Associated factors with mycotoxin exposure in Spanish population.

Human exposure to mycotoxins is a global concern since filamentous fungi can contaminate food and feed from crops to ready-to-eat meals. Human urine biomonitoring is a widely used technique to evaluate mycotoxins exposure, as an alternative to food correlation studies. The aim of this study is to describe human exposure to mycotoxins and to investigate the associated sociodemographic, lifestyle and dietary variables. Participants were 540 women from the Valencia (Spain) cohort of the Spanish Childhood and Environment Project (INMA). A validated multi-mycotoxin method using HPLC-Q-TOF-MS was applied to determine the concentration of ten selected mycotoxins: Enniatin A, Enniatin B, Enniatin A1, Enniatin B1, Beauvericine, Aflatoxin B1, Aflatoxin B2, Aflatoxin G1, Aflatoxin G2 and Ochratoxin A. A simultaneous untargeted screening of mycotoxins and their metabolites has been performed. Mycotoxins associations were assessed by bivariate and multivariate regression models using participants' sociodemographic, lifestyle and dietary data collected through questionnaires. Mycotoxins were detected in 81% of urine samples. The method quantified mycotoxins concentrations in up to 151 samples. Most quantified mycotoxins were: Enniatin B [% of detection (concentration range)] = 26% (1.0-39.7 ng/mg) and Enniatin B1 = 7% (0.5-14.4 ng/mg). Besides the ten-targeted mycotoxins, other mycotoxins and metabolites were studied, and higher incidence was observed for Deepoxy-deoxynivalenol (45%), Ochratoxin B (18%) and Ochratoxin α (17%). Higher mycotoxins concentrations were associated with rural areas as well as with participants belonged to lower social class, beer, light sodas and fruit juice consumers. On the contrary, higher processed meat intake was related to lower mycotoxins' levels. Studies are required to better evaluate the exposure to mycotoxins from food and their environmental relationships.

Inactivation of African swine fever virus inoculated in liquid plasma by spray drying and storage for 14 days at 4°C or 20°C.

African swine fever virus (ASFV) is a dsDNA virus that can cause high mortality in pigs of all ages. Spray-dried porcine plasma (SDPP) is a highly digestible ingredient used in feed because it benefits performance, gut function and immunity. The objectives were to test if the spray-drying (SD) conditions along with post-drying storage of product for 14 days can inactivate ASFV inoculated in liquid plasma. Fresh liquid porcine plasma was inoculated with ASFV (BA71V) to a final concentration of 105.18 ±0.08 TCID50/mL of liquid plasma. Triplicate 2-L samples of spiked plasma were SD in a lab drier set at an outlet temperature of 80°C or 71°C. The final dried samples were stored at 4°C or 20°C for 14 d. Liquid and SD samples were analyzed for ASFV infectivity in two mirror 24-well plaques containing VERO cells monolayers. Wells were inoculated with different dilutions of SDPP dissolved 1:9 in PBS. One plaque was immediately frozen at -80°C and the other was incubated at 37°C for 3 d. Each dilution was replicated 9 times. After incubation both plaques were analyzed for ASFV by qRT-PCR. Results indicated that the SD process inactivated between 3.2 to 4.2 Logs ASFV TCID50/mL and 2.53 to 2.75 Logs TCID50/mL when the outlet temperature were 80°C and 71°C respectively. All SD samples stored at 4°C or 20°C for 14 d were absent of infectious ASFV. The combination of SD and post drying storage at both temperatures for 14 d was able to inactive >5.18 ±0.08 Log10 of ASFV inoculated in liquid porcine plasma, demonstrating that the manufacturing process for SDPP can be considered safe regarding ASFV.

Effect of incubation and analysis temperatures on sperm kinematics and morphometrics during human semen analysis / Efecto de las temperaturas de incubación y análisis sobre la cinemática y la morfometría de los espermatozoides durante el análisis de semen humano

Introduction: Human semen analysis must be performed after the liquefaction of the ejaculate. This takes place about 30min after ejaculation and samples must be maintained in the lab during this time. The temperatures for this incubation and the final analysis of motility are crucial but seldom taken into account. This study aims to examine the effect of these temperatures on various sperm parameters both manually (sperm count, motility, morphology, viability, chromatin condensation and maturation and DNA fragmentation) and CASA (kinematics and morphometrics, using an ISAS®v1 CASA-Mot and CASA-Morph systems, respectively) analyzed. Methods: Seminal samples from thirteen donors were incubated for 10min at 37°C followed by additional 20min at either room temperature (RT, 23°C) or 37°C and then examined following WHO 2010 criteria. Results: The data obtained show that there were no significant differences (P>0.05) in the subjective sperm quality parameters with incubation temperature. On the other hand, the head sperm morphometric parameters were significantly higher after room temperature incubation showing, in addition, lower ellipticity (P<0.05). Furthermore, kinematic parameters were evaluated both at RT and 37°C for the two incubation temperatures. In general, the four temperature combinations showed that kinematic parameters followed this order: RT-RT Conclusions: Our results showed that temperature control during both incubation and analysis is needed for accurate semen analysis, recommending the use of 37°C during the entire process. (AU)

Introducción: El análisis de semen humano debe realizarse después de la licuefacción del eyaculado. Esto ocurre aproximadamente a los 30minutos después de la eyaculación. Las temperaturas para esta incubación y el análisis final de la motilidad son cruciales, pero rara vez se tienen en cuenta. Este estudio tiene como objetivo examinar el efecto de estas temperaturas en varios parámetros de los espermatozoides tanto de forma manual (recuento de espermatozoides, motilidad, morfología, viabilidad, condensación y maduración de la cromatina y fragmentación del ADN) como CASA (cinemática y morfometría, utilizando un CASA-Mot ISAS®v1 y Sistemas CASA-Morph, respectivamente) analizados. Métodos: Las muestras seminales de 13 donantes se incubaron durante 10minutos a 37°C, seguidas de 20minutos adicionales a temperatura ambiente (TA, 23°C) o a 37°C y luego se examinaron siguiendo los criterios de la OMS 2010. Resultados: Los datos obtenidos muestran que no hubo diferencias significativas (p>0,05) en los parámetros subjetivos de calidad del esperma con la temperatura de incubación. Por otro lado, los parámetros morfométricos de la cabeza de los espermatozoides fueron significativamente más altos después de la incubación a temperatura ambiente, mostrando, además, una elipticidad más baja (p<0,05). Además, los parámetros cinemáticos se evaluaron tanto a temperatura ambiente como a 37°C para las dos temperaturas de incubación. En general, las cuatro combinaciones de temperatura mostraron que los parámetros cinemáticos siguieron este orden: RT-RT < RT-37 < 37-37 < 37-RT (temperaturas de incubación y análisis, respectivamente). Conclusiones: Nuestros resultados mostraron que el control de la temperatura durante la incubación y el análisis es necesario para un análisis de semen preciso, recomendando el uso de 37°C durante todo el proceso. (AU)

Effect of incubation and analysis temperatures on sperm kinematics and morphometrics during human semen analysis.

INTRODUCTION: Human semen analysis must be performed after the liquefaction of the ejaculate. This takes place about 30min after ejaculation and samples must be maintained in the lab during this time. The temperatures for this incubation and the final analysis of motility are crucial but seldom taken into account. This study aims to examine the effect of these temperatures on various sperm parameters both manually (sperm count, motility, morphology, viability, chromatin condensation and maturation and DNA fragmentation) and CASA (kinematics and morphometrics, using an ISAS®v1 CASA-Mot and CASA-Morph systems, respectively) analyzed. METHODS: Seminal samples from thirteen donors were incubated for 10min at 37°C followed by additional 20min at either room temperature (RT, 23°C) or 37°C and then examined following WHO 2010 criteria. RESULTS: The data obtained show that there were no significant differences (P>0.05) in the subjective sperm quality parameters with incubation temperature. On the other hand, the head sperm morphometric parameters were significantly higher after room temperature incubation showing, in addition, lower ellipticity (P<0.05). Furthermore, kinematic parameters were evaluated both at RT and 37°C for the two incubation temperatures. In general, the four temperature combinations showed that kinematic parameters followed this order: RT-RT

Optimization of human semen analysis using CASA-Mot technology.

The purpose of this study is to investigate the optimal framerate (FR) and the use of different counting chambers for improving CASA-Mot technology use in Andrology. Images were captured at 500 fps, then segmented and analyzed in several ranges of FRs (from 25 to 250) to define the asymptotic point that as an optimal FR. This work was replicated using counting chambers based in capillarity (disposable) or drop displacement (reusable) to study their effects on the motility results and kinematic values of the samples under the different experimental conditions. The α value (asymptote corresponding to FRo) of the exponential curve was 150.23 fps, corresponding to a VCL of 130.58 mm/s, far from the value of 98.89 mm/s corresponding to 50 fps (the highest FR used by most current CASA-Mot systems). Our results have shown that, when using reusable counting chambers, type and depth have influence. In addition, different results were obtained depending on the area of image captured inside the different counting chamber types. To have reliable results in human sperm kinematic studies, almost 150 fps should be used for capturing and analyzing and differences between chambers should be considered by sampling from different areas, to obtain a representative value of the whole sample.

Clinical, Pathological and Virological Outcomes of Tissue-Homogenate-Derived and Cell-Adapted Strains of Porcine Epidemic Diarrhea Virus (PEDV) in a Neonatal Pig Model.

Porcine epidemic diarrhea virus (PEDV) is characterized by diarrhea, vomiting, dehydration, and high mortality rates in neonatal piglets. Two distinct genogroups, S-INDEL (G1a, G1b) and non-S INDEL (G2a, G2b, and G2c), circulate worldwide and are characterized by varying degrees of virulence. Here, we compared the early pathogenesis of a PEDV S-INDEL strain obtained from intestine homogenate (CALAF-HOMOG) or adapted to cell culture by 22 passages (CALAF-ADAP) and a virulent non-S INDEL strain (PEDV-USA) in newborn piglets. After orogastric inoculation of PEDV strains, body weight, temperature and clinical signs were monitored for 48 hpi. Pathological studies were performed at 48 hpi and RNA extracts from jejunal content (at 48 hpi) and rectal swabs (at 0 and 48 hpi) were tested for the presence of PEDV RNA as well as sequenced and compared to the inoculum. Piglets inoculated with PEDV-USA and CALAF-HOMOG isolates showed more severe weight loss, diarrhea, villi fusion and atrophy compared to CALAF-ADAP inoculated piglets. The viral load of rectal swabs was higher in the PEDV-USA inoculated group, followed by CALAF-HOMOG and CALAF-ADAP isolates. Similarly, viral RNA load in jejunal content was comparable among PEDV-USA and CALAF-HOMOG inoculated piglets and higher than that of CALAF-ADAP ones. The comparison of three full PEDV sequences of the inocula with the corresponding ones of pigs after 48 hpi yielded a nucleotide identity >99.9%. This study highlights variations in virulence among S-INDEL and non-S INDEL strains and between S-INDEL isolates obtained from homogenate and cell culture.

Development and Validation of LC-Q-TOF-MS Methodology to Determine Mycotoxin Biomarkers in Human Urine.

Mycotoxin contamination of foodstuffs is a health concern worldwide and monitoring human exposure to mycotoxins is a key concern. Most mycotoxins and their metabolites are excreted in urine, but a reliable detection method is required, considering the low levels present in this biological sample. The aim of this work is to validate a sensitive methodology capable of simultaneously determining ten targeted mycotoxins as well as detecting untargeted ones by using Liquid Chromatography coupled to Quadrupole Time of Flight Mass Spectrometry (LC-Q-TOF-MS). The targeted mycotoxins were: enniatin A, B, A1, and B1, beauvericine, aflatoxin B1, B2, G1 and G2, and ochratoxin A. Several extraction procedures such as liquid-liquid extraction, dilute and shoot, and QuEChERS were assessed. Finally, a modified simple QuEChERS extraction method was selected. Creatinine adjustment and matrix-matched calibration curves are required. The limit of detection and limit of quantification values ranged from 0.1 to 1.5 and from 0.3 to 5 ng/mL, respectively. Recoveries achieved were higher than 65% for all mycotoxins. Later, the method was applied to 100 samples of women's urine to confirm the applicability and determine their internal exposure. The untargeted mycotoxins most found were trichothecenes, zearalenones, and ochratoxins.

Human kinematic and morphometric sperm subpopulation analysis using CASA technology: A new approach to spermatozoa classification / Análisis cinemático y morfométrico de subpoblaciones de espermatozoides humanos utilizando tecnología CASA: un nuevo enfoque para la clasificación de los espermatozoides

Introduction: Semen analysis is a clinical method aimed at determining the fertility of a male individual. The traditional subjective method lacks the reliability that can be achieved by computer-assisted sperm analysis (CASA) technology. Unfortunately, this technology has only been used when taking into consideration individually different sperm characteristics. The aim of this work is to present an integrative mathematical approach that considers different seminal variables to establish human sperm subpopulations. Methods: Samples were obtained from thirteen volunteers via masturbation and were analyzed by the routine subjective method and two objective systems, CASA Motility (CASA-Mot) and CASA Morphology (CASA-Morph). Results: Seminogram variables were reduced to three principal components (PC) showing two subpopulations. Kinematics and morphometric variables each rendered three PCs for four subpopulations. Conclusions: These results lay the foundations for future studies including different geographical, social, ethnic and age range conditions with the aim of achieving a definitive view of the human semen picture. (AU)

Introducción: El análisis de semen es el método clínico para determinar la fertilidad masculina. El método subjetivo tradicional carece de la fiabilidad, que se puede obtener con el uso de la tecnología del análisis de semen asistido por ordenador (CASA). Desafortunadamente, esta tecnología se ha venido utilizando únicamente teniendo en cuenta de forma independiente las diversas características de los espermatozoides. El objetivo del presente estudio es presentar una aproximación matemática que incluye diversas variables seminales para definir las posibles subpoblaciones espermáticas. Métodos: Las muestras se obtuvieron por masturbación de 13 voluntarios, que se analizaron de forma subjetiva, así como con 2 sistemas objetivos, para el análisis de la movilidad (CASA-Mot) y la morfología (CASA-Morph). Resultados: Tanto las variables cinemáticas como las morfométricas rindieron 3 componentes principales y 4 subpoblaciones. Conclusión: Estos resultados sientan las bases para estudios futuros que incluyan diferencias geográficas, sociales, étnicas o de rango de edad con el ánimo de obtener una definición concluyente sobre las características seminales de la especie humana. (AU)

Human kinematic and morphometric sperm subpopulation analysis using CASA technology: A new approach to spermatozoa classification.

INTRODUCTION: Semen analysis is a clinical method aimed at determining the fertility of a male individual. The traditional subjective method lacks the reliability that can be achieved by computer-assisted sperm analysis (CASA) technology. Unfortunately, this technology has only been used when taking into consideration individually different sperm characteristics. The aim of this work is to present an integrative mathematical approach that considers different seminal variables to establish human sperm subpopulations. METHODS: Samples were obtained from thirteen volunteers via masturbation and were analyzed by the routine subjective method and two objective systems, CASA Motility (CASA-Mot) and CASA Morphology (CASA-Morph). RESULTS: Seminogram variables were reduced to three principal components (PC) showing two subpopulations. Kinematics and morphometric variables each rendered three PCs for four subpopulations. CONCLUSIONS: These results lay the foundations for future studies including different geographical, social, ethnic and age range conditions with the aim of achieving a definitive view of the human semen picture.

Immune response does not prevent homologous Porcine epidemic diarrhoea virus reinfection five months after the initial challenge.

The aim of the present study was to evaluate the duration of protective immunity against Porcine epidemic diarrhoea virus (PEDV). To do so, a two phases study was performed. In the first phase, 75 four-week-old pigs (group A) were orally inoculated (0 days post-inoculation; dpi) with a European PEDV G1b strain and 14 were kept as controls (group B). The second phase started five months later (154 dpi), when animals in group A were homologous challenged and animals in group B were challenged for first time. Clinical signs, viral shedding and immune responses were evaluated after each inoculation, including the determination of antibodies (ELISA and viral neutralization test, IgA and IgG ELISPOTs using peripheral blood mononuclear cells and lymph node cells) and the frequency of interferon-gamma (IFN-γ) secreting cells. During the first phase, loose stools/liquid faeces were observed in all group A animals. Faecal shedding of PEDV occurred mostly during the first 14 days but, in some animals, persisted until 42 dpi. All inoculated animals seroconverted for specific-PEDV IgG and IgA, and for neutralizing antibodies (NA). At 154 dpi, 77% of pigs were still positive for NA. After that, the homologous challenge resulted in a booster for IgG, IgA, NA, as well as specific-PEDV IgG, IgA and IFN-γ secreting cells. In spite of that, PEDV was detected in faeces of all pigs from group A, indicating that the immune response did not prevent reinfection, although the duration of the viral shedding and the total load of virus shed were significantly lower for previously challenged pigs (p < .05). Taken together, the results indicated that, potentially, maintenance of PEDV infection within an endemic farm may occur by transmission to and from previously infected animals and also indicates that sterilizing immunity is shorter than the productive life of pigs.

Microfabrication of poly(acrylamide) hydrogels with independently controlled topography and stiffness.

The stiffness and topography of a cell's extracellular matrix (ECM) are physical cues that play a key role in regulating processes that determine cellular fate and function. While substrate stiffness can dictate cell differentiation lineage, migration, and self-organization, topographical features can change the cell's differentiation profile or migration ability. Although both physical cues are present and intrinsic to the native tissues in vivo, in vitro studies have been hampered by the lack of technological set-ups that would be compatible with cell culture and characterization. In vitro studies therefore either focused on screening stiffness effects in cells cultured on flat substrates or on determining topography effects in cells cultured onto hard materials. Here, we present a reliable, microfabrication method to obtain well defined topographical structures of micrometer size (5-10 µm) on soft polyacrylamide hydrogels with tunable mechanical stiffness (3-145 kPa) that closely mimic the in vivo situation. Topographically microstructured polyacrylamide hydrogels are polymerized by capillary force lithography using flexible materials as molds. The topographical microstructures are resistant to swelling, can be conformally functionalized by ECM proteins and sustain the growth of cell lines (fibroblasts and myoblasts) and primary cells (mouse intestinal epithelial cells). Our method can independently control stiffness and topography, which allows to individually assess the contribution of each physical cue to cell response or to explore potential synergistic effects. We anticipate that our fabrication method will be of great utility in tissue engineering and biophysics, especially for applications where the use of complex in vivo-like environments is of paramount importance.

On the equivalence between homogeneously prepared sources and sources prepared by seeding in layers for different geometries, energies and matrix parameters.

Measuring the radioactive content of environmental samples requires the use of appropriate reference materials with the same composition and density as the matrices to be measured. If they are not available, ad hoc artificially spiked reference materials are an alternative. Spiking in layers requires a detailed study of the drop distribution, as energy and decay scheme of the component radionuclides must be taken into account to produce a reference material that represents, in efficiency terms, a real sample. A method based on Monte Carlo simulation has been developed to find the optimal distribution of drops in layers for the combination of two typical soil samples and four radionuclides. Results have been validated by comparison with samples prepared by two techniques: methanol bath and spiking in layers.

Malestares en femenino: itinerarios terapéuticos de seis mujeres con fibromialgia / Malaise in feminine: therapeutic itineraries of six women with fibromyalgia

El objetivo principal ha sido explorar la experiencia de mujeres con fibromialgia en relación a su itinerario terapéutico, a la vivencia del diagnóstico y al día a día con la enfermedad. METODOLOGÍA: estudio fenomenológico y constructivista de seis narrativas, obtenidas gracias a entrevistas semiestructuradas de corte biográfico y analizadas mediante el análisis de contenido. RESULTADOS: Las narrativas se organizaron siguiendo estos ejes: (a) problema, que surge con la aparición de los síntomas y la inserción de la persona en el sistema sanitario, (b) nudo, basado en la búsqueda de un diagnóstico y en las alteraciones del mundo de la vida cotidiana que provoca el dolor, y (c) desenlace, el tratamiento. CONCLUSIONES: Para comprender la fibromialgia es necesario aplicar paradigmas menos medicalizadores y emplear modelos de corte bio-psico-social que permitan situar la experiencia individual con y en la enfermedad. Consideramos necesario debatir los modelos y roles de género que provocan sufrimiento en las mujeres

The main objective has been to explore the experience of women with fibromyalgia in relation to their therapeutic itinerary, to the experience of the diagnosis and the coexistence with the disease. METHODOLOGY: phenomenological and constructivist study about six narratives, obtained through semi-structured interviews of a biographical nature and analyzed through content analysis. RESULTS: The narratives were organized following these axes: (a) the problem referring to the first symptoms and the insertion of the person in the health system problem, with the appearance of symptoms and the insertion of the person in the health system, (b) the crux, based on the search for a diagnosis and alterations of the world of daily life that promotes the pain and (c) outcome, treatment. CONCLUSIONS: To understand fibromyalgia, it is necessary to apply less medicalizing paradigms and to use bio-psycho-social models that allow the individual experience with and in the disease. We consider it necessary to debate the models and gender roles that cause suffering in women

Evaluation of the effectiveness of the SurePure Turbulator ultraviolet-C irradiation equipment on inactivation of different enveloped and non-enveloped viruses inoculated in commercially collected liquid animal plasma.

The objective of this study was to evaluate the effectiveness of the SurePure Turbulator ultraviolet-C (UV-C, 254 nm wavelength) irradiation equipment on inactivation of different enveloped and non-enveloped viruses in commercially collected liquid animal plasma. Specifically, Pseudorabies virus (PRV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine epidemic diarrhea virus (PEDV), Bovine viral diarrhea virus (BVDV), Classical swine fever virus (CSFV), Swine influenza virus (SIV) as enveloped viruses and Porcine parvovirus (PPV), Swine vesicular disease virus (SVDV), Porcine circovirus type 2 (PCV-2) and Senecavirus A (SVA) as non-enveloped viruses, were inoculated in bovine or porcine plasma and subjected to different UV-C irradiation doses (0, 750, 1500, 3000, 6000 and 9000 J/L) using an UV-C device developed for opaque liquid working under turbulent flow. The enveloped viruses tested were inactivated at < 3000 J/L of UV-C, being the dose needed to inactivate 4 log TCID50 (4D) of 1612 J/L for PRV,1004 J/L for PRRSV, 1953 J/L for PEDV, 1639 J/L for SIV, 1641 J/L for CSFV and 1943 J/L for BVDV. The non-enveloped viruses tended to have higher 4D values: 2161 J/L for PPV, 3223 J/L for SVA and 3708 J/L for SVDV. Because the initial viral concentration was <4.0 Log for PCV-2, it was not possible to calculate the 4D value for this virus. In conclusion, these results demonstrated that the SurePure Turbulator UV-C treatment system is capable of inactivating significant levels of swine viruses inoculated in commercially collected porcine or bovine plasma. It was concluded that irradiation with UV-C can provide an additional redundant biosafety feature in the manufacturing process of spray-dried animal plasma.

Ovarian torsion and spontaneous ovarian hyperstimulation syndrome in a twin pregnancy: A case report.

INTRODUCTION: Ovarian hyperstimulation syndrome (OHSS) is extremely rare in spontaneous pregnancies. Spontaneous OHSS can result from glycoprotein hormones stimulating follicle-stimulating hormone receptors (FSHR). PRESENTATION OF CASE: We report a twin pregnancy in which ovarian torsion and hemoperitoneum complicating OHSS were treated with left adnexectomy and aspiration. The only trigger for spontaneous OHSS in this case was high levels of chorionic gonadotropin hormone. DISCUSSION: Multiple pregnancy, gestational trophoblastic disease, primary hypothyroidism, thyroid-stimulating hormone/gonadotropin-secreting adenomas, and mutations of the FSHR gene may trigger spontaneous OHSS. CONCLUSION: Spontaneous OHSS should be included in the differential diagnosis of acute abdomen in pregnant women; if spontaneous OHSS is diagnosed, the etiology should be determined in order to focus the treatment and avoid future complications.

No transmission of hepatitis E virus in pigs fed diets containing commercial spray-dried porcine plasma: a retrospective study of samples from several swine trials.

BACKGROUND: Hepatitis E virus (HEV) has been reported in the human population and pigs are a recognized reservoir for HEV and a possible source of HEV transmission to humans. Spray-dried porcine plasma (SDPP) is an ingredient commonly used in feed for pigs around the world. Even though processing conditions used to produce SDPP should be adequate to inactivate HEV, it was of interest to analyze commercial SDPP samples for presence of genome and antibodies (AB) against HEV and to retrospectively analyze serum samples collected from pigs used in past experiments that had been fed diets containing either 0% or 8% SDPP to detect potential transmission of HEV as determined by seroconversion. RESULTS: Eighty-five commercial SDPP samples were analyzed by ELISA and 100% of them contained AB against HEV, while 22.4% (11 of 49 samples analyzed) were positive for HEV RNA. Frozen sera samples (n = 140) collected from 70 pigs used in past experiments that had been fed diets containing either 0% or 8% commercial SDPP was analyzed by ELISA for AB against HEV. Age of pigs at sera sampling ranged from 3 to 15 weeks and feeding duration of diets ranged from approximately 4 to 9 weeks. One lot of SDPP used in one experiment was analyzed and confirmed to contain HEV RNA. Regardless of the diet fed, some sera samples collected at the beginning of an experiment contained AB titer against HEV. These sera samples were collected from weaned pigs prior to feeding of the experimental diets and the HEV titer was probably from maternal origin. However, by the end of the experiments, HEV titer was not detected or had declined by more than 50% of the initial titer concentration. CONCLUSIONS: To our knowledge, this is the first study reporting presence of HEV AB titer and RNA in SDPP. Retrospective analysis of serum collected from pigs fed diets with SDPP revealed no indication of seroconversion to HEV. The results indicate that feeding SDPP in diets for pigs does not represent a risk of transmitting HEV, even though HEV genome may be detected in SDPP.

Characterization of homologous and heterologous adaptive immune responses in porcine reproductive and respiratory syndrome virus infection.

The present study characterized the homologous and heterologous immune response in type-I porcine reproductive and respiratory syndrome virus (PRRSV) infection. Two experiments were conducted: in experiment 1, eight pigs were inoculated with PRRSV strain 3262 and 84 days post-inoculation (dpi) they were challenged with either strain 3262 or strain 3267 and followed for the next 14 days (98 dpi). In experiment 2, eight pigs were inoculated with strain 3267 and challenged at 84 dpi as above. Clinical course, viremia, humoral response (neutralizing and non-neutralizing antibodies, NA) and virus-specific IFN-γ responses (ELISPOT) were evaluated all throughout the study. Serum levels of IL-1, IL-6, IL-8, TNF-α and TGF-ß were determined (ELISA) after the second challenge. In experiment 1 primo-inoculation with strain 3262 induced viremia of ≤ 28 days, low titres of homologous NA but strong IFN-γ responses. In contrast, strain 3267 induced longer viremias (up to 56 days), higher NA titres (≤ 6 log2) and lower IFN-γ responses. Inoculation with 3267 produced higher serum IL-8 levels. After the re-challenge at 84 dpi, pigs in experiment 1 developed mostly a one week viremia regardless of the strain used. In experiment 2, neither the homologous nor the heterologous challenge resulted in detectable viremia although PRRSV was present in tonsils of some animals. Homologous re-inoculation with 3267 produced elevated TGF-ß levels in serum for 7-14 days but this did not occur with the heterologous re-inoculation. In conclusion, inoculation with different PRRSV strains result in different virological and immunological outcomes and in different degrees of homologous and heterologous protection.

Evaluation of the efficacy of commercial vaccines against bluetongue virus serotypes 1 and 8 in experimentally infected red deer (Cervus elaphus).

Red deer (Cervus elaphus) is a widespread and abundant species susceptible to bluetongue virus (BTV) infection. Inclusion of red deer vaccination among BTV control measures should be considered. Four out of twelve BTV antibody negative deer were vaccinated against serotype 1 (BTV-1), and four against serotype 8 (BTV-8). The remaining four deer acted as unvaccinated controls. Forty-two days after vaccination (dpv), all deer were inoculated with a low cell passage of the corresponding BTV strains. Serological and virological responses were analyzed from vaccination until 28 days after inoculation (dpi). The vaccinated deer reached statistically significant (P<0.05) higher specific antibody levels than the non vaccinated deer from 34 (BTV-8) and 42 (BTV-1) dpv, maintaining stable neutralizing antibodies until 28 dpi. The non vaccinated deer remained seronegative until challenge, showing neutralizing antibodies from 7 dpi. BTV RNA was detected in the blood of the non vaccinated deer from 2 to 28 dpi, whereas no BTV RNA was found in the vaccinated deer. BTV was isolated from the blood of non vaccinated deer from 7 to 28 dpi (BTV-1) and from 9 to 11 dpi (BTV-8). BTV RNA could be identified by RT-PCR at 28 dpi in spleen and lymph nodes, but BTV could not be isolated from these samples. BT-compatible clinical signs were inapparent and no gross lesions were found at necropsy. The results obtained in the present study confirm that monovalent BTV-1 and BTV-8 vaccines are safe and effective to prevent BTV infection in red deer. This finding indicates that vaccination programs on farmed or translocated red deer could be a useful tool to control BTV.

Immunochemical determination of oxytetracycline in fish: comparison between enzymatic and time-resolved fluorometric assays.

An indirect competitive enzyme-linked immunosorbent assay (ELISA) with photometric detection of horseradish peroxidase (HRP) activity, was developed in plate to detect oxytetracycline (OTC) in Gilthead sea bream (Sparus aurata) samples. The results were compared to those obtained by time-resolved fluoroimmunoassay (TR-FIA) using a secondary antibody with coproporphyrin of platinum (II) (PtCP) as marker. The limits of detection obtained in fish extract were 16 and 0.08 microg kg(-1) for photometric and fluorometric detections, respectively; therefore, they were suitable for fish quality control according to the maximum residue level established by the European Union. An extraction procedure using methanol:water 70:30 (v/v)+1 mL EDTA 0.1 M, and different clean-up procedures based on solid-phase extraction (C(18), polymeric reversed phase, SCX, Si) was assayed. The matrix effects were overcome by means of an average tetracycline-free fish extract calibration curve used for quantification. The OTC optimized ELISA can also be applied to determine tetracycline and chlortetracycline residues with good results. Thus, the developed immunoassay could be considered as a generic assay for the most used tetracyclines in aquaculture antibiotic treatments. In order to confirm the utility of the developed immunoassay as a semi-quantitative methodology, fish samples obtained from different supermarkets were analyzed. Results correlate well with those obtained with a reference HPLC method.

Controlled delivery systems using antibody-capped mesoporous nanocontainers.

This paper describes the design of new controlled delivery systems consisting of a mesoporous support functionalized on the pore outlets with a certain hapten able to interact with an antibody that acts as a nanoscopic cap. The opening protocol and delivery of the entrapped guest is related by a displacement reaction involving the presence in the solution of the antigen to which the antibody is selective. As a proof-of-the-concept, the solid MCM-41 was selected as support and was loaded with the dye [Ru(bipy)(3)]Cl(2). Then a suitable derivative of the hapten 4-(4-aminobenzenesulfonylamino)benzoic acid was anchored on the outer surface of the mesoporous support (solid S1). Finally the pores were capped with a polyclonal antibody for sulfathiazole (solid S1-AB). Delivery of the dye in the presence of a family of sulfonamides was studied in phosphate-buffered saline (PBS; pH 7.5). A selective uncapping of the pores and dye delivery was observed for sulfathiazole. This delivery behavior was compared with that shown by other solids that were prepared as models to assess the effect of the hapten and its interaction with antibody in the dye delivery control in the presence of the antigen.