The Zymoseptoria tritici white collar-1 gene, ZtWco-1, is required for development and virulence on wheat.

The fungus Zymoseptoria tritici causes Septoria Tritici Blotch (STB), which is one of the most devastating diseases of wheat in Europe. There are currently no fully durable methods of control against Z. tritici, so novel strategies are urgently required. One of the ways in which fungi are able to respond to their surrounding environment is through the use of photoreceptor proteins which detect light signals. Although previous evidence suggests that Z. tritici can detect light, no photoreceptor genes have been characterised in this pathogen. This study characterises ZtWco-1, a predicted photoreceptor gene in Z. tritici. The ZtWco-1 gene is a putative homolog to the blue light photoreceptor from Neurospora crassa, wc-1. Z. tritici mutants with deletions in ZtWco-1 have defects in hyphal branching, melanisation and virulence on wheat. In addition, we identify the putative circadian clock gene ZtFrq in Z. tritici. This study provides evidence for the genetic regulation of light detection in Z. tritici and it open avenues for future research into whether this pathogen has a circadian clock.

Comparison of Two Multilocus Sequence Typing Schemes for Mycoplasma bovis and Revision of the PubMLST Reference Method.

Mycoplasma bovis causes pneumonia, pharyngitis, otitis, arthritis, mastitis, and reproductive disorders in cattle and bison. Two multilocus sequence typing (MLST) schemes have been developed for M. bovis, with one serving as the PubMLST reference method, but no comparison of the schemes has been undertaken. Although the PubMLST scheme has proven to be highly discriminatory and informative, the recent discovery of isolates missing one of the typing loci, adh-1, raises concern about its suitability for continued use. The goal of our study was to compare the performance of the two MLST schemes and identify a new reference scheme capable of fully typing all isolates. We evaluated 448 isolates from diverse geographic and anatomic sites that collectively represent cattle, bison, deer, and a goat. The discrimination indexes (DIs) for the PubMLST and the alternative scheme are 0.909 (91 sequence types [STs]) and 0.842 (77 STs), respectively. Although the PubMLST scheme outperformed the alternative scheme, the adh-1 locus must be retired from the PubMLST scheme if it is to be retained as a reference method. The DI obtained using the six remaining PubMLST loci (0.897, 79 STs) fails to reach the benchmark recommended for a reference method (0.900), mandating the addition of a seventh locus. Comparative analysis of genome sequences from the isolates used here identified the dnaA locus from the alternative scheme as the optimal replacement for adh-1 This revised scheme, which will be implemented as the new PubMLST reference method, has a DI of 0.914 and distinguishes 88 STs from the 448 isolates evaluated.

Prevalence and phylogeny of Chlamydiae and hemotropic mycoplasma species in captive and free-living bats.

BACKGROUND: Bats are hosts for a variety of microorganisms, however, little is known about the presence of Chlamydiales and hemotropic mycoplasmas. This study investigated 475 captive and free-living bats from Switzerland, Germany, and Costa Rica for Chlamydiales and hemotropic mycoplasmas by PCR to determine the prevalence and phylogeny of these organisms. RESULTS: Screening for Chlamydiales resulted in a total prevalence of 31.4%. Positive samples originated from captive and free-living bats from all three countries. Sequencing of 15 samples allowed the detection of two phylogenetically distinct groups. These groups share sequence identities to Chlamydiaceae, and to Chlamydia-like organisms including Rhabdochlamydiaceae and unclassified Chlamydiales from environmental samples, respectively. PCR analysis for the presence of hemotropic mycoplasmas resulted in a total prevalence of 0.7%, comprising free-living bats from Germany and Costa Rica. Phylogenetic analysis revealed three sequences related to other unidentified mycoplasmas found in vampire bats and Chilean bats. CONCLUSIONS: Bats can harbor Chlamydiales and hemotropic mycoplasmas and the newly described sequences in this study indicate that the diversity of these bacteria in bats is much larger than previously thought. Both, Chlamydiales and hemotropic mycoplasmas are not restricted to certain bat species or countries and captive and free-living bats can be colonized. In conclusion, bats represent another potential host or vector for novel, previously unidentified, Chlamydiales and hemotropic mycoplasmas.

Systemic infection in European perch with thermoadapted virulent Aeromonas salmonicida (Perca fluviatilis).

In non-salmonid fish, Aeromonas salmonicidacan cause local infections with severe skin ulcerations, known as atypical furunculosis. In this study, we present a systemic infection by a virulent A. salmonicidain European perch (Perca fluviatilis).This infection was diagnosed in a Swiss warm water recirculation aquaculture system. The isolate of A.  salmonicida encodes a type three secretion system (TTSS) most likely located on a plasmid similar to pAsa5/pASvirA, which is known to specify one of the main virulence attributes of the species A. salmonicida. However, the genes specifying the TTSS of the perch isolate show a higher temperature tolerance than strains isolated from cold-water fish. The function of the TTSS in virulence was verified in a cytotoxicity test using bluegill fry and epithelioma papulosum cyprinid cells.

Sensitive and Specific Recombinase Polymerase Amplification Assays for Fast Screening, Detection, and Identification of Bacillus anthracis in a Field Setting.

Four isothermal recombinase polymerase amplification (RPA) assays were developed for fast in-field identification of Bacillus anthracis The RPA assays targeted three specific sequences (i.e., the BA_5345 chromosomal marker, the lethal factor lef [from pXO1], and the capsule-biosynthesis-related capA [from pXO2]) and a conserved sequence in the adenylate cyclase gene (adk) for the Bacillus cereus group. B. anthracis-specific RPA assays were tested first with purified genomic DNAs (n = 60), including 11 representatives of B. anthracis, and then with soil (n = 8) and white powder (n = 8) samples spiked with inactivated B. anthracis spores and/or other biological agents. The RPA assays were also tested in another laboratory facility, which blindly provided DNA and lysate samples (n = 30, including 20 B. anthracis strains). RPA assays displayed 100% specificity and sensitivity. The hands-off turnaround times at 42°C ranged from 5 to 6 min for 102 genomic copies. The analytical sensitivity of each RPA assay was ∼10 molecules per reaction. In addition, the BA_5345 and adk RPA assays were assessed under field conditions with a series of surface swabs (n = 13, including 11 swabs contaminated with B. thuringiensis spores) that were blindly brought to the field laboratory by a chemical, biological, radiological, and nuclear (CBRN) sampling team. None of the 13 samples, except the control, tested positive for B. anthracis, and all samples that had been harvested from spore-contaminated surfaces tested positive with the adk RPA assay. All three B. anthracis-specific RPA assays proved suitable for rapid and reliable identification of B. anthracis and therefore could easily be used by first responders under field conditions to quickly discriminate between a deliberate release of B. anthracis spores and a hoax attack involving white powder.IMPORTANCE In recent decades, particularly following the 11 September 2001 and Amerithrax attacks, the world has experienced attempts to sow panic and chaos in society through thousands of white-powder copycats using household powders to mimic real bioterrorism attacks. In such circumstances, field-deployable detection methods are particularly needed to screen samples collected from the scene. The aim is to test the samples directly using a fast and reliable assay for detection of the presence of B. anthracis While this would not preclude further confirmatory tests from being performed in reference laboratories, it would bring useful, timely, and relevant information to local crisis managers and help them make appropriate decisions without having to wait for quantitative PCR results (with turnaround times of a few hours) or phenotypic identification and sequencing (with turnaround times of a few days). In the current investigation, we developed a set of isothermal RPA assays for the rapid screening and identification of B. anthracis in powders and soil samples, with the purpose of discriminating a deliberate release of B. anthracis spores from a hoax attack involving white powder; this would also apply to dispersion by spraying of aerosolized forms of B. anthracis Further work is now ongoing to confirm the first observations and validate the on-site use of these assays by first responders.

Mycoplasma bovis co-infection with bovine viral diarrhea virus in bovine macrophages.

Several studies suggest that synergisms between Mycoplasma bovis and other microorganisms might exacerbate disease outcome of bovine mycoplasmosis. Screening several bovine cell types to assess their potential use as in vitro infection models for M. bovis, it was observed that a widely used cell line of bovine macrophages (Bomac cells) is in fact persistently infected with bovine viral diarrhea virus (BVDV). The cell line was first cured of this virus allowing comparative studies between both cell lines. Subsequently, uptake and co-culture of two M. bovis strains of different clonal complexes with Bomac cells contaminated with BVDV and in BVDV-free Bomac cells were assessed. Additionally, cell viability, cytotoxicity and induction of apoptosis after infection with M. bovis were evaluated. No differences in the levels of uptake and growth in co-culture were observed between the two Bomac cell types and both M. bovis strains. Cytotoxicity was increased after infection of BVDV-free cells with one of the two strains, while apoptotic cell death was slightly induced by this strain in both cell lines. Overall, the presence or absence of BVDV in Bomac cells did not grossly change the parameters tested upon infection with M. bovis. Nevertheless, this cell model is very useful when studying viral co-infections with bacteria and could also be used for multiple co-infections. Considering the broad contamination of cell cultures with BVDV, careful screening for this virus should routinely be performed as its presence might be relevant depending on the molecular mechanisms being investigated.

Tularemia among free-ranging mice without infection of exposed humans, Switzerland, 2012.

The animals primarily infected by Francisella tularensis are rapidly consumed by scavengers, hindering ecologic investigation of the bacterium. We describe a 2012 natural tularemia epizootic among house mice in Switzerland and the assessment of infection of exposed humans. The humans were not infected, but the epizootic coincided with increased reports of human cases in the area.

Identification of amino acid substitutions with compensational effects in the attachment protein of canine distemper virus.

The hemagglutinin (H) gene of canine distemper virus (CDV) encodes the receptor-binding protein. This protein, together with the fusion (F) protein, is pivotal for infectivity since it contributes to the fusion of the viral envelope with the host cell membrane. Of the two receptors currently known for CDV (nectin-4 and the signaling lymphocyte activation molecule [SLAM]), SLAM is considered the most relevant for host susceptibility. To investigate how evolution might have impacted the host-CDV interaction, we examined the functional properties of a series of missense single nucleotide polymorphisms (SNPs) naturally accumulating within the H-gene sequences during the transition between two distinct but related strains. The two strains, a wild-type strain and a consensus strain, were part of a single continental outbreak in European wildlife and occurred in distinct geographical areas 2 years apart. The deduced amino acid sequence of the two H genes differed at 5 residues. A panel of mutants carrying all the combinations of the SNPs was obtained by site-directed mutagenesis. The selected mutant, wild type, and consensus H proteins were functionally evaluated according to their surface expression, SLAM binding, fusion protein interaction, and cell fusion efficiencies. The results highlight that the most detrimental functional effects are associated with specific sets of SNPs. Strikingly, an efficient compensational system driven by additional SNPs appears to come into play, virtually neutralizing the negative functional effects. This system seems to contribute to the maintenance of the tightly regulated function of the H-gene-encoded attachment protein. Importance: To investigate how evolution might have impacted the host-canine distemper virus (CDV) interaction, we examined the functional properties of naturally occurring single nucleotide polymorphisms (SNPs) in the hemagglutinin gene of two related but distinct strains of CDV. The hemagglutinin gene encodes the attachment protein, which is pivotal for infection. Our results show that few SNPs have a relevant detrimental impact and they generally appear in specific combinations (molecular signatures). These drastic negative changes are neutralized by compensatory mutations, which contribute to maintenance of an overall constant bioactivity of the attachment protein. This compensational mechanism might reflect the reaction of the CDV machinery to the changes occurring in the virus following antigenic variations critical for virulence.

Invasion and persistence of Mycoplasma bovis in embryonic calf turbinate cells.

Mycoplasma bovis is a wall-less bacterium causing bovine mycoplasmosis, a disease showing a broad range of clinical manifestations in cattle. It leads to enormous economic losses to the beef and dairy industries. Antibiotic treatments are not efficacious and currently no efficient vaccine is available. Moreover, mechanisms of pathogenicity of this bacterium are not clear, as few virulence attributes are known. Microscopic observations of necropsy material suggest the possibility of an intracellular stage of M. bovis. We used a combination of a gentamicin protection assay, a variety of chemical treatments to block mycoplasmas entry in eukaryotic cells, and fluorescence and transmission electron microscopy to investigate the intracellular life of M. bovis in calf turbinate cells. Our findings indicate that M. bovis invades and persists in primary embryonic calf turbinate cells. Moreover, M. bovis can multiply within these cells. The intracellular phase of M. bovis may represent a protective niche for this pathogen and contribute to its escape from the host's immune defense as well as avoidance of antimicrobial agents.

Cyto-adherence of Mycoplasma mycoides subsp. mycoides to bovine lung epithelial cells.

BACKGROUND: Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a respiratory disease of cattle, whereas the closely related Mycoplasma mycoides subsp. capri (Mmc) is a goat pathogen. Cyto-adherence is a crucial step in host colonization by mycoplasmas and subsequent pathogenesis. The aim of this study was to investigate the interactions between Mmm and mammalian host cells by establishing a cyto-adherence flow cytometric assay and comparing tissue and species specificity of Mmm and Mmc strains. RESULTS: There were little significant differences in the adherence patterns of eight different Mmm strains to adult bovine lung epithelial cells. However, there was statistically significant variation in binding to different host cells types. Highest binding was observed with lung epithelial cells, intermediate binding with endothelial cells and very low binding with fibroblasts, suggesting the presence of effective adherence of Mmm on cells lining the airways of the lung, which is the target organ for this pathogen, possibly by high expression of a specific receptor. However, binding to bovine fetal lung epithelial cells was comparably low; suggesting that the lack of severe pulmonary disease seen in many infected young calves can be explained by reduced expression of a specific receptor. CONCLUSIONS: Mmm bound with high efficiency to adult bovine lung cells and less efficiently to calves or goat lung cells. The data show that cyto-adherence of Mmm is species- and tissue- specific confirming its role in colonization of the target host and subsequent infection and development of CBPP.

A genomic perspective on a new bacterial genus and species from the Alcaligenaceae family, Basilea psittacipulmonis.

BACKGROUND: A novel Gram-negative, non-haemolytic, non-motile, rod-shaped bacterium was discovered in the lungs of a dead parakeet (Melopsittacus undulatus) that was kept in captivity in a petshop in Basel, Switzerland. The organism is described with a chemotaxonomic profile and the nearly complete genome sequence obtained through the assembly of short sequence reads. RESULTS: Genome sequence analysis and characterization of respiratory quinones, fatty acids, polar lipids, and biochemical phenotype is presented here. Comparison of gene sequences revealed that the most similar species is Pelistega europaea, with BLAST identities of only 93% to the 16S rDNA gene, 76% identity to the rpoB gene, and a similar GC content (~43%) as the organism isolated from the parakeet, DSM 24701 (40%). The closest full genome sequences are those of Bordetella spp. and Taylorella spp. High-throughput sequencing reads from the Illumina-Solexa platform were assembled with the Edena de novo assembler to form 195 contigs comprising the ~2 Mb genome. Genome annotation with RAST, construction of phylogenetic trees with the 16S rDNA (rrs) gene sequence and the rpoB gene, and phylogenetic placement using other highly conserved marker genes with ML Tree all suggest that the bacterial species belongs to the Alcaligenaceae family. Analysis of samples from cages with healthy parakeets suggested that the newly discovered bacterial species is not widespread in parakeet living quarters. CONCLUSIONS: Classification of this organism in the current taxonomy system requires the formation of a new genus and species. We designate the new genus Basilea and the new species psittacipulmonis. The type strain of Basilea psittacipulmonis is DSM 24701 (= CIP 110308 T, 16S rDNA gene sequence Genbank accession number JX412111 and GI 406042063).

Comparison of microscopic and metagenomic approaches to identify cereal pathogens and track fungal spore release in the field.

Wheat is one of the main staple food crops, and 775 million tonnes of wheat were produced worldwide in 2022. Fungal diseases such as Fusarium head blight, Septoria tritici blotch, spot blotch, tan spot, stripe rust, leaf rust, and powdery mildew cause serious yield losses in wheat and can impact quality. We aimed to investigate the incidence of spores from major fungal pathogens of cereals in the field by comparing microscopic and metagenomic based approaches for spore identification. Spore traps were set up in four geographically distinct UK wheat fields (Carnoustie, Angus; Bishop Burton, Yorkshire; Swindon, Wiltshire; and Lenham, Kent). Six major cereal fungal pathogen genera (Alternaria spp., Blumeria graminis, Cladosporium spp., Fusarium spp., Puccinia spp., and Zymoseptoria spp.) were found using these techniques at all sites. Using metagenomic and BLAST analysis, 150 cereal pathogen species (33 different genera) were recorded on the spore trap tapes. The metagenomic BLAST analysis showed a higher accuracy in terms of species-specific identification than the taxonomic tool software Kraken2 or microscopic analysis. Microscopic data from the spore traps was subsequently correlated with weather data to examine the conditions which promote ascospore release of Fusarium spp. and Zymoseptoria spp. This revealed that Zymoseptoria spp. and Fusarium spp. ascospore release show a positive correlation with relative humidity (%RH). Whereas air temperature (°C) negatively affects Zymoseptoria spp. ascospore release.

Bovine Bacillus anthracis in Cameroon.

Bovine Bacillus anthracis isolates from Cameroon were genetically characterized. They showed a strong homogeneity, and they belong, together with strains from Chad, to cluster Aß, which appears to be predominant in western Africa. However, one strain that belongs to a newly defined clade (D) and cluster (D1) is penicillin resistant and shows certain phenotypes typical of Bacillus cereus.

Direct detection of Mycoplasma bovis in milk and tissue samples by real-time PCR.

Bovine mastitis caused by Mycoplasma bovis is of great economic importance to the beef and dairy industry. Here we describe a new specific real-time PCR assay targeting the uvrC gene that was developed to directly detect M. bovis from milk and tissue samples without laborious DNA purification.

Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples.

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

Identification of Francisella tularensis cluster in central and western Europe.

We conducted a molecular analysis of Francisella tularensis strains isolated in Switzerland and identified a specific subpopulation belonging to a cluster of F. tularensis subsp. holarctica that is widely dispersed in central and western continental Europe. This subpopulation was present before the tularemia epidemics on the Iberian Peninsula.

Coinfection of Swiss cattle with bovine parainfluenza virus 3 and Mycoplasma bovis at acute and chronic stages of bovine respiratory disease complex.

Viral agents such as bovine respiratory syncytial virus (BRSV) and bovine parainfluenza virus 3 (BPIV-3) are considered primary infectious agents in bovine respiratory disease complex (BRDC). Information regarding the pathogenesis of BRDC is scarce, especially at an advanced chronicity stage, in addition to ongoing coinfection with other primary agents such as Mycoplasma bovis. Based on a retrospective review of histology slides from 104 autopsy cases, we classified cases according to type of pneumonia and chronicity. We performed immunohistochemistry (IHC) for BRSV, BPIV-3, and M. bovis as well as real-time PCR (rtPCR) for M. bovis on lung tissue of all 104 cases and correlated results with the morphologic type of pneumonia. Histomorphologically, 79 cases were classified as bronchopneumonia, 16 as bronchointerstitial pneumonia, and 9 as interstitial pneumonia. In 89 cases, at least 1 of the investigated agents was detected by IHC; 44 of these cases had a coinfection. BPIV-3 was the predominant agent present, as a single infection in 39 cases, and in coinfection with M. bovis in 39 cases. Comparing the detection methods for M. bovis, rtPCR was more specific and sensitive than IHC. The combination of both methods provided a good visual tool for assessing severity and distribution of M. bovis antigen within the tissue. Unlike BRSV, BPIV-3 and M. bovis persisted in chronic BRDC, suggesting ongoing impairment of defense mechanisms in the lung.

Large-Scale Analysis of the Mycoplasma bovis Genome Identified Non-essential, Adhesion- and Virulence-Related Genes.

Mycoplasma bovis is an important pathogen of cattle causing bovine mycoplasmosis. Clinical manifestations are numerous, but pneumonia, mastitis, and arthritis cases are mainly reported. Currently, no efficient vaccine is available and antibiotic treatments are not always satisfactory. The design of new, efficient prophylactic and therapeutic approaches requires a better understanding of the molecular mechanisms responsible for M. bovis pathogenicity. Random transposon mutagenesis has been widely used in Mycoplasma species to identify potential gene functions. Such an approach can also be used to screen genomes and search for essential and non-essential genes for growth. Here, we generated a random transposon mutant library of M. bovis strain JF4278 containing approximately 4000 independent insertion sites. We then coupled high-throughput screening of this mutant library to transposon sequencing and bioinformatic analysis to identify M. bovis non-essential, adhesion- and virulence-related genes. Three hundred and fifty-two genes of M. bovis were assigned as essential for growth in rich medium. Among the remaining non-essential genes, putative virulence-related factors were subsequently identified. The complete mutant library was screened for adhesion using primary bovine mammary gland epithelial cells. Data from this assay resulted in a list of conditional-essential genes with putative adhesion-related functions by identifying non-essential genes for growth that are essential for host cell-adhesion. By individually assessing the adhesion capacity of six selected mutants, two previously unknown factors and the adhesin TrmFO were associated with a reduced adhesion phenotype. Overall, our study (i) uncovers new, putative virulence-related genes; (ii) offers a list of putative adhesion-related factors; and (iii) provides valuable information for vaccine design and for exploring M. bovis biology, pathogenesis, and host-interaction.

Molecular epidemiology of Bacillus anthracis: determining the correct origin.

We analyzed and compared strains of Bacillus anthracis isolated from husbandry and industrial anthrax cases in Switzerland between 1952 and 1981 with published data using multiple-locus variable-number tandem repeat analysis. Strains isolated from autochthonous cases of anthrax in cattle belong to genotype B2, together with strains from continental Europe, while human B. anthracis strains clustered with genotype A4. These strains could be traced back to outbreaks of human anthrax that occurred between 1978 and 1981 in a factory processing cashmere wool from the Indian subcontinent. We interpret the worldwide occurrence of B. anthracis strains of cluster A4 to be due to the extensive global trade of untreated cashmere wool during the last century.

Rapid diagnosis and quantification of Francisella tularensis in organs of naturally infected common squirrel monkeys (Saimiri sciureus).

Francisella tularensis, a small Gram-negative facultative intracellular bacterium, is the causative agent of tularaemia, a severe zoonotic disease transmitted to humans mostly by vectors such as ticks, flies and mosquitoes. The disease is endemic in many parts of the northern hemisphere. Among animals, the most affected species belong to rodents and lagomorphs, in particular hares. However, in the recent years, many cases of tularaemia among small monkeys in zoos were reported. We have developed a real-time PCR that allows to quantify F. tularensis in tissue samples. Using this method, we identified the spleen and the kidney as the most heavily infected organ containing up to 400 F. tularensis bacteria per simian host cell in two common squirrel monkeys (Saimiri sciureus) from a zoo that died of tularaemia. In other organs such as the brain, F. tularensis was detected at much lower titres. The strain that caused the infection was identified as F. tularensis subsp. holarctica biovar I, which is susceptible to erythromycin. The high number of F. tularensis present in soft organs such as spleen, liver and kidney represents a high risk for persons handling such carcasses and explains the transmission of the disease to a pathologist during post-mortem analysis. Herein, we show that real-time PCR allows a reliable and rapid diagnosis of F. tularensis directly from tissue samples of infected animals, which is crucial in order to attempt accurate prophylactic measures, especially in cases where humans or other animals have been exposed to this highly contagious pathogen.