Multitarget and suspect screening of antimicrobials in soil and manure by means of QuEChERS - liquid chromatography tandem mass spectrometry.

The present work aimed to develop an accurate analytical method for the simultaneous analysis of twenty-four antimicrobials in soil:compost and animal manure samples by means of ultra-high performance liquid chromatography coupled to a triple-quadrupole mass spectrometer (UHPLC-QqQ). For this purpose, the effectiveness of two extraction techniques (i.e. focused ultrasound solid-liquid extraction (FUSLE) and QuEChERS (quick, easy, cheap, effective, rugged and safe)) was evaluated, and the clean-up step using solid-phase extraction (SPE) was also thoroughly studied. The method was successfully validated at 10 µg·kg-1, 25 µg·kg-1, and 50 µg·kg-1 showing adequate trueness (70-130%) and repeatability (RSD < 30%), with few exceptions. Procedural limits of quantification (LOQPRO) were determined for soil:compost (0.45 to 7.50 µg·kg-1) and manure (0.31 to 5.53 µg·kg-1) samples. Pefloxacin could not be validated at the lowest level since LOQPRO ≥ 10 µg·kg-1. Sulfamethazine (7.9 ± 0.8 µg·kg-1), danofloxacin (27.1 ± 1.4 µg·kg-1) and trimethoprim (4.9 ± 0.5 µg·kg-1) were detected in soil samples; and tetracycline (56.8 ± 2.8 µg·kg-1), among other antimicrobials, in the plants grown on the surface of the studied soil samples. Similarly, sulfonamides (SAs), tetracyclines (TCs) and fluoroquinolones (FQs) were detected in sheep manure in a range of 1.7 ± 0.3 to 93.3 ± 6.8 µg·kg-1. Soil and manure samples were also analysed through UHPLC coupled to a high-resolution mass-spectrometer (UHPLC-qOrbitrap) in order to extend the multitarget method to suspect screening of more than 22,281 suspects. A specific transformation product (TP) of sulfamethazine (formyl-sulfamethazine) was annotated at 2a level in manure samples, among others. This work contributes to the efforts that have been made in the last decade to develop analytical methods that allow multitarget analysis of a wide variety of antimicrobials, including TPs, which is a complex task due to the diverse physicochemical properties of the antimicrobials.

Tick-borne pathogens in ticks from urban and suburban areas of north-western Spain: Importance of Ixodes frontalis harbouring zoonotic pathogens.

To identify the questing tick populations in urban and suburban areas from the city of Lugo (NW Spain), ticks were collected monthly by flagging. The presence of Borrelia spp., Rickettsia spp. and Anaplasma phagocytophilum also was determined by polymerase chain reaction (PCR) and sequence analysis. Overall, 342 questing ticks were collected; the tick abundance was higher in suburban (95.9%) than in urban areas (4.1%). Ixodes frontalis was the most abundant (86.5%); 88.5% were larvae, 11.1% nymphs and 0.3% adults. All development stages of I. ricinus (7.3%) and adults of Rhipicephalus sanguineus sensu lato (5.8%) and Dermacentor reticulatus (0.3%) were found. Rickettsia spp. (31.9%) was more prevalent than Borrelia spp. (2.7%); no ticks were positive to A. phagocytophilum. Six Rickettsia species were identified (R. slovaca, R. monacensis, R. massiliae, R. raoultii, R. sibirica subsp. mongolitimonae and R. aeschielmanii); Candidatus Rickettsia rioja and two novel Rickettsia species also were detected. In addition, Borrelia turdi (1.8%) and B. valaisiana (0.9%) were identified in Ixodes ticks. This is the first report of R. slovaca in R. sanguineus s.l. and of R. monacensis, R. raoultii, R. slovaca, R. sibirica subsp. mongolitimonae and Ca. R. rioja in I. frontalis. Since most of the pathogens detected are zoonotic, their presence in these areas may have implications for public health.

Primary Cutaneous Lymphoma Registry of the Spanish Academy of Dermatology and Venereology (AEDV): Data for the First 5 Years. / Registro de linfomas cutáneos primarios (RELCP) de la AEDV: datos tras 5 años de funcionamiento.

BACKGROUND AND OBJECTIVE: Primary cutaneous lymphomas (PCL) are uncommon. Observations based on the first year of data from the Spanish Registry of Primary Cutaneous Lymphomas (RELCP, in its Spanish abbreviation) of the Spanish Academy of Dermatology and Venereology (AEDV) were published in February 2018. This report covers RELCP data for the first 5 years. PATIENTS AND METHODS: RELCP data were collected prospectively and included diagnosis, treatments, tests, and the current status of patients. We compiled descriptive statistics of the data registered during the first 5 years. RESULTS: Information on 2020 patients treated at 33 Spanish hospitals had been included in the RELCP by December 2021. Fifty-nine percent of the patients were men; the mean age was 62.2 years. The lymphomas were grouped into 4 large diagnostic categories: mycosis fungoides/Sézary syndrome, 1112 patients (55%); primary B-cell cutaneous lymphoma, 547 patients (27.1%); primary CD30+lymphoproliferative disorders, 222 patients (11%), and other T-cell lymphomas, 116 patients (5.8%). Nearly 75% of the tumors were registered in stage I. After treatment, 43.5% achieved complete remission and 27% were stable at the time of writing. Treatments prescribed were topical corticosteroids (1369 [67.8%]), phototherapy (890 patients [44.1%]), surgery (412 patients [20.4%]), and radiotherapy (384 patients [19%]). CONCLUSION: The characteristics of cutaneous lymphomas in Spain are similar to those reported for other series. The large size of the RELCP registry at 5 years has allowed us to give more precise descriptive statistics than in the first year. This registry facilitates the clinical research of the AEDV's lymphoma interest group, which has already published articles based on the RELCP data.

Basics for the potential use of saliva to evaluate stress, inflammation, immune system, and redox homeostasis in pigs.

The use of saliva as a biological sample has many advantages, being especially relevant in pigs where the blood collection is highly stressful and painful, both for the animal and the staff in charge of the sampling. Currently one of the main uses of saliva is for diagnosis and detection of infectious diseases, but the saliva can also be used to measure biomarkers that can provide information of stress, inflammation, immune response and redox homeostasis. This review will be focused on the analytes that can be used for such evaluations. Emphasis will be given in providing data of practical use about their physiological basis, how they can be measured, and their interpretation. In addition, some general rules regarding sampling and saliva storage are provided and the concept of sialochemistry will be addressed. There is still a need for more data and knowledge for most of these biomarkers to optimize their use, application, and interpretation. However, this review provides updated data to illustrate that besides the detection of pathogens in saliva, additional interesting applicative information regarding pigs´ welfare and health can be obtained from this fluid. Information that can potentially be applied to other animal species as well as to humans.

Molecular detection and identification of piroplasms (Babesia spp. and Theileria spp.) and Anaplasma phagocytophilum in questing ticks from northwest Spain.

Anaplasma phagocytophilum and some piroplasm species are pathogens mainly transmitted by Ixodes ricinus. Considering that this tick species is predominant in north-western Spain, individual specimens (652 nymphs, 202 females and 202 males) and 23 larval pools were processed to determine the prevalence of these pathogens in questing I. ricinus from that region. Additionally, Dermacentor marginatus, Dermacentor reticulatus, Ixodes frontalis and Ixodes acuminatus were individually analysed. The groESL operon as well as the 16S rRNA and msp2 genes of Anaplasma were analysed. Similarly, piroplasms were identified at the 18S rRNA gene and the ITS1 of Babesia spp. and Theileria spp. Babesia venatorum (1.5%), A. phagocytophilum (0.7%), Babesia microti (0.3%) and Theileria sp. OT3 (0.2%) were detected in I. ricinus. A single I. frontalis (8.3%) tested positive to A. phagocytophilum. Although a low percentage of I. ricinus were infected with A. phagocytophilum and piroplasms, a potentially human pathogenic variant of A. phagocytophilum was detected, and both Babesia species found were zoonotic. Since the vector of Theileria sp. OT3 remains unknown, further investigations are needed to unravel the role of I. ricinus in the transmission of this piroplasm.

Thrombotic thrombocytopenia following ChAdOx1 nCov-19 vaccination.

Serious thromboembolic events with concurrent thrombocytopenia, sometimes accompanied by bleeding, have occurred very rarely following administration of the ChAdOx1 nCoV-19 vaccine. We report the case of a 59-year-old male with an unremarkable medical history who presented to the emergency department with increasing breathlessness five days after receiving the first dose of ChAdOx1 nCov-19. The patient's blood results showed mild thrombocytopenia and a very high D-dimer, and a pulmonary embolism was confirmed through a CT pulmonary angiogram, which led to a provisional diagnosis of vaccine-induced immune thrombotic thrombocytopenia. The condition was then treated with immunoglobulin and intravenous argatroban in line with the guidance from the Expert Haematology Panel focussed on Vaccine-induced Thrombosis and Thrombocytopenia before conversion to apixaban.

Temporal and spatial spread of Hypoderma actaeon infection in roe deer from peninsular Spain determined by an indirect enzyme-linked immunosorbent assay.

The host switching of Hypoderma actaeon (Diptera: Oestridae), a specific parasite of red deer (Cervus elaphus), towards roe deer (Capreolus capreolus) has been recently reported in Spain. To provide information about the temporal and spatial spreading of H. actaeon infection in roe deer, 244 serum samples from animals hunted in Spain between 2013 and 2018 were analysed by an indirect enzyme-linked immunosorbent assay. The overall seropositivity was 13.9%. Seropositivity was higher in continental (27.7%) and mountainous (12%) areas from central Spain, followed by southern-Mediterranean (11.2%) and northern-oceanic regions (3.5%). Differences were significant between central-continental and northern-oceanic regions (P = 0.003). No differences were found according to the sex and age of roe deer (P > 0.05). In 2013, all seropositive animals were concentrated in two distant areas in central and southern Spain, suggesting that the host switch could have occurred independently in both regions. Changes in the pattern of distribution of red deer and roe deer could have favoured the spreading of this myiasis towards roe deer, indicating that roe deer may become infested by H. actaeon in areas where both cervids coexist at high densities.

In vitro assessment of the combination of cylindrospermopsin and the organophosphate chlorpyrifos on the human neuroblastoma SH-SY5Y cell line.

Cylindrospermopsin (CYN) is a cyanotoxicant which occurrence is increasing due to climate change. Cylindrospermopsin is able to exert damage in the organism at several levels, among them, in the nervous system. Moreover, it is important to take into account that it is not usually present isolated in nature, but in combination with some other pollutants, being the case of the pesticide chlorpyrifos (CPF). Thus, the aim of the present work was to assess the effects of the interaction of CYN in combination with CPF in the human neuroblastoma cell line SH-SY5Y by evaluating cytotoxicity and mechanistic endpoints. The mixtures 0.25 + 21, 0.5 + 42, 1 + 84 µg/mL of CYN + CPF based on cytotoxicity results, were evaluated, and the isobologram method detected an antagonistic effect after 24 and 48 h of exposure. Moreover, although no alterations of reactive oxygen species were detected, a significant decrease of glutathione levels was observed after exposure to both, CPF alone and the combination, at all the concentrations and times of exposure assayed. In addition, CYN + CPF caused a marked decrease in the acetylcholinesterase activity, providing similar values to CPF alone. However, these effects were less severe than expected. All these findings, together with the morphological study results, point out that it is important to take into account the interaction of CYN with other pollutants. Further research is required to contribute to the risk assessment of CYN and other contaminants considering more realistic exposure scenarios.

Association of Ureaplasma diversum with reproductive disease in cattle.

Aims: To examine the association between the detection of Ureaplasma diversum in vaginal swabs from dairy cows in north western Spain with the diagnosis of granular vulvovaginitis (GVV) and reproductive performance, and the association with subclinical endometritis (SE) in slaughterhouse material. The presence of this microorganism in cases of abortion was also investigated. Methods: From 106 dairy farms in the province of Lugo, 40 herds were randomly selected. Vaginal swabs were obtained from 10 randomly selected cows per farm, then pooled for analysis to detect the presence of U. diversum by quantitative real-time PCR (qPCR). In five of these herds samples from the 10 animals were individually tested for U. diversum, and the presence of GVV lesions and their reproductive efficiency (number of inseminations to achieve pregnancy over two subsequent pregnancies) were determined. Vaginal swabs from uteri of cattle obtained at a slaughterhouse (n = 100) were tested for U. diversum and the presence of SE, defined as >5% polymorphonuclear cells in cytobrush smears, was determined. Sixteen farms with abortion problems submitted samples for culture and PCR testing including for U. diversum. Results: Of the 40 herds, 39 (98%) tested positive for U. diversum. On the five farms, 25/50 (50%) cows tested positive for U. diversum, and more cows with GGV-lesions (16/25; 64%) tested positive than cows without lesions (9/25; 36%) (p = 0.047). There were more cows with poor reproductive efficacy that tested positive (8/11; 57%) than tested negative (3/17; 18%) for U. diversum (p = 0.029). Of the 100 uteri, five tested positive for U. diversum and there were more uteri with SE that tested positive (3/19; 16%) than uteri without SE (2/81; 2%) (p = 0.036). U. diversum was also diagnosed in 4/16 farms with abortion problems and liver appeared to be the best tissue for detecting U. diversum DNA in the fetuses analysed. Conclusions and Clinical Relevance: Infection with U. diversum was present in most of herds investigated and it was statistically associated with GVV, SE and poor reproductive performance. It was also detected in abortions and the liver may also be an additional tissue to be considered in the diagnosis of U. diversum abortion by PCR. The possible association with different diseases in the same area suggests that different presentations should be considered when studying the implications of U. diversum on the reproductive diseases of cattle.

Molecular characterisation and risk factor analysis of Cryptosporidium spp. in calves from Italy.

To provide up-to-date information on the occurrence of Cryptosporidium in pre-weaned calves from Sardinia (Italy), the species implicated and their zoonotic potential, 147 faecal samples from 22 cattle herds were microscopically examined for Cryptosporidium oocysts; positive isolates were molecularly characterised. A questionnaire was developed to identify risk factors for Cryptosporidium infection. Overall, the percentage of positive calves and farms was 38.8 and 68.2%, respectively. The SSU rRNA-based PCR identified two Cryptosporidium species, Cryptosporidium parvum (95.8%) and C. bovis (4.2%). Sequence analyses of the glycoprotein (gp60) gene revealed that all C. parvum isolates belonged to the subtype family IIa (IIaA15G2R1 and IIaA16G3R1), with the exception of three isolates that belonged to the subtype family IId (IIdA20G1b and IIdA20). Mixed logistic regression results indicated that calves aged 15-21 days were more likely to be Cryptosporidium-positive. The risk of being positive was also significantly higher in herds from Central Sardinia and in farms using non-slatted flooring. In addition, the application of disinfectants and milk replacers was significantly associated with higher Cryptosporidium prevalence. In contrast, the risk of being positive was significantly reduced in halofuginone-treated calves. Our results reveal that a significant percentage of suckling calves are carriers of zoonotic subtypes of C. parvum. Thus, both healthy and diarrhoeic calves younger than 1 month may represent a risk for the transmission of cryptosporidiosis in humans and animals.

The clinical and immunological effects of Pru p 3 sublingual immunotherapy on peach and peanut allergy in patients with systemic reactions.

BACKGROUND: The peach non-specific lipid transfer protein, Pru p 3, is the primary sensitizer in fruits and responsible for severe reactions in the Mediterranean area. Peach allergy is frequently associated with other allergies such as peanut. Therefore, it is important to assess how specific immunotherapy to Pru p 3 could affect both peach and peanut tolerance. OBJECTIVES: To evaluate peach and peanut desensitization and immunological changes after 1 year of Pru p 3 sublingual immunotherapy (SLIT) in patients with systemic allergic reactions to peach and/or peanut. METHODS: Forty-eight peach allergic patients, 36 treated with SLIT and 12 non-treated, were monitored for 12 months. Treated patients were subclassified as peanut allergic (Group A), sensitized (Group B) or tolerant (Group C). SLIT effect was evaluated by skin prick test (SPT) reactivity and food challenge. Immunological changes were evaluated by monitoring sIgE and sIgG4 levels and basophil reactivity. RESULTS: After 1 year of SLIT, the weal area in SPT significantly decreased and a significant increase in peach threshold in treated patients was observed (P < 0.001). Patients in Group A showed a significant decrease in peanut SPT weal area and an increase in peanut threshold (P < 0.001). Immunological changes were observed in treated patients only, with a significant decrease in sIgE and a parallel increase in sIgG4, sIgG4/sIgE and basophil reactivity for both Pru p 3 and Ara h 9. CONCLUSIONS AND CLINICAL RELEVANCE: After 1 year, Pru p 3 SLIT induces both desensitization and immunological changes not only for peach but also for other food allergens relevant in the induction of severe reactions such as peanut.

Natural evolution in patients with nonsteroidal anti-inflammatory drug-induced urticaria/angioedema.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most frequent triggers of drug hypersensitivity with NSAIDs-induced urticaria/angioedema (NIUA) the most common phenotype. Loss of hypersensitivity has been reported for IgE-mediated reactions; however, it has not been assessed in nonimmunological reactions such as NIUA. We evaluated NSAID-hypersensitivity over time in NIUA patients. METHODS: Patients confirmed as NIUA by positive drug provocation test (DPT) with acetylsalicylic acid (ASA) during 2005-2012 (V1) were included (n=38). Subjects were prospectively re-evaluated by DPT with ASA/other NSAIDs at two time points between 2013 and 2015 (V2 and V3). Atopy was assessed by skin prick test (SPT) using inhalant and food allergens. RESULTS: Patients were evaluated at V1 and re-evaluated after 60 months (V2; IR:48-81) and a further 18 months (V3; IR:14-24). At V2, the majority (24; 63.15%) tolerated ASA and other NSAIDs (Group A) while 14 (36.84%) still reacted (Group B). At V3, all Group A patients remained tolerant; all Group B patients remained hypersensitive. The number of previous episodes reported at V1 and the percentage of reactions induced by ASA/ibuprofen were significantly lower in Group A (P=.005 and P=.006, respectively). Group A patients developed tolerance 72 months (IR:45-87) after their last evaluated reaction (V1); this interval was shorter in nonatopics (P=.003), patients who experienced reactions over 1 hour after NSAIDs administration (P=.001), and those who experienced isolated urticaria after NSAID intake (P=.024). CONCLUSIONS: NIUA patients may develop tolerance to NSAIDs over time, a process that seems to be influenced by atopy and type of clinical reaction.

Influence of the bacterial growth phase on the magnetic properties of magnetosomes synthesized by Magnetospirillum gryphiswaldense.

BACKGROUND: The magnetosome biosynthesis is a genetically controlled process but the physical properties of the magnetosomes can be slightly tuned by modifying the bacterial growth conditions. METHODS: We designed two time-resolved experiments in which iron-starved bacteria at the mid-logarithmic phase are transferred to Fe-supplemented medium to induce the magnetosomes biogenesis along the exponential growth or at the stationary phase. We used flow cytometry to determine the cell concentration, transmission electron microscopy to image the magnetosomes, DC and AC magnetometry methods for the magnetic characterization, and X-ray absorption spectroscopy to analyze the magnetosome structure. RESULTS: When the magnetosomes synthesis occurs during the exponential growth phase, they reach larger sizes and higher monodispersity, displaying a stoichiometric magnetite structure, as fingerprinted by the well defined Verwey temperature. On the contrary, the magnetosomes synthesized at the stationary phase reach smaller sizes and display a smeared Verwey transition, that suggests that these magnetosomes may deviate slightly from the perfect stoichiometry. CONCLUSIONS: Magnetosomes magnetically closer to stoichiometric magnetite are obtained when bacteria start synthesizing them at the exponential growth phase rather than at the stationary phase. GENERAL SIGNIFICANCE: The growth conditions influence the final properties of the biosynthesized magnetosomes. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editors: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.

Hypoderma actaeon: an emerging myiasis in roe deer (Capreolus capreolus).

Subcutaneous larvae of Hypoderma spp. (Diptera: Oestridae) were detected in the dorsal region in 10 roe deer, Capreolus capreolus (Artiodactyla: Cervidae), hunted in central Spain between January and March 2016. All larvae were found in the inner side of the hide during the skinning of the animals. The study of the morphological features of eight larvae of different stages collected from two animals allowed the identification of Hypoderma actaeon Brauer. The small size (4-5 mm) of some of the first instars suggests that the internal lifecycle of H. actaeon may be exclusively subcutaneous. This is the first confirmation of H. actaeon in roe deer; however, further studies to assess the spread of the parasite and to follow the evolution of this myiasis in roe deer are needed.

Occurrence and Fate of Organic and Organometallic Pollutants in Municipal Wastewater Treatment Plants and Their Impact on Receiving Waters (Adour Estuary, France).

To achieve a "Good Environmental Status by 2015," as demanded by the water framework directive, monitoring programs are needed to furnish data on target compounds. In this study, a first evaluation of influents and main emissions of 3 local wastewater treatment plants (WWTP) in the Adour estuary (southwest of France) was performed for 23 pollutants (10 musk fragrances, 5 alkylphenols, and 8 organometallics), as well as receiving estuarine water from the same area. High frequency of occurrence of these compounds was found in influents samples (musks: 22-100%; alkylphenols 11-100%; organometallics 0-100%) and effluents (musks: 0-100%; alkylphenols 0-100%; organometallics 0-100%). The removal efficiencies were calculated and varied from negative values up to 98% with the lowest values for synthetic musk compounds. Temporal variability of the target compounds also was studied, and a few tendencies were observed. Estimation of the daily output of each WWTP into the estuary also showed that galaxolide, nonylphenol, monobuthyltin, and inorganic mercury were the compounds discharged into the environment at the highest concentrations. Finally, the occurrence of these compounds in estuarine waters was evaluated; most of them were present at concentrations below the limits of quantification (musks: 0.53-41.5 ng/L; alkylphenols 3.4-410 ng/L; organometallics 0.02-0.70 ng/L) suggesting a low impact in the resulting receiving waters.

The expanding toolbox for hepatitis C virus research.

Hepatitis C virus is a major global health concern with 170 million people chronically infected. Despite the availability of potent antiviral agents targeting multiple HCV proteins and cure rates above 90%, global treatment availability, the likelihood of emerging drug-resistant viral variants and the unavailability of a protective vaccine underline the many unresolved questions remaining to be answered. Model systems allowing the dissection of individual HCV life cycle steps have previously been developed and span noninfectious and infectious means of assessing HCV entry and replication, multiple cellular systems enabling host/pathogen interaction studies as well as in vivo model systems for basic as well as translational HCV research. This review provides an overview of available systems and a comparative summary of assays and models.

A collaborative system for endovascular treatment of acute ischaemic stroke: the Madrid Stroke Network experience.

BACKGROUND AND PURPOSE: The complexity and expense of endovascular treatment (EVT) for acute ischaemic stroke (AIS) can present difficulties in bringing this approach closer to the patients. A collaborative node was implemented involving three stroke centres (SCs) within the Madrid Stroke Network to provide round-the-clock access to EVT for AIS. METHODS: A weekly schedule was established to ensure that at least one SC was 'on-call' to provide EVT for all those with moderate to severe AIS due to large vessel occlusion, >4.5 h from symptom onset, or within this time-window but with contraindication to, or failure of, systemic thrombolysis. The time-window for treatment was 8 h for anterior circulation stroke and <24 h in posterior stroke. Outcomes measured were re-canalization rates, modified Rankin Scale (mRS) score at 3 months, mortality and symptomatic intra-cranial haemorrhage (SICH). RESULTS: Over a 2-year period (2012-2013), 303 candidate patients with AIS were considered for EVT as per protocol, and 196 (65%) received treatment. Reasons for non-treatment were significant improvement (14%), spontaneous re-canalization (26%), clinical worsening (9%) or radiological criteria of established infarction (31%). Re-canalization rate amongst treated patients was 80%. Median delay from symptom onset to re-canalization was 323 min (p25; p75 percentiles 255; 430). Mortality was 11%; independence (mRS 0-2) was 58%; SICH was 3%. CONCLUSIONS: Implementation of a collaborative network to provide EVT for AIS is feasible and effective. Results are good in terms of re-canalization rates and clinical outcomes.