Characterization of Antimicrobial Resistance Determinants and Class 1 and Class 2 Integrons in Salmonella enterica spp., Multidrug-Resistant Isolates from Pigs

[EN] Antimicrobial resistance (AMR) and Salmonella spp., are primary concerns in public health. The present study characterizes the AMR determinants of 62 multi-drug resistant (MDR) Salmonella enterica spp., isolates from swine, which were obtained between 2004-2006, a major source of human salmonellosis. The AMR determinants were investigated by PCR, checking the presence of class 1 and class 2 integrons and 29 resistance genes. Genes sul1, blaTEM1-like, aadA2, tet(A), and dfrA12 were more prevalent (p < 0.05) within the determinants that were checked for each of these antimicrobials. Co-existence of different genes conferring resistance to the same antimicrobial was common. No differences in AMR determinants prevalence were observed between Salmonella Typhimurium and other serovars from the study. Class 1 integrons were detected in 48 of 62 isolates, again with no differences being linked to any serovar. Nine different variable regions were observed, 1000 bp/aadA2-1200 bp/blaPSE-1 (13 isolates) and blaOXA-like/aadA1 (eight isolates) were the most common. Four isolates, including S. Typhimurium (2), Salmonella Bredeney (1), and Salmonella Kapemba (1) harboured a class 2 integron 2300 bp estX-sat2-aadA1. Results from the study highlight the importance of class 1 integrons and certain genes in MDR swine Salmonella isolates. The information is of relevance for monitoring in the forthcoming scope of reduction of antibiotic usage in swine productionSIThe authors would like to express their gratitude to the Department Biological Safety, German Federal Institute for Risk Assessment, Berlin, Germany. In particular to all technicians who helped in the laboratory analysis and to the researchers Reiner Helmuth, Bukhard Malorny and Istvan Szabo who provided their advice selflessly. HA is granted by the Spanish Ministry of Economia and Competitividad IJCI-2016-3079

Rapid Selective Detection of Potentially Infectious Porcine Epidemic Diarrhea Coronavirus Exposed to Heat Treatments Using Viability RT-qPCR

Copyright © 2020 Puente, Randazzo, Falcó, Carvajal and Sánchez. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms[EN] Coronaviruses (CoVs) cause severe respiratory, enteric, and systemic infections in a wide range of hosts, including humans and animals. Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, is the etiological agent of porcine epidemic diarrhea (PED), a highly contagious intestinal disease affecting pigs of all ages. In this study, we optimized a viability real-time reverse transcriptase polymerase chain reaction (RT-qPCR) for the selective detection of infectious and heat-inactivated PEDV. PEMAX™, EMA™, and PMAxx™ photoactivable dyes along with PtCl4 and CDDP platinum compounds were screened as viability markers using two RT-qPCR assays: firstly, on PEDV purified RNA, and secondly on infectious and thermally inactivated virus suspensions. Furthermore, PMAxx™ pretreatment matched the thermal inactivation pattern obtained by cell culture better than other viability markers. Finally, we further optimized the pretreatment by coupling viability markers with Triton X-100 in inoculated serum resulting in a better estimation of PEDV infectivity than RT-qPCR alone. Our study has provided a rapid analytical tool based on viability RT-qPCR to infer PEDV infectivity with potential application for feed and feed ingredients monitoring in swine industry. This development would allow for greater accuracy in epidemiological surveys and outbreak investigations.SIThis study was supported by the program of the National Institute of Agricultural and Food Research and Technology (INIA project E-RTA2015-0003-C02-02) of Spanish Government. HP was supported by FPU17/00466 predoctoral grant funded by Spanish Government. WR was supported by APOSTD/2018/150 postdoctoral grant funded by Generalitat Valenciana

Coronavirus

Las enfermedades causadas por coronavirus son conocidas y han sido estudiadas en veterinaria muchos años antes que en medicina humana por su importancia y por sus efectos en distintas especies de aves y de mamíferos. La primera vacuna contra una enfermedad por coronavirus en animales, la bronquitis infecciosa aviar, se desarrolló en 1950 mientras que el primer coronavirus humano se describióen 1965. En el presente trabajo se hace una revisión histórica de los aspectos que pueden ser más interesantes de las enfermedades causadas por coronavirus, aportando algunas experiencias propias con enfermedades causadas por coronavirus en el cerdo, algunas de ellas emergentes e ilustrativas de lo que son estas enfermedades. Se resaltan las características epidemiológicas de las zoonosis causadas por coronavirus, su origen y el carácter preocupantemente emergente de las mismas en medicina humana; es por esto que el conocimiento de los antecedentes de la pandemia actual causada por SARS-CoV-2 resultan muy elocuentes a la hora de interpretar la actual emergencia sanitaria, que aunque sorpresiva no resulta un hecho inesperado

Intralaboratory Validation of a Kinetic Turbidimetric Assay Based on Limulus Amebocyte Lysate (LAL) for Assessing Endotoxin Activity in Cow Milk

[EN] Mastitis, one of the most common diseases in dairy cattle, causes severe losses in the dairy sector worldwide and affects animal welfare. The disease is characterized by an inflammatory reaction of the mammary gland and is mainly caused by bacterial infections, including both Gram-negative and Gram-positive bacteria. The release of endotoxins associated to bacterial lysis is a weighty factor in the clinical course of Gram-negative associated mastitis and should be taken into consideration when using antibiotics in the treatment of these infections. Therefore, endotoxin detection in milk samples would be of help in the management of bovine mastitis. With this aim, we have validated a kinetic turbidimetric assay based on Limulus amebocyte lysate (LAL) for the quantification of endotoxins in milk samples. The assay was adapted to this particular matrix by incorporating filtration and dilution of the milk samples in the procedure. Our results demonstrate the robustness and usefulness of the assay, which allows the identification of coliform mastitis in milk samples from affected cows and the quantification of endotoxin activity in bulk and commercial milk samples. Further studies are required to evaluate the performance of the assay in mastitis milk samples associated to Gram-negative bacteria other than Escherichia coli as well as during the clinical course of these Gram-negative mastitis or after their treatment with antibiotics.S

In vitro Susceptibility and Evaluation of Techniques for Understanding the Mode of Action of a Promising Non-antibiotic Citrus Fruit Extract Against Several Pathogens

Copyright © 2019 de Nova, Carvajal, Prieto and Rubio. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.[EN] The screening for alternatives to antibiotics is an urgent need for the pharmaceutical industry. One of these alternatives seems to be the citrus fruit extracts, which are showing a significant antibacterial activity against Gram-negative and Gram-positive bacteria. One of these citrus extracts, named BIOCITRO®, is assessed in this study to elucidate its bacteriostatic and bactericidal effect and its mode of action on the important pathogens Campylobacter coli, C. jejuni, Escherichia coli, Salmonella enterica ssp. enterica, Clostridium difficile, C. perfringens, and Staphylococcus aureus. For most of the strains tested of these bacteria the product was bactericidal as well as bacteriostatic at the same concentration, and the minimum bactericidal concentrations ranged from 16 to 256 μg/mL. Regarding the mode of action, important changes in the permeability, structure, composition and morphology of the bacterial envelope were evidenced using flow cytometry, Fourier transform infrared spectroscopy and scanning electron microscopy. The main effect of the product was found over carbohydrates and polysaccharides, inducing the release of microvesicles by the cells in addition to other specific effects. During the study, the techniques used were evaluated to clarify their contribution to the knowledge of the mode of action of the product. The survival test elucidated whether the modifications displayed using other techniques affected the viability of the cells or on the contrary, the cells remained viable even with evident changes in their structure, composition or morphology. Flow cytometry showed that for some strains the proportion of cells detected with altered membrane permeability were higher than the number of non-viable cells, and therefore the damage did not affect the viability of some cells. On the contrary, some cells observed using scanning electron microscopy with no apparent damage, were demonstrated non-viable using the survival test, making this technique indispensable in studies of the mode of action of antimicrobials to make a correct interpretation of the data from other techniques.SIThe research was partly financed by PROBENA (2015/00119/001), the company commercializing BIOCITRO® in the EU, but without any influence over the experimental procedures neither results

Detection and Genetic Diversity of Porcine Coronavirus Involved in Diarrhea Outbreaks in Spain

[EN]Porcine enteric coronaviruses include some of the most relevant viral pathogens to the swine industry such as porcine epidemic diarrhea virus (PEDV) or porcine transmissible gastroenteritis virus (TGEV) as well as several recently identified virus such as swine enteric coronavirus (SeCoV), porcine deltacoronavirus (PDCoV) or swine enteric alphacoronavirus (SeACoV). The aim of this study is the identification and characterization of enteric coronaviruses on Spanish pig farms between 2017 and 2019. The study was carried out on 106 swine farms with diarrhea outbreaks where a viral etiology was suspected by using two duplex RT-PCRs developed for the detection of porcine enteric coronaviruses. PEDV was the only coronavirus detected in our research (38.7% positive outbreaks, 41 out of 106) and neither TGEV, SeCoV, PDCoV nor SeACoV were detected in any of the samples. The complete S-gene of all the PEDV isolates recovered were obtained and compared to PEDV and SeCoV sequences available in GenBank. The phylogenetic tree showed that only PEDV of the INDEL 2 or G1b genogroup has circulated in Spain between 2017 and 2019. Three different variants were detected, the recombinant PEDV-SeCoV being the most widespread. These results show that PEDV is a relevant cause of enteric disorders in pigs in Spain while new emerging coronavirus have not been detected so far. However, the monitoring of these virus is advisable to curtail their emergence and spread.SIThis work was supported by the program from the National Institute of Agricultural and Food Research and Technology (INIA project E-RTA2015-0003-C02-02) of Spanish Government. HP, ÓM-A, and HA were supported by Spanish Government (FPU17/00466, FPU16/03485, and BEAGAL-18-106, respectively) and MG-G by Junta de Castilla y León (LE131-18)

Management Models Applied to the Human-Wolf Conflict in Agro-Forestry-Pastoral Territories of Two Italian Protected Areas and One Spanish Game Area

[EN]Our work shows that, despite the persistence of persecutory actions, conservation activity has proved successful for the return of numerous wild mammals to different habitats, including the wolf. The human-wolf conflict is still described in all countries where the wolf is present. This is evidenced by the high number of damages on livestock, and the corpses of wolves found both in protected areas and in those where hunting is permitted. The diagnosis of road accidents, together with poisoning and poaching, are major causes of mortality. Although hunting records the highest percentage of kills in Spain, the demographic stability reported by the censuses suggests that this activity does not have a consistent influence on the Iberian wolf population’s survival. In Italy, where wolf hunting is prohibited, wolf populations are to be increasing. In some Italian situations, wolf attacks on horses seem to cause unwanted damage to foals, but they represent a very precious source of information about the habits of carnivores. A simple management plan would be sufficient to help the coexistence between the productive parts and the ecosystem services ensured by the presence of the wolf. The presence of hybrids is a negative factor.S

Management models applied to the human-wolf conflict in agro-forestry-pastoral territories of two italian protected areas and one spanish game area

Our work shows that, despite the persistence of persecutory actions, conservation activity has proved successful for the return of numerous wild mammals to different habitats, including the wolf. The human-wolf conflict is still described in all countries where the wolf is present. This is evidenced by the high number of damages on livestock, and the corpses of wolves found both in protected areas and in those where hunting is permitted. The diagnosis of road accidents, together with poisoning and poaching, are major causes of mortality. Although hunting records the highest percentage of kills in Spain, the demographic stability reported by the censuses suggests that this activity does not have a consistent influence on the Iberian wolf population’s survival. In Italy, where wolf hunting is prohibited, wolf populations are to be increasing. In some Italian situations, wolf attacks on horses seem to cause unwanted damage to foals, but they represent a very precious source of information about the habits of carnivores. A simple management plan would be sufficient to help the coexistence between the productive parts and the ecosystem services ensured by the presence of the wolf. The presence of hybrids is a negative factor

Regulatory role of microRNA in mesenteric lymph nodes after Salmonella Typhimurium infection

© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.[EN] Salmonellosis is a gastrointestinal disease caused by non-typhoidal Salmonella serovars such as Salmonella Typhimurium. This pathology is a zoonosis, and food animals with subclinical infection constitute a vast reservoir for disease. After intestinal colonization, Salmonella Typhimurium reaches mesenteric lymph nodes (MLN), where infection is controlled avoiding systemic spread. Although the molecular basis of this infection has been extensively studied, little is known about how microRNA (miRNA) regulate the expression of proteins involved in the Salmonella-host interaction. Using small RNA-seq, we examined expression profiles of MLN 2 days after infection with Salmonella Typhimurium, and we found 110 dysregulated miRNA. Among them, we found upregulated miR-21, miR-155, miR-150, and miR-221, as well as downregulated miR-143 and miR-125, all of them previously linked to other bacterial infections. Integration with proteomic data revealed 30 miRNA potentially regulating the expression of 15 proteins involved in biological functions such as cell death and survival, inflammatory response and antigenic presentation. The inflammatory response was found increased via upregulation of miRNA such as miR-21 and miR-155. Downregulation of miR-125a/b, miR-148 and miR-1 were identified as potential regulators of MHC-class I components PSMB8, HSP90B1 and PDIA3, respectively. Furthermore, we confirmed that miR-125a is a direct target of immunoproteasome component PSMB8. Since we also found miR-130 downregulation, which is associated with upregulation of HSPA8, we suggest induction of both MHC-I and MHC-II antigen presentation pathways. In conclusion, our study identifies miRNA that could regulate critical networks for antigenic presentation, inflammatory response and cytoskeletal rearrangements.SIThis work was supported by the Spanish Ministry of Economy and Competitiveness (AGL2011-28904 and AGL2014-54089). JHU is a predoctoral researcher supported by the FPI Research Program of the Spanish Ministry of Economy and Competitiveness (BES-2012-058642). SZL is a postdoctoral researcher supported by the Postdoctoral Trainee Program of the Spanish Ministry of Economy and Competitiveness (FPDI-2013-15619), and a postdoctoral contract co-funded by the XXI University of Cordoba Intramural research Program and the European Regional Development Funds (FEDER).We thank Reyes Alvarez for their technical assistance, and the Functional Genomics Core of the Institute for Research in Biomedicine (IRB) Barcelona for performing the library preparation and small RNA sequencin

Genomic Insights into the Mobilome and Resistome of Sentinel Microorganisms Originating from Farms of Two Different Swine Production Systems

[EN] Antimicrobial resistance (AMR) is a threat to public health due to long-term antimicrobial use (AMU), which promotes the bacterial acquisition of antimicrobial resistance determinants (ARDs). Within food-producing animals, organic and extensive Iberian swine production is based on sustainable and eco-friendly management systems, providing an excellent opportunity to evaluate how sustained differences in AMU impact the development and spread of AMR. Here, through a whole-genome sequencing approach, we provide an in-depth characterization of the resistome and mobilome and their interaction in 466 sentinel bacteria, namely, Escherichia coli, Enterococcus spp., Campylobacter coli, and Staphylococcus spp., recovered from 37 intensive and organic-extensive pig farms. Both ARDs and mobile genetic elements (MGEs) were primarily taxon-associated, with higher similarities among bacteria which were closely phylogenetically related. E. coli exhibited the most diverse resistome and mobilome, with 85.4% mobilizable ARDs, 50.3% of which were plasmid-associated. Staphylococcus spp. exhibited a broad repertoire of ARDs and MGEs, with 52.3% of its resistome being mobilizable. Although Enterococcus spp. carried the highest number of ARDs per isolate and its plasmidome was similar in size to that of E. coli, 43.7% of its resistome was mobilizable. A narrow spectrum of ARDs constituted the C. coli resistome, with point mutations as its main AMR driver. A constrained AMU, as observed in organic-extensive herds, determined a reduction in the quantitative composition of the resistome and the complexity of the resistome-mobilome interaction. These results demonstrate taxon-associated AMR-MGE interactions and evidence that responsible AMU can contribute to reducing AMR pressure in the food chainSIWe acknowledge the excellent technical assistance provided by Diana Molina, Lucía Pérez, and Clara Vega, the help with data management provided by Laura Uelze, and the help by Katharina Thomas and Beatrice Baumann in sequencing library preparation. We also thank the veterinary practitioners and farmers for their willingness to collaborate and, in particular, Álvaro Fernández-Blanco for his support in contacting the farms. O.M.-A. received a grant from the Spanish Government (Ministerio de Universidades), no. EST 19/00806, for a short stay at the German Federal Institute for Risk Assessment. O.M.-A. also holds a grant from the Spanish Government (Ministerio de Educación y Formación Profesional), no. FPU 16/03485. H.A. is financially supported by the “Beatriz Galindo” Program from the Spanish Government (Ministerio de Educación y Formación Profesional), no. BEAGAL-18-106. Study design was performed by A.C., H.A. and A.A.-O. Samples were prepared by O.M.-A. M.B. performed the whole-genome sequencing. C.D. performed the computational analyses, with contribution from J.F.C.-D. Statistical analyses were performed by O.M.-A. with contributions from C.D. and J.F.C.-D. B.M., H.A. and A.A.-O. provided technical and scientific support on the analysis. O.M.-A., H.A., A.C., A.A.-O., B.M., M.B. and C.D. participated in writing the manuscript or contributed to its revision. All authors revised the manuscript and approved the final versio