Occurrence of lipophilic marine toxins in shellfish from Galicia (NW of Spain) and synergies among them.

Lipophilic marine toxins pose a serious threat for consumers and an enormous economic problem for shellfish producers. Synergistic interaction among toxins may play an important role in the toxicity of shellfish and consequently in human intoxications. In order to study the toxic profile of molluscs, sampled during toxic episodes occurring in different locations in Galicia in 2014, shellfish were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), the official method for the detection of lipophilic toxins. The performance of this procedure was demonstrated to be fit for purpose and was validated in house following European guidelines. The vast majority of toxins present in shellfish belonged to the okadaic acid (OA) group and some samples from a particular area contained yessotoxin (YTX). Since these toxins occur very often with other lipophilic toxins, we evaluated the potential interactions among them. A human neuroblastoma cell line was used to study the possible synergies of OA with other lipophilic toxins. Results show that combination of OA with dinophysistoxin 2 (DTX2) or YTX enhances the toxicity triggered by OA, decreasing cell viability and cell proliferation, depending on the toxin concentration and incubation time. The effects of other lipophilic toxins as 13-desmethyl Spirolide C were also evaluated in vitro.

Dinophysis toxins: causative organisms, distribution and fate in shellfish.

Several Dinophysis species produce diarrhoetic toxins (okadaic acid and dinophysistoxins) and pectenotoxins, and cause gastointestinal illness, Diarrhetic Shellfish Poisoning (DSP), even at low cell densities (<103 cells·L⁻¹). They are the main threat, in terms of days of harvesting bans, to aquaculture in Northern Japan, Chile, and Europe. Toxicity and toxin profiles are very variable, more between strains than species. The distribution of DSP events mirrors that of shellfish production areas that have implemented toxin regulations, otherwise misinterpreted as bacterial or viral contamination. Field observations and laboratory experiments have shown that most of the toxins produced by Dinophysis are released into the medium, raising questions about the ecological role of extracelular toxins and their potential uptake by shellfish. Shellfish contamination results from a complex balance between food selection, adsorption, species-specific enzymatic transformations, and allometric processes. Highest risk areas are those combining Dinophysis strains with high cell content of okadaates, aquaculture with predominance of mytilids (good accumulators of toxins), and consumers who frequently include mussels in their diet. Regions including pectenotoxins in their regulated phycotoxins will suffer from much longer harvesting bans and from disloyal competition with production areas where these toxins have been deregulated.

Practical Principles of Palliative Care in Veterinary Oncology: Alleviating the Suffering of the Animal, Owner, and Veterinarian.

Background: Palliative care for pets is a developing area that encompasses animals with cancer and other chronic and degenerative diseases. Objectives: To elucidate the guiding principles of palliative care in veterinary oncology. Methods: This article was structured from an extensive literature review and addresses the growing need for improved palliative care in veterinary medicine. Methods of quality of life assessment in animals with cancer, owner education, and the importance of an interdisciplinary team approach are discussed. Results: Palliative care aims to improve the animal's quality of life, alleviating its physical, emotional, and social suffering. Through attentive communication, palliative care also seeks to alleviate owners suffering from the moment of diagnosis to mourning the patient's death. Conclusions: The importance of offering palliative care alongside anticancer techniques and treatments should not be underestimated and should ideally use a multidisciplinary team approach.

Evaluation of passive samplers as a monitoring tool for early warning of Dinophysis toxins in shellfish.

From June 2006 to January 2007 passive samplers (solid phase adsorbing toxin tracking, SPATT) were tested as a monitoring tool with weekly monitoring of phytoplankton and toxin content (liquid chromatography-mass spectrometry, LC-MS) in picked cells of Dinophysis and plankton concentrates. Successive blooms of Dinophysis acuminata, D. acuta and D. caudata in 2006 caused a long mussel harvesting closure (4.5 months) in the Galician Rías (NW Spain) and a record (up to 9246 ng·g resin-week-1) accumulation of toxins in SPATT discs. Best fit of a toxin accumulation model was between toxin accumulation in SPATT and the product of cell densities by a constant value, for each species of Dinophysis, of toxin content (average) in picked cells. Detection of Dinophysis populations provided earlier warning of oncoming diarrhetic shellfish poisoning (DSP) outbreaks than the SPATT, which at times overestimated the expected toxin levels in shellfish because: (i) SPATT accumulated toxins did not include biotransformation and depuration loss terms and (ii) accumulation of toxins not available to mussels continued for weeks after Dinophysis cells were undetectable and mussels were toxin-free. SPATT may be a valuable environmental monitoring and research tool for toxin dynamics, in particular in areas with no aquaculture, but does not provide a practical gain for early warning of DSP outbreaks.

Cellular Immune Response in Patients Immunized with Three Vaccine Doses of Different Vaccination Schemes Authorized by the Chilean Ministry of Health in January 2022.

In December 2019, a case of atypical pneumonia was reported in Wuhan, China. It was named COVID-19 and caused by SARS-CoV-2. In a few months, scientific groups around the world developed vaccines to reduce the disease's severity. The objective was to evaluate the humoral and cellular immune response post immunization with three different vaccination schedules administered in Chile until January 2022. Sixty volunteers were recruited with a three-dose schedule, who had no history of infection nor close contact with a positive patient. IgG against the spike antigenic domain was detected, and the neutralization capacity against two groups of variants, Original/Alpha and Beta/Gamma, was also measured. Finally, the cellular response with interferon release was measured through IGRA. Results showed that there were significant differences in the neutralizing antibodies for the original and alpha variant when comparing three Comirnaty doses with Coronavac and Vaxzevria. A high number of reactive subjects against the different SARS-CoV-2 variants, alpha, gamma, and delta, were observed, with no significant differences between any of the three schemes, confirming the existence of a cellular immune response against SARS-CoV-2. In conclusion, the three vaccine schemes generated a cellular immune response in these volunteers.

Preliminary study for rapid determination of phycotoxins in microalgae whole cells using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Rapid and sensitive methods for identification of several phycotoxins produced by microalgae species such as yessotoxins (YTXs) for Protoceratium reticulatum, okadaic acid (OA) and pectenotoxins (PTXs) for Prorocentrum spp. and Dinophysis spp., Palytoxins (PLTXs) for Ostreopsis spp., ciguatoxins (CTXs) for Gambierdiscus spp. or domoic acid (DA) for Pseudo-nitzschia spp. are of great importance to the shellfish and fish industry. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to detect several phycotoxins in whole cells of some microalgae which are known as toxin producers. To achieve an appropriate MALDI matrix and a sample preparation method, several matrices and solvent mixtures were tested. The most appropriate matrix system for toxin detection was obtained with 10 µg µL(-1) of DHB in 0.1% TFA/ACN (3:7, v/v) by mixing the intact cells with the matrix solution directly on the MALDI target (dried-droplet technique). Toxin detection by this procedure is much faster than current procedures based on solvent extraction and chromatographic separation. This method allowed the rapid detection of main phycotoxins in some dinoflagellate cells of genus Ostreopsis, Prorocentrum, Protoceratium, Gambierdiscus, Dinophysis and diatoms from Pseudo-nitzschia genus.

Tongue nodules in an atypical canine leishmaniasis in Brazil.

We aimed to report a case of canine leishmaniasis with the only visible clinical sign being the presence of nodules in the lateral region of the tongue. The bitch was treated for a mandibular fracture, when multiple small nodules were observed on the tongue. We identify nodular glossitis with the presence of structures compatible with amastigote forms of Leishmania. The bitch was positive by ELISA, RIFI and PCR assays. Clinical re-evaluation after one year of treatment for leishmaniasis showed clinical improvement, but there was maintenance of antibody titers and infectivity. Lingual nodules as the only clinical sign of the disease is rare, especially in endemic areas, but should be included as differential diagnosis for leishmaniasis in the country.

Detoxification of paralytic shellfish poisoning toxins in naturally contaminated mussels, clams and scallops by an industrial procedure.

Paralytic shellfish poisoning (PSP) episodes cause important economic impacts due to closure of shellfish production areas in order to protect human health. These closures, if are frequent and persistent, can seriously affect shellfish producers and the seafood industry, among others. In this study, we have developed an alternative processing method for bivalves with PSP content above the legal limit, which allows reducing toxicity to acceptable levels. A modification of the PSP detoxifying procedure stablished by Decision 96/77/EC of the European Union in Acanthocardia tuberculata, was developed and implemented for PSP elimination in other bivalves species. The procedure was applied to 6 batches of mussels, 2 batches of clams and 2 batches of scallops, achieving detoxification rates of around 85%. A viable industrial protocol which allows the transformation of a product at risk into a safe product was developed. Although a significant reduction was obtained, in a sample circa 9000 µg STX diHCl equiv/kg, the final toxin level in these highly toxic mussels did not fall below the European limit. The processing protocol described may be applied efficiently to mussels, clams and scallops and it may be a major solution to counteract the closure of shellfish harvesting areas, especially if persistent.

Occurrence of Tetrodotoxin in Bivalves and Gastropods from Harvesting Areas and Other Natural Spaces in Spain.

Tetrodotoxin (TTX) is a potent neurotoxin that is receiving increasing interest in the European Union because it has been found in different fishery products (fish, bivalves and gastropods) captured in European waters. Since available information is scarce, further analytical data regarding the incidence of this toxin in European fishery products is needed in order to perform an appropriate risk assessment devoted to protecting consumers' health. Hence, samples of bivalves and gastropods were collected at different points of the Spanish coast and analyzed by high-performance hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) to evaluate the presence of TTX. None of the analyzed samples showed TTX above an internal threshold of 10 µg/kg or even showed a peak under it. Our results on TTX occurrence obtained in bivalve molluscs and gastropods did not show, at least in the studied areas, a risk for public health. However, taking into account previous positive results obtained by other research groups, and since we did not detect TTX in our samples, a more completed study increasing sampling frequency is needed to ensure proper risk evaluation towards the food safety of these products.

First detection of Pectenotoxin-11 and confirmation of OA-D8 diol-ester in Dinophysis acuta from European waters by LC-MS/MS.

During November 2005, a dense bloom of Dinophysis spp. dominated (>97%) by Dinophysis acuta in the Galician Rías Baixas (NW Spain), provided a unique opportunity to describe the full toxin profile - including toxins that represent a low percentage and escape detection in analyses of single-cell isolates - in plankton concentrates rich in this species. Detection and identification of toxins were carried out by liquid chromatography coupled to mass spectrometry (LC-MS/MS), a method that is based on their retention times (RT) and the fragmentation patterns of their mass spectra. OA-D8 diol-ester and PTX11 were detected in co-occurrence with okadaic acid analogues (OA, DTX2) and PTX2 in plankton concentrates dominated by D. acuta. The presence of a PTX11-isomer, which was suggested to be PTX13 or a novel PTX11-isomer, released in the sea water, was also confirmed in samples obtained after deployment of passive samplers (SPATT) in situ at the time of the D. acuta bloom maximum. The amount of PTX11 per cell of D. acuta, estimated as PTX2 equivalents, ranged between not detected and 2.2pg cell(-1), and represented a maximum of 2.9% of the total toxin content. The variation in PTX11 content per cell of D. acuta, during a daily cycle, followed the same pattern than that of PTX2, with maxima at 21:00 and 03:00h (dark hours), but the amounts per cell were one order of magnitude lower. This is the first report of PTX11, together with the confirmation of OA-D8 diol-ester in D. acuta populations from Europe.

Yessotoxins, a group of marine polyether toxins: an overview.

Yessotoxin (YTX) is a marine polyether toxin that was first isolated in 1986 from the scallop Patinopecten yessoensis. Subsequently, it was reported that YTX is produced by the dinoflagellates Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulax spinifera. YTXs have been associated with diarrhetic shellfish poisoning (DSP) because they are often simultaneously extracted with DSP toxins, and give positive results when tested in the conventional mouse bioassay for DSP toxins. However, recent evidence suggests that YTXs should be excluded from the DSP toxins group, because unlike okadaic acid (OA) and dinophyisistoxin-1 (DTX-1), YTXs do not cause either diarrhea or inhibition of protein phosphatases. In spite of the increasing number of molecular studies focused on the toxicity of YTX, the precise mechanism of action is currently unknown. Since the discovery of YTX, almost forty new analogues isolated from both mussels and dinoflagellates have been characterized by NMR or LC-MS/MS techniques. These studies indicate a wide variability in the profile and the relative abundance of YTXs in both, bivalves and dinoflagellates. This review covers current knowledge on the origin, producer organisms and vectors, chemical structures, metabolism, biosynthetic origin, toxicological properties, potential risks to human health and advances in detection methods of YTXs.

Identification of 19-epi-okadaic acid, a new diarrhetic shellfish poisoning toxin, by liquid chromatography with mass spectrometry detection.

Okadaic acid (1) (OA) and its congeners are mainly responsible for diarrhetic shellfish poisoning (DSP) syndrome. The presence of several OA derivatives have already been confirmed in Prorocentrum and Dinophysis spp. In this paper, we report on the detection and identification of a new DSP toxin, the OA isomer 19-epi-okadaic acid (2) (19-epi-OA), isolated from cultures of Prorocentrum belizeanum, by determining its retention time (RT) and fragmentation pattern using liquid chromatography coupled with mass spectrometry (LC-MS/MS).

Yessotoxins profile in strains of Protoceratium reticulatum from Spain and USA.

Seven strains of Protoceratium reticulatum isolated from Spain and the USA were cultured in the laboratory. Yessotoxins (YTXs) quantification and toxin profile determination were performed by LC-FLD and LC-MS/MS. The four Spanish strains were found to produce YTX and known YTX analogs, however, YTX was not detected in any of the three USA strains. Among the strains that produced YTXs, toxin production ranged between 2.9 and 28.6pg/cell. The YTX profile was substantially different between strains, in three out of the four Spanish strains YTX was the main toxin and in the fourth homoYTX was the prominent toxin. This work demonstrates that YTX is not always the main toxin in P. reticulatum and a high variability in YTX amounts and profile found in other locations is confirmed.

Characterisation of okadaic acid related toxins by liquid chromatography coupled with mass spectrometry.

In the Diarrhetic Shellfish Poisoning (DSP) phenomena, the parent toxins, namely okadaic acid (OA) and/or dinophysistoxin-2 (DTX2), are predominantly found esterified. Therefore, a toxicity assessment of a sample can only be performed after an alkaline hydrolysis step in order to recover the parent molecules in their free form. The presence of several OA diol esters has already been confirmed in Prorocentrum lima and Prorocentrum belizeanum cultures. This paper reports on the analysis of OA diol esters using liquid chromatography coupled with mass spectrometry (LC-MS/MS), and establishes a method for their detection and identification based upon their retention times (RT) and the fragmentation patterns of their mass spectra.

Identification and characterization of DTX-5c and 7-hydroxymethyl-2-methylene-octa-4,7-dienyl okadaate from Prorocentrum belizeanum cultures by LC-MS.

The main toxins produced by the dinoflagellate Prorocentrum belizeanum, DTX5c and 7-hydroxymethyl-2methylene-octa-4,7 dienyl okadaate, were studied by liquid chromatography-coupled with ion trap mass spectrometry (LC-MS/MS). Their retention times (RTs) and fragmentation patterns were established, in particular those of DTX5c, as there is a lack of data about these water soluble OA sulphated derivatives. As an application of the LC-MS methodology, a sample of the toxic P. belizeanum culture was analysed for these metabolites. Both metabolites were detected in cells as majority compounds, whereas levels of these compounds in the culture media were undetectable.

Práticas autorreferidas de enfermeiros sobre gerenciamento de antimicrobianos / Self-report of nurses' antimicrobial stewardship practices / Prácticas autoinformadas de los enfermeros sobre el manejo de antimicrobianos

Objetivo: investigar as práticas autorreferidas de enfermeiros no Programa de Gerenciamento de Antimicrobianos (PGA). Método: estudo transversal, descritivo realizado no período de março a abril de 2021, com 40 enfermeiros de uma instituição privada de ensino superior da cidade de São Paulo. Os dados foram coletados por meio de instrumento estruturado e analisados por estatística descritiva. Resultados: Dentre as 14 práticas de enfermagem esperadas nos PGA, os participantes referiram aplicar oito (57,1%) delas frequentemente: administrar antibiótico na dose e horário corretos, coletar amostras laboratoriais para cultura antes do início do antibiótico, checar o histórico de alergia, instituir precauções específicas, fazer a reconciliação medicamentosa, educar os pacientes/ familiares sobre o uso de antibióticos, monitorar a ocorrência de eventos adversos e participar da tomada de decisão sobre a adequação do paciente para receber antibiótico endovenoso em serviço ambulatorial. Conclusão: os enfermeiros praticam parcialmente as atividades previstas nos PGA. Faz-se necessário avançar na abordagem dessa temática em sessões educacionais e durante a graduação para que as atividades sejam efetivamente incorporadas na prática profissional do enfermeiro. (AU).

Objective: to investigate nurses'self-reported practices about the Antimicrobial Stewardship Program (ASP). Methods: cross-sectional, descriptive study, carried out from March to April 2021, with 40 nurses from a private institution of higher education in the city of São Paulo. Data were collected using a structured instrument and analyzed by descriptive statistics. Results: Among the 14 practices expected by nurses in the ASP, participants reported applying eight (57.1%) frequently: giving antibiotics at the correct dose and time, collecting laboratory samples for culture before starting antibiotics, assessing allergy history, initiating specific precautions, performing medication reconciliation, educating patients/family members about antibiotic use, monitoring the occurrence of adverse events, and participating in decision making about a patient's suitability to receive intravenous antibiotics in outpatient settings. Conclusion: nurses partially practice the activities established in the ASP. It is necessary to advance in the approach of this theme in educational sessions and during graduation so that the activities are effectively incorporated into the professional practice of nurses. (AU).

Objetivo: investigar las prácticas autoinformadas de los enfermeros sobre el Programa para Optimizar el Uso de Antimicrobianos (PROA). Métodos: Estudio transversal, descriptivo, realizado de marzo a abril de 2021, con 40 enfermeros de una institución privada de enseñanza superior de la ciudad de San Pablo. Los datos se recolectaron por medio de un instrumento estructurado y se analizaron mediante estadísticas descriptivas. Resultados: De las 14 prácticas de enfermería esperadas en el PROA, los participantes informaron que realizan ocho (57,1%) con frecuencia: administrar el antibiótico en la dosis y el horario correctos, recolectar muestras de laboratorio para cultivo antes de iniciar el antibiótico, comprobar los antecedentes de alergia, adoptar precauciones específicas, realizar la conciliación de medicación, educar a los pacientes/familiares sobre el uso de los antibióticos, monitorear la aparición de eventos adversos y participar en la toma de decisiones sobre la posibilidad de que el paciente reciba antibióticos intravenosos en el servicio ambulatorio. Conclusión: Los enfermeros realizan parcialmente las actividades previstas en los PROA. Es necesario avanzar en el abordaje de este tema en las sesiones educativas y durante el cursado de la carrera para que los enfermeros incorporen efectivamente las actividades en la práctica profesional. (AU).

Potent neurotoxic action of the shellfish biotoxin yessotoxin on cultured cerebellar neurons.

Yessotoxin (YTX) and its analogues are disulphated polyether compounds of increasing occurrence in seafood. The biological effects of these algal toxins on mammals and the risk associated to their ingestion have not been clearly established. We have used primary cultures of rat cerebellar neurons to investigate whether YTX affected survival and functioning of central nervous system neurons. Exposure to YTX (> or =25 nM) caused first (approximately 8 h) weakening, granulation, and fragmentation of neuronal network, and later (approximately 48 h) complete disintegration of neurites and extensive neuronal death, with a significant decrease in the amount of filamentous actin. The concentration of YTX that reduced by 50% the maximum neuronal survival (EC50(48)) was approximately 20 nM. Lower toxin concentrations (approximately 15 nM) also caused visible signs of toxicity affecting neuronal network primarily. Removal of YTX after 5 h exposure delayed the onset of neurotoxicity but did not prevent neuronal degeneration and death. YTX induced a two-fold increase in cytosolic calcium that was prevented by the voltage-sensitive calcium channel antagonists nifedipine and verapamil. These antagonists were, however, completely ineffective in reducing neurotoxicity. Voltage-sensitive sodium channel antagonists saxitoxin and nefopam, and the NMDA receptor antagonist MK-801 also failed to prevent YTX neurotoxicity. Neuronal death by YTX involved typical hallmarks of apoptosis and required the synthesis of new proteins. Our data suggest neuronal tissue to be a vulnerable biological target for YTX. The potent neurotoxicity of YTX we report raises reasonable concern about the potential risk that exposure to YTX may represent for neuronal survival in vivo.

Detection and identification of glycoyessotoxin A in a culture of the dinoflagellate Protoceratium reticulatum.

The toxin composition of a culture of the dinoflagellate Protoceratium reticulatum was investigated using LC-FLD, after derivatization with DMEQ-TAD (4-(2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalimylethyl)-1,2,4-triazoline-3,5-dione)). Besides yessotoxin (YTX), the new YTX analogue, glycoyessotoxin A (G-YTXA) was detected in culture medium as well as in cells. The conditions for extraction were optimized and the production profile established. Retention time of the resulting fluorescent G-YTXA adduct was identified by comparison of the appropriate standard. Additionally, both G-YTXA and the DMEQ-TAD-G-YTXA adduct were confirmed by LC-MS showing ion peaks at m/z 1273 [M-2Na+H](-) and m/z 1618 [M-2Na+H](-), respectively. The LC-MS(n) displayed a fragmentation pattern similar to that of the YTX series.

Study of the effect of temperature, irradiance and salinity on growth and yessotoxin production by the dinoflagellate Protoceratium reticulatum in culture by using a kinetic and factorial approach.

A complete first order orthogonal plan was used to optimize the growth and the production of yessotoxin (YTX) by the dinoflagellate Protoceratium reticulatum in culture by controlling salinity, temperature and irradiance. Initially, an approach to the kinetic data of cellular density and YTX production for each one of the experimental design conditions was performed. The P. reticulatum growth and YTX production were fitted to logistical equations and to a first-order kinetic model, respectively. The parameters obtained from this adjustment were used as dependent variables for the formulation of the empirical equations of the factorial design tested. The results showed that in practically all the cases for both, P. reticulatum growth and YTX production, irradiance is the primary independent variable and has a positive effect in the range 50-90 micromol photons m(-2) s(-1). Additionally, in certain specific cases, temperature reveals significant positive effects when maintained between 15 and 23 degrees C and salinity in the range of 20-34 displays negative effects. Despite the narrow ranges used in the work, results showed the suitability of factorial analysis to evaluate the optimal conditions for growth and yessotoxin production by the dinoflagellate P. reticulatum.

Production and release of yessotoxins by the dinoflagellates Protoceratium reticulatum and Lingulodinium polyedrum in culture.

The presence of YTX was confirmed in Protoceratium reticulatum cultures and detected for the first time in Lingulodinium polyedrum cultures, mainly in the cells but also, to a lesser extent, dissolved in the culture medium. The production of yessotoxins (YTXs) by cultures of different strains of P. reticulatum and L. polyedrum was studied with liquid chromatography coupled to fluorometric detection using the dienophile reagent DMEQ-TAD and liquid chromatography-mass spectrometry. When comparing toxin production at different stages of culture growth, larger amounts of toxins were observed in the cellular fraction and in the culture medium at the last stage of the culture (day 21) in both species. Although YTX was detected in culture medium, with this study it was not possible to explain which is the release mechanism of the toxin in the medium.