What does the freshwater clam, Corbicula largillierti, have to tell us about chlorothalonil effects?

Chlorothalonil (CLT) is a broad spectrum, and non-systemic fungicide applied in foliar structures to prevent and treat pathogens. This compound reaches to aquatic environments and affects the biota. In this context, the main goal of this study was to assess the effects of CLT at biochemical, tissular, and individual levels of biological organization using the invasive bivalve Corbicula largillierti as a bioindicator species. Clams were exposed to different sublethal concentrations (0, 10, 20 and 50 µg. L-1 CLT) for 96 h. At biochemical level, the enzymatic activity (Glutathione-s-Transferase, Catalase, Acetyl-, Butiryl- and Carboxyl-esterases) and lipid peroxidation were measured in gills and the visceral mass. Also, the digestive gland morphometry through quantitative histological indexes was registered at the tissular level. Finally, filtering activity and burial behavior at the individual level were measured. At the highest CLT concentration, the most significant changes were observed in enzymatic activity (except for butyrylcholinesterase), lipid peroxidation and in digestive gland morphometry. It was also registered increases of the filtering activity and the latency time to burial. Most of the biomarkers assessed showed significant responses under CLT exposure. Therefore, taking into account that C. largillierti was affected by CLT, it can be expected that other species could be in a potential risk if this fungicide is present in freshwater systems.

A multilevel response approach reveals the Asian clam Corbicula largillierti as a mirror of aquatic pollution.

The endobenthic bivalves are widely used as a bioindicators since they inhabit the sediment-water interface and are able to accumulate a different kind of contaminants. In the present work, we evaluated wild Corbicula largillierti (Phillippi, 1844) as a bioindicator of water quality in the central region of Argentina. The responses at different levels of the biological organization were used. We measured organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) levels in water and clams tissues. The biomarkers selected were enzymatic activities (Glutathione S-Transferase, Catalase, Acetyl-, Butyryl-cholinesterase, and Carboxylesterase) morphometry of the digestive gland, condition index and morphology of valves. In order to integrate all the responses a multivariate analysis and integrated stress index were applied. Our results showed the presence of contaminants along the studied river and the ability of C. largillierti to bioaccumulate them. All the biomarkers selected varied according to the water quality gradient, although there was no specific correlation with OCPs and PCBs levels. At the most polluted sites, the detoxification and oxidative stress enzymes, the morphometric analysis of the digestive gland and the variation in the morphology of the valves indicated the water quality degradation. The multivariate analyses allowed to discriminate the sites according to the different biomarker responses. The IBR index also showed a variation pattern according to the environmental quality gradient along the basin. According to the responses shown by C. largillierti we suggest this species as an useful bioindicator of aquatic pollution.

Evaluation of the Health Status of the Silverside (Odontesthes bonariensis) at a RAMSAR Site in South America.

The objective of this work was to evaluate the health status of an economic and ecologically important fish species from Mar Chiquita Lake, a RAMSAR site located in Cordoba, Argentina, relative to the levels of selected persistent organic pollutants (POPs) in lake water and fish tissues. Odontesthes bonariensis was used as a model species, and its health was estimated by means of histological indices in gills and liver. Sampling was performed according to rainy and dry seasons (i.e. dry, rainy and post-rainy). Gill and liver histopathology were evaluated by semi-quantitative indices and morphometric analysis. Although epithelial lifting in gills and lipid degeneration in liver were frequently registered, they are considered as reversible if environmental conditions improve. During rainy and post-rainy seasons fish presented significantly higher scores of liver and total indices. These higher index scores were correlated with increased levels of POPs in gill and liver tissue. Therefore, preventive measures are needed to mitigate the entry of these compounds into the lake.

Multimatrix measurement of persistent organic pollutants in Mar Chiquita, a continental saline shallow lake.

RAMSAR sites are determined by specific characteristics of the environment in terms of ecological productivity as well services for human development, but they are also one of the most threatened ecosystems. Thus, the objective of this work was to evaluate the dynamic of Persistent Organic Pollutants (POPs) in different biotic and abiotic matrixes of the RAMSAR site (wetlands with international importance), Mar Chiquita Lake. Sampling was performed according to land use (agricultural, urban, and industrial) at two stations: Laguna del Plata and Campo Mare. POPs were analyzed in superficial water (Sw), suspended particulate material (SPM), bottom sediment (Bs) and fish tissues (Odontesthes bonariensis). Organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) were analyzed by GC-ECD. HCHs, Endosulfans, DDTs, PCBs and PBDEs were found in all matrixes at both stations. The high persistence and transport processes are responsible for the occurrence of HCHs, DDTs and PCBs in Bs, SPM and fish tissues, even many years after their prohibition. PBDEs showed lower levels according to the scarcity of punctual sources in the area. Endosulfan showed variable amounts in agreement with application periods since this pesticide was used until a few years ago in this area. Finally, PCB levels overpassed the acceptable daily intake for human consumption being a risk for human health Thus, the present report confirms the occurrence of POPs in Mar Chiquita lake, alerting on the contribution of agricultural and urban pollutants in a RAMSAR site. Current results also raise concerns on biomagnification processes through the food web.

Antioxidant, phase II and III responses induced by lipoic acid in the fish Jenynsia multidentata (Anablapidae) and its influence on endolsulfan accumulation and toxicity.

Antioxidants like lipoic acid (LA) are known to trigger augmented antioxidant and phase II and III responses. This study aimed to evaluate the effect of LA in P-glycoprotein (Pgp) expression, glutathione-S-transferase (GST) activity, total antioxidant competence, levels of lipid peroxides (TBARS) and accumulation of the organochlorine insecticide endosulfan (Endo: α-, ß-isomers and sulfate metabolite) in different organs of the fish Jenynsia multidentata. One hundred and twenty females (1.55±0.07 g) were fed during 8 days with (n=60) or without (n=60) a LA enriched ration (6000 mg/kg). Four experimental groups were defined: -LA/-Endo; +LA/-Endo; -LA/+Endo; and +LA/+Endo. Endo groups were exposed during 24 h to 1.4 µg of insecticide/L. Results showed that only LA induced a significant increment in liver Pgp expression. GST activity was augmented in liver after exposure to LA or Endo. TBARS levels were lowered in liver and gills after LA pre-treatment. Total antioxidant capacity was lowered in liver of Endo exposed fish, a result that was reversed by LA pre-treatment. It is concluded that LA induced the expected effects in terms of Pgp expression, GST activity and reduced TBARS levels although favored α-Endo accumulation in brain. However, the Endo metabolism to the more persistent endosulfan sulfate was not facilitated by LA pre-treatment.

Prevención de la enfermedad cardiovascular en atención primaria: comparación de la función SCORE para países europeos de bajo riesgo con la tabla calibrada para España / Prevention of cardiovascular disease in primary care: a comparison of the function SCORE for low risk European countries with the calibrated SCORE for Spain

Introducción. Estudio comparativo de 2 ecuaciones de valoración del riesgo de mortalidad cardiovascular (RCV): función SCORE para países europeos de bajo riesgo y función SCORE calibrada para España, para conocer el perfil de riesgo de los pacientes de nuestro centro de salud y valorar las diferencias entre ambos métodos. Material y métodos. Estudio descriptivo transversal, de un cupo de pacientes de un centro de salud, seleccionándose los de edades comprendidas entre los 35-74 años y sin evento cardiovascular (n=398 pacientes). Se calculó el RCV mediante las 2 ecuaciones, se evaluaron las diferencias de clasificación obtenidas con ambas tablas de manera continua y la distribución de pacientes en cada grupo de riesgo. Resultados. La muestra estudiada presenta un perfil global de RCV bajo. Ambos métodos de estimación de riesgo presentaron una buena correlación (coeficiente de Pearson de 0,975, p<0,001). El RCV promedio estimado por la función SCORE calibrada para España fue superior al RCV estimado por la función SCORE europea (2,04 frente a 1,46%, p<0,001). El SCORE calibrado para España clasificó con un riesgo alto (mortalidad ≥ 5% en 10 años) al 12,9% de los pacientes (frente al 7% del SCORE europeo de países de bajo riesgo). Conclusiones. Aunque ambos métodos de estimación de RCV presentan buena correlación, el SCORE calibrado para España clasifica a los pacientes con un RCV un 28% superior al SCORE europeo. Deben realizarse más estudios de poblaciones locales para una correcta estimación del RCV (AU)

Introduction. This is a comparative Study of two cardiovascular risk (CVR) functions; the SCORE for European countries of low risk and the calibrated SCORE for Spain and the objective is to determine the risk profile and evaluate the differences between both methods. Material and methods. This is a descriptive cross-sectional study of a group of patients in our healthcare area. We selected those with ages between 35-74 years and without any previous cardiovascular event (n=398 patients). The CVR was calculated by both equations, evaluating the differences of classification obtained with both methods. Results. The studied sample had a low CVR profile. Both methods of estimation of risk correlated well (Pearson's coefficient of 0.975, P<.001). The average CVR estimated by the function SCORE calibrated for Spain was higher than the CVR estimated by the European SCORE (2.04 vs. 1.46%, P<.001). The Spanish calibrated SCORE predicted a high risk (mortality risk ≥5% in 10 years) for 12.9% of the patients (vs. 7% of the European SCORE). Conclusions. Although both methods of CVR estimation had a good correlation, the calibrated SCORE for Spain obtained a CVR 28% higher than the European SCORE. More studies of local populations must be performed for a correct estimation of the CVR (AU)

Uptake, tissue distribution and metabolism of the insecticide endosulfan in Jenynsia multidentata (Anablepidae, Cyprinodontiformes).

The study reports the accumulation, distribution and metabolism of technical endosulfan in Jenynsia multidentata. Adult females were exposed to acute sublethal concentrations (0.072, 0.288 and 1.4 µg L⁻¹). After 24 h, fish were sacrificed and gills, liver, brain, intestine and muscle were removed. Results show that both isomers of technical-grade endosulfan (α- and ß-) are accumulated in fish tissues and biotransformation to endosulfan sulfate occurs at all concentrations tested. Significantly differences in endosulfan accumulation were only found at 1.4 µg L⁻¹ but not between the lowest concentrations. However a similar distribution pattern was observed at all exposure levels where liver, intestine and brain had the highest levels of α-, ß-endosulfan and endosulfan sulfate. Moreover, liver and brain showed the highest endosulfan sulfate:α-endosulfan ratios due to high biotransfomation capacity. J. multidentata demonstrated to be a sensitive species under exposure to technical endosulfan and, therefore, could be used to assess aquatic pollution.

Endosulfan induces changes in spontaneous swimming activity and acetylcholinesterase activity of Jenynsia multidentata (Anablepidae, Cyprinodontiformes).

We assessed changes in spontaneous swimming activity and acetylcholinesterase (AchE) activity of Jenynsia multidentata exposed to Endosulfan (EDS). Females of J. multidentata were exposed to 0.072 and 1.4 microg L(-1) EDS. Average speed and movement percentage were recorded during 48 h. We also exposed females to EDS at five concentrations between 0.072 and 1.4 microg L(-1) during 24 h, and measured the AchE activity in brain and muscle. At 0.072 microg L(-1) EDS swimming motility decreased relative to the control group after 45 h, while at 1.4 microg L(-1) EDS swimming motility decreased after 24 h. AchE activity significantly decreased in muscle when J. multidentata were exposed to EDS above 0.072 microg L(-1), while no significant changes were observed in brain. Thus, changes in swimming activity and AchE activity in muscle are good biomarkers of exposure to EDS in J. multidentata.

Oxidative stress responses in different organs of Jenynsia multidentata exposed to endosulfan.

We evaluate antioxidant responses of Jenynsia multidentata experimentally exposed to sublethal concentrations of endosulfan (EDS). The main goal was to determine differences in the response between different organs to assess which one was more severely affected. Thus, we exposed females of J. multidentata to EDS during 24h, measuring the activity of GST, GR, GPx, CAT and LPO in brain, gills, liver, intestine and muscle of both exposed fish and controls. GST activity was inhibited in gills, liver, intestine and muscle of exposed fish but was induced in brain. GR and GPx activities were increased in brain and gills at 0.014 and 0.288 microg L(-1), respectively. GPx activity was inhibited in liver and muscle at all studied concentrations whereas inhibition was observed in the intestine above 0.288 microg L(-1). Exposure to 1.4 microg L(-1) EDS caused CAT inhibition and increase of LPO levels in liver. LPO was also increased in brain at almost all concentrations tested. We find that the brain was the most sensitive organ to oxidative damage. Thus, J. multidentata could be used as a suitable bioindicator of exposure to EDS measuring activities of antioxidant enzymes in brain and liver as biomarkers.

LAF1, a MYB transcription activator for phytochrome A signaling.

The photoreceptor phytochrome (phy) A has a well-defined role in regulating gene expression in response to specific light signals. Here, we describe a new Arabidopsis mutant, laf1 (long after far-red light 1) that has an elongated hypocotyl specifically under far-red light. Gene expression studies showed that laf1 has reduced responsiveness to continuous far-red light but retains wild-type responses to other light wavelengths. As far-red light is only perceived by phyA, our results suggest that LAF1 is specifically involved in phyA signal transduction. Further analyses revealed that laf1 is affected in a subset of phyA-dependent responses and the phenotype is more severe at low far-red fluence rates. LAF1 encodes a nuclear protein with strong homology with the R2R3-MYB family of DNA-binding proteins. Experiments using yeast cells identified a transactivation domain in the C-terminal portion of the protein. LAF1 is constitutively targeted to the nucleus by signals in its N-terminal portion, and the full-length protein accumulates in distinct nuclear speckles. This accumulation in speckles is abolished by a point mutation in a lysine residue (K258R), which might serve as a modification site by a small ubiquitin-like protein (SUMO).

Characterization of the sialic acid binding activity of transmissible gastroenteritis coronavirus by analysis of haemagglutination-deficient mutants.

Transmissible gastroenteritis coronavirus (TGEV) agglutinates erythrocytes of several species by virtue of sialic acid binding activity of the surface protein S. We have isolated and characterized five haemagglutination-defective (HAD) mutants. In contrast to the parental virus, the mutants were unable to bind to porcine submandibulary mucin, a substrate rich in sialic acid. Each of the mutants was found to contain a single point mutation in the S protein (Cys155Phe, Met195Val, Arg196Ser, Asp208Asn or Leu209Pro), indicating that these amino acids are affecting the sialic acid binding site. In four of the HAD mutants a nearby antigenic site is affected in addition to the sialic acid binding site, as indicated by reactivity with monoclonal antibodies. The parental virus was found to have an increased resistance to the detergent octylglucoside compared to the HAD mutants. This effect depended on cellular sialoglycoconjugates bound to the virion. If the binding of sialylated macromolecules was prevented by neuraminidase treatment, the parental virus was as sensitive to octylglucoside as were the HAD mutants. We discuss the possibility that the sialic acid binding activity helps TGEV to resist detergent-like substances encountered during the gastrointestinal passage and thus facilitates the infection of the intestinal epithelium. An alternative function of the sialic acid binding activity - accessory binding to intestinal tissues - is also discussed.

Isolation of hemagglutination-defective mutants for the analysis of the sialic acid binding activity of transmissible gastroenteritis virus.

The surface protein S of transmissible gastroenteritis virus (TGEV) has a sialic acid binding activity that enables the virus to agglutinate erythrocytes. A protocol is described that has been successfully applied to the isolation of hemgglutination-defective mutants. The potential of these mutants for the characterization of the sialic acid-binding site and the function of the binding activity is discussed.

Two amino acid changes at the N-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism.

To study the molecular basis of TGEV tropism, a collection of recombinants between the PUR46-MAD strain of transmissible gastroenteritis coronavirus (TGEV) infecting the enteric and respiratory tracts and the PTV strain, which only infects the respiratory tract, was generated. The recombinant isolation frequency was about 10(-9) recombinants per nucleotide and was 3.7-fold higher at the 5'-end of the S gene than in other areas of the genome. Thirty recombinants were plaque purified and characterized phenotypically and genetically. All recombinant viruses had a single crossover and had inherited the 5'- and 3'-halves of their genome from the enteric and respiratory parents, respectively. Recombinant viruses were classified into three groups, named 1 to 3, according to the location of the crossover. Group 1 recombinants had the crossover in the S gene, while in Groups 2 and 3 the crossovers were located in ORF1b and ORF1a, respectively. The tropism of the recombinants was studied. Recombinants of Group 1 had enteric and respiratory tropism, while Group 2 recombinants infected the respiratory, but not the enteric, tract. Viruses of both groups differed by two nucleotide changes at positions 214 and 655. Both changes may be in principle responsible for the loss of enteric tropism but only the change in nucleotide 655 was specifically found in the respiratory isolates and most likely this single nucleotide change, which leads to a substitution in amino acid 219 of the S protein, was responsible for the loss of enteric tropism in the closely related PUR46 isolates. The available data indicate that in order to infect enteric tract cells with TGEV, two different domains of the S protein, mapping between amino acids 522 and 744 and around amino acid 219, respectively, are involved. The first domain binds to porcine aminopeptidase N, the cellular receptor for TGEV. In the other domain maps a second factor of undefined nature but which may be the binding site for a coreceptor essential for the enteric tropism of TGEV.

Transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (N-glycolylneuraminic acid) binding activity.

The hemagglutinating activity of transmissible gastroenteritis virus (TGEV), an enteric porcine coronavirus, was analyzed and found to be dependent on the presence of alpha-2,3-linked sialic acid on the erythrocyte surface. N-Glycolylneuraminic acid was recognized more efficiently by TGEV than was N-acetylneuraminic acid. For an efficient hemagglutination reaction the virions had to be treated with sialidase. This result suggests that the sialic acid binding site is blocked by virus-associated competitive inhibitors. Porcine respiratory coronavirus (PRCV), which is serologically related to TGEV but not enteropathogenic, was found to be unable to agglutinate erythrocytes. Incubation with sialidase did not induce a hemagglutinating activity of PRCV, indicating that the lack of this activity is an intrinsic property of the virus and not due to the presence of competitive inhibitors. Only monoclonal antibodies to an antigenic site that is absent from the S protein of PRCV were able to prevent TGEV from agglutinating erythrocytes. The epitope recognized by these antibodies is located within a stretch of 224 amino acids that is missing in the S protein of PRCV. Our results indicate that the sialic acid binding activity is also located in that portion of the S protein. The presence of a hemagglutinating activity in TGEV and its absence in PRCV open the possibility that the sialic acid binding activity contributes to the enterotropism of TGEV.

Molecular bases of tropism in the PUR46 cluster of transmissible gastroenteritis coronaviruses.

Transmissible gastroenteritis coronavirus (TGEV) infects both, the enteric and the respiratory tract of swine. S protein, that is recognized by the cellular receptor, has been proposed that plays an essential role in controlling the dominant tropism. The genetic relationship of S gene from different enteric strains and non-enteropathogenic porcine respiratory coronaviruses (PRCVs) was determined. A correlation between tropism and the genetic structure of the S gene was established. PRCVs, derived from enteric isolates have a large deletion at the N-terminus of the S protein. Interestingly, two respiratory isolates, attenuated Purdue type virus (PTV-ATT) and Toyama (TOY56) have a full-length S gene. PTV-ATT has two specific amino acid differences with the S protein of the enteric viruses. One is located around position 219, within the deleted area, suggesting that alterations around this amino acid may result in the loss of enteric tropism. To study the role of different genes in tropism, a cluster of viruses closely related to PUR46 strain was analyzed. All of them have been originated by accumulating point mutations from a common, virulent isolate which infected the enteric tract. During their evolution these viruses have lost, virulence first, and then, enteric tropism. Sequencing analysis proved that enteric tropism could be lost without changes in ORFs 3a, 3b, 4, 6, and 7, and in 3'-end untranslated regions (3'-UTR). To study the role of the S protein in tropism recombinants were obtained between an enteric and a respiratory virus of this cluster. Analysis of the recombinants supported the hypothesis on the role in tropism of S protein domain around position 219.

Evolution and tropism of transmissible gastroenteritis coronavirus.

Transmissible gastroenteritis coronavirus (TGEV) is an enteropathogenic coronavirus isolated for the first time in 1946. Nonenteropathogenic porcine respiratory coronaviruses (PRCVs) have been derived from TGEV. The genetic relationship among six European PRCVs and five coronaviruses of the TGEV antigenic cluster has been determined based on their RNA sequences. The S proteins of six European PRCVs have an identical deletion of 224 amino acids starting at position 21. The deleted area includes the antigenic sites C and B of TGEV S glycoprotein. Interestingly, two viruses (NEB72 and TOY56) with respiratory tropism have the S protein with a similar size to the enteric viruses. NEB72 and TOY56 viruses have 2 and 15 specific amino acid differences with the enteric viruses, respectively. Four of the residues changed are located within the deletion present in the PRCVs and may influence the enteric tropism of TGEV in vivo. A receptor binding site (RBS) used by the virus to infect ST and other cell types might be located between sites A and D of the S glycoprotein, since monoclonal antibodies (MAbs) specific for these sites inhibit the binding of the virus to ST cells. An evolutionary tree relating 13 enteric and respiratory isolates has been proposed. According to this tree, a main virus lineage evolved from a recent progenitor which was circulating around 1941. From this, secondary lineages originated PUR46, NEB72, TOY56, MIL65, BRI70, and the PRCVs, in this order. Least squares estimation of the origin of TGEV-related coronaviruses showed a significant constancy in the mutation fixation rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen selection and presentation to protect against transmissible gastroenteritis coronavirus.

The antigenic structure of the S glycoprotein of transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) has been determined and correlated with the physical structure. Four antigenic sites have been defined (A, B, C, and D). The sites involved in the neutralization of TGEV are: A, D, and B, sites A and D being antigenically dominant for TGEV neutralization in vitro. These two sites have specific properties of interest: site A is highly conserved and is present in coronaviruses of three animal species, and site D can be represented by synthetic peptides. Both sites might be relevant in protection in vivo. PRCV does not have sites B and C, due to a genomic deletion. Complex antigenic sites, i.e., conformation and glycosylation dependent sites, have been represented by simple mimotopes selected from a library expressing recombinant peptides with random sequences, or by anti-idiotypic internal image monoclonal antibodies. An epidemiological tree relating the TGEVs and PRCVs has been proposed. The estimated mutation fixation rate of 7 +/- 2 x 10(-4) substitutions per nucleotide and year indicates that TGEV related coronaviruses show similar variability to other RNA viruses. In order to induce secretory immunity, different segments of the S gene have been expressed using a virulent forms of Salmonella typhimurium and adenovirus. These vectors, with a tropism for Peyer's patches may be ideal candidates in protection against TGEV.

[Audit of clinical records: clinico-microbiologic study of urinary infections in a team of primary care]. / Auditoría de historias clínicas: estudio clinicomicrobiológico de infecciones urinarias en un equipo de atención primaria.

To carry out an audit of clinical records in our center for the evaluation of the quality of care before the introduction of protocols, several prevalent conditions were selected, and among them urinary tract infections (UTI). Another aim of the study was to evaluate the autochthonous flora responsible for UTI and its resistence to commonly used antimicrobials. A series of acceptable criteria and standards were set as quality controls, and the real index was found below the preselected one in all cases. The most commonly isolated organism was E. coli, followed by Proteus, which were resistent to trimetoprim-sulfamethoxazole in 56% and 71.4% of cases, respectively. Problems of organization and knowledge, and a high resistence rate to common antimicrobials were detected; the following were suggested as measures for improvement: introduction of a protocol, need for continuing education, reduction in the care demand, health education and improvement in the antibiotic policy.