Alberto Valencia
email: valencia@insa-toulouse.fr

Claude Le Men

Christophe Ellero

Christine Lafforgue-Baldas

Philippe Schmitz

Jeffrey F Morris

Claude Lemen

Direct observation at the microscale of particle deposition during the first stage of the microfiltration process

Keywords: Micro-filtration, Membrane fouling, Cake formation, Microsystem, Pore protection 1

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

Introduction

Membrane filtration as a separation process is widely used in many industries and various other human activities [START_REF] Charcosset | Procédés membranaires à application pharmaceutique et biotechnologique[END_REF]- [START_REF] Vasileva | Hydrolysis of whey lactose by immobilized β-galactosidase in a bioreactor with a spirally wound membrane[END_REF]. To optimize performance, it is natural to seek effective solutions to filtration and separation problems [START_REF] Kudra | FILTERS AND FILTRATION HANDBOOK 4th Edition[END_REF]. One important hindering feature regarding membrane filtration is the fouling phenomenon, which is mainly related to particle accumulation and the consequent permeate flux decline [START_REF] Schmitz | Particle aggregation at the membrane surface in crossflow microfiltration[END_REF]. Various approaches have been explored to overcome these issues. Some of the current research topics are: membrane surface modification, the flow hydrodynamic conditions, the geometry and membrane reconditioning. The work of Xue et al. [START_REF] Xue | A catechol-based biomimetic strategy combined with surface mineralization to enhance hydrophilicity and anti-fouling property of PTFE flat membrane[END_REF] provides guidance to an efficient method for improvement of membrane wettability and water flux, through surface coating and mineralization. The potential for improvement based on alteration of the hydrodynamic conditions has been studied as well [START_REF] Liu | Saw-tooth spacer for membrane filtration: Hydrodynamic investigation by PIV and filtration experiment validation[END_REF]. Different geometries change the flow patterns and may induce larger shear stress, a factor that tends to hinder cake formation. Studies have also been made to elucidate how filtration parameters may be varied to optimize the process. Pressure step changes have been shown to have an influence on the cake structure of kaolin slurry and nanosilica sol [START_REF] Iritani | Compression and expansion properties of filter cake accompanied with step change in applied pressure in membrane filtration[END_REF]. When the pressure is increased stepwise, the cake structure at the initial pressure tends to approach that of a cake formed under the later pressure; nonetheless in the case of stepwise pressure decrease, the initial compressed cake does not expand to the corresponding later pressure state. In addition, to avoid cake build-up, methods to clean and recondition the membrane performance to its initial state are major research topics [START_REF] Mendret | Dead-end ultrafiltration and backwash: dynamic characterisation of cake properties at local scale[END_REF], [START_REF] Mores | Direct visual observation of yeast deposition and removal during microfiltration[END_REF].

Other approaches addressing cake structure and the influence of particle interactions have been applied in order to better understand the mechanism involved in the filtration phenomenon [START_REF] Altmann | Particle deposition and layer formation at the crossflow microfiltration[END_REF], [START_REF] Schluep | Initial transient effects during cross flow microfiltration of yeast suspensions[END_REF]. The use of laser triangulometry gave an important breakthrough for in-situ non-invasive data acquisition; however, there remains a need to enhance resolution of this method. Understanding the filtration mechanisms at the microscale is crucial for process optimization and hardware design, as well as to model the cake structure, morphology and porosity distribution to predict filtration performances under different operating conditions. Direct observation techniques are especially valuable in this regard, as these allow in-situ, non-invasive data acquisition. The direct observation through membrane (DOTM) experimental approach, first proposed by Hodgson et al. [START_REF] Hodgson | Visual study of crossflow microfiltration with inorganic membranes: resistance of biomass and particulate cake[END_REF] and further developed by Li et al. [START_REF] Li | Direct observation of particle deposition on the membrane surface during crossflow microfiltration[END_REF], has been used to observe particle deposition on the membrane surface during crossflow microfiltration, in work which considered flowrates both below and above the critical flux. The main drawbacks of this technique remain the limited optical depth and the focal plane of the observation. The configuration used by Li et al. allows observation of the top of the cake, which is convenient to study the fractional membrane coverage but is less suited for probing other cake properties. Inspired by this experimental configuration, Zhang et al. [START_REF] Zhang | Determination of critical flux by mass balance technique combined with direct observation image analysis[END_REF] developed a mass balance method combined with the direct observation technique using image processing. This method gave a more accurate representation of deposition of particles. The DOTM technique has also been coupled with confocal laser scanning microscopy (CLSM), which uses the fluorescence principle to obtain 3D information at different depths of the studied sample. The work by Ben Hassan et al. [START_REF] Ben Hassan | In situ 3D characterization of monodispersed spherical particle deposition on microsieve using confocal laser scanning microscopy[END_REF] characterized particle deposition and cake structure using laser excitation and fluorescent cells and particles. The pore protection phenomenon and its dependence on membrane geometry were observed. The main drawback of this technique is that it relies on the very thin optical depth associated with the laser penetration. This limits the observable cake thickness and requires the filtration to be run stepwise, which yields a non-continuous analysis. The 2007 study by Le Clech et al. [START_REF] Le-Clech | Visualisation of polysaccharide fouling on microporous membrane using different characterisation techniques[END_REF] used direct observation from the side to study fouling formation on a hollow fiber membrane. This configuration revealed a concentration gradient during filtration deposition of alginate/bentonite solution, but the resolution of the acquired images did not allow examination at the particle level. More recent work by Laar et el. [START_REF] Van De Laar | Transition-state theory predicts clogging at the microscale[END_REF] used direct observation of clogging phenomenon in dead-end filtration. They used different membrane configurations and coupled the observation with particle image velocimetry (PIV); this work showed it was possible to provide insight on the flow through the pores and its effect on particle clogging mechanisms.

In this paper the focus is on the validation of a new microsystem for characterizing the fouling phenomenon in filtration and particularly cake properties using the direct observation from the side. The system allows the insitu real-time acquisition of images, from which the concentration field and mean velocity were estimated. This system operates under dead-end filtration condition. This approach is accurate for analyzing the first layers of cake and the first stage of cake formation; even under crossflow operation conditions it is well known that some particles accumulate at the membrane surface as the flow direction in the vicinity of the membrane is perpendicular to the filtration surface [START_REF] Ben Hassan | Coupling of local visualization and numerical approach for particle microfiltration optimization[END_REF]. In addition, dead-end operating condition is particularly adequate for performing the mass balance, which is the basis of the further quantitative analysis. The mass balance has been a source of uncertainty in previous studies on crossflow operation mode, which pointed out the difficulty to assess the deposited mass thorough the filtration [START_REF] Mendret | Mise au point de méthodes de caractérisation non destructives du colmatage de membranes : application à la caractérisation in situ d'un dépôt particulaire en ultrafiltration frontale en lien avec les performances du procédé[END_REF].

The designed filtration membrane is composed of consecutives slots aligned with different spacing obtained by plasma photo-etching. The system has been validated by analyzing the behavior of rigid spherical particle suspension near the membrane during the early stages of membrane blocking, deposition of the first cake layers, and the transition to a fully-developed cake. Particle deposition in the early filtration is studied for different membrane geometries and its effect in later cake growth is examined and compared to other studies of the literature. The cake first layers structure is qualitatively described and then simulated to compare the simulation results with the experimental measurements. The analysis method using image processing is explained and results obtained for model micro particles are discussed.

Materials and methods

2.1

Experimental set-up and protocol An original direct observation (DO) experimental set-up was constructed so that particle deposition could be characterized in-situ and in real time from the side. Micro-filtration cells were specially designed and crafted using photolithography and plasma etching techniques in collaboration with the laboratory LAAS (Laboratoire d'Analyse et d'Architecture des Systèmes). The dead-end filtration cell consists of a straight channel with a filtering structure at the middle. This structure is composed of consecutive slots arranged in different configurations (Fig. 1). These configurations were selected to explore the effect of slot width and distance (miming pore size and pore density in usual membranes) on permeability and cake formation : C is the slot width and Pe is the slot distance that is related to slot periodicity or pore density. The channel is 11.5 mm long, 1 mm in width and 30 µm in depth. Finally, the cell is covered with a glass slip of 170 µm thickness allowing in-situ observation. The very fine microstructure of the membrane for C2Pe5 and C4Pe7 units was an important challenge as the finest walls of the membrane (pore distance) are 3 µm wide and 30 µm deep, conditions for which the plasma etching becomes more difficult to perform and the wall structure is more fragile.

A microscope objective attached to a camera (Basler acA 1920-155µm) is used for real-time imaging and data acquisition (Fig. 2a). A syringe pump (Harvard Apparatus 33) provides suspension flow-rates in the range of 1 to 20 µl/min resulting in linear velocity of 0.5 -15 mm/s at the filter. A digital pressure gauge (Type E2 -Sika) is used to measure pressure.

A 78.9% water -21.1% glycerin mixture (by volume) is used as suspending fluid to match the particle density of 1.055 g/cm 3 and provide neutrally buoyant condition. In order to avoid the presence of dust particles in the system, the water-glycerin solution is filtered (0.2 µm pore diameter). Before preparing the suspension, dissolved gas is extracted from solution and the suspension prior to each experiment to avoid the formation of gas bubbles due to pressure drop when the fluid passes through the membrane; a vacuum pump (Knf, Laboport) is used for this purpose. After dust and dissolved gas are removed, the system is filled with water-glycerin mixture and characterized for the pure fluid flow. After the stationary flow condition is reached, the particle suspension is injected. The filtration starts and real-time imaging and data acquisition is launched when the first particles arrive to the observation field. The choice of the camera frame rate is in agreement with range of velocities related to the flowrate. Working on low Reynolds number conditions (< 1), Stokes flow is assumed. As it is intended to study the actual particle deposition it is compulsory to have an acquisition rate fast enough to capture particle movement. The neutrally buoyant condition of the experiments made gravity and inertial forces negligible relative to viscous forces. This results in particles following closely the streamlines with negligible relative (slip or migration) velocity to the fluid.

Particle suspension

The suspension used for the experiments consists of latex beads (Micro Particles GmbH PS-R-8.4) of mean diameter dp = 8.4 µm and standard deviation 0.09 µm suspended in the noted water -glycerin mixture. The concentration C ~ 0.1% (V/V) is according to the clogging capacity of the system and the duration of the experiments.

2.3

Image processing A specialized Python code is used to analyze the acquired images. This code is based on three modules: the particle velocimetry module (PVM), the particle concentration module (PCM), and the cake growth monitoring module (CGM). All modules use an identification function and a dedicated algorithm to extract the desired information, namely the velocity, concentration or the deposited mass.

The identification function detects contours and allows assessment of their properties. The size, shape and the position of an identified object are used to differentiate the particles from the cake. These parameters are calibrated for a sample of 100 objects in order to determine the best fit. For both particles and the cake, the image is first binarized and voids are filled to allow the contours to be better identified. Having the contours and the properties of the detected objects, filters are applied to classify them and to perform the analysis (Fig. 3). The PCM identifies particles above the cake zone, allowing calculation of the vertical concentration profile and the instantaneous mean concentration in the imaged zone. The PVM goes beyond identification by using a correlation function to track particle trajectories. This correlation function is also used to calculate the mean velocity from the displacement of the vertical concentration profile in the time-lapse of two consecutive images. This information allows analyzing the cake growth (surface, mean height, heterogeneity) and correlate it to particle concentration and velocity.

2.3.1

Cake growth monitoring module After contours are identified, the CGM uses the position filter to classify a contour as part of the cake. When the cake is found, the coordinates of the surface are extracted and then the cake mean height and heterogeneity are determined.

2.3.2

Particle concentration module Once the contours are classified, there are two approaches to calculate the concentration. In the first the number of contours is related to individual spherical particles which have an associated volume. Then dividing the particles cumulative volume by the channel volume provides an estimate of the concentration. The second approach consists of inspecting each pixel line and evaluating the particle presence as a 1D signal. Then this 1D information is transformed into a spherical particle cross section. The ratio of pixels occupied by particles to the total number of pixels in the cross section to channel cross section yields a measure of the vertical concentration profile.

2.3.3

Particle velocimetry module For the PVM the algorithm is developed according to previous studies [START_REF] Honkanen | Recognition of highly overlapping ellipse-like bubble images[END_REF], [START_REF] Acuña | Tracking velocity of multiple bubbles in a swarm[END_REF]. This function allows tracking several particles at consecutive instants and hence re-constructing their individual trajectories. Knowing the time interval Δt between acquisition of sequential images, it is also possible to calculate particle velocities.

For this purpose, the image fraction corresponding to the individual particle is extracted as a reference matrix R(N,N) determined from the image pixel values. Then the image fraction of the particle vicinity at the immediately next image is also extracted in a target matrix T(M,M). The autocorrelation matrix is then calculated for R(N,N) and T(M,M):

, = | + , + - , | , Eq. 1
The minimum value of the correlation matrix reveals the position (i,j) of R in T that corresponds to the coordinate of the particle in the immediately next instant. This algorithm is designed to characterize flow irregularities and heterogeneity in membrane permeability. However, while individual tracking of particles is possible, it could take long calculation time. This method is mainly used to study particle trajectories when a preferential flow in a certain zone of the membrane is observed. Another approach to study flow is based on the vertical concentration profile calculated previously using the PCM. The correlation between the concentration profiles of two consecutive images is used to determine the velocity at which the concentration front is driven towards the cake. This provides an estimation of the mean velocity. For each method, a sampling is done to compare the code results to the values calculated directly from the images.

2.4

Data analysis From the concentration obtained from the images as the volume represented by the particles identified within the observation window to the corresponding channel volume the mass balance can be established. Comparing the observed cake height using the CGM, to the integration of the concentration extracted by the PCM.yields:

1 -ℎ = ℎ ! Eq. 2
where Q (µm³/s) is the flow rate, C (% V/V) the concentration computed using the PCM, S (µm²) the area of the channel cross section, " the porosity and the right side of the equation corresponds to the mean height of the forming cake obtained using the CGM. The porosity " is obtained fromEq. 2, in which it is the only unknown parameter. Then the porosity previously found is compared to the one obtained from the analysis of the pressure data using the Kozeny-Carman expression for the cake resistance in Darcy's law,

= ∆$ % & ' + ℎ ( ) * + ,1 -",* - + ",* . / Eq. 3
with ΔP (Pa) the trans-membrane pressure, µ (Pa•s) the dynamic viscosity, Rm (m -1 ) the membrane hydraulic resistance, ℎ ( (m) the cake mean height, Sp (m -1 ) the specific surface of the particles, the porosity and KK = 5 for spherical particles [START_REF]Fluid flow though granular beds[END_REF]. The term in parentheses corresponds to the total hydraulic resistance model 0 = ' + 1 . This approach is accurate since the whole channel depth remains within the optical scope of observation, and hence the information lost in depth when calculating the concentration and the cake height remains negligible.

2.5

Numerical simulations In order to characterize the hydrodynamics in the system and the cake building process the system was analyzed using the commercially available finite element code COMSOL Multiphysics TM . The simulations are focused on the microfiltration cell performance as well as the early behavior of the cake formation. The stationary laminar flow of pure fluid was solved for the (i) entire system without the membrane, (ii) with the membrane and (iii) the membrane-cake interaction simplified geometry (Fig. 4). These geometries were studied in order to analyze the different contributions to the total pressure drop in the system: the channel alone (Fig. 4a), the channel-membrane (Fig. 4b) and the membrane blocking geometry (Fig. 4c); these were also related to the hydraulic resistance contributions from the Darcy equation Rm and Rc. The geometry for the membrane-cake interaction when the first particles arrive was simulated based on the real observation of the slot blocking configurations depending on the different membrane geometries (Fig. 1) and supposing an optimal membrane coverage. The stationary laminar flow was simulated in order to find the pressure drop and the individual hydraulic resistance for comparing to the experimental values.

2.5.1

Initial conditions A reference pressure of 0 Pa is set at the outlet (blue zone in Fig. 4c). The initial velocity is calculated based on the experimental flow rate of 10 µl/min divided by the corresponding channel section and set as the normal velocity at the inlet (red zone in Fig. 4c). The fluid is set as 20° C water/glycerin mixture and has a density of 1060 kg/m 3 and a viscosity of 2.0E-3 Pa.s.

2.5.2

Flow model The Navier-Stokes equations simplified to the Stokes equations for the laminar regime Re<<1 are considered for the simulations. The three dimensional domain (Fig. 4) is modeled taking into account the wall effects as the channel depth is small. Navier-Stokes momentum and continuity equations are simplified to: 0 = -∇4 + ∇. ,% ∇6 + ∇6 7 -∇. 6 = 0

Eq. 4

with p is the pressure and u the flow velocity

The boundary conditions (Fig. 4a andb) were set as no slip on the walls, along with the inlet and the outlet conditions. For the membrane-cake interaction from the top view in Fig. 4d, the bottom and top boundaries (edges) were considered walls while the side boundaries were considered symmetric to simulate a periodic domain. This is a fair assumption as the membrane length ratio to the channel depth is much larger than unity. The inlet corresponds to the red highlighted surface at the top and the outlet corresponds to the bottom blue surface in Fig. 4c.

3

Results and discussion

The strategy described was used in filtration experiments to characterize the system and study the influence of membrane geometry on cake construction. From the instantaneous mean height of the cake, mean concentration above the cake surface and the particle-flow velocity it is possible to monitor the cake growth, perform a mass balance and describe the cake structure related to the porosity. The permeability and the Kozeny coefficient of the resulting deposits are analyzed. Using Eq. 3 the estimated porosity is checked.

3.1

Qualitative analysis

The first stages of the filtration exhibit behavior strongly dependent on the membrane geometry. As shown in Fig. 5, after the first particles reach the membrane, the relation between the particle diameter and the slot distance induces different particle cake structures. The characteristic Stokes flow corresponds to Re < 1 [START_REF]Chapter 9 -Boundary Layers and Related Topics[END_REF] which is the case for the filtration experiments. The viscous forces are higher than the inertial forces; as a result, particles in the dilute suspension follow closely the fluid flow streamlines with negligible particle-fluid relative velocities. Consequently, particles are guided by the flow and they flow preferentially to the slots. The slot distribution plays a role in the in the organization of the particles in the first layers; once the slots are blocked, the following particles approaching a blocked slot are driven to the membrane surface zone corresponding to the walls separating the slots. Depending on the distance between slots, a certain number of particles can deposit on these zones, causing the particles to arrange differently in order to fill the whole membrane surface. Fig. 5 a,c,e) First stage of cake formation for C4Pe7, C4Pe10 and C4Pe20 configurations, respectively. b,d,f) Sketches of the typical structure of particle arrangement for the corresponding membrane configuration For C4Pe7 units, dp is small compared to the diameter of the particles (8.4 µm) and the slot width is C = 4 µm; with dp = 3 µm, particles are not able to lay on the walls, moreover dp is too small and their arrangement results in a moderate slot blocking since they cannot be closely aligned over a slot. According to this arrangement, particles centers are not aligned on the same axis following the membrane length direction (x), hence causing a shift of the particles in the depth direction (y). This causes the overlaying highlighted by the circles in Fig. 5a, as some particles are displaced in y, some of them cannot be observe in the focus plane. Fig. 5b illustrates the previous analysis: for the C4Pe7 configuration, particles are spread over all the membrane with large spaces between each other and in a limited depth, thus large voids are formed in the first layers resulting in high porosity. This phenomenon has been reported as the pore protection phenomenon and it yields a more porous blocking layer [START_REF] Ben Hassan | In situ 3D characterization of monodispersed spherical particle deposition on microsieve using confocal laser scanning microscopy[END_REF]. The membrane blocking remains moderate and this slot configuration induces a pyramidal arrangement as it is possible to see in Fig. 5a andb.

In the case of C4Pe10 dp = 6 µm which corresponds almost to the space and it allows particle centers to align on the membrane length axis (x). In C4Pe10 membranes, the "pore protection phenomenon" is no longer detected, as due to membrane configuration there is no particle shift in the (y) direction. The distance between slots dp allows particles to align in the x direction, however it is not enough for particle to reach the walls of the membrane surface. The resulting packing for C4Pe10 membrane appears higher than for the case of C4Pe7 units. The membrane surface is blocking is accentuated, the structure of the first layer tends to be regularly organized in a pyramidal structure similar to the body-centered cubic packing (Fig. 5c andd). The filtration cake formed for C4Pe10 units results consistently in less voids and a less porous cake first layers.

For the C4Pe20 units, particle distribution over the slots is similar to that observed for C4Pe10 units with no "pore protection phenomenon". The slot distance dp = 16 µm is large enough and particles are optimally organized on the slots with their centers aligned in the membrane length axe. In addition, particles can deposit on the walls due to the larger distance between pores that allows the particles to arrange in random structures. This leads to more disorganized layers as shown in Fig. 5e andf. Since the structure is less conditioned by the membrane configuration, the hydrodynamics play a more important role in the arrangement of particles. The first arriving particles mainly block the slots and the following are driven by the fluid to the zones of the slots that are still open to the flow. This leads to a more complex and disorganized structure that could result in more compact cake layers.

The pore protection phenomenon and the existence of preferential arrangements depending on slots (or pore) distance were previously described. In the work by Ben Hassan et al. [START_REF] Ben Hassan | In situ 3D characterization of monodispersed spherical particle deposition on microsieve using confocal laser scanning microscopy[END_REF] using CLSM for direct observation of a thin cake (~35µm), it was possible to obtain evidence of particle organization patterns related to the ratio of particle size to distance between pores. Even if the membrane geometry is different in the current work, particles are initially and preferentially driven by the flow to the slots. The slot distribution affects how the membrane is blocked since particles with a size large compared to the distance between slots, protect neighboring slots from being blocked. It is thus evident that different membrane geometries affect pore blocking and particle arrangement. These effects persist to a certain degree into the later cake formation but they are attenuated as the system dynamics are later dominated by the cake growth rather than the membrane and the membrane geometry and the membrane-cake interaction (blocking) which explains the decreasing behavior of the porosity shown in Fig. 6.

3.2

Quantitative analysis The filtration phenomenon is studied from the slot when particle blocking takes place, to the transition state where the dynamics are controlled by the membrane and the first layers of the deposit-. The pressure difference across the cake alone and the porosity evolution as a function of the deposited mass per unit of membrane surface throughout the filtration are represented in Fig. 6. The particle deposit formed on the C4Pe7 membrane exhibits a lower pressure difference than the cakes formed with the other filtration units. The C4Pe7 cake also undergoes a change of the pressure slope around m = 55 g/m². This change corresponds to the moment at which C4Pe7 and C4Pe10 porosity curves almost overlap and could suggest a rearrangement of the C4Pe7 cake structure into a more stable and compact organization. This possible particle rearrangement is consistent with the higher void fraction in the first layers previously discussed (Fig. 5 a andb). These voids could suffer a partial collapse, which may lead to the rise observed in the pressure drop. This could explain why the pressure values obtained for C4Pe7 membrane approach the values observed for the cake formed using C4Pe10 units. The pressure evolution corresponds is consistent with the qualitative description of the induced cake structure regarding the membrane geometry. As both cakes obtained with C4Pe7 and C4Pe10 membranes exhibit a similar pyramidal structure in the first layers, it is not surprising the agreement of the two curves with the collapse of the initial voids. The cake formed on the C4Pe10 membrane shows an intermediate behavior between the C4Pe7 and the C4Pe20. The pressure rise slope could suggest that the particle organization in the deposit is more stable than that obtained for C4Pe7 membrane and there is not a significant rearrangement of the particles as the pressure increases. A consistently higher pressure drop is observed for the C4Pe20 membrane cake. In this case, a more random particle arrangement within the first layers of the deposit could result in a more compact structure. The fluid drives particles to the remaining open filtering zones in the slots instead of spreading over the walls; therefore, for a deposited mass, the amount of particles blocking the flow through the slots can be higher for C4P20 membranes resulting in lower porosities and the elevated pressure drop.

It is also interesting to consider the later behavior when the cake layer growth is larger for the different slot width. Pressure drop is consistently lower for the cake formed on C4Pe7 membrane and higher for C4Pe20. This difference suggests that the organized arrangement of particles in the first layers combined with the pore blocking have an effect on later cake structure and the mean porosity values. Membrane influence remains throughout the filtration experiments up to 70 g/m 2 ; nevertheless, further cake formation out of the studied range should drive the system dynamics to be determined by the cake. Fig. 6 Pressure drop across the cake alone and cake porosity behavior as a function of the deposited mass per surface unit.

The porosity values obtained in these experiments are comparable to the values usually reported for spherical particles. The work of Ouchiyama and Tanaka [START_REF] Ouchiyama | Porosity estimation for random packings of spherical particles[END_REF] used a simplified model to estimate the porosity of randomly packed spheres and found typical values of 0.43. In the work by Ben Hassan et al. [START_REF] Ben Hassan | In situ 3D characterization of monodispersed spherical particle deposition on microsieve using confocal laser scanning microscopy[END_REF] using different types of membranes, the effect of pore size and spacing on the cake porosity for a ~4 -5 layers (~25 µm) was demonstrated. Porosity values of 0.41 and 0.49 were found for cakes of monodispersed particles (4.8 µm diameter) and for two different membranes. It was concluded that for smaller pore distance the pore protection phenomenon is enhanced and thus porosity is higher. Comparing with theoretical packing models, the porosity evolution for C4Pe7 and C4Pe10 membranes varies from loosest regular packing (cubic lattice: 6 points of contact per sphere) to loose random packing with theoretical porosities varying from 0.48 -0.40, respectively. On the other hand, in the case of C4Pe20 membrane the porosity varies from loose random packing to close random packing (vibrated beds), corresponding to theoretical porosities of 0.40 -0.36, respectively [START_REF] Dullien | Porous Media: Fluid Transport and Pore Structure[END_REF]. This confirms the previous qualitative analysis of the cake structure and particle organization obtained for the different membrane geometries. It can be noticed that in these experiments the theoretical densest regular packing (porosity ~0.26) is never reached. Fig. 7a shows the variations of the cake permeability as a function of the deposited mass per membrane surface unit. The cake permeability () 1 = ℎ / 1 ) is calculated from the cake resistance. The cake hydraulic resistance is obtained from the difference of the total and the membrane resistance ( 1 = 0 -' . The cake resistance allows definition of the permeability resulting from the cake and the membrane blocking. These two resistance components can be observed in the permeability evolution which exhibits an important decrease as the first layers are formed. The initial sharp decrease can be related to the slot blockage phenomenon. This can be confirmed from the variation of cake height as a function of the deposited mass (Fig. 7c) for which 10 g/m 2 corresponds to 15 µm of cake height. With regards to particle size this corresponds to the second layer formation. The later permeability becomes gradually more influenced by the further cake growth and decreases toward a stable value. This corresponds to the previous qualitative explanation, which suggests a possible particle rearrangement in the deposits as the pressure rises. The differences in the initial permeability values are explained by two main features: the partial blocking phenomenon and the induced cake structure. For C4Pe7 membranes the initial permeability is higher and this is related to the geometry and the accentuated slot blockage by the particles for C4Pe10 and C4Pe20 units. The later cake build-up controls the permeability behavior as it approaches a stable value. In the case of C4Pe10 and C4Pe20 units the absence of the "pore protection" phenomenon (Fig. 5c andd) results in a lower permeability than C4Pe7. As the cake formation continues in C4Pe10 units the permeability becomes stable around the same values as found for C4Pe7. This is consistent with the porosity evolution that overlaps for C4Pe7 and C4Pe10 around 55 g/m² and it is also confirmed by a lower permeability corresponding to a lower porosity for C4Pe20 membrane.

To provide a more complete characterization of the fouling mechanisms, the porosity " estimated using Eq. 2 is compared with the porosity * calculated using Eq. 3 with KK = 5. Fig. 7b shows both " and * represented by symbols and solid lines respectively. These two estimates of the porosity exhibit a similar behavior for the cakes formed under the different membrane configurations; however the values differ slightly, with * consistently lower than " . This difference could be explained by the assumption of a constant value for KK when calculating * . Previous work has reported the dependence of the KK on the porosity [START_REF] Knackstedt | Direct evaluation of length scales and structural parameters associated with flow in porous media[END_REF]. Although the Kozeny-Carman formula provides a semiempirical relation for the permeability, analytical development of the equations of continuity and momentum for creeping flow has been made. Snyder and Stewart [START_REF] Snyder | Velocity and pressure profiles for Newtonian creeping flow in regular packed beds of spheres[END_REF] solved creeping flow through a loose cubic packing ( = 0.476) and dense cubic packing ( = 0.26) media of uniform spheres. Their solutions show a fair agreement with the Kozeny-Carman equation, finding KK values of 4.16 and 4.86, for the high and low porosity media respectively. Vidal et al. [START_REF] Vidal | Effect of particle size distribution and packing compression on fluid permeability as predicted by lattice-Boltzmann simulations[END_REF] performed massive parallel Lattice-Boltzmann simulations of flow through monodispersed and highly polydispersed spherical particle packing using Monte-Carlo methods. Their results show a reasonable good agreement with Carman-Kozeny formula. For monodispersed particles, they obtained KK values ranging from 5.5 to 6.0 for high ( = 0.46) and low ( = 0.38) cake porosities respectively. The experimental results for " and the previous literature results show good agreement and validate the current estimation regardless of the slight differences with * .

Considering KK variable as a function of the porosity, Eq. 3 is used to calculate the values for each estimated porosity " . In Fig. 7d, KK varies within a consistent range: for the three geometries they are close to but generally above the typical value of KK = 5 for monodispersed spheres. For C4Pe7 and C4Pe10 filtration cakes, KK exhibits barely an evolution with mean values of 6.0 and 6.6 respectively; for C4Pe20, KK starts at a high value and decreases towards a stabilization around ~7. In the noted prior studies [START_REF] Snyder | Velocity and pressure profiles for Newtonian creeping flow in regular packed beds of spheres[END_REF], [START_REF] Vidal | Effect of particle size distribution and packing compression on fluid permeability as predicted by lattice-Boltzmann simulations[END_REF], lower porosities lead to higher KK, which is also the case for the current experiments.

3.3

Numerical simulation As described in Section 2.5, three geometries were studied in order to analyze membrane blocking when the first cake layers are forming and confirm the experimental results. Following findings previously described in the literature, a uniform cake surface is expected when the filtration has already taken place and the filtration dynamics are controlled predominantly by the cake rather than the membrane; however in the first stages of the filtration, membrane permeability gradients could influence the first layers formation [START_REF] Mendret | Numerical investigations of the effect of nonuniform membrane permeability on deposit formation and filtration process[END_REF]. In the case of these simulations as shown in Section 2.5, an optimal coverage of the membrane is studied, corresponding to the sketches of Fig. 4d, e andf. The results of the qualitative analysis helped to define the simplified model geometries for the simulations (Fig. 5). The pressure drop across the system for the three simulated geometries is calculated, and from this value the contributions to the total resistance are determined(Rm and Rc).

The simulation results allow calculation of the hydraulic resistance of the first layer of particles (blocking layer), which are responsible for the partial blocking of the membrane. In order to compare the hydraulic resistance caused by the first layer with the experimental results obtained for ~3 layers, the mean height of the blocking layer is calculated taking into account the geometry of the membrane (specifically Pe), indeed the number of particle blocking the slots depends on the numbers of slots per unit of area. For this reason, the blocking layer height for the three geometries is not the same and the lowest value of Pe (C4Pe7) yields a larger mean height of the blocking layer; for higher slot distance (C4Pe20) there are fewer pores and hence less particles deposited, and this contributes to make the blocking layer height smaller. It must be pointed out that the mean height of the blocking layer for all the membrane configurations is lower than the particle diameter (8.4 µm). The particle penetration inside the slot also contributes to this feature. As these simulations were run only for the blocking layer, only one pair of data was obtained for each membrane configuration. Fig. 8 Experimental values of the cake-blocking hydraulic resistance for the different membrane geometries when the initial layers are forming (dashed lines with filled symbols). Hollow symbols represent the simulated resistance due to particles blocking the slots.

Fig. 8 shows the comparison of the simulated (1 layer) and the experimental (~ 3 layer) hydraulic resistance caused by the cake and the membrane blocking as a function of the deposit height at the beginning of the filtration. Hollow symbols represent the simulation results for the cake hydraulic resistance when the slots are optimally blocked for each membrane geometry. The values are plotted as a function of the mean height for the corresponding geometry. Note the proximity of the simulation results with the respective experimental curve at the early membrane blocking (1 layer ~3-7 µm height depending on the membrane geometry). This indicates that the experimental data is in a consistent range. There is a difference in the resistance obtained from the simulations for the different geometries. This could be explained by the assumption of an optimal blocking of the slots by the particles; this is not always the case in the real experiments as some flow streamlines are closer to the membrane walls than a particle radius. This promotes particles to deposit on the wall before reaching a pore and is likely a reason why the simulations are displaced from the experimental curves.

Nevertheless, these simulations provide a means of rationally evaluating the qualitative description of the membrane blocking in order to compare with the quantitate analysis of the experiments. Indeed, the qualitative analysis remains subject to the interpretation of 2D images, which leads to an uncertainty in the depth direction. However, as the simulated 3D geometry is based on the qualitative description, the calculations obtained from the simulations confirms the consistency of the approach as there is a fair agreement with the experimental results.

Conclusion

The proposed in-situ and real-time experiment based on the design and construction of a new micro-system combine with the analysis methodology are found to be of significant utility for characterizing the first stage of the filtration process of micrometric particles. The cake growth and its organization are analyzed though the CGM, which tracks the cake surface during filtration. The cake mean height is analyzed with the concentration and velocity calculations to check the mass balance. An additional qualitative analysis is performed to characterize the structure of the first particle layers. The observations are used to describe the cake structure and define the geometry for the simulations.

The filtration performance for the different membrane geometries appear to be quite similar, but the pressure drop can be affected by the distance between slots. The initial particle arrangement is notably conditioned by the membrane geometry that has also an influence on pore blocking and further cake growth, which influences the pressure drop differences. The numerical simulations provide a means of rationally evaluating the role of the membrane geometry and the first blocking particles on the deposit formed at the membrane. The simulation results for the blocking layer hydraulic resistance are in agreement with the experimental data for the current suspension. The qualitative and quantitative analysis conclude on the effect of membrane geometry on "pore blocking" and permeability reduction. The size and the distance between slots as well as the particle diameter play a role on the "pore protection phenomenon". These conclusions can be applied to real industrial applications by characterizing industrial membrane filtration units (cylindrical pores instead of slots) using the crucial parameters pointed out in this study, the size of the slot/pore and the distance between two neighbor slots/pores. This could lead to a better choice of membrane units depending on the material to be retained. The optical set-up designed for this study and analysis techniques developed are interesting tools that could be adapted to provide in-situ and real time information to perform deeper analysis for different filtration conditions, like for example crossflow filtration.

Fig. 1

 1 Fig. 1 Microfiltration cell configurations. Left figure represents the channel configuration with the filtration cell in the middle. The inlet is at the top and outlet at the bottom of the channel. Highlighted at right: zoom view of the corresponding cell membrane, C2Pe5 (units in µm) and the other different geometries.

Fig. 2 a

 2 Fig. 2 a) Scheme of the experimental set-up. 1) Syringe pump, 2) Syringe, 3) Reservoir, 4) Manometer, 5) Cell support, 6) Optical bench 7) Acquisition computer. b) Detail of the microfiltration cell, design and SEM observation.

Fig. 3 a

 3 Fig. 3 a) Raw image of a particle approaching the membrane in a filtration experiment. b) Particle (blue) and cake (green) identification after binarizing and filling.

Fig. 4 a

 4 Fig. 4 a) Front view of the channel geometry without the membrane (Suspension inlet at the top of the channel and permeate outlet at the bottom of the channel). b) Complete geometry of the channel with the membrane. c) 3D simplified geometry for cake-membrane interaction (for C4Pe7 membrane). d,e,f) Top-view of the simplified geometry C4Pe7, C4Pe20, C4Pe10 respectively. g) Detail on the membrane simulated geometry, example for a C4Pe10 unit.

Fig. 7 a

 7 Fig. 7 a) Permeability of the cake. b) Porosity " estimated using Eq. 2 (○ C4Pe7, □ C4Pe10, Δ C4Pe20) and Darcy's equation * (Color solid lines) for the different membranes. c) Mean cake height. d) Estimated Kozeny coefficient. All the graphics are plotted as function of the deposited mass per membrane surface unit.

Table 1 .

 1 Microfiltration cell geometries

	Membrane	Slot width,	Periodicity,	Slot distance
	reference	C (µm)	Pe (µm)	dp wall (µm)
	C2Pe5	2	5	3
	C4Pe7	4	7	3
	C4Pe10	4	10	6
	C4Pe20	4	20	16

Acknowledgments

This work was done in the frame of the research grant program AAP 2015 from Idex UNITI "NEMESIS" conv-ANR-11Idex-0002-02.

The authors thank Pierre Joseph from the « Laboratoire d'analyse et d'architecture des systèmes » (LAAS).


Hacene Medkour

Younes Laidoudi
email: younes.laidoudi@yahoo.com

Eric Athias
email: ericathias@yahoo.fr

Amar Bouam
email: amarbouam@yahoo.fr

Sylvestre Dizoé

Bernard Davoust
email: bernard.davoust@gmail.com

Oleg Mediannikov

Hacène Medkour

Molecular and serological detection of animal and human vector-borne pathogens in the blood of dogs from Côte d'Ivoire

Keywords: Vector-borne pathogens, Côte d'Ivoire, Dog, PCR, Leishmania infantum, Trypanosoma congolense, Babesia vogeli, Filariidae, Ehrlichia canis

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

Introduction

Canine vector-borne diseases (CVBD) are a group of rapidly spreading, globally distributed diseases caused by a range of arthropod-borne pathogens, including ticks, fleas, mosquitoes and phlebotomine sandflies which are the vectors of a rank of bacteria, viruses, protozoa and helminths. In addition to their veterinary importance, some CVBD-causing pathogens are of major zoonotic concern [START_REF] Otranto | Managing canine vector-borne diseases of zoonotic concern: part two[END_REF]. Dogs are sentinels and reservoirs of diseases that are a risk for human population, therefore, the interest to follow CBVD in dog populations remains of greater importance. Ehrlichia canis, Hepatozoon spp., Anaplasma spp., Leishmania infantum, Trypanosoma spp., Babesia spp. and filariae are the most vector-borne pathogens detected in dogs in multiple countries [START_REF] Moreno | Canine leishmaniasis: Epidemiological risk and the experimental model[END_REF][START_REF] Santamaria | Molecular Diagnosis and Species Identification of Ehrlichia and Anaplasma Infections in Dogs from Panama, Central America[END_REF][START_REF] Dahmani | Molecular investigation and phylogeny of Anaplasmataceae species infecting domestic animals and ticks in Corsica, France. Parasites and Vectors[END_REF][START_REF] Marié | Sub-clinical infection of dogs from the Ivory Coast and Gabon with Ehrlichia, Anaplasma, Mycoplasma and Rickettsia species[END_REF][START_REF] Liu | Molecular survey of canine vector-borne diseases in stray dogs in Thailand[END_REF].

Bacteria from the Anaplasmataceae family include numerous dog-associated pathogens vectored by ticks [START_REF] Dahmani | Molecular investigation and phylogeny of Anaplasmataceae species infecting domestic animals and ticks in Corsica, France. Parasites and Vectors[END_REF]. Canine monocytic ehrlichiosis (CME) caused by E. canis and transmitted by the tick Rhipicephalus sanguineus, is the most recognized, and is responsible for severe clinical signs and a high mortality rate in infected dogs. The infection is common in Sub-Saharan Africa, such as Côte d'Ivoire and Gabon [START_REF] Marié | Sub-clinical infection of dogs from the Ivory Coast and Gabon with Ehrlichia, Anaplasma, Mycoplasma and Rickettsia species[END_REF]. Native African dogs are often asymptomatic (immunotolerance). Warm climates seem to be favorable for the infectious agents propagation [START_REF] Liu | Molecular survey of canine vector-borne diseases in stray dogs in Thailand[END_REF].

Canine leishmaniosis, caused by L. infantum and transmitted by a phlebotomine sand fly vector, is the most serious CVBD considering its large distribution (in Central and South America, Asia and several countries of the Mediterranean Basin) and high pathogenicity [START_REF] Attipa | Association between canine leishmaniosis and Ehrlichia canis co-infection : a prospective case-control study[END_REF][START_REF] Medkour | Canine vectorborne protozoa: Molecular and serological investigation for Leishmania spp., Trypanosoma spp., Babesia spp., and Hepatozoon spp. in dogs from Northern Algeria[END_REF]. In Sub-Saharan Africa, data on CanL are rare, except some reports in Senegal [START_REF] Faye | Canine visceral leishmaniasis caused by Leishmania infantum in Senegal: Risk of emergence in humans?[END_REF], Burkina-Faso [START_REF] Sangare | First Detection of Leishmania infantum in Domestic Dogs from Burkina Faso (West Africa)[END_REF]. Anecdotal L. infantum infections in human were also reported in Côte d'Ivoire [START_REF] Kouassi | Leishmaniose viscérale à Abidjan à propos de trois observations (French). Three cases of visceral leishmaniasis in Abidjan, Côte d'Ivoire[END_REF] and Gambia [START_REF] Desjeux | Leishmaniasis in The Gambia. 2. A study of possible vectors and animal reservoirs, with the first report of a case of canine leishmaniasis in The Gambia[END_REF].

Canine trypanosomosis is a disease caused by haemoprotozoan parasites Trypanosoma brucei brucei and T. congolense in Africa [START_REF] Keck | Epidemiological study of canine trypanosomosis in an urban area of Ivory Coast[END_REF]. This disease can also be caused by T. cruzi, the agent of American trypanosomosis known as the Chagas disease in humans. Dogs are also infected by T. b. rhodesiense and T. b. gambiense pathogens for humans [START_REF] Eloy | Canine trypanosomiasis: etiology of infection and implications for public health[END_REF].

Babesiosis is a potentially serious illness caused by intraerythrocytic protozoan parasites of the genus Babesia. B. vogeli, B. gibsoni and B. rossi are the main causative agents of the canine babesiosis vectored by ticks. They may also be transmitted vertically [START_REF] Fukumoto | Fatal experimental transplacental Babesia gibsoni infections in dogs[END_REF] or by blood exchange in fighting dogs [START_REF] Jefferies | Blood, Bull Terriers and Babesiosis: Further evidence for direct transmission of Babesia gibsoni in dogs[END_REF]. Babesia species have a global distribution and have been identified in African dogs [START_REF] Matjila | Confirmation of occurrence of Babesia canis vogeli in domestic dogs in South Africa[END_REF].

Among the vector-borne pathogenic helminths belonging to the order Spiruridae, the suborder Spirurina and the families Filariidae and Onchocercidae: Dirofilaria immitis and D. repens are probably the best known to cause infestations in dogs and humans [START_REF] Otranto | Managing canine vector-borne diseases of zoonotic concern: part two[END_REF][START_REF] Otranto | Changing distribution patterns of canine vector borne diseases in Italy: leishmaniosis vs. dirofilariosis[END_REF], followed by Acanthocheilonema reconditum (Spirurida, Onchocercidae) often infects dogs [START_REF] Brianti | New insights into the ecology and biology of Acanthocheilonema reconditum (Grassi, 1889) causing canine subcutaneous filariosis[END_REF].

Differently from other filarioids transmitted by mosquitoes (e.g., D. immitis and D. repens) or ticks (e.g., Cercopithifilaria spp.) to dogs, A. reconditum is vectored by fleas (i.e., Ctenocephalides canis, C. felis, Pulex irritans, P. simulans, Echidnophaga gallinae) or lice (i.e., Heterodoxus spiniger, Linognathus setosus) with a rate of infestation in fleas of about 5% [START_REF] Brianti | New insights into the ecology and biology of Acanthocheilonema reconditum (Grassi, 1889) causing canine subcutaneous filariosis[END_REF]. Globally, vector-borne diseases account for about 17% of the burden of all infectious diseases and cause millions of dollars in losses to the livestock industry each year, with implications for human and animal health and the global economy [START_REF] Who | The world health report 2004-changing history[END_REF]. Information on filarial infections in dogs in sub-Saharan Africa is scarce. From a review of available literature by [START_REF] Schwan | Filariosis of domestic carnivores in Gauteng, KwaZulu-Natal and Mpumalanga provinces, South Africa, and Maputo province[END_REF] it appeared that all the above mentioned species have been reported from various places on the continent [START_REF] Schwan | Filariosis of domestic carnivores in Gauteng, KwaZulu-Natal and Mpumalanga provinces, South Africa, and Maputo province[END_REF]. survey for filariae in dogs in northern Kenya and in Zambia have later been documented [START_REF] Albrechtová | Occurrence of filaria in domestic dogs of Samburu pastoralists in Northern Kenya and its associations with canine distemper[END_REF] [START_REF] Siwila | Filarial infections in domestic dogs in Lusaka, Zambia[END_REF].

In Côte d'Ivoire, few information is available on the prevalence and the distribution of CVBD. The purpose of this study was to evaluate the occurrence of these pathogens of veterinary and public (for some of them) health importance and their diversity in dogs from the city of Abidjan (kennel dogs) and in a city near Yamoussoukro (village dogs) by using serological and molecular tools including new PCR assays.

Methods

2.1.Study Sample collection and preparation

In April 2018, blood samples were collected from 123 dogs, living in three kennels in Abidjan (N=95) and Kongouanou town (N=28), (7°00'42.0"N 5°22'04.0"W) localized 24 km from Yamoussoukro and 241 km from Abidjan. The largest kennel is located (5°16'54.9"N 3°56'46.0"W) in Koumassi, district of Abidjan and the other two are between five (Cocody) and six (Treichville) kilometers away. Kennel dogs are working dogs used for protection, security and defense. They belong to adapted breeds (ridgeback, Belgian shepherds, etc.).

They receive veterinary care and sometimes external pest control. The village dogs from Kongouanou are medium-sized dogs mostly used for hunting. They live outdoors freely in the village and its surroundings. They do not benefit from preventive veterinary care (32% carried ticks). Data about sanitary status or possible prophylaxis for kennels dogs were collected. A volume of 4 to 5 mL of blood was collected from the radial vein of each dog, into dry and EDTA tubes; serum was recovered after centrifugation (10 min, 3000g) and frozen at -20 °C.

EDTA tubes were directly frozen at -20 °C. Each animal sampled was examined and information about gender, breed and age were collected.

The animals were examined and sampled after obtaining the verbal consent of the persons (owners) responsible for the dogs. The blood samples were taken from radial vein by veterinarians (French and Ivorian) with the ethical responsibility to ensure the animal care in accordance with Articles 433-434; Chapter 2 of the Ivoirian Penal Code.

2.2.Serological assay to detect E. canis and L. infantum specific antibodies

The presence of anti-E. canis and anti-L. infantum promastigote specific antibodies was revealed by indirect fluorescent antibody test (IFAT). Plate wells were sensitized by 10 µL and 20 µL of the commercial antigens of E. canis and L. infantum (Zoetis, France) respectively and assays were assessed following manufacturer's instructions. Both anti-Ehrlichia and anti-Leishmania antibodies were detected by secondary antibodies against rabbits, anti-dog immunoglobulins G conjugated with fluorescein isothiacyanate (Sigma-Aldrich, St Louis, MO, USA). Sera were considered positive when antibodies titers were higher than 1:100 and 1:80 dilutions for Ehrlichia and Leishmania respectively.

2.3.Morphological identification of filariae species

Blood samples were examined for the presence of microfilaria after concentration by a modified Knott test (Knott, 1939) which is the gold standard [START_REF] Mccall | Chapter 4 Heartworm Disease in Animals and Humans[END_REF]. Microfilariae were quantified and identified when existed. When co-infections were found, we performed a serial dilution until we obtained 1 microfilariae/ 200 mL of blood which was used to extract the DNA. The objective was to concentrate only one species of microfilariae and extract its DNA for a possible future molecular identification.

2.4.Molecular assays

DNA extraction

DNA was extracted from 200 µL of blood after digestion with proteinase K at +56°C overnight using a commercial DNA extraction kit (QIAamp DNA Mini Kit®, [Qiagen, Courtaboeuf, France]) and performed on BIOROBOT EZ1 (Qiagen, Qiagen, Courtaboeuf, France) per the manufacturer's instructions. In order to achieve maximum yield, digestion was performed overnight with proteinase K in Qiagen lysis buffer before the QIAamp DNA Mini Kit® was used. DNA was eluted in 200 µl of distilled water and stored at -20°C.

PCR tools design for Leishmania spp. and Trypanosoma spp. screening

We designed primers and probe using the conserved region of encoding ribosomal RNA genes 18S to detect DNA from Leishmania species: a first, qPCR for the screening of all Leishmania targeting a conserved region of the 18S gene; a second, conventional PCR targeting the same gene that produce the 550-bp amplicon for the following confirmation and species identification. In order to detect DNA from trypanosomes, we designed a qPCR system targeting the ribosomal RNA genes 5.8S. To design the group-specific primers and probes, the corresponding genetic sequences of all Leishmania/Trypanosoma species available in the GenBank were multiple aligned. All conserved motifs were visualized using BioEdit software version 7.0.5.3 (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The free web-based Primer3 software, version 4.0 (http://frodo.wi.mit.edu/primer3/) was used to design the primers and probes based on chosen conserved motifs (Table 1).

The specificity and sensitivity of all PCR assays were tested in silico using primer-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and validated in vitro using a DNA panel of cultivated Leishmania species: L. infantum, L. donovani, L. major and DNA of L. guyanensis for Leishmania PCR systems, a DNA panel of cultivated Trypanosoma species: T. congolense IL 3000, T. evansi, T. vivax, T. brucei brucei, T. brucei gambiense and T. brucei gambiense biyiamina groupe II for Trypanosoma qPCR system, and several arthropods of laboratorymaintained colonies as well as human, monkey, donkey, horse, cattle, mouse and dog DNAs for all the PCR systems (Table S1).

Molecular screening for the presence of pathogens DNA

All the DNA samples were screened for Leishmania spp. using two qPCR assays: i) the 18Sbased qPCR was used for the initial screening and the detection of DNA Leishmania spp., ii) the specific L. infantum qPCR assay based on the amplification of kinetoplast minicircle DNA (kDNA), recognized for its high sensitivity which allows the quantification of the parasite load [START_REF] Mary | Quantification of Leishmania infantum DNA by a Real-Time PCR Assay with High Sensitivity[END_REF]. qPCR assays were performed to screen DNA samples using primers and probes for Anaplasmataceae, Trypanosoma spp. and Piroplasmida (Table 1). Sequences of primers and probes and their respective sources used in this study are presented in Table 1. Concerning Filariidae, we used a triplex qPCR to detect D. immitis, D. repens and A. reconditum targeting the Cox1 gene. In order to detect the occult filariosis du to D. immitis we screened the samples using a qPCR targeting ftsZ gene specific to Wolbachia endosymbiont of D. immitis (Laidoudi et al. unpublished).

Reaction mix for qPCR assays contained 5 μl of the DNA template, 10 μl of Eurogentec Master Mix Roche, 0.5 μl of each reverse and forward primers and UDG, 0.5 μl of the FAMlabeled probe (Table 1) and 3 μl of distilled water DNAse and RNAse free, for a final volume of 20 μl. The qPCR amplification was performed in a CFX96 Real-Time system (Bio-rad Laboratories, Foster City, CA, USA) with the following thermal profile: The first step is an incubation at 50°C for two minutes and an initial denaturation step at 95°C for five minutes, followed by 40 cycles of denaturation at 95°C for 5 seconds and annealing-extension at 60°C for 30 seconds. We included the DNA of the target parasites or bacteria as positive controls and master mixtures as a negative control for each test. Samples were considered positive when the cycle threshold (Ct) was lower than 35 Ct for all qPCR assays and lower than 38 Ct for Leishmania 18S-based qPCR.

PCR amplification, sequencing and phylogenic analysis

Positive samples by qPCR for vector borne pathogens were confirmed by standard PCR assays and sequencing for the fragments of the genes 23S for Anaplasmataceae, kinetoplast DNA, ITS II and 18S for Leishmania spp., ITS I for Trypanosoma spp. and 18S for Piroplasmida. Primer pairs are listed in the (Table 1). Positive samples by the triplex qPCR for Filariidae were also confirmed by standard PCR amplifying a 1230 bp from 18S gene of Filariidae species (Laidoudi et al. unpublished).

The standard PCR assays consisted of a volume of 25 µl, including 12.5 µl of Amplitaq Gold Master Mix, 0.75 µl of each primer, 5 μl of DNA model and water. The thermal cycling conditions were: incubation step at 95°C for 15 minutes, 40 cycles of 1 minute at 95°C, 30 annealing at a different hybridization temperature for each PCR assays and one minute at 72°C followed by a final extension for five minutes at 72°C (Table 1 (https://www.megasoftware.net/). Bootstrap analyses were conducted using 100 replicates.

2.5.Statistical analysis

Statistical analysis was conducted using IBM SPSS statistics version 23. After the databases were established, the association between infections and age, sex, breeds and origin (Kennels vs village) of dogs was assessed using the X 2 test. The Fischer's exact test was used when the percentages were low. Statistically significant was consider at P-value <0.05.

Results

3.1.Sensitivity and specificity of PCR assays

Designed PCR assays listed in Table 1 were tested. First, we tested the Leishmania genusspecific 18S rRNA-based qPCR system by screening of positive controls (Table S1). As expected in silico, we amplified all available positive controls: L. infantum, L. donovani, L.

major and L. guyanensis. These controls were also amplified by the Leishmania 18S rRNAbased standard PCR system. In order to validate these news PCR systems and to reproduce the results, Leishmania-positive samples were tested simultaneously by Leishmania genusspecific 18S, L. infantum-specific kDNA qPCR assays and 18S, ITS1, ITS2 standard PCR tools. All of them gave the same result. Secondly, we tested the Trypanosoma 5.8S rRNAbased qPCR system by screening the controls. As tested in silico, it amplified all positive controls (Table S1). The same results were obtained by using Trypanosoma ITS1-based standard PCR. None of the DNA negative controls were amplified by any of the PCR tools.

3.2.Serological and molecular results

A total of 123 dogs were sampled: 91 males and 32 females. Ages were available for all dogs. There was no significant association between sex, age, breed and the presence of vector-borne dog infections in this study (PV>0.05), except for filariasis for which mongrel dogs were more susceptible (PV=0.02). By contrast, dogs from Kongouanou (village dogs) were found to be more infected 60.7% (17/28) than dogs from Abidjan (kennel dogs) 32.6% (31/95) for at least one pathogen (PV= 0.003). Also, Filariidae infections were more frequent in the village dogs 35.7% (10/28) than the kennel dogs 3.2% (3/95) (PV= 0.0000008) (Table 3). We could not sequence the positive sample for D. immitis because it was co-infected with other species. Phylogenetic trees are built on the basis of these genes and show the position of the identified species (Fig. 3).

3.3.Phylogenetic analysis results

Positive

Sequences were submitted in the GenBank database under accession numbers: MK494932-MK494941 for E. canis, MK481041-MK481044 for L. infantum ITS2 sequences, MK770156-MK770158 for L. infantum kDNA sequences, MK495745-MK495747 for T.

congolense, MK495836-MK495837 for Babesia vogeli, MK495727-MK495733 for A.

reconditum, MK495734 and MK495735 for D. repens.

Discussion

This study describes canine vector-borne infections in dogs from Côte d'Ivoire. Our results

showed that pathogens such as E. canis, L. infantum, T. congolense, B. canis vogeli, A.

reconditum, D. repens and D. immitis are endemic in this area.

The Anaplasmataceae family includes several pathogens associated with dogs; they are reported worldwide and transmitted by Ixodidae ticks. [START_REF] Otranto | Managing canine vector-borne diseases of zoonotic concern: part two[END_REF]. In this study, we found a prevalence of 17.1% (21/123) by qPCR 23S. This is not new, as this region is known to be endemic [START_REF] Marié | Sub-clinical infection of dogs from the Ivory Coast and Gabon with Ehrlichia, Anaplasma, Mycoplasma and Rickettsia species[END_REF]. Sequencing analysis reported the presence of one species, E. canis, the causative agent of monocytic ehrlichiosis. We also noted a seroprevalence of 12 (93/111) of the dogs in Gabon were seropositive [START_REF] Roqueplo | Canine ehrlichiosis in the Ivory Coast and Gabon: Alteration of biochemical blood parameters based on Ehrlichia canis serology[END_REF]. Also, 27% of R. sanguineus s.l. ticks collected (16/60) from dogs in Abidjan, were positive for E. canis using quantitative real-time PCR [START_REF] Socolovschi | Ehrlichia canis in Rhipicephalus sanguineus ticks in the Ivory Coast[END_REF]. Since then, we believe that there is a decreasing in the prevalence due to the awareness of veterinarians and dog owners in the. But the area remains endemic because the dog's state of life promotes transmission and persistence of infection (presence of ticks) and not all dog owners are able to treat and protect their dogs. We did not find Anaplasma platys, the agent of canine cyclic thrombocytopenia. However, prevalence of 8.5 and 56.2% has already been reported in Ivorian dogs and ticks collected on these dogs, respectively [START_REF] Matei | Molecular detection of Anaplasma platys infection in free-roaming dogs and ticks from Kenya and Ivory Coast[END_REF]. the high rate of L. infantum on female sand fly species (5.4% of Sergentomyia dubia, 4.2% of S. schwetzi and 3.6% of S. magna) [START_REF] Senghor | Transmission of Leishmania infantum in the Canine Leishmaniasis Focus of Mont-Rolland, Senegal: Ecological, Parasitological and Molecular Evidence for a Possible Role of Sergentomyia Sand Flies[END_REF] strongly suggests that the L. infantum life cycle is well established in this area. In addition, this raises further questions about vectors in sub-Saharan regions and challenges once again the dogma that leishmaniasis is exclusively transmitted by the genus Phlebotomus in the Old World. In Burkina-Faso, CanL prevalence is about 5.9% (5/85) [START_REF] Sangare | First Detection of Leishmania infantum in Domestic Dogs from Burkina Faso (West Africa)[END_REF]. The disease has mainly been described in the north of the African continent, range of results reported for some countries in the Mediterranean Basin using IFAT: Algeria: 27-45% [START_REF] Adel | Estimation of canine Leishmania infection prevalence in six cities of the Algerian littoral zone using a Bayesian approach[END_REF],Tunisia 18-53% [START_REF] Rami | Leishmaniose canine dans le Rif (côte méditerranéenne marocaine) : enquête séro-épidémiologique[END_REF][START_REF] Nejjar | Canine leishmaniasis due to Leishmania infantum MON-1 in northern Morocco[END_REF], Morocco 9-19% [START_REF] Djetchi | The study of trypanosome species circulating in domestic animals in two human African trypanosomiasis foci of Côte d'Ivoire identifies pigs and cattle as potential reservoirs of Trypanosoma brucei gambiense[END_REF][START_REF] Umeakuana | Identification of Trypanosoma brucei gambiense in naturally infected dogs in Nigeria. Parasites and Vectors [Internet[END_REF].

Data about canine leishmaniosis in

To perform molecular screening of Trypanosoma spp. we have developed a new qPCR system to detect all Trypanosoma species. This molecular tool seems to be sensitive and specific and allowed us to found a prevalence of 5.3% (5/95) in dogs from Abidjan. In 2003, Keck et al. reported a prevalence of 30 % in 123 dogs from kennels of Abidjan by using PCR specific primers for T. congolense "forest type" with no difference between age, class, breed and sex [START_REF] Keck | Epidemiological study of canine trypanosomosis in an urban area of Ivory Coast[END_REF]. The fact that we found fewer infected dogs may be due to the sensitization of local veterinarians against this disease. T. congolense "forest type" has been found in other host animals (sheep, goats, cattle, pigs) in Côte d'Ivoire and other forest areas [START_REF] Djetchi | The study of trypanosome species circulating in domestic animals in two human African trypanosomiasis foci of Côte d'Ivoire identifies pigs and cattle as potential reservoirs of Trypanosoma brucei gambiense[END_REF]. However, we did not detect this trypanosome. ITS 1 gene sequencing showed 99% of identity with T.

congolense isolate 237-51-00095-1-60-10 (GenBank: MG283145) identified in Glossina palpalis palpalis in Cameroon (Fig. 2). In Nigeria, nineteen dogs presenting with canine African trypanosomosis were infected with trypanosomes of the T. brucei group [START_REF] Umeakuana | Identification of Trypanosoma brucei gambiense in naturally infected dogs in Nigeria. Parasites and Vectors [Internet[END_REF]. T.

congolense is cyclically transmitted by tsetse flies (Diptera: Glossinidae). Trypanosomosis caused by T. congolense is a major threat to livestock production in Sub-Saharan Africa.

Infected dogs can revealed pale mucous membranes, weak pulse, enlarged lymph nodes, rough hair coat and loss of skin elasticity [START_REF] Ramesh | Diagnosis and Treatment of Canine Trypanosomiasis -a Case Study[END_REF].

We found a prevalence of 1.6% (2/123) infected by B. canis vogeli. To our knowledge, this is the first detection of this pathogen in Côte d'Ivoire. This species has a global distribution [START_REF] Dantas-Torres | The brown dog tick, Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae): From taxonomy to control[END_REF] and has been identified in Africa [17][41]. The presence of ticks Rhipicephalus sanguineus s.l., the main vector of B. canis vogeli [START_REF] Otranto | Arthropod-borne pathogens of dogs and cats: From pathways and times of transmission to disease control[END_REF] on dogs in Côte d'Ivoire [START_REF] Socolovschi | Ehrlichia canis in Rhipicephalus sanguineus ticks in the Ivory Coast[END_REF] Maputo province, Mozambique [START_REF] Schwan | Filariosis of domestic carnivores in Gauteng, KwaZulu-Natal and Mpumalanga provinces, South Africa, and Maputo province[END_REF]. A. reconditum is generally considered non-pathogenic for dogs, it lives in the peritoneal cavity and adipose tissue [START_REF] Magnis | Morphometric analyses of canine blood microfilariae isolated by the Knott's test enables Dirofilaria immitis and D. repens species-specific and Acanthocheilonema (syn. Dipetalonema) genus-specific diagnosis[END_REF]. Recently, it has been reported to induce skin lesions with atopic allergy in a dog [START_REF] Sivagurunathan | A Case of Acanthocheilonema reconditum in a dog[END_REF]. This parasite is transmitted by fleas and chewing lice [START_REF] Albrechtová | Occurrence of filaria in domestic dogs of Samburu pastoralists in Northern Kenya and its associations with canine distemper[END_REF].

We found 13.8% (17/123) of dogs with two or three pathogens and E. canis-L. infantum association was the most observed 4.9% (6/123) co-infection. Co-infections in dogs is common. It is known that infection of the dog with more than one pathogen is associated with high pathogenicity [START_REF] Baxarias | Does co-infection with vector-borne pathogens play a role in clinical canine leishmaniosis ?[END_REF]. The main cases observed are co-infections of dogs with tick-borne pathogens, including E. canis, B. vogeli, A. platys and Hepatozoon canis, that increase anemia for example, more than mono-infections [START_REF] Rojas | Vector-borne pathogens in dogs from Costa Rica: First molecular description of Babesia vogeli and Hepatozoon canis infections with a high prevalence of monocytic ehrlichiosis and the manifestations of co-infection[END_REF]. Co-infection L. infantum-A. reconditum has been found in two dogs (1.6%). Co-infections L. infantum with filariidae are known. In Portugal, 8.3 % of 230 dogs were infected with both L. infantum and D. immitis [START_REF] Maia | Molecular detection of Leishmania infantum, filariae and Wolbachia spp. in dogs from southern Portugal. Parasites and Vectors [Internet][END_REF]. This coinfection has also been observed in dogs from Greece [START_REF] Ntais | Leishmania infantum and Dirofilaria immitis coinfection in dogs in Greece[END_REF]. This is relatively observed in dogs living in areas where vectors competent for different pathogens coexist, which can complicate the diagnosis, treatment and prognosis of CVBD [START_REF] Maia | Molecular detection of Leishmania infantum, filariae and Wolbachia spp. in dogs from southern Portugal. Parasites and Vectors [Internet][END_REF].

In addition to these classical co-infections, we also noted co-infections of E.canis-A. reconditum in 3.3% (4/123) and L. infantum-T. congolense in two dogs. Two dogs had triple infection, one of them was positive for the three filaroid species by modified Knott's test and the triplex Cox1-based qPCR, the other was positive for A. reconditum and D. repens by these same tools and negative for D. immitis, but it was positive on qPCR targeting ftsZ gene for Wolbachia endosymbiont for D. immitis, thus suggesting an occult infection. D. immitis has a severe impact on veterinary medicine, because of the heartworm disease threatening dogs and cats, whereas D. repens, causing subcutaneous infestation in dogs, is the main agent of the human dirofilariosis [START_REF] Otranto | Vector-borne helminths of dogs and humans in Europe[END_REF]. D. immitis has been reported from Guinea-Bissau, Nigeria, Senegal, Sierra-Leone and in Central Africa (Angola, Cameroon and Gabon). In addition, it has been reported that D. repens is the most common filarial worm in Nigerian dogs and cats. This species is also endemic in Central and Eastern Africa [START_REF] Schwan | Filariosis of domestic carnivores in Gauteng, KwaZulu-Natal and Mpumalanga provinces, South Africa, and Maputo province[END_REF]. All these co-infections can lead to overlaps or atypical clinical signs. They should be expected in dogs living in areas that are highly endemic for several vector-borne pathogens, mainly in dogs kept predominantly outdoors and not regularly treated with ectoparacitides [START_REF] Roura | Serological evidence of exposure to Rickettsia, Bartonella and Ehrlichia species in healthy or Leishmania infantuminfected dogs from Barcelona, Spain[END_REF]. The prevalence of CVBD varied according to the dog's origin and Kongouanou dogs were more infected than those in Abidjan.

They also had more filaroid infections. These dogs live freely in the open air in the village and its surroundings. They do not receive preventive veterinary care and most of them were tick carriers, compared to kennel dogs, which receive veterinary care and sometimes external pest control.

Conclusion

To the best of our knowledge, this study is the first to report the prevalence and diversity of vector-borne pathogens of humans and animals in dogs from Côte d'Ivoire. Co-infections with more than one pathogen were found to be common.

  The average age was three years old [4 month-10 years] with 49 juveniles [4 month -2 years], 33 young dogs [2-4 years], 26 adults [4-6 years] and 15 oldies (>6years) dogs. Prevalence of the vector-borne pathogens detected by serology or PCR is presented in the

Fig. 3

 3 Fig. 3 Phylogenetic trees highlighting the position of Ehrlichia spp. (A), Leishmania spp. (B and C), Trypanosoma spp. (D) and Babesia spp. (E) and filaroid species (F) identified in the present study compared to other sequences available on GenBank. The sequences for 23S (A), ITSII and kDNA (B and C), ITSI (D) and 18S (E) and 18S (F) genes, for each of the six trees, were aligned using CLUSTALW, and phylogenetic inferences was conducted in MEGA 7 using the maximum likelihood method based on the Tamura-Nei model for nucleotide sequences. . Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. The GenBank accession numbers are indicated at the beginning. Statistical support for the internal branches of the trees was evaluated by bootstrapping with 1,00 iterations. Bootstrapping under 40 were removed.

  ). PCR amplification was performed in a Peltier PTC-200 model thermal cycler (MJ Research Inc., Watertown, MA, USA). The results of amplification were visualized by electrophoresis on 2% agarose gel. The purification of PCR products was performed using NucleoFast 96 PCR plates (Macherey Nagel EURL, Hoerdt, France) according to the manufacturer's instructions. The amplicons were sequenced using the Big Dye Terminator Cycle Sequencing Kit (Perkin Elmer Applied Biosystems, Foster City, CA, USA) with an ABI automated sequencer (Applied Biosystems).

	The obtained electropherograms were assembled and edited using ChromasPro software
	(ChromasPro 1.7, Technelysium Pty Ltd., Tewantin, Australia) and compared with those
	available	in	the	GenBank	database	by	NCBI	BLAST

(https://blast.ncbi.nlm.nih.gov/Blast.cgi). The DNA sequences obtained for each gene, for each pathogen, from positive samples were aligned with those available in the GenBank database for the same gene. A maximum-likelihood method was used to infer the phylogenetic analyses and tree reconstruction was performed using MEGA software version 7

Table 2 .

 2 Resultsshowed that 39% (48/123) of dogs were positive for at least one of the pathogens by either PCR or serology (Table3). Screening using qPCR assays showed a prevalence of 17.1% (21/123) for Anaplasmataceae infections (E. canis was the only species identified), followed Trypanosoma spp. in 4.1% (5/123) and finally Babesia spp. with 1.6% (2/123). Prevalence by IFAT was of 15.4% (19/123) for L. infantum and 12.2% (15/123) for E. canis (Table2).Regarding mono-infections, E. canis infection was the most prevalent with 9.7% (12/123).

	We found a prevalence 8.9% (11/123) for L. infantum, A. reconditum 4.1% (5/123).
	Trypanosoma spp. 1.6% (2/123) and finally Babesia spp. with 0.8% (1/123).
	Mixed infections 13.8% (17/123) were also found in this study (Fig. 2). Mixed infection of E.

by Filariidae infections 10.6% (13/123) with at least one filaria species, L. infantum in 4.1%

(5/123) with 28.9 Leishmania/mL of blood (Min: 2.1; Max: 117.8) as an average of the parasite load found by the kDNA qPCR, canis-L. infantum 4.9% (6/123) was more frequent than E. canis-A. reconditum with 3.3% (4/123), L. infantum-Trypanosoma spp. and L. infantum-A. reconditum with 1.6% (2/123) for each of them and finally, co-infection with E. canis and B. vogeli with only one case (0.8%). Triple infection with A. reconditim-D. immitis-D. repens were observed in two dogs. One of them was positive by modified Knott's test and the triplex qPCR for the three species. The other one was positive for A. reconditum and D. repens but negative for D. immitis for both tests. By contrast, it was positive by qPCR ftsZ for Wolbachia endosymbiont of D. immitis, suggesting an occult infection by D. immitis.

  Côte d'Ivoire are absent. To the best of our knowledge, our study is the first to report L. infantum in dogs from this area. The prevalence observed is donovani, L. infantum, L. major, L. guyanensis as well as probable new species. Only 4% (5/123) were positive for PCR, which differs significantly (PV=0.0026) with serological results. This is not surprising since PCR assays on blood samples often provide less positive results for leishmaniosis than for other sites (bone marrow and lymph node)[START_REF] Socolovschi | Ehrlichia canis in Rhipicephalus sanguineus ticks in the Ivory Coast[END_REF][START_REF] Matei | Molecular detection of Anaplasma platys infection in free-roaming dogs and ticks from Kenya and Ivory Coast[END_REF], hence the choice of IFAT as gold standard in this study. We achieved species identification by the amplification from the qPCR-positive samples ITS2 gene (Table1) and kinetoplast gene. This was conclusive for L. infantum. All positive dogs were apparently healthy. The level of parasite load found is not important (amount 29 parasite/ mL blood), which may explain an asymptomatic carriage stage. Cases of human visceral leishmaniasis were reported in sub-

15.4% (19/123) by IFAT. We have developed a qPCR test with a wide specificity (Leishmania spp.) capable of detecting the main known pathogens of Leishmania species such as L.

Saharan countries such as Côte d'Ivoire and Gambia

[START_REF] Travi | Canine visceral leishmaniasis : Diagnosis and management of the reservoir living among us[END_REF][START_REF] Paltrinieri | Laboratory tests for diagnosing and monitoring canine leishmaniasis[END_REF]

, but these reports remain anecdotal with no evidence of an autochthonous transmission cycle. Three human cases of visceral leishmaniasis were observed in a cohort of 528 patients in Abidjan (2001-2002)

[START_REF] Kouassi | Leishmaniose viscérale à Abidjan à propos de trois observations (French). Three cases of visceral leishmaniasis in Abidjan, Côte d'Ivoire[END_REF]

. The detection of L. infantum in local dogs (15.4%) provides evidence that this region is endemic for CanL and indicates the possible involvement of domestic dogs in the human infection. Classically, sand flies involved in the Leishmania transmission belong either to the Phlebotomus genus (Old World) or to the Lutzomyia genus (New World). The sand fly species involved in L. infantum transmission cycle in Côte d'Ivoire are still unknown and need further investigations. The results obtained in Senegal, with the molecular confirmation of L. infantum species, high seropositivity in 160 dogs (46.3%) and 133 humans (33.3%)

[START_REF] Faye | Canine visceral leishmaniasis caused by Leishmania infantum in Senegal: Risk of emergence in humans?[END_REF]

,

  may explain our results.Concerning Filariidae, 10.6% (13/123) of dogs were infected by at least one species. A. reconditum was the most detected by modified Knott's test and the triplex qPCR (13/123).

	Obtained sequences for 18S rRNA gene showed 99% identity with Acanthocheilonema viteae
	and Loa loa (DQ094171, XR_002251421). No sequence for the 18S rRNA gene of A.
	reconditum is available in GenBank. A. reconditum has a worldwide distribution [19][21]. In
	Liberia, 56 of 137 dogs (40.9%) and about 9.2% of Nigerian dogs were found positive for
	microfilaria of A. reconditum from morphological identification, 8.8% in South Africa and

  For the first time, we highlighted the presence of L. infantum, B. vogeli, A. reconditum, D. immitis and D. repens in this region.This last remains endemic for E. canis and T. congolense. Ivorian veterinarians should be informed of these results in order to be able to detect clinical cases of canine leishmaniasis and make diagnoses using laboratory tests. This epidemiological situation also requires the implementation of a national program to control vector-borne diseases, especially for zoonotic agent such Leishmania infantum, the agent of human visceral leishmaniasis. Location of the study: down Abidjan kennel dogs, up dogs from Kongouanou village Mixed infections found in dogs surveyed in the present study. The legends represent: Coinfection and its prevalence; number of dogs presented this coinfection; frequency of the coinfection according to the sum of coinfections (N= 17).

	Legends:
	Fig 1. (A.	Côte	d'Ivoire	map,
	https://commons.wikimedia.org/wiki/Atlas_of_C%C3%B4te_d%27Ivoire#/media/File:C%C3
	Fig. 2

Table 1

 1 Sequences of primers used for species detection and identification in this study

	620
	Targeted	Target	Name	Primers (5'-3') and probe	Tm	References
	microorganisms	gene
	Anaplasmataceae	23S	TtAna-F	TGACAGCGTACCTTTTGCAT	-	[4]
			TtAna-R	GTAACAGGTTCGGTCCTCCA
			TtAna-S*	FAM-CTTGGTTTCGGGTCTAATCC-TAMRA
			Ana23S-212f	GTTGAAAARACTGATGGTATGCA	55°C
			Ana23S-753	TGCAAAAGGTACGCTGTCAC
	Leishmania spp.	18S	Leish. F	GGTTTAGTGCGTCCGGTG	-	This study
			Leish. R	CGGCCCATAAGATCC CCAA
			Leish. P*	FAM-CGGCCGTAACGCCTTTTCAACTCA -TAMRA
			Leish. F1	CTGTGACTAAAGAAGCGTGAC	52°C This study
			Leish. R1	AGGCCGAATAGAAAAGATACGT
		kDNA	RV1	CTTTTCTGGTCCTCCGGGTAGG	-	[25]
			RV2	CCACCCGGCCCTATTTTACACCAA
			Probe. Leish*	FAM-TTTTCGCAGAACGCCCCTACCCGC-TAMRA
		ITS II	LGITSF2	GCATGCCATATTCTCAGTGTC	60°C [52]
			LGITSR2	GGCCAACGCGAAGTTGAATTC
		kDNA	RV1	CTTTTCTGGTCCTCCGGGTAGG	59°C [54]
			RV2	CCACCCGGCCCTATTTTACACCAA
	Trypanosoma spp.	5.8S	Tryp 5.8SF	CAACGTGTCGCGATGGATGA	-	This study
			Tryp 5.8SR	ATTCTGCAATTGATACCACTTATC
			Tryp 5.8S P*	FAM-GTTGAAGAACGCAGCAAAGGCGAT-TAMRA
		ITS I	ITS1-CF	CCGGAAGTTCACCGATATTG	58°C [51]
			ITS1-BR	TTGCTGCGTTCTTCAACGAA
	Piroplasmida	5.8S	5.8S-F5	AYYKTYAGCGRTGGATGTC		[53]
			5.8S-R	TCGCAGRAGTCTKCAAGTC
			5.8S-S*	FAM-TTYGCTGCGTCCTTCATCGTTGT-MGB
		18S	piro18S-F1	GCGAATGGCTCATTAIAACA	58°C
			piro18S-R4	TTTCAGMCTTGCGACCATACT
	Abbreviations: Tm: Annealing temperature; *: Probe.

621

Table 3

 3 Single and mixed infection of canine vector-borne pathogens

	626
	Infection	Kennel	dogs	n=95	Village	dogs	n=28	Total	N=123
		(Abidjan)			(Kongouanou)
		No. positive	%	No. positive	%	No. positive %
	Single infection	20		21.1	11		39.3		25.2
	Ehrlichia canis	10		10.5	2		7.1		9.8
	Leishmania infantum	7		7.4	4		14.3		8.9
	Trypanosoma congolense	2		2.1	0		0.0		1.6
	Acanthocheilonema reconditum	1		10.5	4		14.3		4.1
	Babesia vogeli	0		0.0	1		3.6		0.8
	Mixed infection	11		11.6	6		21.4		13.8
	E. canis + L. infantum	6		6.3	0		0.0		4.9
	E. canis + B. vogeli	1		1.1	0		0.0		0.8
	E. canis + A. reconditum	0		0.0	4		14.3		3.3
	L. infantum + T. congolense	2		2.1	0		0.0		1.6
	L. infantum + A. reconditum	0		0.0	2		7.1		1.6
	A. reconditum + D. immitis + D. repens	2		2.10	0		0.0		1.6
	Total positive	31		32.6	17		60.7		39
	Total negative	64		67.4	11		39.3		61

Acknowledgments

We are grateful to Emilien Drancourt, Michel Drancourt, José Gomez, Gaoussou Toure and the dog owners for their valuable help with the field work. We also thank Magali Casanova, Hamadi Karembe, Philippe Vincendeau and Marie Varloud for their help. This study was supported by the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection, the National Research Agency under the program « Investissements d'avenir », reference ANR-10-IAHU-03, the Région Provence-Alpes-Côte d'Azur and European funding FEDER PRIMI.

Abbreviations

Supporting Information

Table S1. Positive and negative control DNA used for sensitivity and specificity determination of designed oligonucleotides.

Conflict interests

The authors declare that they have no competing interests.