A Le Gall

Pascal Hilber

C Chesneau

J Bulla

J Toulouse

M L L Machado

B Philoxene

P F F Smith

Dr Stephane Besnard
email: stephane.besnard@unicaen.fr

Le Gall

Machado Ml

Philoxene

The critical role of vestibular graviception during cognitive-motor development

Keywords: Postnatal days USVs: Ultrasonic vocalizations otolith, early development, B6Ei.GL-Nox3het/J, gravity, vestibular system, autism Highlights

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

1.

Introduction

The vestibular organs located within the inner ear have been explored during the last decades as a sensory system essential for maintaining gaze and postural stabilization based on encoding, in three dimensions, the body's position and motion with respect to earth gravity [START_REF] Berthoz | Neuronal mechanisms underlying eye-head coordination[END_REF]. Numerous investigations have demonstrated their crucial role at the brainstem level and have led to the recognition of the vestibular organs as ensuring the basic function of balance [START_REF] Berthoz | Neuronal mechanisms underlying eye-head coordination[END_REF]. However, the vestibular system is an ancestral sensory organ originating from the protochordates more than 500 million years ago [START_REF] Graf | Le système vestibulaire : anatomie fonctionnelle et comparée, évolution et développement[END_REF], which then links the brain to gravitational force well beyond the brainstem reflex networks. Therefore, this indicates that Earth's gravity comprises two main components acting on any living species, namely, a mechanical loading factor which affects the physiology, and as a form of gravitational sensory stimulation which affects the whole brain. Remarkably, the extent of vestibular function was discovered during the last decade, in particular, its crucial involvement in many high-level brain functions: (i) spatial cognition (including self-perception, sense of direction, verticality perception, internal representation of gravity, orientation, navigation and spatial memory, and mental calculation) [START_REF] Angelaki | A Vestibular Sensation : Probabilistic Approaches to Spatial Perception[END_REF][START_REF] Angelaki | Neurons compute internal models of the physical laws of motion[END_REF][START_REF] Angelaki | Vestibular system: the many facets of a multimodal sense[END_REF][START_REF] Mast | Spatial cognition, body representation and affective processes: the role of vestibular information beyond ocular reflexes and control of posture[END_REF][START_REF] Pfeiffer | The vestibular system: a spatial reference for bodily selfconsciousness[END_REF][START_REF] Brandt | Vestibular loss causes hippocampal atrophy and impaired spatial memory in humans[END_REF][START_REF] Lopez | In the presence of others: Self-location, balance control and vestibular processing[END_REF][START_REF] Besnard | Editorial: The Vestibular System in Cognitive and Memory Processes in Mammalians[END_REF]; (ii) social cognition [START_REF] Deroualle | Toward a vestibular contribution to social cognition[END_REF]; and (iii) emotional regulation [START_REF] Gurvich | Vestibular insights into cognition and psychiatry[END_REF][START_REF] Zheng | Performance in anxiety and spatial memory tests following bilateral vestibular loss in the rat and effects of anxiolytic and anxiogenic drugs[END_REF].

Recent studies have highlighted numerous direct and indirect cortical projections of vestibular inputs to numerous brain areas [START_REF] Hitier | Vestibular pathways involved in cognition[END_REF] including the opercular insular/retro-insular cortex and surrounding regions [START_REF] Angelaki | A Vestibular Sensation : Probabilistic Approaches to Spatial Perception[END_REF][START_REF] Angelaki | Vestibular system: the many facets of a multimodal sense[END_REF][START_REF] Zwergal | Parallel ascending vestibular pathways: anatomical localization and functional specialization[END_REF][START_REF] Dieterich | The bilateral central vestibular system: its pathways, functions, and disorders[END_REF][START_REF] Lopez | The thalamocortical vestibular system in animals and humans[END_REF]. These regions encode an absolute geocentric reference of living species, up to humans, devoted to the crucial role of gravity-related verticality perception [START_REF] Lopez | The vestibular system: balancing more than just the body[END_REF]. This idiothetic referential frame is supplemented by the overlap of egocentric and allocentric frames provided by the somatosensory and visual systems, respectively, and altogether represent the multisensory integration processes [START_REF] Angelaki | A Vestibular Sensation : Probabilistic Approaches to Spatial Perception[END_REF][START_REF] Cullen | The vestibular system: multimodal integration and encoding of self-motion for motor control[END_REF]. All of these highly complex cortical functions, based at least partly on the vestibular perception of Earth's gravity, allow us to appreciate their importance for the individual in the construction and perception of the 'self', in its interaction with the surrounding world and with others. And yet, adult patients suffering from vestibular deficits as well as patients with stroke and epilepsy occurring within the parieto-temporal junction and surrounded regions, report a large number of cognitive (e.g., [START_REF] Maffei | Processing of visual gravitational motion in the peri-sylvian cortex: Evidence from braindamaged patients[END_REF][START_REF] Babyar | Sinusoidal Transcranial Direct Current Versus Galvanic Vestibular Stimulation for Treatment of Lateropulsion Poststroke[END_REF]) and neuro-psychiatric symptoms [START_REF] Gurvich | Vestibular insights into cognition and psychiatry[END_REF]. The vestibular projections of vestibular gravitational information towards areas of consciousness, can result in feelings of depersonalization, projection out of the body, and loss of spatial landmark [START_REF] Gurvich | Vestibular insights into cognition and psychiatry[END_REF]. As mentioned above, all these motor and mental disorders were investigated at adulthood and resulted in a deterioration of a functional system. One can easily envisage that early alteration of the vestibular system (during pre-or post-natal development) could deeply affect the development of individuals, particularly considering its prenatal maturation [START_REF] Graf | Le système vestibulaire : anatomie fonctionnelle et comparée, évolution et développement[END_REF][START_REF] Jeffery | Prenatal growth and development of the modern human labyrinth[END_REF].

The vestibular organ is mature at 4 months of pregnancy in humans and functional from birth [START_REF] Graf | Le système vestibulaire : anatomie fonctionnelle et comparée, évolution et développement[END_REF][START_REF] Jeffery | Prenatal growth and development of the modern human labyrinth[END_REF]. Some authors have suggested indeed that locomotor and postural development are influenced by the effect of gravity [START_REF] Wubbels | Development of sensory motor reflexes in 2 G exposed rats[END_REF][START_REF] Ronca | Mammalian development in space[END_REF][START_REF] Clarac | Role of gravity in the development of posture and locomotion in the neonatal rat[END_REF], which is reinforced by studies conducted in hypergravity environments [START_REF] Jamon | The development of vestibular system and related functions in mammals: impact of gravity[END_REF]. However, the respective roles of the mechanical and the sensory components of gravity on the species studied remain poorly defined [START_REF] Walton | Identification of a critical period for motor development in neonatal rats[END_REF]. The idea that vestibular information has an influence on sensory-motor development was recently raised by Wiener et al. [START_REF] Wiener-Vacher | Vestibular activity and cognitive development in children: perspectives[END_REF] from a few children' data with complete and bilateral vestibular loss, who showed a delay in motor milestones [START_REF] Wiener-Vacher | Vestibular activity and cognitive development in children: perspectives[END_REF], and poorly dapted strategies [START_REF] Franco | Vestibular function in children underperforming at school[END_REF]. They could also have some difficulties in high-level cognitive functions, peer interactions and communication, as reported in some case forms of Autism Spectrum Disorders (ASD) [START_REF] Grzadzinski | DSM-5 and autism spectrum disorders (ASDs): an opportunity for identifying ASD subtypes[END_REF]. This has become all the more meaningful since we know that the vestibular system participates in self-perception in the adult, in our body schema construction and our relationship with the environment and others, as mentioned above.

Recent studies have provided direct evidence that the otoliths, which sense linear acceleration, including linear gravitational acceleration, may contribute to spatial memory.

First, it has been reported that otolith-deficient mice exhibit deficits in spatial memory tasks [START_REF] Machado | Spatial and non-spatial performance in mutant mice devoid of otoliths[END_REF][START_REF] Yoder | Otolithic information is required for homing in the mouse[END_REF][START_REF] Yoder | Otoconia-deficient mice show selective spatial deficits: Impaired Spatial Performance in Otoconia-Deficient Mice[END_REF][START_REF] Manes | Behavioural and neurochemical characterization of the mlh mutant mice lacking otoconia[END_REF][START_REF] Blankenship | Otolith dysfunction alters exploratory movement in mice[END_REF]. Second, otolith deficiency has been reported to result in a degradation of both thalamic head direction cell and hippocampal place cell responses [START_REF] Yoder | Head direction cell activity in mice: robust directional signal depends on intact otolith organs[END_REF][START_REF] Harvey | Linear Self-Motion Cues Support the Spatial Distribution and Stability of Hippocampal Place Cells[END_REF]. Third, in epidemiological studies, cVEMP and oVEMP function, which provide an index of saccular and utricular function, respectively, have been demonstrated to be predictors of cognitive dysfunction as a result of aging (see [START_REF] Agrawal | Vestibular impairment, cognitive decline and Alzheimer's disease: balancing the evidence[END_REF] for a review). Electrical stimulation of the otolith receptors alone in guinea pigs and rats has also been reported to evoke widespread responses throughout the hippocampus, which is well known to be critical for spatial memory [START_REF] Cuthbert | Electrophysiological evidence for vestibular activation of the guinea pig hippocampus[END_REF][START_REF] Smith | Vestibular contributions to hippocampal and striatal function[END_REF].

The otoliths are not the only part of the vestibular system which can sense gravity of course, since the vertical canals can contribute to this; however, they are a major contributor to the vestibular sense of gravity (e.g., [START_REF] Lacquaniti | Multisensory integration and internal models for sensing gravity effects in primates[END_REF]).

Therefore, to further explore the effects of vestibular graviception on cognitive function and in global self-construction, and to examine the existence of a gravity-related vestibular sensory window during early development, we have evaluated the behavioural consequences of a selective otolith-deficient mouse model of Head Tilted mice (Het) [START_REF] Bergstrom | Deletion mapping of the head tilt (het) gene in mice: a vestibular mutation causing specific absence of otoliths[END_REF], i.e. devoid of vestibular graviception attributable to the otoliths, and we have looked for a potential maternal care effect, and visuo-vestibular and somesthetic-vestibular interactions. Our hypothesis was that mice devoid of otoliths would develop abnormally in terms of sensorimotor and spatial memory function.

Methods

General procedures:

Experiments

Sensorimotor developmental milestones tests

Young mice were individually monitored daily during the pre-weaning period for the appearance of developmental landmarks. Each day of the testing sessions, the pups were weighed (to the nearest 0.01g), the global development was observed (head support, pelvis and shoulder support, onset of motion, limb coordination…), but not quantified, in order to detect and exclude animals with potential malformations. The age for the eyes' opening was noted for each pup. The pups were tested from 2 to 10 postnatal days (PND) as detailed below and weight gain was considered as a sign of growth of the pups and indirectly as an indication of appropriate maternal feeding. The development of new-born mice was quantified from a panel of five behavioural tasks (righting reflex test, cliff drop aversion test, negative geotaxis test, whisker stimulation response evaluation, forelimb grasping reflex test) based on the work of Fox [START_REF] Fox | Reflex-ontogeny and behavioural development of the mouse[END_REF] and chosen to evaluate the motor and cognitive development in rodents, especially focused on the cerebellum, sensory systems and vestibular system maturation. These tests measure the typical motor development of the pup involving body righting mechanisms and balance, notably through the perception of verticality and body perception, body orientation, and individual orientation as a result of vestibular information integration.

The righting reflex is a primitive reflex of the pups which allows them to restore a normal orientation of the body i.e., the four limbs on the ground, when a pup is on its back.

This reflex was evaluated by placing the young mouse on its back and by measuring the time required by the animal to turn over to return to a normal position with all four limbs on the ground. This task evaluates upright position and verticality perception from vestibular information.

The negative geotaxis test is based on the natural aversion of mice to have their head and forelimbs in a ventral position compared to the pelvis and hindlimbs and the observation of a righting reflex to restore them to a safe position. In this test, the mouse was placed on a sloping surface/inclined plane at 45° in an upright position with its head tilted down and the time required by the animal to turn around, crawl up the slope and come back to an up-right head position, was noted.

The cliff drop aversion test represents a reflex to avoid a dangerous situation including an empty space and falling. To measure this reflex, the pup was placed on the edge of a cliff (i.e., a turned-over cage) with the tip of its forelimbs and nose over this edge and the time required by the animal to turn and crawl away from the cliff drop in order to escape the void, was measured.

The whiskers' placing response test consists of bilaterally stimulating the pup's face, by placing a solid object (a paintbrush was chosen here) in contact with vibrissae and measuring the reaction of the mouse, i.e., a raising of the head and an extension of the forelimbs in order to grasp the object. The duration to succeed in each test was measured (60s max, success =1, failure = 0).

The forelimb stick grasping reflex consists of bilaterally stimulating the pup's forelimbs,by approaching and putting a solid object (a toothpick was chosen here) in contact with the palmar face of the forelimbs and measuring the reaction of the mouse, i.e., the ability of pups to grasp the object. The duration to succeed in each test was measured (60s max, success =1, failure = 0).

Ultrasonic vocalization (USV)

During the neonatal stage, the ability of pups to respond to maternal separation and isolation by the temporary emission of ultrasonic vocalizations (USVs), constitutes a survival instinct and an indicator of normal development [START_REF] Simola | Ultrasonic vocalizations as a tool in studying emotional states in rodent models of social behaviour and brain disease[END_REF]. The recording and quantification of USV emitted by pups following maternal separation and cold stress is useful to investigate neurodevelopmental disorders in rodents [START_REF] Scattoni | Ultrasonic vocalizations: a tool for behavioural phenotyping of mouse models of neurodevelopmental disorders[END_REF]. Here the earlier USVs of Het pups (n = 10 litters, n = 63 pups) were recorded at baseline using SONOTRACK 2.4.0 (Metris B.V., Netherlands)

at PNDs 2, 4, 6, 8 and 10 during a 5-min session of dam-littermate separation. On each day of testing, each pup was removed from the home cage and placed into an empty plastic container (length 25 cm, width 8 cm, height 10 cm) located inside a sound-attenuating and temperaturecontrolled box, and assessed for USVs during a 5-min session. At the end of the recording session, each pup was replaced in the home cage with dam and littermates. The average success to generate USV emissions, the average of the number of calls per min, the average duration of calls (ms) and the average frequency of the calls (kHz), were measured and analysed.

Home retrieval test (PND 9)

This test was adapted from the Homing test reported by Scattoni et al. [START_REF] Scattoni | Basal forebrain cholinergic lesions in 7day-old rats alter ultrasound vocalisations and homing behaviour[END_REF] and is based on the natural tendency of blind pups to return to the safety of their nest due to olfactory cues, i.e., when motor development allows short distance travelling. On PND 9, individual pups were transferred to a Plexiglas cage (36 cm x22.5 cm, walls 10 cm high). Wood shavings removed from the home cage were evenly spread on one third of the surface of the cage and cotton wool from the nest was dispersed on this dirty litter (12 cm x 22.5 cm, nest area). The rest of the cage was covered with clean bedding (two thirds of the cage, 24 cm x 22.5 cm, clean neutral area).

The pup was placed in the middle of the surface of the Plexiglas cage, i.e., in the clean neutral area at 18 cm from the cage edge, facing the edge for 3 min. Homing performance was scored according to 4 criteria: (a) latency to reach the area containing nest litter; (b) time spent in the area containing nesting litter; (c) percentage of success reaching the nest area; and (d) the average latency to start to move. Furthermore, the locomotor activity of the pups was assessed by qualitative observation.

Maternal care and parental behaviour

Maternal behaviour requires continuous observation of the dam during the first postnatal period. The behaviour of the dam before each test and during the progress of the experiments was monitored as well, but not quantified, in order to detect potential defects inducing biases in the experiments. Pups are extremely vulnerable to predator attack and cold temperature and one of the many roles of the dam is to ensure that pups are in a safe condition, i.e., inside the nest. The retrieval assay generates an instinctive maternal behaviour and provides a measure of the ability of the dam to find pups moved outside the nest. Two tests were performed here in order to evaluate maternal basic instincts: the all pup retrieval test (2 sessions) and the one pup retrieval test (5 sessions) in both groups of dams (n = 5 dam Het -/-versus 5

dam Het-/+).

The all pup retrieval assay (PND 4 & PND 6) was performed at PND 4 and PND 6 in female mice and the littermate's home cage during a 10-min period. To perform the test, the dam was briefly separated from its pups for 30 s in order to generate stress and removed from its home cage while the pups were removed from the nest and placed together in the opposite corner of their cage, always at the same distance from the nest. The dam was then reintroduced into her cage facing the wall and in the empty nest. The behaviour of the dam was observed during a 10-min period and the latency to retrieve the first pup and to take it back to the nest was measured.

The one pup retrieval assay (PND 2 to 10) was performed each day of the testing sessions in female mice and the littermate's home cage during a 3-min period of induced stress. To perform the test, the dam was left in the home cage with her pups while the experimenter removed one pup, randomly chosen, to the nest and placed it in the opposite corner of the cage, always at the same distance from the nest. The latency to retrieve the isolated pup and to take it back to the nest was measured. The dam had 3 min to perform the test and if the dam failed to place the pup inside the nest after the 3 min session test, the pup was placed inside the nest by the experimenter and the test was considered as a failure.

Statistical analysis

All statistical analyses were carried out using the software R 3.3.1 [START_REF]R: a language and environment for statistical computing[END_REF]. First, continuous, numeric responses were analysed using linear mixed models [START_REF] Lindstrom | Nonlinear Mixed Effects Models for Repeated Measures Data[END_REF] and the R package nlme [START_REF] Pinheiro | nlme: Linear and Nonlinear Mixed Effects Models[END_REF]. Second, binary response variables were analysed using generalized linear mixed effects models [START_REF] Bates | Nonlinear Mixed Effects Models for Longitudinal Data[END_REF] and the R package lme4 [START_REF] Bates | Fitting Linear Mixed-Effects Models Using lme4[END_REF]. Third, ordinal responses or numerical responses with only few possible values were analysed using cumulative link mixed models [START_REF] Bates | Fitting Linear Mixed-Effects Models Using lme4[END_REF] and the R package ordinal [START_REF] Tutz | Random effects in ordinal regression models[END_REF]. In the case of non-normally distributed data, Mann-Whitney U tests were used, except for longitudinal data where a non-parametric analysis was implemented in the R package nparLD [START_REF] Christensen | ordinal -Regression Models for Ordinal Data[END_REF]. Frequency data were analysed using a Chi-squared test [START_REF] Christensen | ordinal -Regression Models for Ordinal Data[END_REF]. The level of statistical significance for all analysis was set at p ≤ 0.05.

Results

General developmental observations

The observation of the age of eye opening in pup mice revealed no significant genotype effect (Fig. 1, p-value > 0.05, ns) while the age of eye opening was about 14 days in both Het-/-and Het-/+ groups i.e., within the time window described in the literature for C57BL6 mice.

No differences were detected between males and females; therefore, data were collapsed across sex (p-value > 0.05).

Sensory and motor skills development assessment

Weight measurements in the pup mice (n= 27 Het-/-vs n=36 Het-/+) revealed no group effect: the body weight (Fig. 2A) was similar between Het-/-and Het-/+ control pups (Mann-Whitney test, p-value > 0.05, ns), showing evidence of good physiological growth provided by appropriate maternal feeding. The performance of mice for the grasping reflex was also similar in both Het-/-mice and controls (data not shown, 100% success for all animals).

The progression of the global developmental score (calculated from all rated tasks: righting reflex, cliff drop aversion test, negative geotaxis test and whisker stimulation response), requiring vestibulo-somesthetic and cerebellar integration, was lower in otolithdeficient mice (Het-/-) compared to control (Het-/+) mice (Fig. 2B

Spatial orientation with olfactory guidance

Spatial orientation with olfactory guidance in pups, tested by observing the strong natural tendency of the immature and blind pup to find its way back to the nest, was impaired in Het-/-pups. Het-/-pups took significantly longer to reach the nest area ("Latency to reach the nest", Mann-Whitney test, p ≤ 0.01, Fig. 3A) and spent less time in the nest area than controls (Mann-Whitney test, p-value ≤ 0.01, Fig. 3B) at PND9. The otolith-deficient Het-/-pups showed a lower percentage of success reaching the nest area compared to Het-/+ mice (Chi 2tests for equality of proportions, p-value ≤ 0.01, Fig. 3C), while the latency period before the first movement reaction in pups remained similar in both groups (Mann-Whitney test, p-value > 0.05, ns, Fig. 3D). Two aberrant values were excluded from analysis in the Home retrieval test performed at PND9 (n=25 Het-/-versus 36 Het-/+).

Ultrasonic vocalizations (USV)

The ultrasonic vocalizations were altered in Het-/-mice. The change in probability of success in USV emission in Het-/-pups was different compared to controls: while Het-/+ mice showed a negative age trend (-0.269 ± 0.112, p-value ≤ 0.05), the age trend in Het-/mice showed a significantly different positive progression (0.28 ± 0.144, p-value ≤ 0.001, Fig. 4A). This led to lower values for the average success for the USV emissions of Het-/-mice at PND2 (p-value ≤ 0.05) and higher values at PND10 (p-value ≤ 0.01) compared to controls.

However, the number of vocalizations emitted per min, restricted to animals emitting USVs, was impaired in Het-/-mice (p-value ≤ 0.05, Fig. 4B), while both the average duration of calls and the average frequency of calls showed a statistically significant age effect (p-value ≤ 0.001; Fig. 4C) in both groups, but no significant genotype effect (p-value > 0.05, ns, Fig. 5d) from PND2 to 10.

Maternal care and parental behaviour

The estimation of care for Het-/-and control dams towards their pups was similar (n=5 dam Het-/-versus n=5 dam Het-/+). The first test (one pup retrieval test) analysis revealed no significant genotype effect at each day when the test was performed, of the ability of dams to find and to put the isolated pup in the nest with the littermates (p-value ≤ 0.05; Fig. 5A and5B).

The results obtained in the second test (all pups retrieval test) analysis revealed no genotype effect of the ability of dams to find and to put the isolated pup in the nest with the littermates at PND6 and 8 (p-value > 0.05; Fig. 6A and6B).

Discussion

The absence of difference in body weight kinetics between Het-/-and Het-/+ control pups revealed parallel evidence of good physiological growth provided by appropriate maternal feeding. The lowest level of archaic sensorimotor reflex, devoid of vestibular influence (the primitive grasping reflex), was similar in both Het-/-mice and controls, suggesting that a congenital lack of otoliths did not influence basic sensorimotor loop implementation during gestation. In our study, the progression of the global developmental score requiring vestibulosomesthetic and cerebellar integration was decreased in otolith-deficient mice (Het-/-) compared to control (Het-/+) mice. Het-/+ mice showed a positive age trend whereas the age trend in Het-/-mice was significantly weaker during the early post-natal developmental period from PND2 to PND10. The maximum difference between PND6 and PND10 confirmed the existence of a sensitive developmental period for gravity information. Some studies with animal models of a modified gravity environment corroborate such an hypothesis (see [START_REF] Ronca | Altered gravity effects on mothers and offspring: the importance of maternal behaviour[END_REF] for a review). However, these latter studies where young rats were transiently exposed to altered gravity conditions (hyper-or microgravity) revealed temporary impairment and quick recovery when they were replaced in 1g normal conditions (see [START_REF] Ronca | Altered gravity effects on mothers and offspring: the importance of maternal behaviour[END_REF] for a review). The developmental tests requiring perception of body orientation in space (righting reflex, cliff drop aversion and negative geotaxis test) revealed poorer performance in Het-/-compared to Het-/+ mice. The results of the Het-/-mice, offering a sustainable inhibition of the vestibular perception of gravity by the otoliths while preserving its direct mechanical stimulus, suggest that a large part of the delay in sensorimotor development is related to the vestibular gravisensors, as previously reported in studies performed in microgravity or hypergravity conditions (see [START_REF] Ronca | Altered gravity effects on mothers and offspring: the importance of maternal behaviour[END_REF] for a review). Obviously, the vertical canals also make a contribution to the perception of gravity (e.g., [START_REF] Lacquaniti | Multisensory integration and internal models for sensing gravity effects in primates[END_REF]); however, our results suggest this is not sufficient to compensate for the loss of the otolithic component of graviception.

We are confident that the behavioural effects observed in this study are a consequence of development without functional otoliths rather than simply reflecting the effects of the loss of otoliths in general. First, the heterozygous tilted mice did not exhibit the behavioural abnormalities of the homozygous tilted mice during the equivalent developmental period.

Although selective otolith lesions have never been achieved in experimental behavioural studies in adult animals due to the surgical difficulty of lesioning them alone, without affecting the semi-circular canals, a large literature has demonstrated that complete vestibular lesions result in spatial memory deficits in adult rats but without the other sensorimotor changes observed here (see [START_REF] Besnard | Editorial: The Vestibular System in Cognitive and Memory Processes in Mammalians[END_REF] for a review. It is very likely that the lack of otolith function in the tilted mice was responsible for the developmental sensorimotor function and spatial memory deficits observed in this study. However, we cannot selectively lesion the otoliths in either adult mice nor young and new-born nice in order to compare them with tilted mice due to the surgical limitations or limitations of chemical injections; and even if we could do this the comparison would not be valid because the tilted mice do not have surgical lesions of the otoliths. Essentially, it is impossible to compare the genetic cause of the loss of the otoliths in tilted mice to any adult, unless the mice survived into adulthood; however, this would not control for the developmental effects of the genetic defect.

Spatial orientation with olfactory guidance in pups, was impaired in Het-/-pups at PND 9, suggesting a vestibular otolithic contribution to spatial orientation. This also suggests that the otolithic inputs are involved early in motion planning to reach a goal. Interestingly, we previously reported lasting spatial navigation and spatial memory impairments in Het-/-mice and mice with otolith deficiency in adulthood [START_REF] Machado | Spatial and non-spatial performance in mutant mice devoid of otoliths[END_REF][START_REF] Blankenship | Otolith dysfunction alters exploratory movement in mice[END_REF]. Therefore, one can speculate that the spatial disorientation observed at PND 9 is not compensated by either the visual (allocentric frame) system or the somesthetic system (egocentric frame) at the adult stage, reinforcing the hypothesis that developmental sensory disorders influence long-term spatial cognitive abilities. We may suggest that at the adult stage, alteration might also be due to disorders in building cognitive maps.

The existence of sensory-related critical periods throughout development, i.e. time windows of higher vulnerability to environmental changes, has previously been demonstrated with visual system ontogenesis [START_REF] Imbert | Receptive field characteristics and plastic properties of visual cortical cells in kittens reared with or without visual experience[END_REF]. Early visual deprivation influences the types of spatial representation and strategies of navigation (response strategy versus spatial strategy) [START_REF] Thinus-Blanc | Representation of space in blind persons: vision as a spatial sense?[END_REF]. Here, we showed that development in mice has an early sensory component dependent on the vestibular perception of Earth's gravity, probably combined with its mechanical component.

Similarly, vestibulo-deficient infants suffer from motor impairments [START_REF] Wiener-Vacher | Vestibular activity and cognitive development in children: perspectives[END_REF] characterized by delayed walking, higher risk of falling, and long-term postural instability), varying with age and walking-dependency. Additionally, spatial cognitive processes could be affected, with difficulty in detecting one's own movement in space and in estimating distance of motion, probably through path integration impairment [START_REF] Wiener-Vacher | Vestibular activity and cognitive development in children: perspectives[END_REF]. However, this deficiency remains to be investigated during childhood. Our present observations in mice highlight the possible gravisensory role of vestibular pathology during human development.

The ultrasonic communication to respond to maternal separation was altered in Het-/mice, since the number of emissions was decreased or increased. Unlike control mice, maternal separation and a cold stimulus did not always cause USV emissions at PND2 in Het-/-mice, but their higher rate of response at PND10 revealed a temporal shift in the optimal response rate in this test, highlighting a potential delay in the establishment of mother-pup communication. The delay in USV emissions in Het-/-mice compared to controls may be the result of: (i) a mechanical inability to produce USVs at an early age; (ii) a different level of sensitivity to thermal variations; (iii) a different stress response and management due to maternal separation; or iv) an underlying cause of a social communication deficit and emotional reactivity. Het-/-pups seemed to exhibit a delay in the emissions, with no changes in the profile of USVs. Although the exact role of the USVs temporarily emitted by isolated pups is still debated (social communication, instinctive behaviour of pup nest homing, or physiological response to homeothermic stress) [START_REF] D'amato | Pups call, mothers rush: does maternal responsiveness affect the amount of ultrasonic vocalizations in mouse pups?[END_REF][START_REF] Blumberg | Do infant rats cry?[END_REF], the modulation of isolated emissions due to genetic manipulation and/or environmental conditions is an index of delayed development [START_REF] Simola | Ultrasonic vocalizations as a tool in studying emotional states in rodent models of social behaviour and brain disease[END_REF][START_REF] Branchi | Ultrasonic vocalisation emitted by infant rodents: a tool for assessment of neurobehavioural development[END_REF].

Moreover, maternal care, known to play a critical role in the development of pups, cannot be involved in USV impairments in the present study since the behaviour of Het-/-and control dams towards their pups was similar throughout two control tests.

All of the developmental disorders reported here in Het-/-mice have been documented

in validated mouse models of autism spectrum disorders (ASD), i.e. sensorimotor development, spatial orientation, and social communication impairments [START_REF] Branchi | Ultrasonic vocalisation emitted by infant rodents: a tool for assessment of neurobehavioural development[END_REF][START_REF] De Filippis | Early Behavioural Alterations in Mouse Models of Autism Spectrum Disorders: A Step Forward Towards the Discovery of New Therapeutic Approaches[END_REF][START_REF] Provenzano | Mutant Mouse Models of Autism Spectrum Disorders[END_REF]. Interestingly, Het-/-mice presented a greater response to whisker stimulation during the early stages of development, demonstrating a hypersensitivity to somesthetic stimulation, which is one of the key symptoms in the early diagnosis of ASD in children with social disability [START_REF] Silverman | Behavioural phenotyping assays for mouse models of autism[END_REF][START_REF] Mccormick | Sensory symptoms in children with autism spectrum disorder, other developmental disorders and typical development: A longitudinal study[END_REF][START_REF] Klin | Two-year-olds with autism orient to nonsocial contingencies rather than biological motion[END_REF]. Children with ASD or attention-hyperactivity disorders present body perception impairments, alteration of spatial orientation (particularly body orientation and body scheme construction), a delay in motor development, and impaired behavioural motor responses sometimes supplemented by emotional and social disturbances [START_REF]Diagnostic and statistical manual of mental disorders: DSM-5, 5th ed[END_REF][START_REF] Ament | Evidence for Specificity of Motor Impairments in Catching and Balance in Children with Autism[END_REF][START_REF] Simonoff | Psychiatric disorders in children with autism spectrum disorders: prevalence, comorbidity, and associated factors in a population-derived sample[END_REF][START_REF] Lind | Spatial Navigation Impairments Among Intellectually High-Functioning Adults With Autism Spectrum Disorder: Exploring Relations With Theory of Mind, Episodic Memory, and Episodic Future Thinking[END_REF][START_REF] Ornitz | Childhood Autism-A Review of the Clinical and Experimental Literature[END_REF], including some clinical signs that are not consistent with gravity-related vestibular symptoms. Taken together, these similarities suggest that the gravity-related sensory vestibular component might play a substantial role in the pathophysiology of ASD during an early stage of development. This idea is not new [START_REF] Torres | Editorial for research topic "Autism: the movement perspective[END_REF][START_REF] Lang | Research in Autism Spectrum Disorders sensory integration therapy for autism spectrum disorders : A systematic review[END_REF]; however, the lack of systematic data on vestibular function in patients with ASD make it impossible to draw firm conclusions at this time.

Conclusion

Our work highlights the notion that development has a critical period which is dependent on the vestibular sensory graviception in mice. The brain-gravity interaction on Earth has thus two crucial components: one mechanical and the other sensory. The symptoms expressed by the congenital otolithic-deficient mice are similar to validated mouse models of autism and highlight the significance of vestibular graviception in the pathophysiology of development.

On the other side, our data reinforce the role and the relevance of the protocols of psychomotricity involving vestibular stimulation. Cognitive-motor consequences in vestibulodeficient infants remain to be investigated with the perspective of a better screening, diagnosis and treatment.

Figure 2 .

 2 Figure 2. The Home Retrieval test performed at PND9. No differences were detected

Figure 3 .

 3 Figure 3. Ultrasonic vocalizations (USVs) in pup mice on PND2, PND4, PND6, PND8,

Figure 4 .

 4 Figure 4. The One Pup Retrieval test performed at PND2, PND4, PND6, PND8, and

Figure 5 .

 5 Figure 5. The All Pups Retrieval test performed at PND4 and PND6. 10 females were tested

  were carried out in accordance with the European Communities Council

	and selectively focusing on the role of the sensory otolith component. The time frame of the
	scheduled tests was similar for each animal and chosen in order to minimize the influence of
	the experimenter's manipulations and possible test interference. If no differences were
	Directive 86/6609/EEC as well as French legislation. Mice were housed under constant detected in comparisons of male and female mice in each test, data were collapsed across
	temperature (21±1 •C) and humidity (55±10%) conditions. Mice were kept under a 12:12 h sex. The technical details of each of the test conditions (age, method, size of groups) are
	normal light: dark cycle (lights on at 8:00 AM, progressively increasing to 400 Lux) with food detailed below.
	and water available ad libitum. General parameters like weight and welfare were regularly
	evaluated. The B6Ei.GL-Nox3het/J mice present a mutation on chromosome 17 inhibiting
	NADPH oxidase 3 gene expression [43] and this leads, in the recessive condition (Het -/-), to
	a specific and complete lack of saccular and utricular otoliths within the inner ear [42],
	making the mice unable to detect gravity with those organs. Two couples of homozygote mice
	B6Ei.GL-Nox3het/J (Jackson Laboratory) were cross-bred and the genotype of each animal of
	the colony was checked by polymerase chain reaction (PCR). All tests were individually
	and randomly performed in 27 Het -/-mice and 36 Het +/-mice serving as control in blind
	conditions (CURB, Caen, France, animal ethical agreement received by the CENOXEMA
	0412-01), the phenotype and genotype being determined by PCR investigation after the
	evaluations. Het-/-mice were investigated from birth and compared to heterozygote normal
	littermates (het-/+) serving as controls (n = 27 Het-/-versus 36 Het-/+). All mice were
	tested in all of the tests described hereafter. The sensorimotor development of newborn pups
	was tested from postnatal days (PND) 2 to 10 with a panel of behavioural tasks (righting
	reflex, cliff avoidance, negative geotaxis, whiskers' stimulation response and grasp reflex
	tests) selected for their ability to measure classic reflex acquisition [44]. Because the Het
	strain (type -/+) presented similar developmental milestones to mouse strains free of genetic
	manipulation (C57BL6), we preferred to compare Het-/-(recessive NADPH oxidase 3
	inhibition) with Het-/+ serving as control mice, avoiding development-related strain effects

  value ≤ 0.001; negative geotaxis test, p-value ≤ 0.01). In the righting reflex (Fig.2C), both groups improved day after day (Het-/+, 0.597 ± 0.081, p-value ≤ 0.001, Het-/-

	, 1.402 ± 0.422, p-value ≤ 0.001), with a significant genotype effect (p-value ≤ 0.01). A
	pseudo post-hoc analysis carried out at each age via a Chi-squared test revealed a significant
	difference from PND4 to PND6 (unadjusted p-value ≤ 0.05). Analysis of the cliff drop
	aversion test (Fig. 2D) revealed poorer performance in the Het-/-group compared to Het-/+:
	Het-/+ mice showed a positive age trend (0.4043 ± 0.133, p-value ≤ 0.01), while the age trend
	in Het-/-mice showed a significantly negative progression (-0.1573 ± 0.150, p-value ≤ 0.001).
	significantly weaker (0.099 ± 0.0307, p-value ≤ 0.001) from PND2 to PND10, with a
	difference between PND6 and PND10 (PND6-PND8 p-value ≤ 0.001; PND10 p-value ≤
	0.01).
	The developmental tests requiring perception of body orientation in space (righting

); Het-/+ mice showed a positive age trend (0.242 ± 0.023, p-value ≤ 0.001), whereas the age trend in Het-/-was reflex, cliff drop aversion and negative geotaxis test, Fig.

2C

, 2D and 2E, respectively) revealed a poorer performance in Het-/-compared to Het-/+ mice (righting reflex and cliff drop aversion test, p-The pseudo post-hoc analysis carried out at each age via a Chi-square test revealed a significant difference from PND6 to PND10 (PND6, unadjusted p-value ≤ 0.05; PND8-PND10 unadjusted p-value ≤ 0.01 vs PND8-PND10 adjusted p-value ≤ 0.05).

The negative geotaxis test analysis (Fig.

2E

) revealed poorer performance in the Het-/compared to the Het-/+ group: The Het-/+ group showed a positive age trend (0.834 ± 0.133, p-value ≤ 0.001) while the age trend in Het-/-mice showed a significantly weaker progression (0.386 ± 0.156, p-value ≤ 0.01). A pseudo post-hoc analysis carried out at each age via a Chi-square test revealed that the difference was observed from PND6 to PND10 (adjusted pvalue ≤ 0.001). Interestingly, Het-/-mice presented a greater response to whisker stimulation during the early stages of development compared to controls (p-value ≤ 0.05, Fig.

2F

), particularly at an early stage at PND2 (pseudo post-hoc, unadjusted p-value ≤ 0.05) and at PND6, but this difference was only marginally supported by statistical analyses at age PND6 (p-value ≤ 0.05).

Legends Figure 1. Sensorimotor development evaluation and weight gain of pup mice from PND2

  In the righting reflex, both groups improved day Het-/-mice showed a significantly negative progression (-0.1573 ± 0.150, p-value ≤ 0.001, ***). An additional group effect was observed (p-value ≤ 0.05, *). A pseudo post-hoc analysis carried out at each age via a Chi-square test revealed that the difference observed value ≤ 0.01, **). A pseudo post-hoc analysis carried out at each age via a Chi-squared test revealed that the difference observed was significant from PND6 to PND10 (adjusted pvalue ≤ 0.001, ***). f, Conversely, Het-/-mice results for the response to whisker stimulation were higher overall compared to control mice (genotype effect, p-value ≤ 0.05, *), particularly at an early stage at PND2 (pseudo post-hoc, unadjusted p-value≤ 0.05, *) and at PND6, but this difference was not validated by statistical analyses at age PND6 (p-value = 0.054).

	to PND10 (n=27 Het-/-versus 36 Het-/+). No differences were detected between male and was significant from PND6 to PND10 (PND6, unadjusted p-value ≤ 0.05, *; PND8-PND10
	female, therefore data were collapsed across sex (p-value > 0.05). a, The weight gain of pups unadjusted p-value ≤ 0.01, ** vs PND8-PND10 adjusted p-value ≤ 0.05, *). e, The negative
	from PND2 to PND10 served as controls for physiological development. The follow-up of geotaxis test analysis showed a poorer performance in the Het-/-compared to the Het-/+
	weight in pup mice revealed no group effect: the weight gain was similar in both groups group: The Het-/+ group showed a positive age trend (0.834 ± 0.133, p-value ≤ 0.001, ***)
	arguing, in parallel, for good growth and appropriate maternal feeding (Mann-Whitney test, p-while the age trend in Het-/-showed a significantly weaker progression (0.386 ± 0.156, p-
	after day (Het-/+, 0.597 ± 0.081, p-value ≤ 0.001, ***, Het-/-, 1.402 ± 0.422, p-value ≤ 0.001,
	***), but a genotype effect was observed (p-value ≤ 0.001, ***). A pseudo post-hoc analysis
	carried out at each age via a Chi-squared test revealed that the difference observed was
	significant from PND4 to PND6 (unadjusted p-value ≤ 0.05, *). d, Analysis of the cliff drop

value > 0.05, ns, data +/-sem). b, The global score of development (calculated from all rated tasks) showed a lower level of sensorimotor development in otolith-deficient mice (Het-/-) compared to control Het-/+ mice: Het-/+ showed a positive age trend (0.242 ± 0.0233, p-value ≤ 0.001, ***), whereas the age trend of Het-/-showed a significantly weaker progression (0.099 675 ± 0.0307, p-value ≤ 0.001, ***) from PND2 to PND10 with a maximum difference between PND6 and PND10 (PND6-PND8, p-value ≤ 0.001, ***, PND10, p-value ≤ 0.01, **). The Het-/+ group present similar developmental milestones and global scores compared to mice strains free of genetic manipulation (C57BL6), so we preferred to compare mice of the same strain, i.e., Het-/-and Het-/+ controls. Results revealed that the sensorimotor development in Het-/-mice is delayed and poorer compared to Het-/+ mice and normal development described in the literature. c, aversion test revealed poorer performance in the Het-/-group compared to Het-/+: Het-/+ mice show a positive age trend (0.4043 ± 0.133, p-value ≤ 0.01, **), while the age trend in

Acknowledgments

The authors would like to thank the Centre National de la Recherche Spatiale, the People Programme of the European Union's Seventh Framework Programme FP7/2007-2013/through REA (Grant number: 318980) and the Normandy Region for their financial support. eferences

Authors Contribution

Le Gall, Hilber, Besnard have designed the research, analysed data and wrote the paper,Chesneau and Bulla have performed the statistical analysis, Le Gall, Toulouse, Machado and Philoxène have performed research, Smith has performed the statistical analysis and wrote the paper.

(n = 5 dam Het-/-vs 5 dam Het-/+). a, The latency to retrieving the separated pups was statistically similar in Het-/-and Het-/+ groups at PND4 and PND6 (Nonparametric Analysis of Longitudinal Data, p-value > 0.05, ns, data +/-sem). b, No significant group effect for latency to put the retrieved pups into the home nest was observed at PND4 and PND6 in this test (Nonparametric Analysis of Longitudinal Data, p-value > 0.05, ns, data +/-sem).


Bani-Sadr
email: fbanisadr@chu-reims.fr

Caroline Migault

Lukshe Kanagaratnam

Maxime Hentzien

Aurélien Giltat

Yohan Nguyen

Aurélie Brunet

Magali Thibault

Astrid Legall

Moustapha Drame

Firouzé Bani-Sadr

Prof Firouzé Bani-Sadr

F Bani

Effectiveness of an education health programme about Middle East respiratory syndrome coronavirus tested during travel consultations

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Since the emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) from the Kingdom of Saudi Arabia in 2012, nearly 2,200 cases of MERS have been identified in the Arabian Peninsula and among visitors to the region, with a case fatality rate of around 39% [START_REF] Alsahafi | The epidemiology of Middle East respiratory syndrome coronavirus in the Kingdom of Saudi Arabia, 2012-2015[END_REF][START_REF] Kim | Middle East respiratory syndrome coronavirus (MERS-CoV) outbreak in South Korea, 2015: epidemiology, characteristics and public health implications[END_REF]. Significant epidemics among families and healthcare providers have been reported [START_REF] Alsahafi | The epidemiology of Middle East respiratory syndrome coronavirus in the Kingdom of Saudi Arabia, 2012-2015[END_REF][START_REF] Kim | Middle East respiratory syndrome coronavirus (MERS-CoV) outbreak in South Korea, 2015: epidemiology, characteristics and public health implications[END_REF], and the intra-family transmission rate has been estimated at 4% [START_REF] Hui | Middle East respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission[END_REF]. There is some evidence that education programs could improve knowledge of infectious diseases transmission and increase subsequent engagement in preventive behaviors [START_REF] Wagman | Effectiveness of an integrated intimate partner violence and HIV prevention intervention in Rakai, Uganda: analysis of an intervention in an existing cluster randomised cohort[END_REF][START_REF] Jewkes | Impact of Stepping Stones on incidence of HIV and HSV-2 and sexual behaviour in rural South Africa: cluster randomised controlled trial[END_REF]. As Hajj pilgrims represent a population who are at risk of returning to their country of residence with MERS-CoV, it is important to target this groups for educational initiatives, with a view to minimizing the risk of contracting and spreading MERSCoV. While secondary cases can be prevented by an education program, Hajj pilgrims represent a population who are at risk of returning to their country of residence with MERS-CoV [START_REF] Hui | Middle East respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission[END_REF][START_REF] Gautret | Hajj pilgrims knowledge about Middle East respiratory syndrome coronavirus, August to September 2013[END_REF][START_REF] Althobaity | Knowledge and awareness of Middle East respiratory syndrome coronavirus among Saudi and Non-Saudi Arabian pilgrims[END_REF]. In 2014, the WHO published travel advice on MERS-CoV for pilgrimages and recommended that each country inform pilgrims. In France, the lay press and media have relayed information about MERS-CoV. We aimed to evaluate the level of knowledge of MERS-CoV among Hajj pilgrims before, and after an educational health program delivered during international vaccine consultations.

Methods

A cross-sectional study was performed in the consultation for travel medicine and international vaccination in Reims University Hospital between July 2014 and October 2015. Consecutive adults (>18 years) who attended for pre-Hajj meningococcal vaccination were eligible to complete an anonymous questionnaire with closed answers to evaluate their level of knowledge about MERS-CoV. The study was approved by the institutional ethics committee of Reims University Hospital under the number 2018-01. Participation was voluntary and without compensation. The standardized questionnaire recorded information on the participant's gender, age, place of birth, educational level (high school certificate yes/no). The second part of questionnaire concerned knowledge about MERS-CoV. All questions are listed in Table 1. The second part of questionnaire concerned knowledge about : the geographical area at risk for MERS-CoV contamination, the transmission routes of the disease (direct contact with camels and person-toperson contact), symptoms, severity of the disease, availability of treatment or vaccines, preventive measures to reduce the possibility of contracting and/or spreading illness during travel and after return -for example, in case of fever and respiratory symptoms (e.g. cough or shortness of breath), within 14 days after travelling, cover your mouth with a tissue when you cough or sneeze, call ahead to a healthcare provider and mention the recent travel, and avoid contact with other people until medical care has been received, to reduce the possibility of spreading the illness to others.

In order to evaluate the effectiveness of the information given during the consultation, the same questionnaire was completed by each Hajj pilgrim before and after the consultation where the information about MERS-CoV was provided. The information given to the Hajj pilgrims was provided by a nurse, using an information leaflet. The last one defined MERS-CoV and detailed the geographical area at risk for MERS-CoV contamination, the different transmission routes of the disease, symptoms, severity of the disease, availability of treatment or vaccines, preventive measures to reduce the possibility of contracting and/or spreading illness during travel and after return (annexe 1). All the answers to the questionnaire were given in the information leaflet. Two nurses participated in the study and both received the same instructions.

The questionnaire and the information leaflet were first tested on non-healthcare providers to assess their intelligibility. Additional explanations were also given on top of the routine information usually delivered usual other explanations given during the travel medicine consultation. If the Hajj pilgrim was not able to read or understand French, they were assisted by a healthcare provider and an unofficial translator.

The responses to the two questionnaires (before and after) were then compared. A question was considered to be answered correctly if the correct answer (for questions with a single answer) or all the correct answers (for answers with multiple correct options) were given. Quantitative variables are presented as mean values with standard deviation (SD). Qualitative variables are presented as number and percentage. Responses before and after explanations were compared with the Mc Nemar test. A p-value of 0.05 was considered statistically significant. All analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA).

Results

All 82 adult Hajj pilgrims who attended a travel medicine and international vaccination consultation to receive meningococcal vaccination during the study period were enrolled. No eligible pilgrim refused to participate. Among 82 adult Hajj pilgrims who attended a travel medicine and international vaccination consultation to receive meningococcal vaccination during the study period, no eligible pilgrim refused to participate. The mean age was 54.8 ± 14.5 years; 53% were women. A large majority of the pilgrims (89%) were able to understand French. Sixty eight percent did not have a high level of education.

The responses to the questionnaire before and after receiving information about MERS-CoV are reported in Table 1. At the time they completed the first questionnaire, only 32% of pilgrims namely know MERS-CoV disease, and 52% of them know the geographical area at risk. More than 50% of them were aware of the contagiousness and severity of the illness. In contrast, pilgrims had poorly knowledge of the routes of transmission (4%), the symptoms (7%) and the preventive behaviours to adopt either abroad (22%), in case of fever (17%) and after return (11%).

Pilgrims had a higher rate of correct responses on each question at the time they completed the second questionnaire, as compared to the first, with 11 out of 13 questions answered significantly better after delivery of educational information about MERS-CoV. However, although the rate of correct answers to the questions about routes of transmission, symptoms, preventive behaviours to adopt in case of fever, and time delay between return and potential MERS-CoV occurrence increased significantly after receiving the information, the rates remained below 50%. The absence of available vaccine or specific treatment was also poorly known, with only 23% and 24% of correct answers respectively, increasing to 76% and 67% respectively after the delivery of information.

Finally, the rates of correct answers differs according level of education (having high level of education yes versus no).

For the first questionnaire, pilgrims with high level of education (n=23) had significantly higher correct answers for 3 questions (is it contagious? (70 vs 42%; p=0.03, is it a serious disease? (78 vs 52%; p=0.03 and is this disease respiratory, cardiac or genetic? (70 vs 44%; p=0.04). For the second questionnaire, the pilgrims with high level of education pilgrims had higher correct answer for only one question (On your return :No special measures required; I must take precautions for the period during which I could develop disease symptoms; I can develop the disease 2 weeks after my return? 2 months after my return?) (57 versus 20%; p=0.002).

Discussion

This study shows that educational disease-specific information delivered during travel vaccine consultations can help improve the level of knowledge about MERS-CoV among Hajj pilgrims consulting for vaccination prior to travel.

At the time of the study, the topic of MERS-CoV and its risk of transmission were extensively covered by the media because of the epidemic in Saudi Arabia. Thus, it was expected that a large portion of the pilgrims would have been informed via the traditional media. Although only 32% of the pilgrims stated that they know the exact name of MERS-CoV, most of them knew about the geographical area at risk (52%), the contagiousness (51%), the severity (61%) and the respiratory features (55%) of the illness. This discordance could probably be explained by the fact that pilgrims may have heard of the epidemic in Saudi Arabia even though they did not know the name of the virus. Our results are in line with those of a study performed in 2013 among pilgrims departing from the South of France showing that 65% of them were aware of an ongoing MERS-CoV epidemic in Saudi Arabia [START_REF] Gautret | Hajj pilgrims knowledge about Middle East respiratory syndrome coronavirus, August to September 2013[END_REF]. However, the information delivered by the media was insufficient to give pilgrims a high level of knowledge about the routes of transmission (4%), the symptoms (7%) and the preventive behaviours (22%). Furthermore, we noted from our health information delivery that while the preventive behaviours during travel were understood in 60% of cases, the recommended course of action in case of fever (35%) and after return (30%) were less well understood. Similarly, in a study performed among nursing students in Korea, the authors found a high knowledge level on MERS-CoV (84.4%) but low rates of correct answers regarding preventive behaviours (44.5%) [START_REF] Kim | Middle East respiratory syndrome-related knowledge, preventive behaviours and risk perception among nursing students during outbreak[END_REF].

The unavailability of vaccines was less well known among the pilgrims in our study (24%) than among Saudi Arabian pilgrims (39%) [START_REF] Althobaity | Knowledge and awareness of Middle East respiratory syndrome coronavirus among Saudi and Non-Saudi Arabian pilgrims[END_REF].

This study has some limitations. First, this was a single-centre study and therefore, the study sample may not be representative of Hajj pilgrims in France. Secondly, the rate of knowledge improvement observed in our study could be related in part to the level of education. Indeed, educational achievement is a well known social determinant of health as it is associated with fundamental knowledge and reasoning ability. In our study, a low proportion of pilgrims had a high level of education (32%). Although Hajj pilgrims with a high level of education had significantly higher proportions of correct answers for 3 questions on the first questionnaire, likely linked to better baseline knowledge, they only had more correct answers on one question in the second questionnaire. Furthermore, the rate of correct answers for the recommended course of action in case of fever (48%) and after return (57%) remained low in these pilgrims, despite their level of education. Finally, this remaining low understanding of the recommended course of action in case of fever and after return could be explained by the number of behaviours the pilgrims had to integrate in a short time, regarding both selfmedical care and preventive behaviours to avoid spreading MERS-CoV.

Information targeting the public is the preferred means to implement infection control, and has proven its efficacy in HIV infection, multidrug bacterial resistance, and other infectious disease epidemics [START_REF] Robert | Knowledge of antibiotics and antibiotic resistance in patients followed by family physicians[END_REF][START_REF] Ruiz-Perez | The Effectiveness of HIV Prevention Interventions in Socioeconomically Disadvantaged Ethnic Minority Women: A Systematic Review and Meta-Analysis[END_REF][START_REF] Diclemente | Prevention and control of sexually transmitted infections among adolescents: the importance of a socio-ecological perspective-a commentary[END_REF]. In our study, poor knowledge about MERS-CoV was observed among adult Hajj pilgrims attending a travel medicine and international vaccination consultation. Information given during this consultation significantly increased the level of knowledge of the pilgrims, but specific knowledge about preventive behaviours to adopt in case of fever, and time delay between return and potential MERS-CoV occurrence remained insufficient (below 50%) suggesting potential difficulties in achieving epidemic control in case of MERS-CoV contamination. Therefore,

improved delivery of specific information remains necessary to increase the level of knowledge about targeted aspects of the disease.