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The increasing prevalence of pESI(like)-positive, multidrug-resistant (MDR) S. Infantis in Europe is a cause of major concern. As previously demonstrated, the pESI(like) megaplasmid is not only a carrier of antimicrobial resistant (AMR) genes (at least tet, dfr, and sul genes), but also harbours several virulence and fitness genes, and toxin/antitoxin systems that enhance its persistence in the S. Infantis host. In this study, five prototype pESI(like) plasmids, of either CTX-M-1 or CTX-M-65 ESBL-producing strains, were long-read sequenced using Oxford Nanopore Technology (ONT), and their complete sequences were resolved. Comparison of the structure and gene content of the five sequenced plasmids, and further comparison with previously published pESI(like) sequences, indicated that although the sequence of such pESI(like) ‘mosaic’ plasmids remains almost identical, their structures appear different and composed of regions inserted or transposed after different events. The results obtained in this study are essential to better understand the plasticity and the evolution of the pESI(like) megaplasmid, and therefore to better address risk management options and policy decisions to fight against AMR and MDR in Salmonella and other food-borne pathogens.Graphical representation of the pESI-like plasmid complete sequence (ID 12037823/11). Block colours indicate the function of the genes: red: repB gene; pink: class I integrons (IntI); yellow; mobile elements; blue: resistance genes; green: toxin/anti-toxin systems; grey: mer operon; light green: genes involve in conjugation.

A collection of 177 genomes of Salmonella Typhimurium and its monophasic variant isolated in 2014–2019 from Italian poultry/livestock (n = 165) and foodstuff (n = 12), previously screened for antimicrobial susceptibility and assigned to ST34 and single-locus variants, were studied in-depth to check the presence of the novel mcr-9 gene and to investigate their genetic relatedness by whole genome sequencing (WGS). The study of accessory resistance genes revealed the presence of mcr-9.1 in 11 ST34 isolates, displaying elevated colistin minimum inhibitory concentration values up to 2 mg/L and also a multidrug-resistant (MDR) profile toward up to seven antimicrobial classes. Five of them were also extended-spectrum beta-lactamases producers (blaSHV–12 type), mediated by the corresponding antimicrobial resistance (AMR) accessory genes. All mcr-9-positive isolates harbored IncHI2-ST1 plasmids. From the results of the Mash analysis performed on all 177 genomes, the 11 mcr-9-positive isolates fell together in the same subcluster and were all closely related. This subcluster included also two mcr-9-negative isolates, and other eight mcr-9-negative ST34 isolates were present within the same parental branch. All the 21 isolates within this branch presented an IncHI2/2A plasmid and a similar MDR gene pattern. In three representative mcr-9-positive isolates, mcr-9 was demonstrated to be located on different IncHI2/IncHI2A large-size (∼277–297 kb) plasmids, using a combined Illumina–Oxford Nanopore WGS approach. These plasmids were also compared by BLAST analysis with publicly available IncHI2 plasmid sequences harboring mcr-9. In our plasmids, mcr-9 was located in a ∼30-kb region lacking different genetic elements of the typical core structure of mcr-9 cassettes. In this region were also identified different genes involved in heavy metal metabolism. Our results underline how genomics and WGS-based surveillance are increasingly indispensable to achieve better insights into the genetic environment and features of plasmid-mediated AMR, as in the case of such IncHI2 plasmids harboring other MDR genes beside mcr-9, that can be transferred horizontally also to other major Salmonella serovars spreading along the food chain.

Black rot caused by Alternaria alternata in yellow pitahaya (Selenicereus megalanthus) is a significant postharvest disease that is difficult to control. Sodium bicarbonate (SBC) treatments were tested in order to incorporate them into an integrated disease management program and to reduce postharvest use of synthetic fungicide. The control of black rot on fruit treated with 298 mM (2.5%) sodium bicarbonate (SBC) was superior to that resulting from other SBC treatments on fruit examined after 21 d at 12 °C plus 5 d of shelf-life at 20 °C. Treatment with 298 mM reduced weight loss, retained color and firmness, slowed changes in total soluble solids, titratable acid content, and pH of yellow pitahaya during storage and shelf-life. After storage, the general appearance of fruit treated with 298 mM SBC was significantly better than that of those treated with imazalil or non-treated fruit. Treatment with 298 mM SBC is potentially useful in controlling postharvest black rot in yellow pitahaya without harming its sensory quality.

Avian malaria is a parasitic disease of birds caused by protozoa belonging to the genus Plasmodium, within the order Haemosporida. Penguins are considered particularly susceptible, and outbreaks in captive populations can lead to high mortality. We used a multidisciplinary approach to investigate the death due to avian malaria, occurred between 2015 and 2019, in eight African penguins (Spheniscus demersus) kept in two Italian zoos located in central Italy, and situated about 30 km apart. We also provided information about the presence and circulation of Plasmodium spp. in mosquitoes in central Italy by sampling mosquitoes in both zoos where penguin mortalities occurred. In the eight dead penguins, gross and histopathological lesions were consistent with those previously observed by other authors in avian malaria outbreaks. Organs from dead penguins and mosquitoes collected in both zoos were tested for avian malaria parasites by using a PCR assay targeting the partial mitochondrial conserved region of the cytochrome b gene. Identification at species level was performed by sequencing analysis. Plasmodium matutinum was detected in both dead penguins and in mosquitoes (Culex pipiens), while Plasmodium vaughani in Culex pipiens only. Parasites were not found in any of the PCR tested Aedes albopictus samples. Based on our phylogenetic analysis, we detected three previously characterized lineages: Plasmodium matutinum LINN1 and AFTRU5, P. vaughani SYAT05. In Culex pipiens we also identified two novel lineages, CXPIP32 (inferred morphospecies Plasmodium matutinum) and CXPIP33 (inferred morphospecies P. vaughani). Significantly, LINN1 and AFTRU5 were found to be associated to penguin deaths, although only LINN1 was detected both in penguins (along the years of the study) and in Culex pipiens, while AFTRU5 was detected in a single penguin dead in 2017. In conclusion, in our study Plasmodium matutinum was found to cause avian malaria in captive penguins kept in Europe, with Culex pipiens being its most probable vector. Our results are in agreement with previous studies suggesting that Culex pipiens is one of the main vectors of Plasmodium spp. in Europe and the Northern Hemisphere. Zoos maintaining captive penguins in temperate areas where Culex pipiens is abundant should be well aware of the risks of avian malaria, and should put every effort to prevent outbreaks, in particular during the periods when the number of vectors is higher.

The blaNDM-5-producing E. coli Sequence Type (ST)167 high-risk clone is emerging worldwide in human clinical cases, while its presence in companion animals is sporadic and has never been described in Italy. Using a combined Oxford Nanopore (ONT) long-reads and Illumina short-reads sequencing approach, an E. coli ST167 isolated from a hospitalized dog, was in-depth characterized by WGS and the plasmid containing bla

Salmonella Infantis is one of the five serovars most frequently causing human salmonellosis in Europe, mainly associated with poultry. A clone harbouring a conjugative plasmid of emerging S. Infantis (pESI)-like megaplasmid, carrying multidrug resistant (MDR) and extended-spectrum beta-lactamases (ESBL) genes, has spread in the Italian broiler chicken industry also causing human illness. This work is aimed at elucidating the molecular epidemiology of S. Infantis and pESI-like in Europe using whole-genome sequencing and bioinformatics analysis, and to investigate the genetic relatedness of S. Infantis clones and pESI-like from animals, meat, feed and humans provided by institutions of nine European countries. Two genotyping approaches were used: chromosome or plasmid SNP-based analysis and the minimum spanning tree (MST) algorithm based on core-genome multilocus sequence typing (cgMLST). The European S. Infantis population appeared heterogeneous, with different genetic clusters defined at core-genome level. However, pESI-like variants present in 64.1 % of the isolates were more genetically homogeneous and capable of infecting different clonal lineages in most of the countries. Two different pESI-like with ESBL genes (n=82) were observed: bla CTX-M-1-positive in European isolates and bla CTX-M-65-positive in American isolates (study outgroup). Both variants had toxin-antitoxin systems, resistance genes towards tetracyclines, trimethoprim, sulphonamides and aminoglycosides, heavy metals (merA) and disinfectants (qacEΔ). Worryingly, 66 % of the total isolates studied presented different gyrA chromosomal point mutations associated with (fluoro)quinolone resistance (MIC range 0.125–0.5 mg/L), while 18 % displayed transferable macrolide resistance mediated by mph, mef and erm(B) genes. Proper intervention strategies are needed to prevent further dissemination/transmission of MDR S. Infantis and pESI-like along the food chain in Europe.

Objectives To the best of our knowledge, we describe the first evidence in Europe of an MDR, blaNDM-4-positive Escherichia coli isolated from a food-producing animal, harboured by a novel IncFII plasmid of which we report the complete sequence. Methods One blaNDM-4-positive E. coli isolated in 2019 from the caecal contents of a fattening pig in Italy was in-depth characterized by combined bioinformatic analysis of Oxford Nanopore long reads and Illumina short reads, for in silico typing, determination of the blaNDM-4 genetic context and full reconstruction of the blaNDM-4–carrying plasmid. Results The isolate belonged to ST641 and to the genoserotype O108:H23 and tested positive for different virulence genes and plasmid replicons. The MDR phenotype of resistance to all β-lactams, carbapenems, sulfamethoxazole and trimethoprim was mediated by blaTEM-1B, blaNDM-4, sul1/sul3 and dfrA12, respectively. The blaNDM-4 gene was harboured by a novel 53 043 bp IncFII plasmid (pMOL412_FII) composed of four main genetic regions, including an MDR region (MRR-NDM-4) of 16 kb carrying blaNDM-4 and several antimicrobial resistance genes located in a class 1 integron. pMOL412_FII was closely related to another ∼90.3 kb plasmid (pM109_FII) harbouring blaNDM-4 in an E. coli isolated from a human patient in Myanmar. Conclusions To the best of our knowledge, we have identified for the first time in Europe an NDM-producing Enterobacterales in livestock and resolved the complete sequence of the novel pMOL412_FII plasmid harbouring blaNDM-4 in an MRR. A global One Health approach, comparing genomic data from different sources and geographical areas, may help to trace back and control possible plasmid-borne carbapenemase gene transmission between animals and humans and along the food chain at international level.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) clones other than Clonal Complex (CC)398, as CC1, have been isolated in pigs in some countries, and appeared to be prevalent in Italy. The aim of this study was to evaluate the capability of Sequence Type (ST)1, CC1, LA-MRSA clone to colonize and to be transmitted among piglets. Eighteen caesarean-derived/colostrum-deprived piglets of 35 days of age were assigned randomly to three groups: four seeder piglets were contaminated with a spa type t127, ST1, SCCmec V, MRSA (Group A), 10 MRSA-negative piglets were exposed to Group A after 2 days post-contamination, dpc (Group B) and 4 piglets were used as control group (Group C). Piglets were evaluated until 44 dpc (Group A) or at 42 days post-exposure, dpe (Group B) and then euthanized and necropsied. All nasal and skin cultures of Group A resulted MRSA-positive throughout the experiment starting from two dpc, while Group C tested always MRSA-negative. At first sampling, all Group B piglets became positive and remained positive throughout the experiment. This is the first colonization/transmission study with a CC1 LA-MRSA in pigs. The results add further knowledge on the ability of CC1 LA-MRSA to colonize pigs, and on colonization/transmission patterns, both suggesting good host adaptation.

We investigated the evolution and epidemiology of a novel livestock-associated methicillin-resistant Staphylococcus aureus strain, which colonizes and infects urban-dwelling Danes even without a Danish animal reservoir. Genetic evidence suggests both poultry and human adaptation, with poultry meat implicated as a probable source.

Staphylococcus aureus is involved in a wide variety of diseases in humans and animals, and it is considered one of the most significant etiological agents of intramammary infection in dairy ruminants, causing both clinical and subclinical infections. In this study, the intra-farm prevalence and circulation of methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) were investigated on an Italian dairy sheep farm previously identified as MRSA-positive by testing bulk tank milk (first isolation in 2012). Human samples (nasal swabs, hand skin samples, and oropharyngeal swabs) from 3 persons working in close contact with the animals were also collected, and the genetic characteristics and relatedness of the MRSA isolates from human and animal sources within the farm were investigated. After 2yr from the first isolation, we confirmed the presence of the same multidrug-resistant strain of MRSA sequence type (ST)1, clonal complex (CC)1, spa type t127, staphylococcal cassette chromosome mec (SCCmec) type IVa, showing identical pulsed field gel electrophoresis (PFGE) and resistance profiles at the farm level in bulk tank milk. Methicillin-resistant S. aureus isolates were detected in 2 out of 556 (0.34%) individual milk samples, whereas MSSA isolates were detected in 10 samples (1.8%). The MRSA were further isolated from udder skin samples from the 2 animals that were MRSA-positive in milk and in 2 of the 3 examined farm personnel. All MRSA isolates from both ovine and human samples belonged to ST(CC)1, spa type t127, SCCmec type IVa, with some isolates from animals harboring genes considered markers of human adaptation. In contrast, all MSSA isolates belonged to ruminant-associated CC130, ST700, spa type t528. Analysis by PFGE performed on selected MRSA isolates of human and animal origin identified 2 closely related (96.3% similarity) pulsotypes, displaying only minimal differences in gene profiles (e.g., presence of the immune evasion cluster genes). Although we observed low MRSA intra-farm prevalence, our findings highlight the importance of considering the possible zoonotic potential of CC1 livestock-associated MRSA, in view of the ability to persist over years at the farm level. Biosecurity measures and good hygiene practices could be useful to prevent MRSA spread at the farm level and to minimize exposure in the community and in categories related to farm animal industry (e.g., veterinarians, farmers, and farm workers).

Pandemic methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 97 (CC97) lineages originated from livestock-to-human host jumps. In recent years, CC97 has become one of the major MRSA lineages detected in Italian farmed animals. The aim of this study was to characterize and analyze differences in MRSA and methicillin-susceptible S. aureus (MSSA) mainly of swine and bovine origins. Forty-seven CC97 isolates, 35 MRSA isolates, and 6 MSSA isolates from different Italian pig and cattle holdings; 5 pig MRSA isolates from Germany; and 1 human MSSA isolate from Spain were characterized by macrorestriction pulsed-field gel electrophoresis (PFGE) analysis, multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCC mec ) typing, and antimicrobial resistance pattern analysis. Virulence and resistance genes were investigated by PCR and microarray analysis. Most of the isolates were of SCC mec type V (SCC mec V), except for two German MRSA isolates (SCC mec III). Five main clusters were identified by PFGE, with the German isolates (clusters I and II) showing 60.5% similarity with the Italian isolates, most of which (68.1%) grouped into cluster V. All CC97 isolates were Panton-Valentine leukocidin (PVL) negative, and a few ( n = 7) tested positive for sak or scn . All MRSA isolates were multidrug resistant (MDR), and the main features were erm (B)- or erm (C)-mediated ( n = 18) macrolide-lincosamide-streptogramin B resistance, vga (A)-mediated ( n = 37) pleuromutilin resistance, fluoroquinolone resistance ( n = 33), tet (K) in 32/37 tet (M)-positive isolates, and blaZ in almost all MRSA isolates. Few host-associated differences were detected among CC97 MRSA isolates: their extensive MDR nature in both pigs and dairy cattle may be a consequence of a spillback from pigs of a MRSA lineage that originated in cattle as MSSA and needs further investigation. Measures should be implemented at the farm level to prevent spillover to humans in intensive farming areas.

A Corrigendum onColistin Resistance Mediated by mcr-1 in ESBL-Producing, Multidrug Resistant Salmonella Infantis in Broiler Chicken Industry, Italy (2016–2017)by Carfora, V., Alba, P., Leekitcharoenphon, P., Ballarò, D., Cordaro, G., Di Matteo, P., et al. (2018) Front. Microbiol. 9:1880. doi: 10.3389/fmicb.2018.01880In the original article, there was an error in the Materials and Methods, subsection Isolates. The four S. Infantis isolates originated from broilers (n = 2) and broiler meat samples (n = 2).A correction has been made to Materials and Methods, subsection Isolates: Four multidrug resistant (MDR) S. Infantis, displaying a colistin MIC value ≥ 4 mg/L, were detected among 324 S. Infantis isolates collected in the frame of antimicrobial resistance (AMR) monitoring activities conducted from 2001 to 2017 by the National Reference Laboratory for Antimicrobial Resistance (NRL-AR) and screened for antimicrobial susceptibility. The four S. Infantis isolates originated from broilers (n = 3) and from broiler meat sample (n = 1) (Supplementary Table 1).The authors apologize for this error and state that this does not change the scientific conclusionsof the article in any way.The original article has been updated.

Colistin resistance by mobilisable mcr genes has been described in bacteria of food-animal origin worldwide, which has raised public health concerns about its potential foodborne transmission to human pathogenic bacteria. Here we provide baseline information on the molecular epidemiology of colistin-resistant, mcr-positive Escherichia coli and Salmonella isolates in food-producing animals in Italy in 2014-2015. A total 678, 861 and 236 indicator E. coli, Extended Spectrum Beta-Lactamase (ESBL)/AmpC-producing E. coli, and Salmonella isolates, respectively, were tested for colistin susceptibility. These isolates were collected according to the EU harmonized antimicrobial resistance monitoring program and are representative of at least 90 and 80% of the Italian poultry (broiler chickens and turkeys) and livestock (pigs and bovines < 12 months) production, respectively. Whole genome sequencing by Illumina technology and bioinformatics (Center for Genomic Epidemiology pipeline) were used to type 42 mcr-positive isolates by PCR. Colistin resistance was mainly observed in the ESBL/AmpC E. coli population, and was present in 25.9, 5.3, 6.5, and 3.9% of such isolates in turkeys, broilers, pigs, and bovines, respectively. Most colistin-resistant isolates (141/161, 87.5%) harbored genes of the mcr-1 group. mcr-1 was also detected in a small proportion of Salmonella isolates (3/146, 2.0%) in turkeys. Additional mcr types were mcr-3 in four ESBL-producing E. coli from bovines, and two mcr-4 in ESBL (n = 1) and indicator E. coli (n = 1) from pigs and bovines. We describe notable diversity of mcr variants with predominance of mcr-1.1 and mcr-1.2 on conjugative IncX4 plasmids in E. coli and in Salmonella serovars Typhimurium, Newport, Blockley from turkey. A new variant, mcr-1.13 was detected in the chromosome in E. coli in turkey and pig isolates. Additionally, we describe mcr-3.2 and mcr-4.3 in E. coli from bovines, and mcr-4.2 in E. coli from pigs. These findings elucidate the epidemiology of colistin resistance in food-producing animals in Italy along with its genetic background, and highlight the likelihood of mcr horizontal transfer between commensal bacteria and major food-borne pathogens (Salmonella) within the same type of productions. Thorough action and strategies are needed in order to mitigate the risk of mcr transfer to humans, in a “One Health” perspective.

The ENGAGE project (http://www.engage-europe.eu/) was a collaboration between eight institutions across Europe. The aim was to boost the scientific cooperation to use whole genome sequencing (WGS) analysis in food safety and public health protection. ENGAGE focused on Escherichia coli (commensal E. coli) and different Salmonella spp. serotypes. A total of 3,360 genomes, 778 and 2,582 of E. coli and Salmonella, respectively, were produced. These genomes were stored and shared among partners in a temporary repository to be submitted to the European Nucleotide Archive by the end of the project. Generated genomes were used for benchmarking exercises to assess the possibility of replacing conventional typing with WGS for outbreak investigation. For the analysed strains, the benchmarking exercises showed that SPAdes assembly performed better than Velvet and that, by using different bioinformatics tools, WGS Salmonella serotyping and antimicrobial resistance genes detection, were largely in concordance with phenotypic data. Discrepancies were related to sequence quality and phenotype misclassification rather than to limitations of the bioinformatics tools. All partners were able to infer the expected phylogeny for the Salmonella and Campylobacter isolates in benchmarking exercises. Two WGS proficiency tests (assessing different genomic quality markers) were conducted among partners with satisfactory results. Guidelines including available bioinformatics tools and standard operating procedures (wet and dry lab) were prepared and posted online. Workshops, training courses and twinning programmes were conducted. The training focused on online, Galaxy‐based, and command line bioinformatics tools. To reach out beyond ENGAGE, an e‐learning course (17 videos) was developed and made available online. Several proof of concept projects were run and some outcomes published, e.g. the discovery of colistin resistance gene, mcr‐5. Overall, the project showed that laboratories without previous WGS experience need a period of time to implement and perform WGS for foodborne pathogens routine analysis. All developed material will remain available on the ENGAGE website.

Rational strategies for obtaining malaria vaccine candidates should include not only a proper selection of target antigens for antibody stimulation, but also a versatile molecular design based on ordering the right pieces from the complex pathogen molecular puzzle towards more active and functional immunogens. Classical Plasmodium falciparum antigens regarded as vaccine candidates have been selected as model targets in this study. Among all possibilities we have chosen epitopes of PfCSP, STARP; MSA1 and Pf155/RESA from pre- A nd erythrocyte stages respectively for designing a large 82-residue chimeric immunogen. A number of options aimed at diminishing steric hindrance for synthetic procedures were assessed based on standard Fmoc chemistry such as building block orthogonal ligation; pseudo-proline and microwave-assisted procedures, therefore the large-chimeric target was produced, characterized and immunologically tested. Antigenicity and functional in vivo efficacy tests of the large-chimera formulations administered alone or as antigen mixtures have proven the stimulation of high antibody titers, showing strong correlation with protection and parasite clearance of vaccinated BALB/c mice after being lethally challenged with both P. berghei-ANKA and P. yoelii 17XL malaria strains. Besides, 3D structure features shown by the large-chimera encouraged as to propose using these rational designed large synthetic molecules as reliable vaccine candidate-presenting systems.

A totally effective, antimalarial vaccine must involve sporozoite and merozoite proteins (or their fragments) to ensure complete parasite blocking during critical invasion stages. This Special Report examines proteins involved in critical biological functions for parasite survival and highlights the conserved amino acid sequences of the most important proteins involved in sporozoite invasion of hepatocytes and merozoite invasion of red blood cells. Conserved high activity binding peptides are located in such proteins' functionally strategic sites, whose functions are related to receptor binding, nutrient and protein transport, enzyme activity and molecule-molecule interactions. They are thus excellent targets for vaccine development as they block proteins binding function involved in invasion and also their biological function. © 2017 Future Medicine Ltd.

Los Polisacáridos no Amiláceos (PNA) son un tipo de carbohidratos presentes en antinutricionales con el fin de disminuir los niveles de grasa corporal. Para esta investigación se contó con tres grupos de pollos de engorde de la línea Ross: al primero se le incluyó alcachofa (Cynara scolymus) en la dieta; al segundo, cebada (Hordeum vulgare), y al tercero se le dejó sólo el alimento balanceado comercial. Como resultado final se obtuvo un menor porcentaje de grasa en canal en los pollos del tratamiento 1, seguido por los pollos del tratamiento 2, y los mayores porcentajes de grasa en canal fueron los de los pollos del grupo 3 o control. Como resultado adicional, en este grupo 3 se presentaron muertes por Síndrome Ascítico (SA), con lo cual se plantea la posibilidad de emplear los PNA, con el fin de disminuir la presentación de enfermedades metabólicas.

The importance of CSP- and STARP-derived ? and ? dihedral angles in mHABP structure was analysed by 1H NMR in the search for molecules which can be included as components of a first-line-of-defence Plasmodium falciparum sporozoite multi-epitope vaccine against the most lethal form of human malaria. Most of the aforementioned dihedral angles were left-hand-like polyproline type II (PPIIL) structures whilst others had right-hand-like ?-helix (?R), thus allowing mHABPS to fit better into MHCII molecules and thereby form an appropriate pMHCII complex and also establish the H-bonds which stabilise such complex and by this means induce an appropriate immune response. This information has great implications for vaccine development, malaria being one of them. © 2012 Elsevier Inc.

Photobacterium damselae subsp. damselae (PDD) is a known pathogen of fish, humans and marine mammals. In this study, a Multilocus Sequence Typing (MLST) scheme based on six housekeeping genes (glp, gyrB, metG, pnt, pyrC, and toxR) was developed to better understand the PDD population structure and used to type 73 PDD isolates from cetaceans, mainly striped dolphins (Stenella coeruleoalba) involved in mortality episodes, and from a few marine chelonians. Five reference ATCC strains were also included in the study. Typing allowed the discrimination of groups of PDD strains isolated from different host species, at different times and from different geographic areas, suggesting that a clonal PDD group may have spread in the Tyrrhenian sea at the time of an Unusual Mortality Event (UME) among cetaceans, mainly striped dolphins, occurred in early 2013 along the Italian western coasts.

Fully-protective, long-lasting, immunological (FPLLI) memory against Plasmodium falciparum malaria regarding immune protection-inducing protein structures (IMPIPS) vaccinated into monkeys previously challenged and re-challenged 60 days later with a lethal Aotus monkey-adapted P. falciparum strain was found to be associated with preferential high binding capacity to HLA-DR?1* allelic molecules of the major histocompatibility class II (MHC-II), rather than HLA-DR?3*, ?4*, ?5* alleles. Complete PPIIL 3D structure, a longer distance (26.5 Å ± 1.5 Å) between residues perfectly fitting into HLA-DR?1*PBR pockets 1 and 9, a gauche? rotamer orientation in p8 TCR-contacting polar residue and a larger volume of polar p2 residues was also found. This data, in association with previously-described p3 and p7 apolar residues having gauche+ orientation to form a perfect MHC-II-peptide-TCR complex, determines the stereo-electronic and topochemical characteristics associated with FPLLI immunological memory. © 2016 Elsevier Inc.

SIAP-1 and SIAP-2 are proteins which are implicated in early events involving Plasmodium falciparum infection of the Anopheles mosquito vector and the human host. High affinity HeLa and HepG2 cell binding conserved peptides have been previously identified in these proteins, i.e. SIAP-1 34893 ( 421KVQGLSYLLRRKNGTKHPVY 440) and SIAP-1 34899 ( 541YVLNSKLLNSRSFDKFKWIQ 560) and SIAP-2 36879 ( 181LLLYSTNSEDNLDISFGELQ 200). When amino acid sequences have been properly modified (replacements shown in bold) they have induced high antibody titres against sporozoites in Aotus monkeys (assessed by IFA) and in the corresponding recombinant proteins (determined by ELISA and Western blot). 1H NMR studies of these conserved native and modified high activity binding peptides (HABPs) revealed that all had ?-helical structures in different locations and lengths. Conserved and corresponding modified HABPs displayed different lengths between the residues fitting into MHCII molecule pockets 1-9 and different amino acid orientation based on their different HLA-DR?1 * binding motifs and binding registers, suggesting that such modifications were associated with making them immunogenic. The results suggested that these modified HAPBs could be potential targets for inclusion as components of a fully-effective, minimal sub-unit based, multi-epitope, and multistage anti-malarial vaccine. © 2011 Elsevier Inc..

Background: There is an increasing interest in the study of microorganisms that inhabit extreme environments for reasons that vary from gaining insight into the origin of life to the searching of new biotechnological applications. Aims: In this work, we studied the tolerance of fungi isolated from the Aguas Agrias Stream (AAS; Tharsis, Huelva, Spain), an acidic metal-rich environment, to a culture medium prepared with water from this extreme ecosystem (AASW medium). The ability of some culture collection strains of moulds and yeasts to grow on AASW medium was also assessed. Methods: For moulds, a tolerance index was calculated by dividing the growth diameter of colonies on AASW medium by the diameter in the control medium, and their germinative potential was recorded. For yeasts and yeast-like fungi, the minimum inhibitory concentration of AASW was determined. Results: In general, the fungi isolated from the AAS showed differences in their ability to germinate and grow on AASW medium. Collection strains of the genus Aspergillus could grow on AASW medium, but showed some differences in tolerance when compared to environmental isolates. Conclusions: Extremotolerant fungi can manifest differences in their tolerance to culture media that simulate the conditions of their natural habitat. The results of this work suggest that the ability of fungi to grow in acidic, metal-rich environments might be more widespread than previously thought, and highlight the importance of determining the factors that are responsible for tolerance to these extreme environments.

Streptococcus pyogenes infections remain a health problem in several countries due to poststreptococcal sequelae. We developed a vaccine epitope (StreptInCor) composed of 55 amino acids residues of the C-terminal portion of the M protein that encompasses both T and B cell protective epitopes. The nuclear magnetic resonance (NMR) structure of the StreptInCor peptide showed that the structure was composed of two microdomains linked by an 18-residue α-helix. A chemical stability study of the StreptInCor folding/unfolding process using far-UV circular dichroism showed that the structure was chemically stable with respect to pH and the concentration of urea. The T cell epitope is located in the first microdomain and encompasses 11 out of the 18 α-helix residues, whereas the B cell epitope is in the second microdomain and showed no α-helical structure. The prediction of StreptInCor epitope binding to different HLA class II molecules was evaluated based on an analysis of the 55 residues and the theoretical possibilities for the processed peptides to fit into the P1, P4, P6, and P9 pockets in the groove of several HLA class II molecules. We observed 7 potential sites along the amino acid sequence of StreptInCor that were capable of recognizing HLA class II molecules (DRB1*, DRB3*, DRB4*, and DRB5*). StreptInCor-overlapping peptides induced cellular and humoral immune responses of individuals bearing different HLA class II molecules and could be considered as a universal vaccine epitope.

To block the different stages of Plasmodium falciparum invasion into human hepatocytes and red blood cells, we have focused on those proteins belonging to the pre-erythrocytic stage. One of these proteins is Sporozoite Threonine and Asparagine Rich Protein (STARP), which is a ligand used by P. falciparum parasites to bind Hepatic cells (HepG2). Previous studies on this protein identified two conserved peptides binding with high activity to HepG2 cells (namely 20546 and 20570) with corresponding critical hepatic-cell binding residues and determined an important role for these two peptides in the invasion process. This study shows the results of immunization trials in Aotus monkeys with native STARP peptides and analogues modified in critical hepatic-cell binding residues. The results show that native peptides are not immunogenic but can induce high-antibody titers when their critical residues are replaced by other with similar volume and mass but different polarity. Nuclear Magnetic Resonance (1H NMR) studies revealed that native peptides (non-immunogenic) displayed shorter ?-helical regions compared to their highly immunogenic modified analogues. Binding assays with HLA-DR?1* molecules showed that 20546 modified peptides inducing high-antibody titers (24972, 24320 and 24486) bound to HLA-DR?1*0301 molecules, while the 20570 modified analogue (24322) bound to HLA-DR?1*0101. The results support including these high-immunogenic STARP-derived modified peptides as pre-erythrocytic candidates to be included in the design of a synthetic antimalarial vaccine. © 2010 Elsevier Ltd.

In recent years, several lines of evidence have questioned the asexual nature of Aspergillus fumigatus , showing that this fungus possesses a fully functional sexual reproductive cycle that leads to the production of cleistothecia and ascospores. The presence of a sexual cycle in A. fumigatus could have signifi cant medical implications, as sexual reproduction might contribute to increased virulence or resistance to antifungal agents. In the present work, we studied the relationship between mating type and invasiveness in A. fumigatus . Statistical analysis of the results showed a signifi cant association between the mating type MAT1-1 and an invasive origin of the isolates. Similarly, when the clinical or environmental origin of isolates was considered instead of their invasive or non-invasive origin, a signifi cant association between the mating type MAT1-1 and clinical origin was observed. Finally, the association between mating type MAT1-1 and pathogenicity, measured by an Elastase Activity Index � 1, was signifi cant. Our results suggest a possible association between the MAT1-1 mating type and A. fumigatus invasiveness.

Background: Aspergillus fumigatus, like many other fungal species of clinical relevance, has been traditionally regarded as an asexual organism. However, in last few years several pieces of evidence question this such assumption, suggesting that the sexual state of A. fumigatus may still be undiscovered. These investigations have finally led to the recent discovery of a teleomorph stage of A. fumigatus, which has been named Neosartorya fumigata. Aims: To review the most important findings on A. fumigatus sexuality and discuss the possible implications of such findings on its pathogenicity. Methods: A bibliographic search was performed to find the main works that study the sexuality of fungal pathogens and, especially, of A. fumigatus. Moreover, data from our recent investigations in this field were also introduced to the discussion. Results: The existence of a teleomorph for A. fumigatus could have significant clinical repercussions, as sexual reproduction might produce offspring with increased virulence and/or resistance to antifungal agents. In this sense, the results of our investigations suggest the existence of an association between the MAT1-1 mating type and the invasiveness of A. fumigatus isolates. Conclusions: The study of the sexual reproduction of the fungal pathogens and its possible relationship with virulence will continue to be a topic of interest during the next years, not only because of its basic interest, but also for the possible clinical repercussions.

Clostridium difficile is considered an important uncontrolled cause of neonatal diarrhea. Also, the presence of bacteria in the feces of the animal could represent a zoonosic risk for the contamination of meat products. Therefore, it is necessary to have procedures available for the early detection of C. difficile in animals. The current study describes a new semi-automated procedure for the recovery of C. difficile DNA from pig feces and subsequent amplification by polymerase chain reaction (PCR) of three different sequences: the triose phosphate isomerase gene tpi, specific for this bacterial species, and the tcdA and tcdB genes, which code for the A and B toxins of C. difficile, respectively. Twenty-two fecal samples microbiologically positive for C. difficile were used. The tpi and tcdA genes were amplified in all of them. The internal fragment of tcdB was detected from 21 of these extracts; the negative sample gave a positive result when a different primer pair was used. None of the 10 DNA extracts obtained from culture-negative samples gave a positive result. The method presented in this article eliminates the interference caused by the possible presence of PCR inhibitors. To the authors' knowledge, this is the first description of a PCR procedure for detection of C. difficile DNA from domestic animal feces.

Clostridium difficile is considered to be an important causative agent of porcine neonatal diarrhoea, having taken over from classic bacterial pathogens. However, there are currently no clear data concerning the prevalence of this microorganism in piglets, or about its relative distributions among diarrhoeic and non-diarrhoeic animals. In the present study, we analyzed the presence of C. difficile in rectal swabs from 780 piglets from two age groups (newborn and 1-2-month-old pigs) by means of molecular and microbiological procedures. Furthermore DNA was isolated from the bacteria in order to identify toxin A and toxin B genes.C. difficile was not found in any of the 239 samples taken from 1- to 2-month-old pigs. Bacteria were, however, recovered from 140 out of 541 newborn piglets (25.9%), including both diarrhoeic and non-diarrhoeic animals, and animals from control farms (free of diarrhoeic animals). Genes for the production of both toxins A and B were identified in 132 of the 140 isolates (A(+)B(+)). Only seven isolates, all from the same farm and from non-diarrhoeic animals, lacked both toxin genes (A(-)B(-)), while one isolate from this same group of animals was A(-)B(+).This study provides the first report comparing the prevalence of C. difficile in large numbers of diarrhoeic and non-diarrhoeic animals. There was no clear link between bacterial isolation and neonatal porcine diarrhoea.

In recent years, several lines of evidence have questioned the asexual nature of Aspergillus fumigatus, showing that this fungus possesses a fully functional sexual reproductive cycle that leads to the production of cleistothecia and ascospores. The presence of a sexual cycle in A. fumigatus could have significant medical implications, as sexual reproduction might contribute to increased virulence or resistance to antifungal agents. In the present work, we studied the relationship between mating type and invasiveness in A. fumigatus. Statistical analysis of the results showed a significant association between the mating type MAT1-1 and an invasive origin of the isolates. Similarly, when the clinical or environmental origin of isolates was considered instead of their invasive or non-invasive origin, a significant association between the mating type MAT1-1 and clinical origin was observed. Finally, the association between mating type MAT1-1 and pathogenicity, measured by an Elastase Activity Indexor = 1, was significant. Our results suggest a possible association between the MAT1-1 mating type and A. fumigatus invasiveness.