Jeanne Naudet

Laurent Crespin

Julien Cappelle

Angeli Kodjo

Florence Ayral
email: florence.ayral@vetagro-sup.fr

Circulating serogroups of Leptospira in swine from a 7-year study in France (2011-2017)

Keywords: Leptospira, Pig, Reproductive failure, Microagglutination test, Australis, Icterohaemorrhagiae

Background: Leptospirosis is a widespread zoonotic disease caused by pathogenic Leptospira and is responsible for significant economic porcine livestock losses. Knowledge of Leptospira serogroups and their distributions is important for evaluation of the relevance of leptospirosis management measures, including use of the prophylactic vaccine that was recently made available in France. A retrospective study was conducted to determine the relationships between different circulating Leptospira serogroups. Pigs from across France presenting clinical signs suggestive of leptospirosis were tested with the microagglutination test (MAT) between 2011 and 2017. We used weighted averages to determine serogroup distributions according to MAT results and considering cross-reactions.

Results: A total of 19,395 pig sera, mostly from Brittany, were tested, and 22.7% were found to be positive for at least one Leptospira serogroup. Analysis of the 4,346 seropositive results for which the putative infective serogroup could be defined, revealed that two out of ten serogroups were much more frequent than the others: Australis (48.5%) and Icterohaemorrhagiae (38.2%). Other serogroups, including Autumnalis, Panama, Ballum, Tarassovi, Sejroe, Grippotyphosa, Bataviae, and Pomona, were less common.

Conclusions:

Although diagnostic laboratory data cannot be extrapolated to infer the distribution of Leptospira serogroups at the nationwide scale in France, the analysis of such data can provide an overview of the relationship between circulating Leptospira serogroups in space and time. During the last decade, protection against the serogroups Australis and Icterohaemorrhagiae would have prevented most of the clinical porcine leptospirosis cases in the large number of farms that we studied. In the future, epidemiological information related to circulating Leptospira serogroups should be extracted from data with a standardized approach for use in nationwide or international surveillance and prophylactic strategy support.

Introduction

Leptospirosis is a worldwide zoonotic disease of major importance caused by pathogenic spirochetes of the genus Leptospira. To date, 38 pathogenic Leptospira species have been described (comprising subclades 1 and 2, previously referred to as pathogenic and intermediary Leptospira, respectively) [START_REF] Vincent | Revisiting the taxonomy and evolution of pathogenicity of the genus Leptospira through the prism of genomics[END_REF]. Among the currently known pathogenic Leptospira taxa, more than 300 serovars have been identified and classified into serogroups according to their antigenic similarities [START_REF] Levett | Systematics of Leptospiraceae[END_REF].

In both humans and other animals, Leptospira infection can cause mild or strong clinical signs or be asymptomatic. In swine, acute and chronic infections are described mainly with regard to reproductive impairments (abortion, stillbirth, and perinatal mortality) responsible for economic losses; however, deterioration of the general condition, including haemorrhage, haematuria, renal damage and death, has also been described [START_REF] Bolin | Diagnosis of leptospirosis in swine[END_REF]. Asymptomatic carriage also appears to occur in pigs, allowing for the undetected transmission and maintenance of the bacteria on farms [START_REF]Animal leptospirosis[END_REF].

Transmission generally occurs by contact with urinecontaminated water, in which the bacteria can survive for several months [START_REF] Andre-Fontaine | Waterborne Leptospirosis: survival and preservation of the virulence of pathogenic Leptospira spp. in fresh water[END_REF]. The main hosts of Leptospira bacteria are rodents; however, other wild and domestic mammal species can also be involved in the transmission pathways [START_REF] Ayral | Hedgehogs and mustelid species: major carriers of pathogenic Leptospira, a survey in 28 animal species in France (20122015)[END_REF][START_REF]Animal leptospirosis[END_REF]. Because pig farming is mainly conducted indoors in France, contamination is most likely via the introduction of an infected individual or through contact with commensal rodents. Furthermore, as close contact between animals can promote intraherd transmission of the bacteria, the regulation that has recently mandated that pregnant sows be grouped together (Directive 2008/120/EC) could lead to an increased number of Leptospira-infected sows.

Two stages of leptospirosis generally occur. The first is the acute phase, which occurs in the first weeks of infection, when the hosts may show clinical signs [START_REF]Animal leptospirosis[END_REF]. The second is the immune phase, which generally occurs in the second week of infection, when the host starts to produce antibodies against Leptospira [START_REF]Animal leptospirosis[END_REF]. During the acute phase, polymerase chain reaction (PCR) performed on blood is highly sensitive and can rapidly detect pathogenic Leptospira species [START_REF] Niloofa | Diagnosis of Leptospirosis: comparison between microscopic agglutination rest, IgM-ELISA and IgM rapid immunochromatography test[END_REF][START_REF] Picardeau | Rapid tests for diagnosis of leptospirosis: current tools and emerging technologies[END_REF][START_REF] Musso | Laboratory diagnosis of leptospirosis: a challenge[END_REF]; nonetheless, when treatment is effective, negative results may occur. Culturing is less advantageous than PCR for early diagnosis; the culture method is time-consuming, and the isolation of Leptospira is rare. The microagglutination test (MAT) is a serologic method that detects only antibodies indicating a past or current infection [START_REF] Levett | [END_REF]. Nonetheless, the MAT is the immunological reference standard for experimental leptospirosis diagnosis by the World Organization for Animal Health (OIE) and the World Health Organization (WHO) [START_REF] Anonymous | Leptospirosis[END_REF][START_REF] Anonymous | Human leptospirosis: guidance for diagnosis, surveillance and control[END_REF]. Another supportive immunological test for the detection of antibodies is the enzyme-linked immunosorbent assay (ELISA) [START_REF] Padilha | The use of ErpY-like recombinant protein from Leptospira interrogans in the development of an immunodiagnostic test for swine leptospirosis[END_REF]. However, its diagnostic accuracy has not been completely established [START_REF] Niloofa | Diagnosis of Leptospirosis: comparison between microscopic agglutination rest, IgM-ELISA and IgM rapid immunochromatography test[END_REF]. Among the Leptospira tests, the MAT has the advantage of a higher sensitivity and the potential identification of a particular serogroup using available reference strains. Cross-reactivity between serogroups frequently occurs in microagglutination testing and results from a lack of specificity, especially from predominant nonspecific IgM antibodies at the onset of infection [15]. In these cases, the MAT results involve titres directed against two or more serogroups, thus preventing determination of the infecting serogroup.

The burden of Leptospira on pig farms in Europe remains unknown. However, epidemiological information has been extracted from laboratory MAT data for diagnostic purposes. According to these data, between 19% and 26% of tested pigs were seropositive in Italy, Germany and France [START_REF] André-Fontaine | Leptospirosis in domestic animals in France: serological results from 1988 to 2007[END_REF][START_REF] Bertelloni | Epidemiology of leptospirosis in North-Central Italy: fifteen years of serological data (2002-2016)[END_REF][START_REF] Strutzberg-Minder | Passive surveillance of Leptospira infection in swine in Germany[END_REF][START_REF] Tagliabue | Serological surveillance of Leptospirosis in Italy: two year national data (2010-2011)[END_REF]. However, the inclusion criteria (e.g., the inclusion or exclusion of pigs without clinical suspicion of infection) and the serogroup panels varied among studies, limiting reliable comparisons of the results.

In addition, previous serological surveys do not report how the MAT results, including the cross seroreactivity results, were managed [START_REF] André-Fontaine | Leptospirosis in domestic animals in France: serological results from 1988 to 2007[END_REF][START_REF] Bertelloni | Epidemiology of leptospirosis in North-Central Italy: fifteen years of serological data (2002-2016)[END_REF][START_REF] Strutzberg-Minder | Passive surveillance of Leptospira infection in swine in Germany[END_REF][START_REF] Tagliabue | Serological surveillance of Leptospirosis in Italy: two year national data (2010-2011)[END_REF]. According to previous studies, the interpretation of MAT results is subjective, and possible cross-reactions should be considered [START_REF] Chappel | Impact of proficiency testing on results of the microscopic agglutination test for diagnosis of Leptospirosis[END_REF][START_REF] Miller | Variability in results of the microscopic agglutination test in dogs with Clinical Leptospirosis and Dogs Vaccinated against Leptospirosis[END_REF]. [START_REF] Chappel | Impact of proficiency testing on results of the microscopic agglutination test for diagnosis of Leptospirosis[END_REF] recommended that in the presence of cross-reactions, the serogroup associated with the maximum titre should be considered the predominant serogroup. In addition, [START_REF] Miller | Variability in results of the microscopic agglutination test in dogs with Clinical Leptospirosis and Dogs Vaccinated against Leptospirosis[END_REF] showed that the values of a single dilution titre vary among operators. Thus, a strict difference of two dilutions between the maximum titre and the others should be considered to account for this variability and ensure that the putative infective serogroup is identified with sufficient evidence. As no consensus currently exists in the literature regarding the identification of the predominant serogroup, we suggest the use of weighted averages to evaluate the importance of circulating serogroups from large sets of MAT surveillance data. Weighted averages allow the assignment of higher weights to certain observations considered more reliable or important than others in the computation of the average [START_REF] Sokal | Biometry: the principles and practice of statistics in biological research[END_REF].

Given that several mammalian species may act as reservoirs for swine leptospirosis, disease source control is complex. Vaccination may be a promising way to reduce the health and economic consequences of Leptospira infections. Vaccines provide protection against homologous or closely related but not heterologous serovars; therefore, particular attention should be given to identifying relevant serovar antigens for vaccine development. In France, a swine vaccine that protects against the following serogroups has been commercialized since 2019: Icterohaemorrhagiae, Australis, Grippotyphosa, Pomona and Tarassovi. However, a comprehensive survey of all serogroups circulating in swine herds in France is required to assess the relevance of the inclusion of a given serogroup in a putative vaccine. The last such survey dates back to 2007 [START_REF] André-Fontaine | Leptospirosis in domestic animals in France: serological results from 1988 to 2007[END_REF], and in light of the spatiotemporal variation in serogroup distribution observed in domestic animals [START_REF] Ayral | Distribution of leptospira serogroups in cattle herds and dogs in France[END_REF][START_REF] Stokes | Prevalence of serum antibodies against six Leptospira serovars in healthy dogs[END_REF], updated data are needed. Using a descriptive approach for the analysis of available laboratory data, our paper aims to report the results of circulating Leptospira serogroups from a large dataset obtained from swine herds in France.

Materials and methods

For confirmatory diagnosis, 28,332 swine sera were collected by local veterinarians from pigs that showed clinical signs consistent with leptospirosis. The samples originated from throughout the country. The veterinarians requested the laboratory for MAT depending on their ability to recognize the clinical expression of the disease, which may vary among individuals, and consent of the owners. Thus, the collection of samples from farms with potential leptospirosis varied according to clinical signs, veterinarian experience and owner consent. In addition, because the sensitivity of the MAT may be low in the early infection stage or for specific serogroup infections [START_REF]Animal leptospirosis[END_REF][START_REF] Ellis | Isolation of leptospires from the genital tract and kidneys of aborted sows[END_REF], samples from several pigs are recommended to increase the sensitivity of the diagnosis at farm level. However, no consensus regarding the number of individuals to be included for confirmatory analysis is available, which lead to variability in the number of contributions per farm. A farm was defined as the group of animals from which pig serum was sampled. It is thus an epidemiological unit. Altogether, the available data resulted from convenience sampling, allowing descriptive statistical analysis.

Serum samples were analysed by the Laboratoire des Leptospires (Marcy-l'Étoile, France) between October 2007 and April 2017 using the MAT to detect antibodies against a large panel of serogroups. Over the ten-year duration of the survey, the panel of serogroups varied slightly. However, 19,395 sera sampled between January 2011 and January 2017 were tested for the following serogroups, with their serovars in parentheses: Australis (munchen, australis, and bratislava), Autumnalis (autumnalis and bim), Ballum (ballum), Bataviae (bataviae), Icterohaemorrhagiae (icterohaemorrhagiae and copenhageni), Grippotyphosa (grippotyphosa and vanderhoedoni), Panama (panama and mangus), Pomona (pomona and mozdok), Sejroe (sejroe, saxkoebing, hardjo, and wolffi) and Tarassovi (tarassovi). This test was performed using a serum dilution series from 1:100 to 1:6400, and a titre ≥ 1:100 was considered suggestive of a past or cur- rent infection.

To avoid the limitations of cross-reactions and the subjective nature of MAT interpretation at the individual level [START_REF] Miller | Variability in results of the microscopic agglutination test in dogs with Clinical Leptospirosis and Dogs Vaccinated against Leptospirosis[END_REF], we used a weighted average approach. Weighted averages allowed us to assign higher weights to certain observations considered more reliable than others in the computation of the average [START_REF] Sokal | Biometry: the principles and practice of statistics in biological research[END_REF]. Here, we assumed that a previously or currently infected individual would be associated with MAT seroreactivity directed against a single serogroup or a combination of two or three serogroups because of possible cross-reactions. Therefore, we used the number of serogroups detected in the combination of serogroups as the weight. These weights also ensured that each individual was given the same weight (1.00) in all computations.

More precisely, we applied the following criteria:

(1) A single predominant serogroup was defined by either a titre ≥ 1:100 against a single serogroup or a threefold or greater difference between the highest titre and the other titres. In both cases, a weight value of 1.00, associated with a high certainty of incrimination, was assigned to the predominant serogroup.

(2) Equally predominant serogroups were defined based on the detection of titres ≥ 1:100 against two or three serogroups with equal titres or a less than threefold difference between the highest titre and the next highest titre. The presence of two or three predominant serogroups most likely resulted from cross reactions [START_REF] Kusum | Comparison of leptospiral serovars identification by serology and cultivation in northeastern region, Thailand[END_REF]. Thus, weight values of 0.50 or 0.33 were allotted to the two or three predominant serogroups, respectively.

(3) Serological profiles with more than three predominant serogroups were considered uninformative and were removed from the data analysis, as these profiles likely resulted from cross-reactive IgM antibodies at the onset of infection [15,[START_REF] Miller | Variability in results of the microscopic agglutination test in dogs with Clinical Leptospirosis and Dogs Vaccinated against Leptospirosis[END_REF][START_REF] Lupidi | Serological follow-up of patients involved in a localized outbreak of leptospirosis[END_REF].

The frequency of each serogroup was computed as a weighted average by summing the numbers of positive swine sera per serogroup, weighting by the values defined above, and dividing by the total number of positive sera [START_REF] Sokal | Biometry: the principles and practice of statistics in biological research[END_REF].

A seropositive farm was a farm in which at least one individual tested positive. Each seropositive farm was associated with one or more serogroups depending on the MAT profile obtained from each tested pig. The serogroups computed at the farm level included all seroreactive serogroups regardless of the number of pigs reactive against each serogroup. For instance, a farm with two pigs that were seroreactive against the serogroup Australis would be associated with that serogroup. A farm with one pig seroreactive against the serogroup Australis and one pig seroreactive against the serogroups Icterohaemorrhagiae and Panama would be associated with the serogroups Australis, Icterohaemorrhagiae and Panama.

To describe the spatial serogroup distribution, mainland France was studied at the "département" scale (France is divided into 96 "départements", administrative units similar to counties in the United States). The tested pigs in each "département" were visualized in Quantum GIS (QGIS version 2.18) with the background map from IGN GEOFLA ® . Open access pig density data were used for pig distribution mapping [START_REF] Gilbert | Global pigs distribution in 2010 (5 minutes of arc)[END_REF].

To explore the temporal variability of the serogroups, the numbers of swine sera positive for a given serogroup weighted by the values defined above were retrieved for each year. These counts were analysed using generalized estimated equations (GEEs) for three reasons [START_REF] Liang | Longitudinal data analysis using generalized linear models[END_REF].

First, GEE models were developed from generalized linear models to accommodate correlated data [START_REF] Hardin | Generalized estimating equations[END_REF]. In our study, the primary sampling unit was the farm and so some correlations among the counts of Leptospira circulating serogroups were expected at the farm level. Second, we were not interested in obtaining estimates for a particular farm but rather in describing the year-to-year variability among the relative numbers of serogroups at the scale of all farms (marginal models). Third, a limitation of GEE models is that they rely on the asymptotic normality of estimators for inferences [START_REF] Agresti | Categorical data analysis[END_REF]. With 1,114 farms sampled from 2011 to 2016 (2017 was discarded because the sampling was not complete for this year, with only ten farms sampled), we are confident that this approximation was reasonable for the data analysis. Because the data were count data, we used a log link function with a Poisson distribution. The model also included the number of seropositive individuals per farm as an offset variable. We followed the methods of Hardin & Hilbe [START_REF] Hardin | Generalized estimating equations[END_REF], who recommended an unstructured working correlation matrix for complete datasets with few observations (three herein) by panels. PROC GENMOD was used to fit the GEE models (SAS Institute Inc. 2012. SAS/STAT Software, Version 9.4. Cary, NC).

Results

Of the 19,395 tested swine sera, 4,398 (22.7%) were positive for at least one serogroup, including 2,608 that were positive for a single serogroup, 1,410 that were positive for two serogroups and 328 that were positive for three serogroups. Using the three criteria defined above, 52 MAT results (1.2% of the positive results) were seroreactive against more than three serogroups and were therefore excluded from the data analysis. Laboratory analysis using only the sera for which a single serogroup was unambiguously identified would have been based on 2,608 positive samples (59.3% of the samples testing positive), whereas our data analysis was based on 4,346 positive samples (98.8% of the samples testing positive), corresponding to a 66% increase in sample size. The predominant serogroups were Australis and Icterohaemorrhagiae, which had frequencies of 48.5% and 38.2%, respectively, followed by Autumnalis (6.1%), Panama (5%), Ballum (1.2%), Tarassovi (0.5%), Sejroe (0.2%), Grippotyphosa (0.2%), Bataviae (0.1%), and Pomona (< 0.1%) (Fig. 1).

The numbers of tested animals were higher in four "départements" of the Western France (n = 14,509, or 75% of the samples) than in those of the other regions. As shown in Fig. 2 (top right), Western France includes the vast majority of pigs reared in the country. Seropositivity against the predominant serogroups Australis and Icterohaemorrhagiae was retrieved in 55 and 52 of the 68 tested "départements", respectively (Fig. 2).

Sera originated from a total of 2325 farms across France, among which 1124 (48%) had at least one seropositive individual. These farms were distributed in 87% (n = 59/68) of the tested "départements" in mainland France. The serogroups obtained at the farm level are shown in Table 1. More than 97.5% of the farms (n = 1,087) were associated with at least Australis and/or Icterohaemorrhagiae, and 50% (n = 561) were exclusively associated with Australis and/or Icterohaemorrhagiae. Fig. 2 The spatial distribution of the swine tested and the seropositivity against Australis, Icterohaemorrhagiae per "département" (administrative district) in mainland France

The temporal distribution of the dataset, including seropositive vs. seronegative pigs and farms, is displayed in Table 2. Considering the temporal variability of the serogroups among years (Table 3), the fit of the model was adequate, as shown by the result that only seven Pearson residuals were larger than 3 (6 were less than 3.84 and one was 8.82, out of 3,342 observations) and that there was no overdispersion (normalized Pearson chi-square = 0.61). The relative counts of serogroups varied widely from year to year, as shown by the significant interactions (generalized score for serogroup×year: χ2 (DF = 10) = 203.79, p < 0.0001). The relative counts of Australis and Icterohaemorrhagiae fluctuated from year to year but nevertheless represented approximately 80% of infected individuals each year. The correlations among the serogroups were negative (Table,[START_REF]Animal leptospirosis[END_REF].

Discussion

Employing a retrospective approach, our study shows that antibodies against pathogenic Leptospira serogroups were frequently detected on French pig farms. In addition, two serogroups were detected much more frequently than the others among seropositive pigs included, Australis and Icterohaemorrhagiae, with frequencies of 48.5% and 38.2%, respectively. These high frequencies suggested that among the 4,346 seropositive pigs, most associated Leptospira infections originated from only two serogroups out of the ten detected in this study.

The observed predominant seroreactivity against Leptospira serogroups Australis and Icterohaemorrhagiae in pigs was consistent with previous data reported in France [START_REF] André-Fontaine | Leptospirosis in domestic animals in France: serological results from 1988 to 2007[END_REF]. This suggests the absence of any changes in the epidemiological context of Leptospira strain distribution. A recent study in Italy that included pigs with clinical suspicion of leptospirosis was implemented using an eight-serovar panel; seven serovars were the same as those in our panel, and their study identified Australis and Pomona as the most frequently detected serogroups [START_REF] Tagliabue | Serological surveillance of Leptospirosis in Italy: two year national data (2010-2011)[END_REF]. However, caution is warranted in interpreting this consistency because the previous study do not report how the MAT results with cross-seroreactivity were managed, and the approach may have differed from that in the present study. These observations support the need for a standardized approach for MAT surveillance data analysis.

Previous studies have described a poor correlation between the presence of antibodies and the carrier state [START_REF] Ellis | Isolation of leptospires from the genital tract and kidneys of aborted sows[END_REF][START_REF] Ellis | Boars as carriers of leptospires of the Australis serogroup on farms with an abortion problem[END_REF]. Among 22 Australis-infected sows, six had titres above 1:100, which is the limit of positivity recommended by the OIE for screening and diagnosis [START_REF] Anonymous | Leptospirosis[END_REF][START_REF] Ellis | Isolation of leptospires from the genital tract and kidneys of aborted sows[END_REF]. Our data underestimated the number of seroreactive samples against the serogroup Australis. However, we assume that the underestimation is the same among farms and over time, leading to a lower relative importance of the serogroup Australis in our results.

Regardless of the method used, the simple counts excluding any MAT result with cross reaction or the weighted average method, the results revealed predominance of the serogroups Australis and Icterohaemorrhagiae. The weighted average method allowed a consistent interpretation of the results among data and the inclusion of 66% more MAT results. The weighted average approach was thus useful for providing a more comprehensive overview of the results. In addition, it is an opportunity to standardize output of laboratory MAT results and to compare them among laboratories.

Australis and Icterohaemorrhagiae remained predominant in the farm-level analysis. This predominance was observed over time, even though the relative proportion of Australis and Icterohaemorrhagiae serogroups varied from one year to another. For anonymity reasons, it was not possible to identify farms that were potentially sampled multiple times during the study period; however, this number of farms was assumed negligible considering that our sampling was limited to 10% of the total pig farms in France (n = 22,000) [START_REF] Roguet | Evolution des modèles d'exploitations porcines en France sous l'effet des contraintes économiques et réglementaires depuis[END_REF]. In addition, the number of contributions (number of sampled pigs per farm) varied from one farm to another and may have had an impact on the results. Nonetheless, the contribution variation is unlikely to change the predominance of Australis and Icterohaemorrhagiae.

Regarding MAT, a previous experimental infection study showed that Leptospira-specific IgG (titres above 1:100) could be detected for more than 100 days [START_REF] Ballard | The immunoglobulin response of swine following experimental infection with leptospira interrrogans serovar pomona[END_REF]. Even when a threshold of positivity of 1:100 is applied, recently infected, chronically infected or previously exposed individuals cannot be clearly distinguished [START_REF]Animal leptospirosis[END_REF]. However, because we were chiefly interested in the distribution of the Leptospira serogroups at a large temporal scale, any evidence of past infection was relevant to the description of the serogroup distribution in pigs. In addition, the serogroups Australis and Icterohaemorrhagiae were predominant in each year from 2011 to 2016 in our dataset, which suggests that the misclassification of past or current infections would have limited effects on our results.

Most of the samples were obtained from Brittany, which is an intensive pig farming area compared with other regions in France. However, the serogroup distribution was similar among regions. The samples analysed originated from swine herds with clinical suspicion of leptospirosis; thus, we can conclude that throughout France, herds with reproductive disorders that were exposed to Leptospira were most likely exposed to the Australis and Icterohaemorrhagiae serogroups. Among the pig isolates related to the serogroup Australis, some were more likely to be associated with disease [START_REF] Ellis | Restriction endonuclease analysis as a taxonomic tool in the study of pig isolates belonging to the Australis serogroup of Leptospira interrogans[END_REF], and Australis exposure in pigs should be seriously considered as a potential cause of reproductive failure. A broader survey including nonsymptomatic herds could help clarify the relationship between Leptospira serogroup exposure and reproductive failure. However, given the low MAT titres found in infected sows and boars from farms where reproductive failure had occurred and where the serovar bratislava had been recovered from aborted sows, foetuses and boars, this may not be of value for the Australis serogroup.

Herd management of leptospirosis may rely on animal reservoir control. Rattus norvegicus is a frequent commensal rodent in livestock buildings and a selective carrier of Leptospira of the serogroup Icterohaemorrhagiae [START_REF] Ayral | Distribution of Leptospira interrogans by multispacer sequence typing in Urban Norway Rats (Rattus norvegicus): a survey in France in 2011-2013[END_REF][START_REF] Thiermann | The Norway rat as a selective chronic carrier of Leptospira icterohaemorrhagiae[END_REF]. Thus, rats may play a role to some extent in Icterohaemorrhagiae pig exposure, and this exposure could be reduced through rat management. As no selective carriers of Leptospira of the serogroup Australis have been described among commensal rodents, pigs themselves could be the main reservoir host in the context of pig farming, as suggested by Ellis [START_REF]Animal leptospirosis[END_REF]. Leptospirosis management through the culling of Leptospira carriers is limited by the low specificity and sensitivity of the MAT [38]. According to our results, which showed that more than 50% of the seropositive pigs were exposed to Australis and/or Icterohaemorrhagiae, management options preventing such infections could have greatly reduced the burden of the disease among the pigs.

As the serogroups Australis and Icterohaemorrhagiae are also pathogenic to humans, infected pig populations represent a potential cause of occupational disease, especially for breeders or slaughterhouse staff [START_REF] Mirambo | Seropositivity of Brucella spp. and Leptospira spp. antibodies among abattoir workers and meat vendors in the city of Mwanza, Tanzania: a call for one health approach control strategies[END_REF][START_REF] Thornley | Changing epidemiology of human leptospirosis in New Zealand[END_REF]. Following a One Health approach, leptospirosis management in pig populations may contribute to the protection of human populations and to the mitigation of Leptospira persistence and transmission in the country.

Conclusions

The analysis of data from diagnostic laboratories is useful for obtaining an indication of the circulating Leptospira serogroups in a region or a country and over time. Our results suggested that over the study period, most Leptospira infections in swine in France originated from only two serogroups, Australis and Icterohaemorrhagiae, out of the ten used for the laboratory analysis. This information should be considered to support future prophylactic measures.

In Europe, numerous laboratories have published interpreted MAT results. However, there is no consensus regarding serovars to be included in the panel, the positive threshold and serogroup determination in the case of cross-reactions, leading to the incomparability of results among studies. Therefore, this is an appeal to veterinary laboratories in Western Europe to standardize their approach to MAT surveillance data analysis. Based on our results and previously published results, future MAT surveillance data analysis in Western Europe should be supported by microagglutination testing with a common set of serovars belonging to the serogroups Australis, Ballum, Bataviae, Icterohaemorrhagiae, Grippotyphosa, Panama, Pomona, Sejroe and Tarassovi and using a weighted average approach to determine the serogroup distributions and considering cross-reactions. contributed to the concept and design of the study and critically revised the manuscript. JC was involved in the spatial data analysis. LC was involved in the analysis of temporal variability. JN wrote the first draft of the manuscript and constructed the figures. AK provided the data from the Laboratoire des Leptospires. All authors read and approved the final manuscript.

Fig. 1

 1 Fig. 1 The distribution of Leptospira serogroups among 4,346 swine samples. Australis (AUS), Autumnalis (AUT), Ballum (BAL), Icterohaemorrhagiae (ICT), Panama (PAN)

Table 1

 1 Number of farms having pigs with MAT profiles including Australis, Icterohaemorrhagiae and/or

other serogroups Leptospira serogroups OnlyAustralis Only Icterohaemorrhagiae Onlyothers Australis and Icterohaemorrhagiae Australisand others Icterohaemorrhagiae and others Australis, Icterohaemorrhagiae and others

	Num-	134	94	27	333	66	51	409
	ber of
	farms

Table 2

 2 Features of the dataset (n = 19,343) and distribution of the seropositive and seronegative pigs and farms over time from 2011 to 2017.

	Year	2011 2012 2013 2014 2015 2016 2017
	Total tested samples 795 2770 3089 3965 3715 4846 163
	Number of seroposi-	190	550 597 988 664 1320 37
	tive pigs
	Number of seronega-	605 2,220 2,492 2,977 3,051 3,526 126
	tive pigs
	Total tested farms	100	333 347 497 459 568 21
	Number of tested	70	197 126 236 173 312 10
	positive farms
	Number of tested	30	136 221 261 286 256 11
	negative farms
	Minimum sample	1	1	1	1	1	1 1
	size per farm
	Maximum sample	31	42	31	35	31	31 17
	size per farm
	Median sample size	7	7	8	7	7	8 8
	per farm
	Median number of	2	2	4	4	3	4 3.5
	seropositive samples
	per seropositive farm

Total tested pigs is the sum (in bold) of the two lines below (tested positive and negative). Total tested farms is the sum (in bold) of the two lines below (positive and negative)

Table 3

 3 Weighted number of pigs with MAT profiles including Australis, Icterohaemorrhagiae and/or other serogroups between 2011 and 2017

	Leptospira	Years						Total
	Serogroups
		2011 2012 2013 2014 2015 2016 2017
	Australis	86.67 224.17 242.50 343.50 376.17 802.33 24.00	2,099.33
	Icterohaemor-	82.83 231.67 286.67 529.33 205.17 310.33 5.50	1,651.50
	rhagiae
	Others	20.50 94.17 67.83 115.17 82.67 207.33 7.50	595.17
		190	550	597	988	664	1320 37	4346
	Total

Table 4

 4 Estimated correlations between serogroups from the unstructured working correlation matrix used with the generalized estimating equation model

		Australis	Icterohaemorrhagiae Others
	Australis Icterohaemorrhagiae Others	1.00 -0.66 -0.43	-0.66 1.00 -0.26	-0.43 -0.26 1.00

Acknowledgements

The authors thank the technicians for their technical support in laboratory procedures and microagglutination testing between 2007 and 2017. We are also grateful to VetAgro Sup for funding the study.

Funding

This work was supported by VetAgro Sup, Marcy L'Etoile, France [Agriculture Ministry Funding].

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.

Abbreviations

MAT: Microagglutination test; AUS: Australis.; AUT : Autumnalis; BAT: Bataviae; GRI: Grippotyphosa; ICT: Icterohaemorrhagiae; PAN: Panama; POM: Pomona; PYR: Pyrogenes; TAR : Tarassovi; SJ: Sejroe.

Authors' contributions

FA made substantial contributions to the concept and design of the study, interpreted the results and critically revised the manuscript. LC and JC

Declarations Ethics approval and consent to participate

The present research does not include experimental data, and all laboratory analyses were performed on data from previous examinations of swine for routine diagnosis. The Ethical Committee was consulted, and it confirmed that approval was not required in this case.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Kana Ishisone

Guido Ori

Mauro Boero

Structural, dynamical, and electronic properties of the ionic liquid 1-Ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide

Keywords: First Principles, Molecular Modeling, Ionic Liquids

published or not. The documents may come

Structural, dynamical, and electronic properties of the ionic liquid 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide

at an atomic level of this ionic liquid, so far not yet completely unraveled. Mobilities of the two ionic species are obtained via long-lasting dynamical simulations at finite temperature, allowing for a simultaneous monitoring and quantification of the isomerization occurring in the IL.

Moreover, a thorough analysis of the electronic structure and partial charges distribution characterizing the two components, cation and anion, allow rationalizing the nature of the electrostatic interactions, hydrogen bonding properties of the two ionic counterparts and the infra-red and dielectric response of the system for a full characterization of the IL.

INTRODUCTION

The forefront field of device electronics based on 2D semiconducting (nano)materials such as graphene, h-BN, MoS2, WSe2, and black phosphorous (BP) is becoming a playground for applications in forthcoming electronic and optoelectronic devices. In this type of system, a crucial factor is the possibility of achieving a high interfacial charge density (HICD) crucial to trigger unprecedented and interesting phenomena such as superconductivity [START_REF] Ye | Superconducting Dome in a Gate-Tuned Band Insulator[END_REF][START_REF] Costanzo | Gate-induced superconductivity in atomically thin MoS2 crystals[END_REF] and quantum interference (QI) effects [START_REF] Jia | Quantum interference mediated vertical molecular tunneling transistors[END_REF] . Furthermore, HICD has been indicated as a key factor to improve the performance of emerging memory devices technologies for neuromorphic computing [START_REF] Upadhyay | Emerging Memory Devices for Neuromorphic Computing[END_REF][START_REF] John | Synergistic Gating of Electro-Iono-Photoactive 2D Chalcogenide Neuristors: Coexistence of Hebbian and Homeostatic Synaptic Metaplasticity[END_REF] . In this general scenario, ionic liquids (ILs) are becoming major component to pioneer novel 2D materials-based field-effect transistors, which could greatly benefit from a gating capable of inducing a HICD, as the latter would allow to reduce the operating voltages [START_REF] Ye | Superconducting Dome in a Gate-Tuned Band Insulator[END_REF][START_REF] Costanzo | Gate-induced superconductivity in atomically thin MoS2 crystals[END_REF][START_REF] Jia | Quantum interference mediated vertical molecular tunneling transistors[END_REF][START_REF] Upadhyay | Emerging Memory Devices for Neuromorphic Computing[END_REF][START_REF] John | Synergistic Gating of Electro-Iono-Photoactive 2D Chalcogenide Neuristors: Coexistence of Hebbian and Homeostatic Synaptic Metaplasticity[END_REF][START_REF] Gao | Triboiontronic Transistor of MoS2[END_REF] with clear advantages in terms of energy consumption and sustainability. From a general point of view, ILs are compounds made of a liquid salt containing mobile organic cations and inorganic anions and their use as effective gating media has already been pioneered and fully acknowledged [START_REF] John | Synergistic Gating of Electro-Iono-Photoactive 2D Chalcogenide Neuristors: Coexistence of Hebbian and Homeostatic Synaptic Metaplasticity[END_REF][START_REF] Gao | Triboiontronic Transistor of MoS2[END_REF][START_REF] Kelly | All-printed thin-film transistors from networks of liquid-exfoliated nanosheets[END_REF][START_REF] Park | Transport-map analysis of ionic liquid-gated ambipolar field-effect transistors[END_REF][START_REF] Huang | Large-Area Synthesis of Highly Crystalline WSe2 Monolayers and Device Applications[END_REF] . Under an applied voltage, the ionic moieties composing the IL undergo a separation, and one of the two ionic species, either cations or anions, depending on the local electric field, accumulate on the 2D material surface, forming an electric double layer (EDL). In turn, the charge density that can be induced at the hybrid interface becomes orders of magnitude larger than that of a conventional silicon back gate. Despite intensive experimental efforts, accompanied on a few occasions by atomistic simulations either at a static quantum chemical level to complement experiments [START_REF] Umebayashi | Evidence of Conformational Equilibrium of 1-Ethyl-3-methylimidazolium in Its Ionic Liquid Salts: Raman Spectroscopic Study and Quantum Chemical Calculations[END_REF][START_REF] Dhumal | Electronic Structure and Normal Vibrations of the 1-Ethyl-3-methylimidazolium Ethyl Sulfate Ion Pair[END_REF] or by classical molecular dynamics with ad-hoc force fields [START_REF] Fujii | Liquid Structure of Room-Temperature Ionic Liquid, 1-Ethyl-3-methylimidazolium Bis-(trifluoromethanesulfonyl) Imide[END_REF][START_REF] Liang | Molecular Understanding of Charge Storage in MoS2 Supercapacitors with Ionic Liquids[END_REF][START_REF] Zhao | Adding Solvent into Ionic Liquid-Gated Transistor: The Anatomy of Enhanced Gating Performance[END_REF] key questions remain unanswered about the specific interactions occurring between the ILs and the 2D material. An additional difficulty is the fact that the integration of ILs in real applications is hampered by the lack of fundamental understanding of the interactions occurring at a molecular and atomic level. This is even more crucial in the case of ILs which exist in a liquid phase at room temperature, hence presenting all the operational problems linked to fluidity-related instability. Moreover, ILs also offer an additional advantage as "green" and recyclable solvents for chemical synthesis and can be used to develop of hybrid organic-inorganic materials in which they are confined inside the porous network of an inorganic framework [START_REF] Vioux | From Ionogels to Biredox Ionic Liquids: Some Emerging Opportunities for Electrochemical Energy Storage and Conversion Devices[END_REF][START_REF] Schlaich | Electronic screening using a virtual Thomas-Fermi fluid for predicting wetting and phase transitions of ionic liquids at metal surfaces[END_REF][START_REF] Ori | Structure and Dynamics of Ionic Liquids Confined in Amorphous Porous Chalcogenides[END_REF] . As such, in view of the concerns about sustainability, ILs are prone to have a moderate environmental impact in terms of toxicity and biodegradability. 18,19. By using a first-principles molecular dynamics (FPMD), empowered by free energy sampling techniques, when necessary, we provide a microscopic insight into the IL system, 1-Ethyl-3methylimidazolium bis(trifluoromethylsulfonyl)imide [EMIM] [TFSI], currently targeted for forefront applications [START_REF] Klein | Potential dependent capacitance of[END_REF][START_REF] Yuan | Preparation and characterization of a novel ionic conducting foam-type polymeric gel based on polymer PVdF-HFP and ionic liquid [EMIM][TFSI][END_REF][START_REF] Höfft | Electronic Structure of the Surface of the Ionic Liquid [EMIM][Tf2N] Studied by Metastable Impact Electron Spectroscopy (MIES), UPS, and XPS[END_REF][START_REF] Reichert | Solid-State Analysis of Low-Melting 1,3-Dialkylimidazolium Hexafluorophosphate Salts (Ionic Liquids) by Combined X-ray Crystallographic and Computational Analyses[END_REF] . To date, a limited number of studies [START_REF] Bühl | Ab Initio Molecular Dynamics of Liquid 1,3-Dimethylimidazolium Chloride[END_REF][START_REF] Wendler | Short Time Dynamics of Ionic Liquids in AIMD-Based Power Spectra[END_REF] have been published exploiting FPMD to tackle this class of systems. This is mainly due to the associated computational workload needed to model a realistic IL at the thermodynamic conditions of typical experiments. By resorting to long-lasting dynamical simulations, we aim at unraveling the structural and dynamical properties of this IL, so far not yet investigated at this level of accuracy. Our study provides a convincing picture of the relative distances of the moieties composing the IL and their evolution as a result of the electrostatic and hydrogen bond (H-bond) interaction regulating the behavior of the liquid. The 100 ps time scale of our simulations allows a thorough analysis of the electronic structure and partial charges distribution of the two components, cation and anion, represent an insight into the nature of the electrostatic interactions and H-bonding properties not accessible to experiments, yet important for boosting this innovative research line and to unravel all the fundamental microscopic features escaping experimental probes.

COMPUTATIONAL METHODS

Our computational approach makes use of the FPMD within the density functional theory [START_REF] Kohn | Self-Consistent Equations Including Exchange and Correlation Effects[END_REF] (DFT) framework and according to the Car-Parinello 27 method as implemented in the developer's version of the CPMD 28 code. The valence-core interaction was described by normconserving numerical pseudopotential of the Martins-Trouiller type [START_REF] Troullier | Efficient pseudopotentials for plane-wave calculations[END_REF][START_REF] Troullier | Efficient pseudopotentials for plane-wave calculations. II. Operators for fast iterative diagonalization[END_REF] and the exchange and correlation functional of Becke [START_REF] Becke | Density-functional exchange-energy approximation with correct asymptotic behavior[END_REF] and Lee-Yang-Parr 32 (BLYP) was complemented by a first principles evaluation of the van der Waals dispersion forces based on the Wannier functions centers [START_REF] Silvestrelli | Van der Waals interactions in DFT made easy by Wannier functions[END_REF] (WFCs) boosted by a propagation scheme [START_REF] Ikeda | Role of van der Waals corrections in first principles simulations of alkali metal ions in aqueous solutions[END_REF] to reduce the computational burden.

Valence electrons are treated explicitly and their wavefunctions are expanded on a plane-wave (PW) basis set with a cut-off of 100 Ry. This value was accurately tuned based on the convergence of the stress tensor (pressure) as a function of the number of PWs done in the preparation phase before starting the production run. Canonical NVT simulations were done by controlling the temperature via a Nosé-Hoover [START_REF] Nosé | A molecular dynamics method for simulations in the canonical ensemble[END_REF][START_REF] Nosé | A unified formulation of the constant temperature molecular dynamics methods[END_REF][START_REF] Hoover | Canonical dynamics: Equilibrium phase-space distributions[END_REF] thermostat and for the numerical integration of the Car-Parrinello equations of motion, a fictitious electron mass of 400.0 au and a time step of 3.0 au (0.0726 fs) ensured good numerical control of the constants of motion.

Our [EMIM] [TFSI] simulated system started from a liquid in a periodically repeated cubic supercell of lateral size equal to L=20.8597 Å, amounting to 680 atoms and 1360 electrons. This system size includes twenty formula units of [EMIM] [TFSI] (see Figure 1), amounting to 20

[EMIM] cations and 20 [TFSI] anions. is shown in the right panel and the atoms color code adopted here and in the following is white for H, gray for C, blue for N, red for O, yellow for S and cyan for F. The atomic labeling and colors will be the same in the following figures.

The initial construction of the simulation cell started from the experimental density of 1.5183 g/cm [START_REF] Jia | Quantum interference mediated vertical molecular tunneling transistors[END_REF] , as reported in the literature [START_REF] Jacquemin | Density and viscosity of several pure and water-saturated ionic liquids[END_REF][START_REF] Fredlake | Thermophysical Properties of Imidazolium-Based Ionic Liquids[END_REF] . On these grounds, before moving to FPMD, a classical MD simulation was used to prepare the initial configuration employing the classical force field developed by Canongia Lopes and coworkers [START_REF] Lopes | Modeling Ionic Liquids using a Systematic All-Atom Force Field[END_REF] , with its updated parameterization by Köddermann et al. [START_REF] Köddermann | Molecular Dynamic Simulations of Ionic Liquids: A Reliable Description of Structure, Thermodynamics and Dynamics[END_REF] .

An analysis of the internal pressure during NVT dynamics at different cell sizes allowed to optimize the reported value, which is in excellent agreement (1.5140 g/cm [START_REF] Jia | Quantum interference mediated vertical molecular tunneling transistors[END_REF] ) with the experimental density and leaves the simulated system with a residual pressure oscillating around -0.4 GPa (see Fig. S1 in the Supporting Information), compatible with the accuracy allowed by DFT simulations [START_REF] Magyar | Equations of state for mixtures: results from density-functional (DFT) simulations compared to high accuracy validation experiments on[END_REF][START_REF] Rousseau | Ab initio Simulation of Phase Transitions and Dissociation of H2S at High Pressure[END_REF] . Additional computational details will be given in the next paragraphs whenever needed to support the discussion.

RESULTS AND DISCUSSION

Structural properties

The complexity of this IL system makes FPMD simulations computationally demanding. The large steric volume occupied by each ion and the non-trivial molecular structure require long simulation times to reach the thermal equilibrium (see Figure S1 in the Supporting Information for additional details). For this reason, we performed a ~130 ps long NVT dynamics at the target temperature of 300 K, being the typical room temperature at which many experiments are performed [START_REF] Jia | Quantum interference mediated vertical molecular tunneling transistors[END_REF][START_REF] Upadhyay | Emerging Memory Devices for Neuromorphic Computing[END_REF][START_REF] John | Synergistic Gating of Electro-Iono-Photoactive 2D Chalcogenide Neuristors: Coexistence of Hebbian and Homeostatic Synaptic Metaplasticity[END_REF][START_REF] Gao | Triboiontronic Transistor of MoS2[END_REF][START_REF] Kelly | All-printed thin-film transistors from networks of liquid-exfoliated nanosheets[END_REF][START_REF] Park | Transport-map analysis of ionic liquid-gated ambipolar field-effect transistors[END_REF][START_REF] Huang | Large-Area Synthesis of Highly Crystalline WSe2 Monolayers and Device Applications[END_REF] , apart in the case of superconductivity, and at which devices based in ILs are expected to operate. We recall that the melting point of [EMIM][TFSI] is as 256 K [START_REF] Rotnicki | Phase transitions, molecular dynamics and structural properties of 1-Ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ionic liquid[END_REF] , which means that the system it is in liquid phase at room temperature. Starting from an overall analysis of the IL as a whole, all along the trajectory generated by our long-lasting dynamics, we monitored the pair correlation functions (PCFs) of the IL using the standard formula

( ) = 〈∑ ∑ ( -)〉 (1)
being N the number of molecules contained in the supercell of volume V = L 3 and rij = |Ri -Rj | the distance between the center of mass Ri of each [EMIM] cation and/or [TFSI] anion (each center of mass position is sketched in Figure S2 of the Supporting Information). Thus, we focus here on the cation-cation, anion-anion, and cation-anion distribution, leaving the analysis of the specific atoms composing the two moieties to a later analysis of the intra-molecular structure.

The result obtained is summarized in Figure 2 and provides an insightful average picture of the IL structure. Namely, the two ionic moieties keep a minimal distance of about 3.9 Å all along the dynamics with a major distribution in the range 4.9-6.7 Å where the first shoulder and the most pronounced peak of the corresponding PCF are located (Figure 2). This is mainly regulated by the mutual electrostatic interaction and by the presence of hydrogen bonds (Hbonds) as more extensively discussed in the forthcoming section dedicated to the electronic This result is sketched in Figure 3, whereas other relevant interatomic distances distributions are shown in Figure S4 of the Supporting Information. As it can be seen from the PCF of Figure 3, rather strong H-bonds ranging from 1.6 to 3 Å (first peak of the PCF) are formed between the two ionic species and these contribute to the structure of the liquid which, indeed, is not kept together exclusively by simple Coulomb interactions between more or less distant point-like charges. This seems to be due to the fact that a trans configuration has more degrees of freedom to form H-bonds with nearby [EMIM] cation surrounding the anion in the liquid state, without being constrained to be bound the [EMIM] molecules shown both in Figure 3 and 4 and representing the only two possibilities in a vacuum [START_REF] Dhumal | Electronic Structure and Normal Vibrations of the 1-Ethyl-3-methylimidazolium Ethyl Sulfate Ion Pair[END_REF] . Indeed, a full optimization of isolated

[EMIM] cis-[TFSI] and [EMIM] trans-[TFSI] performed within our computational scheme in a vacuum has given a total energy difference between the two configurations of E = 2.68 kcal/mol with the trans isomer energetically located above the cis one. Yet, this is due to the reduced number of H-bonds that such an isolated system can realize as a stand-alone molecular pair but does not reflect the actual situation in an extended liquid, where a wealth of possibilities in forming H-bonds (sketched in Figure 3 as dashed lines pointing outside the shown pair) exist, thus conferring a higher stability to the trans conformer, reflected in the distribution in Figure 4.

This result can be ascribed to both the wide variety of H-bonding formation and to the minimization of the steric repulsion between the symmetric sulphonyl and trifluoromethyl groups promoted by the cis conformation [START_REF] Ori | Ionic liquid confined in silica nanopores: molecular dynamics in the isobaric-isothermal ensemble[END_REF] . For an IL like the one targeted in this study, 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide, an ∼75% content of trans conformer was determined in the bulk IL using Raman spectroscopy and ab initio calculations [START_REF] Lassègues | Raman and ab initio study of the conformational isomerism in the 1-ethyl-3-methyl-imidazolium bis(trifluoromethanesulfonyl)imide ionic liquid[END_REF] . Note that in a confined environment more efficient packing of TFSI anions is promoted by the cis conformation as found in IL confined in silica and semiconducting chalcogenide pores [START_REF] Ori | Structure and Dynamics of Ionic Liquids Confined in Amorphous Porous Chalcogenides[END_REF][START_REF] Ori | Ionic liquid confined in silica nanopores: molecular dynamics in the isobaric-isothermal ensemble[END_REF][START_REF] Holbrey | Crystal structures of imidazolium bis(trifluoromethanesulfonyl)imide 'ionic liquid' salts: the first organic salt with a cis-TFSI anion conformation[END_REF] .

The

For times t sufficiently long, thus being the mean square displacement MSD = <(Ri(t) -Ri(0)) [START_REF] Costanzo | Gate-induced superconductivity in atomically thin MoS2 crystals[END_REF] > the one computed only in the last part of the plot in Figure 5. Indeed, we observed that at least 10 ps are needed to approach the caging regime due to the local vibrations of the ionic species. After the equilibration stage, represented by the initial slope, a mobility regime is reached and used to compute the ionic mobilities. Details are given in the text.

The estimated mobility coefficients are found to be close to the available experimental measurements [START_REF] Noda | Pulsed-Gradient Spin-Echo 1 H and 19 F NMR Ionic Diffusion Coefficient, Viscosity, and Ionic Conductivity of Non-Chloroaluminate Room-Temperature Ionic Liquids[END_REF][START_REF] D'agostino | Diffusion, Ion Pairing and Aggregation in 1-Ethyl-3-Methylimidazolium-Based Ionic Liquids Studied by 1 H and 19 F PFG NMR: Effect of Temperature, Anion and Glucose Dissolution[END_REF] done with spin-echo technique. Specifically, we obtained mobility coefficients equal to 0.0070 Å 2 /ps for the [EMIM] cation and of 0.0061 Å 2 /ps for the [TFSI] anion, to be compared with the values of 0.007 and 0.004 Å 2 /ps for the cation and the anion, respectively, measured [START_REF] Noda | Pulsed-Gradient Spin-Echo 1 H and 19 F NMR Ionic Diffusion Coefficient, Viscosity, and Ionic Conductivity of Non-Chloroaluminate Room-Temperature Ionic Liquids[END_REF][START_REF] D'agostino | Diffusion, Ion Pairing and Aggregation in 1-Ethyl-3-Methylimidazolium-Based Ionic Liquids Studied by 1 H and 19 F PFG NMR: Effect of Temperature, Anion and Glucose Dissolution[END_REF] at room temperature. This is also an indication of the time scale required to get a realistic FPMD simulation of this class of systems able to reproduce with acceptable accuracy slowly varying dynamical quantities. By looking into the details of the atomic structure of both the cation and the anion, we remark that, as shown in Figure 4, a predominance of trans conformers of the [TFSI] has been detected. Now, by analyzing the dynamics of the single molecules along the trajectory, we can see that a same molecule does not keep its configuration (either trans or cis) all along the motion of the IL but sometime undergoes spontaneous switch between the two isomers. A typical example representative of this trend is shown in Figure 6, where the isomers are discriminated based on the C-S-S-C torsion angle, as specified in paragraph 3.1, and indicate that there is only a small barrier separating the two isomers. Indeed, the system can overcome it with simple dynamical fluctuations during the 300 K NVT dynamics. As evidenced by this BME analysis, a free energy barrier of about 3.1 kcal/mol separates the trans from the cis configuration, sufficiently small to be overcome during our regular (unconstrained) NVT dynamics by simple statistical fluctuations. In fact, by computing from the free energy profile F the corresponding probability as exp(-F/kBT), being kB the Boltzmann constant and T the simulation temperature, we get a curve that can be superposed to the statistical distribution almost exactly.

Electronic properties

Being the electronic structure of the system available simultaneously with the atomic configuration in a FPMD approach, we computed the Kohn-Sham eigenstates of the average configuration of the liquid at 300 K. More precisely, in the Hamiltonian diagonalization procedure, to the occupied states, regularly updated in a FPMD approach, we added the lowest twelve unoccupied states. The result is sketched in Figure 8. To quantify the partial charges of each atom, we exploited the electronic density, available on the fly all along the dynamics, and computed for several uncorrelated configurations the value of the Bader charges [START_REF] Bader | A quantum theory of molecular structure and its applications[END_REF][START_REF] Henkelman | A fast and robust algorithm for Bader decomposition of charge density[END_REF] of each atom composing the cation and the anion of the IL. The result of this analysis is reported in Figure 10. In this case three WFCs remain as lone pairs in proximity of the F site, whereas one doubly occupied WFC is located along the C-F bond (see Figure 11) but strongly displaced toward the F site (see Figure S11 in Supporting Information), thus accounting for a negative partial charge of F, as opposed to a positive one on C, in perfect agreement with the Bader charges analysis.

The details of other WFCs are reported in Figure S12 of the Supporting Information.

The availability of the WFCs is also a useful tool to compute the local dipole moment p of each

[EMIM][TFSI] pair of the IL as a simple sum of point-like charges

= ∑ -∑ (3)
where ZI is the valence charge of the I th atom, RI its cartesian position in the simulation cell, fi the occupation number of the i th Wannier orbital or center (fi =2 in a spin restricted approach or 1 in a spin unrestricted calculation) and ri WFC the position of the i th Wannier function center. We recall that the only gauge invariant dipole that has a physical meaning [START_REF] Kiefer | Experimental Vibrational Study of Imidazolium-Based Ionic Liquids: Raman and Infrared Spectra of 1-Ethyl-3-methylimidazolium Bis(trifluoromethylsulfonyl)imide and 1-Ethyl-3-methylimidazolium Ethylsulfate[END_REF] has the advantage that has been experimentally detected [START_REF] Kiefer | Experimental Vibrational Study of Imidazolium-Based Ionic Liquids: Raman and Infrared Spectra of 1-Ethyl-3-methylimidazolium Bis(trifluoromethylsulfonyl)imide and 1-Ethyl-3-methylimidazolium Ethylsulfate[END_REF] , thus allowing for a direct comparison, and computed within quantum chemical approaches comparable to the one used here [START_REF] Eilmes | Explicit Solvent Modeling of IR and UV-Vis Spectra of 1-Ethyl-3-methylimidazolium Bis(trifluoromethylsulfonyl)imide Ionic Liquid[END_REF] . Equally important is the dielectric function 1() of the ionic liquid [START_REF] Huang | Static Relative Dielectric Permittivities of Ionic Liquids at 25 o C[END_REF] , since it is a key parameter determining the solvation properties of the IL and a probe of the response of the system to an applied electric field, thus directly useful for the realization of devices. To extract this information, we make use of the well-known Kramers-Kronig relations 62,63

( ) = 1 + ∫ • ( ) (5)
The computed IR spectrum and accompanying dielectric function are shown in Figure 13.

Consistently with the experimental outcome [START_REF] Kiefer | Experimental Vibrational Study of Imidazolium-Based Ionic Liquids: Raman and Infrared Spectra of 1-Ethyl-3-methylimidazolium Bis(trifluoromethylsulfonyl)imide and 1-Ethyl-3-methylimidazolium Ethylsulfate[END_REF][START_REF] Eilmes | Explicit Solvent Modeling of IR and UV-Vis Spectra of 1-Ethyl-3-methylimidazolium Bis(trifluoromethylsulfonyl)imide Ionic Liquid[END_REF] , our computed IR spectrum (lower panel in Figure 13) is dominated by the contribution of [TFSI] and we are able to reproduce all the sharp peaks between 500 and 1500 cm -1 , attributed to all the ring modes as detailed in Ref. 60.

Analogously, the small peak between 2800 and 3100 cm -1 , originating from the C-H stretching modes, are just a small contribution. To disentangle the contributions of each vibrational mode to the IR spectrum, we performed a normal modes analysis of an [EMIM]-cis[TFSI] dimer. These results are summarized in Table S1 in Supporting Information. We can observe that a slight shift toward lower frequencies, affecting particularly this part of our IR spectrum, is not surprising and represent a typical limit in DFT-based calculations of IR response in polar liquids [START_REF] Schmidt | Water Solvation Properties: An Experimental and Theoretical Investigation of Salt Solutions at Finite Dilution[END_REF] . The dielectric function, on the other hand, provides useful information about the frequency range in which the material is operational and allows to tune the current flow upon an applied electric field, a key issue in the fabrication of field-effect transistors exploiting ILs.

CONCLUSIONS

We undertook a thorough analysis of structural, dynamical, electronic and spectroscopical properties of the ionic liquid [EMIM][TFSI], representing a current target system for the realization of next-generation electronic devices. By resorting to long-lasting FPMD simulations, we unravel the highly dynamical yet tunable features of the system, providing a microscopic picture not accessible to experimental probes. In particular, we evidence how the diffusion is a slow process, whereas fast conformational changes occur at room temperature without affecting the overall structure of the polar liquid, which indeed presents pair correlation functions that do not undergo significant change on the simulation time scale afforded here.

The efficient H-bond network realized inside the IL, is responsible for an enhancement of the local dipole moment of the [EMIM][TFSI] pairs that, in turn, confers a remarkable stability to this peculiar liquid phase. This is encouraging in view of the use of these liquid material in devices that are expected to operate at room temperature. The electronic structure is characterized by a rather large band gap, ensuring absence of undesired HOMO-LUMO transitions at the typical working conditions. On the other hand, an analysis of the IR response of the system and the related dielectric function provides insightful information on the best suited range of frequencies where the material can be safely employed, contributing to an ecoefficient realization of nanotechnological devices.

Figure 1 .

 1 Figure 1. Simulated [EMIM] [TFSI] system (left) and detail of the ionic pair constituting the

  properties. A closer inspection of the curious double peak at ~5.2 Å and ~6.1 Å present in the PCF of the [EMIM] [TFSI] has shown that indeed two different conformers contribute separately to this distribution (see Figure S3 in the Supporting Information).

Figure 2 .

 2 Figure 2. Pair correlation functions for the [EMIM] [TFSI] system. The brown line indicates the

Figure 3 .

 3 Figure 3. Trans and cis configurations spontaneously assumed by the [TFSI] in the ionic liquid

Figure 4 .

 4 Figure 4. Distribution of the dihedral angle C-S-S-C of the [TFSI] anion, characterizing the cis-

  distribution of the number of [EMIM] molecules having H---O H-bonds with either a cis or a trans [TFSI] in the liquid are shown in Fig. S5 of the Supporting Information. The trans conformer has a slightly larger number of surrounding [EMIM] molecules with respect to the cis conformer, consistent with our discussion above. Contrary to the anion, which has a very flexible (switchable) structure, the cation presents a moderate molecular flexibility, characterized by a broad distribution of the torsion angle of the C-N bond connecting the ethyl group and the imidazolium ring (2 angle), with a small peak at around 100 o (out-of-plane conformer) (Fig. S6 in Supporting Information). Conversely, the rigid structure of the ethyl group (Ceth2-Ceth1-N2 angle) is underscored by the sharp distribution of the 1 angle peaked at ~110 o in the same figure.

3. 2 5 .〈

 25 Dynamical propertiesBy exploiting the 130 ps long dynamics, we focused on the mobility of the two ionic species in the IL. As done for the overall pair correlation functions in Figure2, the centers of mass Ri(t) of both the [EMIM] cation and the [TFSI] anion were monitored, and the result is shown in FigureWeobserve that the Fickian regime, namely the one in which the mean square displacement MSD scales linearly with time t, is not fully reached even in the last time window of the 130 ps simulation. This makes difficult a reliable estimation of the self-diffusivity of the cations and anions. Yet, at least an indication of the diffusivities can be attempted for both the ionic 3D mobilities (D * ) according to the typical Einstein's formula considering the slope of the MSD plot to have reached the beginning of the diffusion regime toward the end of the simulation * = lim →

Figure 5 .

 5 Figure 5. (a) Sketch of the trajectories of the center of mass of [EMIM] cations (orange) and

Figure 6 .

 6 Figure 6. Evolution of the configuration of a typical [TFSI] molecule during the dynamics. The

Figure 7 .

 7 Figure 7. The left panel show the free energy profile along the S-N-S-C torsion angle (reaction

Figure 8 .

 8 Figure 8. Band diagram and related electronic density of states (DOS) for the simulated

Figure 9 .

 9 Figure 9. Typical highest occupied orbital (HOMO) and lowest unoccupied one (LUMO) in the

Figure 10 .

 10 Figure 10. Schematic representation of the Bader charge of the atoms of the [EMIM] and

Figure 11 .

 11 Figure 11. Wannier functions centers (orange spheres) and their distribution around the S-O

  in a globally neutral simulation cell is the one between the EMIM cation and the TFSI anion. We performed this analysis on our system by computing the dipole moments between each [EMIM]-[TFSI] pair in our simulation cell. According to the PCF between [EMIM] and [TFSI] (Figure2), the first coordination shell has an average radius of ~9.3 Å. On these grounds, the dipole moments of [EMIM]-[TFSI] dimers were calculated according to the criterion that its center-of-mass distance is lower than 9.3 Å. A representative snapshot of an [EMIM] and one surrounding [TFSI] in the first coordination shell is shown in panel (a) of Figure 12 and the related coordination number in this first coordination number is around 7~8 (see Figure S13 in Supporting Information). Panel (b) of Figure 12 shows the distribution of the dipole moment in the ionic liquid. The average value of the dipole moment is 33.0 D, significantly larger than the values computed for an isolated dimer [EMIM][TFSI] in the gas phase (16.1 D for [EMIM]-cis [TFSI] and 20.3 D for [EMIM]-trans [TFSI] dimers). This enhanced value is not an unexpected effect in an extended polar liquid, where the presence of H-bonds of surrounding ions enhances the electrostatic interaction between [EMIM] and [TFSI].

Figure 12 .

 12 Figure 12. (a) Typical geometry of one [EMIM] and its surrounding [TFSI] in the first

Figure 13 .

 13 Figure 13. The real part of refractive index (ε1) and the IR absorption spectrum (α) of the

We thank the HPC Center at the University of Strasbourg funded by the Equipex Equip@Meso project (Programme Investissements d'Avenir) -CPER Alsacalcul/Big Data, and the Grand Equipement National de Calcul Intensif (GENCI) under allocation DARI-A0100906092. We are grateful to Iréné Amiehe Essomba for his kind help in the preparation of the ILs system, and to Emanuele Orgiu and Carlo Massobrio for fruitful discussions and insightful suggestions.

Notes

The authors declare no competing financial interest. ACKNOWLEDGMENT