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<br><em><strong>Background:</strong></em><i>Anisakis simplex</i> is a fish parasite responsible for gastrointestinal and allergic symptoms in humans. The Ani s 11-like protein has been proposed as... more
<br><em><strong>Background:</strong></em><i>Anisakis simplex</i> is a fish parasite responsible for gastrointestinal and allergic symptoms in humans. The Ani s 11-like protein has been proposed as an <i>Anisakis</i> allergen because its primary structure is similar to that of Ani s 11. The aims of this work were to analyse the frequency of detection of the Ani s 11-like protein and assess its diagnostic value. <b><i>Methods:</i></b> rAni s 11-like protein, rAni s 5 and rAni s 4 were expressed in <i>Escherichia coli</i> and rAni s 1 was produced in <i>Pichia pastoris</i>. Recombinant allergen detection patterns in 37 <i>Anisakis</i>-sensitised patients were determined. The stability to pepsin digestion and heat treatment of rAni s 11-like protein was also analysed by IgE immunoblotting. <b><i>Results:</i></b> Ani s 11-like protein is a major allergen detected by 78% of <i>Anisakis</i>-allergic patients, and 13.5% of patients detect only the rAni s 11-like allergen. This allergen is heat stable because it retains its capability of binding IgE after boiling for 30 min and it is resistant to pepsin digestion for 120 min. <b><i>Conclusions:</i></b> These data indicate that the Ani s 11-like protein is a pepsin- and heat-resistant major allergen (Ani s 11.0201) of <i>Anisakis</i> spp. and a valuable tool for <i>Anisakis</i> allergy component-resolved diagnosis
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Bursaphelenchusxylophilus, the pine wilt nematode (PWN), is a quarantine organism with a high potential to damage conifer forests in Europe. This has driven the European Union to protect forest stands from the propagation and dispersion... more
Bursaphelenchusxylophilus, the pine wilt nematode (PWN), is a quarantine organism with a high potential to damage conifer forests in Europe. This has driven the European Union to protect forest stands from the propagation and dispersion of this pathogen. However, the current control measures have been unsuccessful in preventing the dispersion of this organism, as it continues to be detected in wood material through commercial trade. Timber is a material treated with specific products against wood decay organisms, and is now, additionally submitted to thermo-treatment in accordance with International Standards for Phytosanitary Measures No. 15 (ISPM-15) to control PWN. The industrial sector is concerned whether this treatment is sufficient and the current anti-decay wood treatments are effective against this nematode. For this reason, the objective of this work was to evaluate the efficacy of the permitted wood products against B.xylophilus in blue-stained and non-stained green and dry wood in Pinus pinaster Ait, the most sensitive Pinus species. The results show that the anti-sapstain products tested are effective against PWN in green sound wood, but they do not present the same protective behavior in blue-stained wood. The results for other products employed on dry wood (stained and non-stained) show that almost all of them are effective against this nematode. In consequence, timber protected by means of commercialized wood preservatives might be a good way to control PWN in the wood industry, even being a valid alternative and in some situations preventing the application of ISPM-15.
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Fundacion Banco Santander, Sociedad de Amigos del Museo Nacional de Ciencias Naturales, Fundacion Espanola para la Ciencia y Tecnologia, Asociacion Espanola de Cine e Imagen Cientificos, Sociedad Espanola de Biologia Evolutiva
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This work studied the performance of the artificial digestion method in terms of recovery and viability of Anisakis simplex third-stage larvae (L3) when previous treatments given to the infected fish muscle may accidentally render viable... more
This work studied the performance of the artificial digestion method in terms of recovery and viability of Anisakis simplex third-stage larvae (L3) when previous treatments given to the infected fish muscle may accidentally render viable larvae. For that: a) hake mince was spiked with 10 L3/75g mince, frozen at −10, −15, −20, and −30 °C and immediately thawed, or stored for 12 or 24 h, and subjected to pepsin digestion; b) the mince was spiked under the same conditions, frozen at the above temperatures and thawed immediately. After manual recovery, L3 were assessed for viability, used to spike again the minced fish and subjected to pepsin digestion; c) the mince was spiked with 10 L3 which were: i) living (i.e. chilled), ii) freeze-surviving (live L3 had been previously recovered after freezing at −10 °C), or iii) dead (frozen at −30 °C or − 80 °C), and then subjected to pepsin digestion. Results showed that the artificial digestion method kills a significant number of larvae that m...
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Abstract Anisakis third-stage larvae (L3) is moderately tolerant to heat and, to mitigate the risk of live L3 intake in cooked seafood, it is important to define with precision at which point after heat treatment the parasite is no longer... more
Abstract Anisakis third-stage larvae (L3) is moderately tolerant to heat and, to mitigate the risk of live L3 intake in cooked seafood, it is important to define with precision at which point after heat treatment the parasite is no longer infective. We aimed to find thermal patterns that allowed to classify fish sandwiches spiked with Anisakis L3 into “low” (100% probability of mortality), “intermediate” and “high” risk groups. For that, experiments with varying set temperatures and heating times have been performed in conditions of different external heating temperatures. Decision points to classify the samples into terminal nodes associated to different risk groups have been obtained with decision tree analyses and then confirmed with linear discriminant analysis. Separation into two (i.e. low vs high + intermediate risk) or three (i.e. low, intermediate, and high risk) distinct thermal patterns (98% and 95.9% correct classifications by cross-validation respectively) was achieved. These results refine heating conditions reported in the EU Regulation, since reaching 60 °C for 1 min in the thermal centre is not sufficient to kill all L3. However, when factors such as relative temperature of heating or time to reach the set temperature are taken into account, other thermal conditions are found that are equally safe in terms of Anisakis L3 inactivation. This, together with the description of “intermediate” and “high” risk groups can help in the risk identification and management, as well as in providing clearer recommendations to consumers.
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The increasing prevalence of nosocomial infections produced by multidrug-resistant (MDR) or extensively drug-resistant (XDR) Pseudomonas aeruginosa is frequently linked to widespread international strains designated as high-risk clones.... more
The increasing prevalence of nosocomial infections produced by multidrug-resistant (MDR) or extensively drug-resistant (XDR) Pseudomonas aeruginosa is frequently linked to widespread international strains designated as high-risk clones. In this work we attempted to decipher the interplay between resistance profiles, high-risk clones and virulence, testing a large (n=140) collection of well characterized P. aeruginosa isolates from different sources (bloodstream infections, nosocomial outbreaks, cystic fibrosis and environment) in a Caenorhabditis elegans infection model. Consistently with previous data, we documented a clear inverse correlation between antimicrobial resistance and virulence in the C. elegans model. Indeed, lowest virulence was linked to XDR profiles, which were typically linked to defined high-risk clones. However, virulence varied broadly depending on the involved high-risk clone; it was high for ST111 and ST235 but very low for ST175. The highest virulence of ST23...
Research Interests: Microbiology, Caenorhabditis elegans, Medical Microbiology, Biology, Medicine, and 11 moreVirulence, Humans, Animals, Pseudomonas aeruginosa, Anti-Bacterial Agents, Genotype, Bacteremia, Microbial Sensitivity Tests, Pseudomonas Aeruginosa, Pharmacology and pharmaceutical sciences, and Cross Infection
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A common drawback in evolutionary science is the fact that the evolution of organisms occurs in geological timing, completely out of the time scale of laboratory experimental work. For this reason, some relevant hypotheses on evolution of... more
A common drawback in evolutionary science is the fact that the evolution of organisms occurs in geological timing, completely out of the time scale of laboratory experimental work. For this reason, some relevant hypotheses on evolution of Metazoans are based on correlations more than on experimental data obtained for testing the robustness of those hypotheses. In the current work, we implement an experimental methodology to analyze the role of infections as a driving force in the evolution of Metazoans (Haldane's hypothesis). To that goal, we have used simple models of virulence with short reproduction times, large populations, and that are easily testable in the laboratory. Using the bacteriovirus nematode Caenorhabditis elegans as a model organism under evolution and their infection by the environmental opportunistic bacterial pathogen Pseudomonas aeruginosa as the selective force, we have demonstrated that bacterial infection selects an evolved nematode lineage resistant to i...
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A survey was carried out to determine the distribution of Tylenchulus semipenetrans in the most important citrus production region of Spain. A total of 230 samples from 150 geographic points were processed and analyzed. The samples... more
A survey was carried out to determine the distribution of Tylenchulus semipenetrans in the most important citrus production region of Spain. A total of 230 samples from 150 geographic points were processed and analyzed. The samples included the most representative types of soil of the region. Thirty-six biotic and abiotic parameters were examined in relation to abundance of females and juveniles of T semipenetrans. Results indicate that the abundance of T. semipenetrans is not related to such parameters, nor the ecological structure that is defined by them in the crop. A gradient of geographic variation of T. semipenetrans was observed that was useful to characterize its distribution pattern. This study is considered a first step in the epidemiologic evaluation of the nematode in the region.
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Background: L3 larvae of anisakid nematodes are an important problem for the fisheries industry and pose a potential risk for human health by acting as agents causing allergies and as potential vectors of pathogens microrganisms. In spite... more
Background: L3 larvae of anisakid nematodes are an important problem for the fisheries industry and pose a potential risk for human health by acting as agents causing allergies and as potential vectors of pathogens microrganisms. In spite of the close bacteria-nematode relationship very little is known of the Anisakids microbiote. Fresh fish could be contaminated by bacteria vectored in the cuticle or in the intestine of Anisakids when the L3 larvae migrate through the muscles. As a consequence the bacterial inoculum will be spread, affecting the quality of the fish, and possible clinical effects cannot be discardedResults: A total of 2,689,113 16S rRNA gene sequences from a total of 113 L3 individuals obtained from fish captured along the FAO 27 area were studied. Bacteria were taxonomically characterized through 1803 representative OTUsequences. Fourteen Phyla, 31 Classes, 52 Orders, 129 Families and 187 genera were unambiguously identified. We have found as part of microbiome an ...
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The total proteomes of Anisakis simplex s.s., A. pegreffii and their hybrid genotype have been compared by quantitative proteomics (iTRAQ approach), which considers the level of expressed proteins. Comparison was made by means of two... more
The total proteomes of Anisakis simplex s.s., A. pegreffii and their hybrid genotype have been compared by quantitative proteomics (iTRAQ approach), which considers the level of expressed proteins. Comparison was made by means of two independent experiments considering four biological replicates of A. simplex and two each for A. pegreffii and hybrid between both species. A total of 1811 and 1976 proteins have been respectively identified in the experiments using public databases. One hundred ninety-six proteins were found significantly differentially expressed, and their relationships with the nematodes’ biological replicates were estimated by a multidimensional statistical approach. Results of pairwise Log2 ratio comparisons among them were statistically treated and supported in order to convert them into discrete character states. Principal component analysis (PCA) confirms the validity of the method. This comparison selected thirty seven proteins as discriminant taxonomic biomark...
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In plant and animal nematode parasites, proteins derived from esophageal gland cells have been shown to be important in the host-nematodes relationship but little is known about the allergenic potential of these proteins in the genus... more
In plant and animal nematode parasites, proteins derived from esophageal gland cells have been shown to be important in the host-nematodes relationship but little is known about the allergenic potential of these proteins in the genus Anisakis. Taking into account the increase of anisakiasis and allergies related to these nematodes, immunoreactive properties of gland cell proteins were investigated. Two hundred ventricles were manually dissected from L3 stage larvae of Aniskakis simplex s.s. to allow direct protein analysis. Denaturing gel electrophoresis followed by monochromatic silver staining which revealed the presence of differential (enriched) proteins when compared to total nematode extracts. Such comparison was performed by means of 1D and 2D electrophoresis. Pooled antisera from Anisakis spp.-allergic patients were used in western blots revealing the presence of 13 immunoreactive bands in the ventricular extracts in 1D, with 82 spots revealed in 2D. The corresponding protei...
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Pseudomonas aeruginosa, a major cause of nosocomial and chronic infections, is considered a paradigm of antimicrobial resistance development. However, the evolutionary trajectories of antimicrobial resistance and the impact of mutator... more
Pseudomonas aeruginosa, a major cause of nosocomial and chronic infections, is considered a paradigm of antimicrobial resistance development. However, the evolutionary trajectories of antimicrobial resistance and the impact of mutator phenotypes remain mostly unexplored. Therefore, whole genome sequencing (WGS) was performed in lineages of wild-type and mutator (ΔmutS) strains exposed to growing concentrations of relevant antipseudomonal agents. WGS provided a privileged perspective of the dramatic effect of mutator phenotypes on the accumulation of random mutations, most of which were transitions as expected. Moreover, a frameshift mutagenic signature, consistent with error-prone DNA polymerase activity consequence of SOS system induction, was also seen. This effect was evidenced for all antibiotics tested, but it was higher for fluoroquinoles than for cephalosporins or carbapenems. Analysis of genotype vs phenotype confirmed expected resistance evolution trajectories but also reve...
Research Interests: Microbiology, Medical Microbiology, Mutation, Cephalosporins, Membrane transport proteins, and 12 morePseudomonas aeruginosa, Anti-Bacterial Agents, Dna Gyrase, Penicillin-Binding Proteins, Carbapenems, Microbial Sensitivity Tests, Pseudomonas Aeruginosa, Base Sequence, Mutation Rate, Fluoroquinolones, Beta Lactamases, and Pharmacology and pharmaceutical sciences
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Ingestion of fish parasitized with Anisakis larvae can produce infestation and/or allergy in consumers. Technological and food processing treatments have been applied to parasitized fish in order to kill the larvae and avoid the... more
Ingestion of fish parasitized with Anisakis larvae can produce infestation and/or allergy in consumers. Technological and food processing treatments have been applied to parasitized fish in order to kill the larvae and avoid the infestation; however, their influence on allergenicity has not been studied. Four lots of hake (Merluccius merluccius) steaks artificially parasitized with Anisakis larvae were subjected to two storage chilling (5 degrees C +/- 1 degrees C) and freezing (-20 degrees C +/- 1 degrees C) treatments and two food processing treatments of heat (final temperature 86.3 degrees C) and microwave (final temperature 66.9 degrees C) and studied by scanning electron microscopy, environmental scanning electron microscopy (ESEM) (acid [pH = 2] and water preparations), and emission of fluorescence. Anisakis larvae were resistant to acid conditions, remaining alive after treatment. Larvae in the heat- and microwave-treated lots presented coagulated and disrupted zones in the ...
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The washing operation of fish muscle is one of the key steps in the production of surimi. The aim of this study was to assess in parasitised minced fish the effect of the washing steps on the allergen removal of Anisakis simplex and on... more
The washing operation of fish muscle is one of the key steps in the production of surimi. The aim of this study was to assess in parasitised minced fish the effect of the washing steps on the allergen removal of Anisakis simplex and on protein yield during surimi processing. Experimentally infected hake (Merluccius merluccius) (50 Anisakis simplex s.s L3 larvae per 100 g of muscle) underwent three successive washing steps with water, phosphate buffer (20 mmol L(-1) ), sodium bicarbonate (60 mmol L(-1) ), or sodium hypochlorite (0.27 mmol L(-1) ) in the surimi processing (4 kg muscle, 1:4 w/v for each solution). Total protein concentration and A. simplex antigens and allergens were evaluated in each waste fraction. The highest removal of Ani s 4 and A. simplex antigens was achieved by using phosphate buffer, together with a good protein yield in the raw surimi. Decrease of the concentration of allergens and antigens as a function of the washing steps rendered a linear trend (R(2) = 0.95 and 0.98 for Ani s 4 and A. simplex antigens, respectively). The conditions for an optimal removal of Anisakis allergens can be established and calculated as a function of the washing steps. This approach opens a line to utilise parasitised fish in a safer way. © 2014 Society of Chemical Industry.
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There are few papers that deal specifically with evolutionary studies and proteomics. However, applying proteomics to these studies promises to open new perspectives apropos the construction of phylogenetic trees and the detection of... more
There are few papers that deal specifically with evolutionary studies and proteomics. However, applying proteomics to these studies promises to open new perspectives apropos the construction of phylogenetic trees and the detection of evolutionary changes. Principles and methods of phylogenetic systematics could be used to compare and evaluate proteomes. This would permit the detection and characterization of specific proteins that have evolutionary value in defining monophyly, paraphyly, and polyphyly.
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The parasite species complex Anisakis simplex sensu lato (Anisakis simplex sensu stricto; (A. simplex s.s.), A. pegreffii, A. simplex C) is the main cause of severe anisakiasis (allergy) worldwide and is now an important health matter. In... more
The parasite species complex Anisakis simplex sensu lato (Anisakis simplex sensu stricto; (A. simplex s.s.), A. pegreffii, A. simplex C) is the main cause of severe anisakiasis (allergy) worldwide and is now an important health matter. In this study, the relationship of this Anisakis species complex and their allergenic capacities is assessed by studying the differences between the two most frequent species (A. simplex s.s., A. pegreffii) and their hybrid haplotype by studying active L3 larvae parasiting Merluccius merluccius. They were compared by 2D gel electrophoresis and parallel Western blot (2DE gels were hybridized with pools of sera from Anisakis allergenic patients). Unambiguous spot differences were detected and protein assignation was made by MALDI-TOF/TOF analysis or de novo sequencing. Seventy-five gel spots were detected and the corresponding proteins were identified. Differentially expressed proteins for A. simplex s.s., A. pegreffii, and their hybrid are described and results are statistically supported. Twenty-eight different allergenic proteins are classified according to different families belonging to different biological functions. These proteins are described for the first time as antigenic and potentially new allergens in Anisakis. Comparative proteomic analyses of allergenic capacities are useful for diagnosis, epidemiological surveys, and clinical research. All MS data have been deposited in the ProteomeXchange with identifier PXD000662 (http://proteomecentral.proteomexchange.org/dataset/PXD000662).
Research Interests: Proteomics, Fish Diseases, Western blotting, Biological Sciences, Animals, and 11 moreAnisakis, Tandem Mass Spectrometry, Allergens, High Performance Liquid Chromatography, High Pressure Liquid Chromatography, Larva, Species Specificity, Two dimensional Gel Electrophoresis, Proteome, Anisakiasis, and Medical and Health Sciences
Abstract We describe 2 methods for extraction of DNA-binding proteins from root-knot nematode feeding sites (ie, galls). DNA-binding activity was assayed by electrophoretic mobility shift assays using fragments from the root-knot... more
Abstract We describe 2 methods for extraction of DNA-binding proteins from root-knot nematode feeding sites (ie, galls). DNA-binding activity was assayed by electrophoretic mobility shift assays using fragments from the root-knot nematode-responsiveLEMMI9 and ...
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A plant-parasitic nematode survey was undertaken in mountainous pastures of southern Spain. Sixty-three species of plant-parasitic nematodes distributed over 25 genera were associated with pastures and grasslands in southern Spain.... more
A plant-parasitic nematode survey was undertaken in mountainous pastures of southern Spain. Sixty-three species of plant-parasitic nematodes distributed over 25 genera were associated with pastures and grasslands in southern Spain. Paratylenchus species (P. microdorus, P. similis, P. nanus and P. ciccaronei) were the most abundant and prevalent plant-parasitic nematodes found. Pratylenchus neglectus and P.thornei were widely distributed in 55 and 41% of sites, respectively. Amplimerlinius globigerus, Helicotylenchus digonicus, H. dihystera, H. tunisiensis, Merlinius brevidens, M. microdorus, Rotylenchus unisexus and Scutylenchus quadrifer were found in more than 10% of the fields, on occasion at densities of more than 200 per 100 cm³ of soil. A further 49 species of plant-parasitic nematodes were identified and reported. A pot experiment, using a range of grasses and forage legumes adapted to the zone, ascertained the host status of the most abundant plant-parasitic nematodes. Helic...
Research Interests: Zoology and Nematology
Root-knot nematodes (Meloidogyne) are one of the most damaging agricultural pests. The polyploid apomictic M. arenaria, M. javanica and M. incognita are particularly ubiquitous and exhibit an extreme polyphagy. The taxonomic position of... more
Root-knot nematodes (Meloidogyne) are one of the most damaging agricultural pests. The polyploid apomictic M. arenaria, M. javanica and M. incognita are particularly ubiquitous and exhibit an extreme polyphagy. The taxonomic position of these three species remains unclear, as does the phylogenetic relationships between them. To characterise phenotype variants among these three species, allozyme electrophoresis (α-EST, MDH, CAT, GPI, DIA, GOT, and SOD) was performed in populations from the Iberian Peninsula. A total of 50 multilocus enzyme electrophoresis (MLEE) phenotype variants were detected of which, according to the esterase pattern, 24 corresponded to M. arenaria, 15 to M. javanica and 11 to M. incognita. The phylogenetic relationships of these 50 MLEE variants were studied following the Neighbour-Joining (NJ) distance method based on an allelic frequencies matrix built using two different methodologies. In addition, Maximum-Parsimony (MP) and Maximum-Likelihood (ML) phylogenet...
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A survey of Meloidogyne in La Vera (Spain) was carried out during October 1995 and 1997. Isozyme electrophoresis (A-EST, MDH, CAT, GPI, DIA, GOT and SOD) was performed for each nematode isolate, each of which was cultured from an egg... more
A survey of Meloidogyne in La Vera (Spain) was carried out during October 1995 and 1997. Isozyme electrophoresis (A-EST, MDH, CAT, GPI, DIA, GOT and SOD) was performed for each nematode isolate, each of which was cultured from an egg mass. In 1995 seven isolates of M. incognita, two of M. javanica and 75 of M. arenaria were found, while in 1997 one isolate of M. javanica and 72 of M. arenaria were found. Taxonomic relationships were established among 151 isolates using enzyme phenotypes by means of Multiple Correspondence and UPGM analysis. Twenty-six Multilocus Enzyme Electrophoresis (MLEE) phenotype variants were detected in ten significant taxonomic units: M. arenaria (seven units), M. incognita (two) and M. javanica (one). A total of 13 loci were identified and allelic frequencies for MLEE phenotype variants were estimated. Genetic distances among them suggested that M. arenaria is a poly- or paraphyletic group formed by at least two or three monophyletic lines of different orig...
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ABSTRACT
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The influence of some environmental factors on the distribution of Xiphinema diversicaudatum and X. pachtaicurn in Continental Spain has been studied taking into account the allopatric distribution of both species in Europe. The... more
The influence of some environmental factors on the distribution of Xiphinema diversicaudatum and X. pachtaicurn in Continental Spain has been studied taking into account the allopatric distribution of both species in Europe. The relationships that X. diversicaudaturn and X. pachtaicum ...