the abandoned Tharsis mine. The aim of the present work is to study the hydrochemical variations in this reservoir, in order to define potential stratification processes in metal load and sulphates. A stratified sampling from the surface, with one meter deep intervals to the bottom of the dam, was performed. The results show a clear stratification of temperature, pH, electric conductivity, dissolved oxygen, metal and sulphate loads associated with depth. There is an increase of metal loads at the bottom of the reservoir, though previous studies only detect iron. The proximity between pH and aluminium suggests that water chemistry is strongly influenced by aluminium precipitation processes. This indicates the buffer effect that aluminium exercises, which precipitates as amorphous or low crystalline phases, introducing hydrogen ions to the system, while alkalinity input tends to raise pH.

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.

Measles outbreak in Andalusia, Spain, January to August 2011.

On 7 January 2011, a six year-old child living in a Roma community near Seville, southern Spain, was hospitalised with measles. Contact tracing identified a probable index case with onset of symptoms on 20 December 2011 and several unreported cases among children under the age of 15 years in the same town. The outbreak initially spread in districts in the city of Seville with a high proportion of Roma residents, and later to other cities and towns in Andalusia. While some towns experienced wide spread of the disease with significant clusters of cases, most of the affected locations saw non-clustered cases or very few secondary cases. The outbreak resulted in 1,759 confirmed or probable cases of which 393 (19%) required hospitalisation. Measles virus of genotype D4 was diagnosed in more than half of the cases. Significant differences (p<0.0001) by age group were found between clustered and non-clustered cases. The highest proportion of clustered cases occurred in the age group of 5-14 yearolds, while the highest proportion of non-clustered cases was seen in those older than 29 years. The last confirmed case related to this outbreak was reported on 20 August 2011.

Remote ischemic postconditioning: does it protect against ischemic damage in percutaneous coronary revascularization? justification and design of a randomized placebo-controlled clinical trial.

OBJECTIVE: Myocardial damage that is associated with percutaneous coronary intervention (PCI) partially affects the results of the procedure, and is related to medium-term cardiovascular death. Remote postischemic conditioning might reduce the myocardial lesions that are associated with PCI, but perhaps less so in diabetics. The aim of this study was to evaluate the protective effect of remote postischemic conditioning in patients undergoing elective PCI for stable angina or non-ST elevation acute coronary syndrome with troponin <1 ng/ml at the time of randomization. METHODS: This randomized single-blinded single-center clinical trial involved 320 patients undergoing elective PCI who were randomized to either receive three 5-min cycles of ischemia by inflation of a cuff on the non-dominant arm to 200 mm Hg (remote postischemic conditioning) or to placebo (uninflated cuff). The primary outcome variable was the maximum increase in troponin in the first 24 h. The secondary outcome variable was readmission due to heart failure or cardiovascular mortality after 1 year of follow-up. In addition, a diabetic population was studied. CONCLUSIONS: This clinical trial evaluated the possible reduction in intervention-related myocardial damage that was attributable to remote postischemic conditioning.

Treatment of advanced-stage canine nasal carcinomas with toceranib phosphate: 23 cases (2015-2020).

OBJECTIVE: To determine the median survival time (MST) of dogs with nasal carcinoma treated with toceranib phosphate. MATERIAL AND METHODS: The databases of four Spanish referral hospitals were retrospectively searched for dogs with a diagnosis of nasal tumours presented between January 2015 and October 2020. Dogs treated with radiotherapy or other chemotherapies prior toceranib were excluded. RESULTS: Twenty-three dogs with a confirmed nasal carcinoma treated with toceranib phosphate and with a CT scan for initial staging according to Adams Modified Staging System were included. Nine dogs had a stage III nasal carcinoma whereas 14 dogs had a stage IV nasal carcinoma. No dog had stages I and II nasal carcinoma. The median overall survival time was 139 days. The difference between the MST between dogs with stages III and IV was not statistically significant [P = 0.6, 140 days for stage III (range 46-401) vs 120 days for stage IV (range 23-600)]. Overall, dogs with epistaxis achieved a longer median survival (166 days) than dogs without epistaxis (83 days). Toceranib phosphate was generally well tolerated. Most dogs had an initial clinical benefit followed by progressive disease. SIGNIFICANCE: This is the first study to report the MST in dogs with stages III and IV nasal carcinoma treated with toceranib phosphate. This retrospective study showed that toceranib phosphate decreases the clinical signs associated with nasal carcinomas.

Tolerability and outcome of palliative treatment for metastatic pulmonary carcinoma in cats.

OBJECTIVES: Pulmonary carcinoma is uncommon in cats and reporting of outcomes following medical treatment is limited, especially in presence of metastases. The aim of this study was to describe the outcome of cats affected by metastatic primary pulmonary carcinoma and to evaluate the tolerability of palliative treatment in this patient population. MATERIALS AND METHODS: Medical records were searched for cats with a cytological or histopathological diagnosis of primary pulmonary carcinoma and evidence of metastatic disease. Cats were treated with antineoplastic agents, corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs) or received no systemic treatment. Cases in which thoracic CT was not performed, and those lacking definitive diagnosis by cytology or histopathology or receiving curative-intent surgery were excluded. RESULTS: Thirty-four cats were identified: 18 were treated with antineoplastic agents and 16 received corticosteroids, NSAIDs or no treatment. Presenting clinical signs included coughing (53%), tachypnoea (26%), gastrointestinal signs (35%) and lethargy (18%). CT scan identified metastases to the lung parenchyma in all cases and additional metastatic lesions in 10 of 34 (59%) cases; pleural effusion was detected in 11 cases (32%). The overall median survival time for all cats was 64 days [range 1-1352 days; 95% confidence interval (CI) 48-164]. Presence of respiratory signs at presentation was the only factor influencing survival in the multivariable analysis. CLINICAL SIGNIFICANCE: Medical treatment was well tolerated and appeared to palliate clinical signs in cats with metastatic pulmonary carcinoma, albeit with a modest duration and short overall survival. The role and benefit of chemotherapy/antineoplastic agents versus conventional palliative drugs in this setting remains unclear.

Experience in neuromuscular diseases in children and adolescents and their comorbidities in a tertiary hospital.

INTRODUCTION: Neuromuscular diseases include a large group of heterogeneous and rare pathologies that affect different components of the motor unit. It is essential to optimize resources to know the prevalence of comorbidities in the most frequent groups to establish an early multidisciplinary approach in a specialized setting. PATIENTS AND METHODS: Retrospective descriptive study of pediatric and adolescent patients with neuromuscular diseases (NMDs). The Inclusion criteria were NMDs patients with motor neuron involvement divided into three groups, depending on the affected component of the motor unit. Group I: involvement of the motor neuron; Group II: peripheral neuropathies; Group III: myopathies. Demographic variables, association with comorbidities, need for respiratory support, and rehabilitative treatment were collected in each group. RESULTS: Ninety-six patients who met the inclusion criteria were studied. In group I, when compared to the other two groups, a higher incidence of scoliosis (68.3%, p = 0.011), deformity of the rib cage (31.3%, p = 0.0001), chronic respiratory insufficiency (62.5%, p = 0.001) and bronchial aspiration (12.5%, p = 0.03) was detected. In this group, 50%of the patients required non-invasive mechanical ventilation (p = 0.0001). The in-hospital requirement for respiratory physiotherapy was higher in group I (75%, p = 0.001). We observed a higher incidence of scoliosis in Group III compared to Group II. CONCLUSIONS: Neuromuscular diseases with motor neuron involvement present more comorbidities and require an early approach after diagnosis to improve prognosis.

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.

Molecular intraspecific characterization of Photobacterium damselae ssp. damselae strains affecting cultured marine fish.

AIMS: The aim of this study was to analyse the intraspecific variability of Photobacterium damselae ssp. damselae strains isolated from different cultured marine fish species using molecular typing methods. METHODS AND RESULTS: Twenty P. damselae ssp. damselae strains isolated from marine fish species were used in this study. Phenotypic characterization of the strains was carried out using standard microbiological methods. Genetic characterization was conducted using three PCR-based methods [random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and repetitive extragenic palindromic-PCR (REP-PCR)]. Dice coefficient and the unweighted pair group method with average linkage were used for numerical analyses of banding patterns. At phenotypic level, the strains analysed showed seven different profiles, which could not be related to the host fish species, geographic area or outbreak of disease. Isolates were grouped into nine and eight clusters using the RAPD technique with primers 5 and 4, respectively. In both cases, the main cluster grouped 45% of strains. The techniques ERIC-PCR and REP-PCR were more discriminatory, both resulting in 14 different clusters, which grouped 15-20% of the isolates. CONCLUSIONS: In this study, the techniques tested are confirmed as good tools for molecular typing, because they allow discrimination between P. damselae ssp. damselae strains isolated within the same outbreak. In addition, ERIC-PCR and REP-PCR methods were more adequate for rapid typing of P. damselae ssp. damselae than RAPD, allowing the discrimination at strain level. SIGNIFICANCE AND IMPACT OF THE STUDY: The results, in agreement with previous studies, confirmed the high intraspecific variability among isolated P. damselae ssp. damselae strains at both phenotypic and genetic levels. This suggests the existence of different clonal lineages that coexist in the same geographic area, within a short period of time (2-3 years). The discrimination at strain level can be useful to study the traceability of infections.

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.