Xuwei Liu
email: xuwei.liu@inrae.fr

Catherine M G C Renard
email: catherine.renard@inrae.fr

Agnès Rolland-Sabaté
email: agnes.rolland-sabate@inrae.fr

Carine Le Bourvellec
email: carine.le-bourvellec@inrae.fr

Exploring interactions between pectins and procyanidins: Structure-function relationships

Keywords: polyphenol, condensed tannins, polysaccharide, homogalacturonan rhamnogalacturonan, methylation, association HPSEC-MALLS, High Performance Size-Exclusion Chromatography combined with Multi-Angle Laser Light Scattering, AIS, Alcohol Insoluble Solids, Isothermal Titration Calorimetry, ITC, [η] z, z-average intrinsic viscosity, R Hz, z-average hydrodynamic radius, R Hw, weight-average hydrodynamic radius, w, weight-average molar mass, ̅ , weight-average apparent molecular density

During fruit and vegetable processing, procyanidins interact with cell walls and form complexes, which further impact their potential health effects. Among cell wall polysaccharides, pectins have the highest affinity for procyanidins. Binding of two procyanidin fractions with twelve pectins of different linearity and size was investigated by ITC, UV-Visible spectrophotometry and HPSEC-MALLS. Pectins interacted preferentially with highly polymerized procyanidins except beet pectins probably because of steric hindrance due to abundant feruloylated arabinans. Linear pectins had higher affinity for procyanidins: this was verified both for comparison between botanical origins (kiwifruit > apple > beet pectins) and between extraction conditions. Debranched pectins, extracted at pH 2.0, had higher affinity and aggregation capacities with procyanidins than those extracted at other pHs. However, the factors affecting pectins of different origins seemed to be different. High molar mass, intrinsic viscosity and hydrodynamic radius contributed more to increased adsorption of procyanidins by apple and beet pectins. Highly linear kiwifruit pectins, with high homogalacturonan content and lower branching ratio bound preferentially to procyanidins. The enthalpy/entropy proportion of the interaction between kiwifruit pectins and procyanidins was higher than that of apple and beet pectins, which suggested more hydrogen bonding. Predominance of homogalacturonan regions and high degree of methylation thus appeared key structural features of pectins for high affinity for procyanidins, while high degree of branching was detrimental. These findings provide the structural foundation for selectivity of interactions in molecular-level.

Introduction

In plant-based food systems (such as fruits, vegetables, and grains), secondary metabolites (e.g., polyphenols) and macromolecules (e.g., proteins and polysaccharides) coexist in strictly compartmented parts of the plant cells and commonly come in contact with each other during processing, mastication and digestion (Le Bourvellec et al., 2019;Le Bourvellec & Renard, 2012).

Structure-function relationships of the main non-digestible biologically active components in plant-based functional foods, polysaccharides and polyphenols, may be regulated by their interactions [START_REF] Dobson | Impact of molecular interactions with phenolic compounds on food polysaccharides functionality[END_REF][START_REF] Kardum | Polyphenols and their interactions with other dietary compounds: implications for human health[END_REF].

Dietary polysaccharide-polyphenol interactions might affect the physical properties of polysaccharides, e.g., texture and stability, in food systems [START_REF] Jin | Study on the coupling progress of thermo-induced anthocyanins degradation and polysaccharides gelation[END_REF][START_REF] Li | Ferulic acid pretreatment alleviates the decrease in hardness of cooked Chinese radish (Raphanus sativus L. var. longipinnatus Bailey)[END_REF][START_REF] Liu | Interactions between cell wall polysaccharides and polyphenols: Effect of molecular internal structure[END_REF][START_REF] Tudorache | Phenolic compounds mediate aggregation of water-soluble polysaccharides and change their rheological properties: Effect of different phenolic compounds[END_REF][START_REF] Tudorache | Gallic acid reduces the viscosity and water binding capacity of soluble dietary fibers[END_REF] as well as their biological activity (Le Bourvellec et al., 2019). In addition, the bioaccessibility, bioavailability and bioefficacy of polyphenols depends on their interaction with other food ingredients, e.g., dietary fiber in particular (Ribas-Agustí, Martín-Belloso, [START_REF] Ribas-Agustí | Food processing strategies to enhance phenolic compounds bioaccessibility and bioavailability in plant-based foods[END_REF]. Such dietary fibers, e.g., apple matrix, apple cell wall (Le Bourvellec et al., 2019;[START_REF] Monfoulet | Effects of the apple matrix on the postprandial bioavailability of flavan-3-ols and nutrigenomic response of apple polyphenols in minipigs challenged with a high fat meal[END_REF] and pure cellulose [START_REF] Phan | Independent fermentation and metabolism of dietary polyphenols associated with a plant cell wall model[END_REF], can be used as a carrier for polyphenols to transport these antioxidants to the human gut microbiota and further produce beneficial physiological effects after their fermentation in small phenolic compounds [START_REF] Loo | Modulation of the human gut microbiota by phenolics and phenolic fiber-rich foods[END_REF][START_REF] Saura-Calixto | Dietary fiber as a carrier of dietary antioxidants: An essential physiological function[END_REF].

Plant polyphenols have at least one aromatic ring and one or more hydroxyl groups (phenol group), ranging from simple phenolic acids to complex flavonoids. Procyanidins, also known as condensed tannins, are a type of proanthocyanidins and in particular they have a high affinity for polysaccharides. They are polymers and oligomers of catechin/epicatechin (flavan-3-ol units) and most commonly connected by B-type bonds, namely C4-C8 or C4-C6 interflavanic linkages with a number average degree of polymerization ( ) varying between 2 and more than 100 [START_REF] Guyot | Thiolysis -HPLC characterization of apple procyanidins covering a large range of polymerization states[END_REF]. The interactions between macromolecules and procyanidins are dependent on their structural characteristics, i.e. molar mass, interflavanic bonds, the presence of galloyl groups and conformation (Le Bourvellec & Renard, 2019;[START_REF] Liu | Interactions between cell wall polysaccharides and polyphenols: Effect of molecular internal structure[END_REF]. In general, their affinity for polysaccharides increases with their and molar mass (Le [START_REF] Bourvellec | Non-covalent interaction between procyanidins and apple cell wall material: Part I. Effect of some environmental parameters[END_REF] Le [START_REF] Bourvellec | Non-covalent interaction between procyanidins and apple cell wall material. Part II: Quantification and impact of cell wall drying[END_REF], 2012;[START_REF] Mamet | Interactions between highly galloylated persimmon tannins and pectins[END_REF][START_REF] Renard | Interactions between apple cell walls and native palle polyphenols/quantification and some consequences[END_REF]. For example, procyanidins have been demonstrated to bind strongly to pectins, a major constituent of most fruit and vegetable plant cell walls (Le [START_REF] Bourvellec | Non-covalent interaction between procyanidins and apple cell wall material. Part II: Quantification and impact of cell wall drying[END_REF][START_REF] Liu | Interactions between cell wall polysaccharides and polyphenols: Effect of molecular internal structure[END_REF][START_REF] Renard | Interactions between apple cell walls and native palle polyphenols/quantification and some consequences[END_REF]. Increasing the level of galloylation of procyanidins also enhances their affinity with pectins due to the increased number of hydroxyl groups and aromatic rings (Le [START_REF] Bourvellec | Non-covalent interaction between procyanidins and apple cell wall material: Part I. Effect of some environmental parameters[END_REF][START_REF] Mcmanus | Polyphenol interactions. Part 1. Introduction; some observations on the reversible complexation of polyphenols with proteins and polysaccharides[END_REF][START_REF] Tang | Structure-Activity Relationships in the Hydrophobic Interactions of Polyphenols with Cellulose and Collagen[END_REF]. (+)-Catechin and polymers composed mainly of (+)-catechin units bind more to polysaccharides than (-)-epicatechin and polymer composed mainly of (-)-epicatechin due to the stereochemistry of flavan-3-ols pyran rings (Le [START_REF] Bourvellec | Non-covalent interaction between procyanidins and apple cell wall material: Part I. Effect of some environmental parameters[END_REF].

and are acid hetero-polysaccharides. The pectic polysaccharides contain three main structural units as follows. Homogalacturonan (HG) is a long and smooth chains of linear α-1,4-linked D-galacturonic acid, in which most of the C-6 carboxyl groups are methyl-esterified and some of the secondary alcohols at the O-2 and/or the O-3 positions may be acetyl-esterified [START_REF] Caffall | The structure, function, and biosynthesis of plant cell wall pectic polysaccharides[END_REF][START_REF] Mohnen | Pectin structure and biosynthesis[END_REF][START_REF] Ridley | Pectins: Structure, biosynthesis, and oligogalacturonide-related signaling[END_REF]. Rhamnogalacturonan-I (RG-I) is composed of a backbone of repeating units of galacturonic acid and rhamnose linked by α-1,2 and α-1,4

glycosidic bonds: [→4)-α-D-GalpA-(1→2)-α-L-Rhap-(1→] and may be substituted at the O-2 and/or the O-3 positions of α-L -rhamnose residues [START_REF] Mohnen | Pectin structure and biosynthesis[END_REF][START_REF] Voragen | Pectin, a versatile polysaccharide present in plant cell walls[END_REF]. Rhamnogalacturonan II (RG-II), making up ~10% of pectins, is the most structurally complex and branched polysaccharide in this family of pectic polysaccharides [START_REF] Caffall | The structure, function, and biosynthesis of plant cell wall pectic polysaccharides[END_REF][START_REF] Ridley | Pectins: Structure, biosynthesis, and oligogalacturonide-related signaling[END_REF]. The highly complex polymer structure of RG-II is composed of an HG backbone of seven to nine (and most probably more) 1,4-linked α-D-Gal-A residues including four side chains clearly consisting of 12 different kinds of monosaccharides through more than 20 different linkages [START_REF] Pérez | A complex plant cell wall polysaccharide: Rhamnogalacturonan II. A structure in quest of a function[END_REF]. Pectins with high degree of methylation (DM) display the strongest affinity for procyanidins due to hydrophobic interactions, while highly branched pectins have more limited interactions with procyanidins, probably due to steric hindrance [START_REF] Watrelot | Interactions between pectic compounds and procyanidins are influenced by methylation degree and chain length[END_REF], 2014). Pectins, especially HG, are susceptible to various enzymatic and non-enzymatic conversion reactions during processing of plant-based products, modifying their structure and, hence, their physicochemical properties [START_REF] Dongowski | Enzymatic degradation studies of pectin and cellulose from red beets[END_REF][START_REF] Fraeye | Influence of pectin properties and processing conditions on thermal pectin degradation[END_REF][START_REF] Renard | Pectins in mild alkaline conditions: β-elimination and kinetics of demethylation[END_REF], which in turn may affect their interaction with procyanidins (Le [START_REF] Bourvellec | Impact of processing on the noncovalent interactions between procyanidin and apple cell wall[END_REF].

Interactions of procyanidins has been studied with pectins of different commercial origins (apple or citrus) or with pectin structural units. Higher affinities were recorded for citrus pectins or for highly methylated homogalacturonans [START_REF] Watrelot | Interactions between pectic compounds and procyanidins are influenced by methylation degree and chain length[END_REF], while type I rhamnogalacturonans with different side-chains [START_REF] Watrelot | Neutral sugar side chains of pectins limit interactions with procyanidins[END_REF] or arabinans [START_REF] Fernandes | Interactions of arabinan-rich pectic polysaccharides with polyphenols[END_REF] had lower affinities. Also, different pectin components will interact in a complex manner determining the fine structure of the biopolymer (e.g., hydrogen, hydrophobic, or ionic bonding) and its spatial conformation [START_REF] Janaswamy | Polysaccharide structures from powder diffraction data: Molecular models of arabinan[END_REF][START_REF] Pérez | The three-dimensional structures of the pectic polysaccharides[END_REF] that will further affect their interaction with procyanidins [START_REF] Watrelot | Neutral sugar side chains of pectins limit interactions with procyanidins[END_REF]. However, there is little systematic information on the effect of native pectin's structural features (both composition and spatial conformation) on their interaction with procyanidins, particularly involving combinations of light scattering and spectroscopy techniques, and together with thermodynamics. Therefore, the determination of the impact of conformational properties and composition of pectins is crucial. To do this, pectins differing for their main structural features, i.e. molar mass, size, proportion of branching, length and content of HG, HG / RG ratio and degrees of methylation and acetylation, were used to gain in-depth understanding of the structure/function relationships that govern their interactions with procyanidins.

For this, a series of twelve pectins with different HG / RG ratios, side-chains and esterifications was prepared by extraction from apple, beet and kiwifruit (two maturities for kiwifruit) cell walls at pH 2.0, 3.5, and 6.0. These twelve different pectins were incubated with procyanidins solutions of intermediate and high , DP9 and DP79, respectively. Interactions were characterized by aggregates formation using UV-visible spectroscopy and by isothermal titration calorimetry. The pectin macromolecular characteristics before and after interactions were determined by size-exclusion chromatography coupled with multi-angle laser light scattering and viscometric detections to better understand the selectivity of their interactions with procyanidins. A deep understanding of the molecular mechanisms that drive the interaction between pectins and procyanidins can enable us to better bridge the gap between food processing and the bioavailability of commensal microbiota fermentation products of pectin and procyanidins. Further, this promotes the design of more rational processing conditions and healthier and more nutritious foods.

Materials and methods

Standards and Chemicals

Ethanol and acetone were provided from Fisher Scientific (Strasbourg, France).

Acetonitrile, methanol of HPLC grade were obtained from VWR International (Radnor, USA). Hexane was from Merck (Darmstadt, Germany). Sugar standards (arabinose, fucose, galactose, xylose, mannose and rhamnose) were from Fluka (Buchs, Switzerland). Methanol-d3 was from Acros Organics (Geel, Belgium). Formic acid, chlorogenic acid, benzyl mercaptan, sodium carbonate, sodium hydroxide, NaBH4, N-methylimidazole, acetic anhydride, toluene-α-thiol, (+)-catechin and (-)-epicatechin were from Sigma-Aldrich (Saint Quentin Fallavier, France).

4-Coumaric acid was obtained from Extrasynthese (Lyon, France). Phloridzin was obtained from Fluka (Buchs, Switzerland).

Extraction, purification and characterization of procyanidins

Plant material

Apple fruits (Malus × domestica Borkh.) from the 'Marie Menard' and 'Avrolles' cider cultivars were harvested at maturity (after starch regression) in the experimental orchard of the Institut Français des Productions Cidricoles (Se és, Orne, France). Fruits were mechanically cored and a formic acid solution (10 mL/L) was sprayed on the fresh material to avoid phenolic oxidation. Cortex tissues were then frozen, freeze-dried, and stored at -20 °C until used.

Procyanidin extraction and purification

Apple polyphenols were extracted from the freeze-dried apple powder (150 g) sequentially by hexane, methanol and aqueous acetone according to a procedure described by [START_REF] Guyot | Thiolysis -HPLC characterization of apple procyanidins covering a large range of polymerization states[END_REF]. Hexane and methanol extracts were discarded as they did not contain the required procyanidin fractions and to eliminate lipids, sugars, organic acids and phenolic compounds of low molar mass. Aqueous acetone extracts containing procyanidins were pooled and concentrated on a rotary evaporator prior to freeze-drying. The freeze-dried aqueous acetone extracts were dissolved (100 g/L) in water acidified with formic acid (99.9:0.1, v/v), centrifuged (16 800 x g, 15 min) and then filtered. They were injected on a 20×5 cm column of LiChrospher 100 RP-18 (12 μm, Merck, Darmstadt, Germany) and purified as described by [START_REF] Brahem | Pear ripeness and tissue type impact procyanidin-cell wall interactions[END_REF]. Procyanidin fractions were concentrated on a rotary evaporator then freeze-dried and stored under vacuum at -80 °C until used.

The purified procyanidin fractions are designated as DP9 (from 'Marie Me ńard') and DP79 (from 'Avrolles').

Procyanidin characterizations

Procyanidins were analyzed by high-performance liquid chromatography (HPLC)

with diode array detection (DAD) with or without thioacidolysis as described by [START_REF] Guyot | Thiolysis -HPLC characterization of apple procyanidins covering a large range of polymerization states[END_REF]. Analysis were performed using the ultra-fast liquid chromatography and controlled by LC Solution software (Shimadzu Prominence system, Kyoto, Japan). The system was operated by two LC-20AD pumps

Prominence LC UFLC, a DGU-20A5 Prominence degasser, a SIL-20ACHT

Prominence autosampler, a CTO-20AC Prominence column oven, an SPD-M20A

Prominence diode array detector and a CBM-20A Prominence communication bus module. Separations were achieved as described in Le [START_REF] Bourvellec | Phenolic and polysaccharidic composition of applesauce is close to that of apple flesh[END_REF]. The average degree of polymerization of procyanidins ( ) was calculated as the molar ratio of all flavan-3-ol units (thioether adduct plus terminal units minus (+)-catechin and (-)-epicatechin naturally present in the samples and determined by analysis of the samples without thiolysis) to (+)-catechin and (-)-epicatechin corresponding to the terminal units minus (+)-catechin and (-)-epicatechin naturally present in the samples and determined by analysis of the samples without thiolysis.

Preparation of pectin fractions

Pectins from apple (A-), beet (B-) and kiwifruits (K-) (ripe R-and overripe O-)

were prepared as described by [START_REF] Liu | Modification of apple, beet and kiwifruit cell walls by boiling in acid conditions: common and specific responses[END_REF]. Cell walls were isolated from the parenchyma of the different edible plant materials as alcohol insoluble solids. Subsequently, pectins were extracted from each cell wall material by boiling for 20 min in a citrate-phosphate solution (0.1 M) at three pH values: 2.0, 3.5 and 6.0. Thus, twelve pectin fractions were obtained. That is, apple, beet, kiwifruit (ripe) and kiwifruit (overripe) pectins extracted at pH 2.0/3.5/6.0 were designated AP2/3/6, BP2/3/6, KPR2/3/6 and KPO2/3/6, respectively. The purpose of this step is to obtain pectins of different compositions and structures.

Initial and free pectin macromolecular characteristics

The pectins (2.5 g/L) were analyzed by High Performance Size-Exclusion Chromatography coupled with Multi-Angle Laser Light Scattering (HPSEC-MALLS) after being filtered as described by [START_REF] Liu | Modification of apple, beet and kiwifruit cell walls by boiling in acid conditions: common and specific responses[END_REF]. Samples (100 μL) were injected and the mobile phase was 0.1 M citrate/phosphate buffer with pH 3.8, and eluted at 0.6 mL/min. The system comprised three HPSEC columns (PolySep-GFC-P3000, P5000 and P6000, 300 ×7.8 mm) and a guard column from Phenomenex (Le Pecq,

France) maintained at 40 °C, a MALLS detector (DAWN HELEOS 8+ fitted with a K5 flow cell and a GaAs laser (λ = 660 nm), a Viscostar III viscometer, both from Wyatt Technology Corporation (Santa Barbara, CA, USA), a diode array detector (SPD-M20A) and a fluorescence detector (RF-20A) set at 360 nm (280 nm excitation) and a refractive index detector (RID-10A) from Shimadzu (Shimadzu Prominence system, Kyoto, Japan).

Mi and RGi, the molar mass and the radius of gyration at each slice of the chromatogram, was determined using the concentration (calculated from the refractometric signal) and the light scattering signal from 5 angles (from 20.4° to 90°)

and data extrapolation to zero angle using the Zimm formalism with a one order polynomial fit (Rolland-Sabaté, Colonna, Potocki-Véronèse, Monsan, & Planchot, 2004) using ASTRA® software from Wyatt Technology Corporation (version 7.1.4 for PC). Rhi, the viscometric hydrodynamic radius at each slice of the chromatogram for the equivalent sphere, was calculated by combining viscosity and molar mass measurements using the following equation derived from the Einstein and Simha relation [START_REF] O;2-C Einstein | Eine neue Bestimmung der Moleküldimensionen[END_REF][START_REF] Einstein | Berichtigung zu meiner Arbeit: Eine neue Bestimmung der Moleküldimensionen[END_REF][START_REF] Simha | The Influence of Brownian Movement on the Viscosity of Solutions[END_REF]:

[η] M = γN V = N R (1)
where [η] and Vhi are the intrinsic viscosity and the hydrodynamic volume at each slice of the chromatogram, γ = 2.5 for spheres and NA the Avogadro number.

The z-average intrinsic viscosity ([η] z), z-average and weight average viscometric hydrodynamic radii ( R Hz and R Hw) and weight-average molar mass ( w) were established using the averaging described in Rolland-Sabaté et al. [START_REF] Rolland-Sabaté | Elongation and insolubilisation of α-glucans by the action of Neisseria polysaccharea amylosucrase[END_REF][START_REF] Rolland-Sabaté | Online determination of structural properties and observation of deviations from power law behavior[END_REF] on the whole peaks. A value of 0.146 mL/g was used as the refractive index increment (dn/dc) for glycans and the normalization of photodiodes was achieved using a low molar mass pullulan standard (P20) from Showa Denko K.K. (Tokyo, Japan). The average apparent molecular density ( ̅ ) was calculated using the following equation:

̅ = (4 3) * R ⁄ ⁄ (2)
The log-log plot of hydrodynamic radius versus the molar mass and the Mark-Houwink-Sakurada plot were established for each sample by using the data taken at each slice of the chromatogram. The power law exponent (α) can be calculated according to the following equations:

[η] = K & M ' (3)
where Ka is a constant and α is the hydrodynamic coefficient which depends on the polymer shape in the solvent.

Phase Diagram

The formation of aggregates was analyzed by spectrophotometry during the pectin-procyanidin interactions as described by [START_REF] Watrelot | Interactions between pectic compounds and procyanidins are influenced by methylation degree and chain length[END_REF]. All measurements were done in duplicates. The turbidity measurements were carried out with a SAFAS flx-Xenius XM spectrofluorimeter (SAFAS, Monaco) at 650 nm on a 96-well microplate at 25 °C. A serial procyanidin solutions (0, 0.03, 0.06, 0.12, 0.24, 0.47, 0.94, 1.875, 3.75, 7.5, 15 and 30 mmol/L (-)-epicatechin equivalent for 'Avrolles'; 0, 0.06, 0.12, 0.24, 0.46, 0.94, 1.875, 3.75, 7.5, 15, 30 Scattering (HPSEC-MALLS). (ΔMw = S2-S1A) and (ΔDPn = S2-S1B) were used to define qualitative changes in weight-average molar mass of pectins and in number average degree of polymerization of procyanidins, respectively.

Isothermal Titration Calorimetry (ITC)

The entropy and enthalpy changes caused by the interactions between procyanidins and pectins were determined by ITC, using TAM III microcalorimeter (TA instruments, New Castle, USA). Purified procyanidins (60 mmol/L in (-)-epicatechin equivalent) and pectins (7.5 mmol/L galacturonic acid equivalent)

were dissolved in the same citrate/phosphate buffer pH 3.8, 0.1 M ionic strength. The reference cell was filled by water. All solutions were degassed prior to measurements.

The pectin solution was placed in the 850 mL sample cell of the calorimeter and the Experiments were carried out in duplicates.

Statistical analysis

Results were expressed as mean values, and their reproducibility was presented as the pooled standard deviation (Pooled SD). For each series of repeated samples, the pooled SDs were calculated using the sum of their respective variances multiplied by their respective degrees of freedom [START_REF] Box | Statistics for Experimenters, an Introduction to Design, Data Analysis and Model Building[END_REF]. Principal

Component Analysis (PCA) was realized using the functions of the library FactoMineR and Factoextra in R statistical software (R Core Team., 2014). Heatmap was performed with Python 3.5 software using the Seaborn package [START_REF] Waskom | Seaborn: Statistical Data Visualization[END_REF].

Results

Structure and composition of fractions

Procyanidins

The two apple varieties 'Marie Ménard' and 'Avrolles' were chosen to obtain two purified fractions of intermediate and high degree of polymerization, respectively (Le [START_REF] Bourvellec | Interactions between apple (Malus x domestica Borkh.) polyphenols and cell walls modulate the extractability of polysaccharides[END_REF]. The composition of isolated apple phenolic fractions is shown in Table 1. The purified extracts from 'Marie Ménard' and 'Avrolles' contained ca. 700 mg/g of polyphenols, mainly procyanidins plus traces of flavan-3-ol monomer i.e. (-)-epicatechin, hydroxycinnamic acids, i.e.

5′-caffeoylquinic acid and p-coumaroylquinic acid, dihydrochalcones, i.e. phloretin xyloglucoside and phloridzine, and a mix of flavonols. 'Marie Ménard' and 'Avrolles' procyanidins were characterized by = 9 and 79, respectively. Both were constituted by more than 98 % (-)-epicatechin units and contained a homologous structure differing by their degree of polymerization. All the results were consistent with Le Bourvellec et al. ( 2004) and (2012).

Pectins

Detailed compositions of the pectins are available in [START_REF] Liu | Modification of apple, beet and kiwifruit cell walls by boiling in acid conditions: common and specific responses[END_REF] and the sugar ratios based on the sugar content and macromolecular characteristic for pectins are calculated in Table 1. HPSEC chromatograms and molar mass distributions of the pectins are presented in Fig. 1. Structural diversity was obtained on two major parameters, namely (i) the nature and composition of side-chains and (ii) macromolecular characteristics, which varied independently in this series of pectins (as described by principal component analysis loading and sample plots in Supplementary Fig. 1). Concerning the neutral sugar side chain abundance and composition, apple pectins were characterized by high xylose, signaling presence of xylogalacturonans [START_REF] Schols | A xylogalacturonan subunit present in the modified hairy regions of apple pectin[END_REF]. Only beet pectins contained detectable ferulic acid, and they had the highest content of neutral sugars, notably arabinose, and highest degree of acetylation. Kiwifruit pectins appeared to be the richest in homogalacturonans, with the lowest arabinose content and arabinose to galactose ratio. All these characteristics were further modulated by the pH used for pectin solubilization, with pectins solubilized at pH 2.0 displaying the most extreme characteristics for their respective origins. Unsaturated double bonds resulting from β-elimination have been detected in AP6, BP6, KPR6 and KPO6 [START_REF] Liu | Modification of apple, beet and kiwifruit cell walls by boiling in acid conditions: common and specific responses[END_REF], leading to reduction of the HG chain length and its potential binding sites after pH 6.0 treatment.

Further information on pectin conformations in solution, calculated by plotting the molar mass versus the intrinsic viscosity obtained with HPSEC-MALLS, is given in Table 2. Two regions were used to determine the Mark-Houwink-Sakurada conformation parameters at different peaks. In the main peak, the exponent α varied between 0.96 and 1.32 indicating an organization close to stiff coils in a good solvent [START_REF] Flory | Molecular configuration of polyelectrolytes[END_REF], with various chain flexibility in agreement with literature data [START_REF] Fishman | Solvent effects on the molecular properties of pectins[END_REF], excepted for AP2 (α = 0.55) which exhibited a value corresponding to random coil conformation in a θ solvent. Most pectins (except AP2) also presented a less important fraction (generally shoulder in the chromatogram from 20 -23 mL, Fig. 1) exhibiting a spheroidal or denser conformation (Table 2) (α between 0 and 0.41) that could correspond to branched aggregates or more folded conformation [START_REF] Alba | Pectin Conformation in Solution[END_REF][START_REF] Lopez-Torrez | Acacia senegal vs. Acacia seyal gums -Part 1: Composition andstructure of hyperbranched plant exudates[END_REF]. This minor fraction represented a higher proportion in AP6 and KPRs (Fig. 1) and seemed to correspond to the main peak in AP2. The lower values of the exponent α (0.55) thus obtained for the main peak in AP2 could be due to a more folded molecule, and this intermediate value (between the sphere and the rod) was most probably caused by the presence of two populations under the main peak, which produced artificially one lower exponent instead of two exponents. Meanwhile, the population of these peaks will undergo some modifications after interaction with procyanidins. Detailed information will be given in the interaction section.

Good diversities and variabilities were obtained in this sample set for pectin linearity, length of side chains, arabinans / galactans ratio, degree of acetylation, molar mass and conformation. No pectins with a low degree of methylation (DM < 30)

were present. This should not affect the investigation of the subsequent interactions as it has been proved that the affinity of low methylated pectins to procyanidins is very low [START_REF] Watrelot | Interactions between pectic compounds and procyanidins are influenced by methylation degree and chain length[END_REF].

Interactions with procyanidins of DP9

Isothermal titration calorimetry

Thermodynamic parameters from ITC titration of pectins by procyanidins DP9 are shown in Table 3A. Typical thermograms were obtained for AP2/3, BP2, KPR2/3/6 and KPO2 (7.5 mM galacturonic acid equivalent) titrated by procyanidin DP9 (60 mM (-)-epicatechin equivalent) with strong exothermic peaks (data not shown). In contrast, no titration could be observed for AP6, BP3/6 and KPO3/6 by procyanidin DP9. Stoichiometry (defined as ratio of (-)-epicatechin/galacturonic acid) was ca. 0.1 for AP2/3, BP2 and KPO2 (1 molecule of (-)-epicatechin bound 10 units of galacturonic acid) and ca. 0.14 for KPR2 (1 molecule of (-)-epicatechin bound 7 units of galacturonic acid) using a one-site model.

The association constant ranged between 2.0 × 10 3 M -1 and 1.2 × 10 4 M -1 and increased in the following order: AP3 ≈ BP2 < KPR3 ≈ KPR6 ≈AP2 << KPR2 <<< KPO2 (Table 3A). KPO2 with the highest Gal A/Rha ratio (91) had the highest affinity for procyanidin DP9, showing a strong positive impact of pectin linearity on their ability to interact with procyanidins. Analysis of the thermodynamic contributions (ΔG = ΔH -TΔS) related to the exothermic reactions indicated a strong entropy contribution (-TΔS from -18 to -14 kJ/mol) showing that the interactions were mostly driven by entropy, i.e. by hydrophobic interactions and the release of water molecules (Le Bourvellec & Renard, 2012;[START_REF] Poncet-Legrand | Interactions between flavan-3-ols and poly(L-proline) studied by isothermal titration calorimetry: Effect of the tannin structure[END_REF][START_REF] Watrelot | Interactions between pectic compounds and procyanidins are influenced by methylation degree and chain length[END_REF][START_REF] Watrelot | Neutral sugar side chains of pectins limit interactions with procyanidins[END_REF]. The enthalpy contributions were very limited for AP3, AP2 and KPR3 (ΔH from -1 to -4 kJ/mol) and higher for AP2, KPR2/6 and KPO2 (ΔH from -6 to -5 kJ/mol) indicating that interactions also involved hydrogen bonds. The proportion of enthalpy for KPR/Os was significantly higher than that of AP3 and BP2 (Table 3A) due to hydrogen bonds which increased their affinity for procyanidin DP9.

Phase diagram

Fig. 2A, B present the heat-map of turbidity at 650 nm. Turbidity of all pectin mixtures with procyanidins DP9 rose with increasing pectin concentrations (Fig. 2A).

This increase was more marked at 30 mM galacturonic acid equivalent for KPR2 and KPO2, with absorbance values of 1.03 and 0.92, respectively, than for other pectins (absorbance from 0.21 to 0.48) at the same concentration. These results were consistent with the results of ITC. The interactions between highly linear pectins and procyanidin DP9 led to formation of aggregates with marked turbidity. For APs, BP3/6, KPR2/3 and KPO2/3, the absorbances at 650 nm increased slightly at a concentration of 30 mM galacturonic acid equivalent. After 3.75 mM pectin concentration, the absorbance of BP2 stabilized around at 0.2. However, AP6, BP3/6 and KPO3/6 also showed increased turbidity while no interactions had been detected

by ITC. This result may indicate that the resulting released or absorbed heat during the interaction stayed below the limit of detection of the nanocalorimetric method.

Absorbance of all pectins at 30 mM (-)-galacturonic acids equivalent rose with increased procyanidins DP9 concentrations (Fig. 2B), which was consistent with the trend of Fig. 2A.

Characterization of unbound pectin and procyanidins

The free pectins and procyanidins remaining in the wells after interactions with 21 30 mM galacturonic acid and 60 mM (-)-epicatechin equivalents were investigated to further understand the impact of the interactions on their macromolecular structure (Fig. 1, Table 4). Fig. 1 showed the chromatograms obtained by HPSEC-MALLS of the initial pectic compounds in buffer (S1A) and the free pectin compounds (at 30 mM in galacturonic acid equivalent) after binding with procyanidins (S2, i.e. pectic compounds that had not formed aggregates). Table 4 showed the corresponding pectin macromolecular characteristics.

After interaction, only 2% -5% of pectins remained in the supernatant. More pectin remained free for AP6, BP6 and KPO/R6 than for AP2/3, BP2/3 and KPO/R2/3, respectively (Table 4). On the one hand, the w of free pectins after binding with procyanidins was lower than the w of initial pectin sample (Table 4). Procyanidin DP9 may have strong selectivity for the bigger molecules of pectin fractions, especially AP2 (ΔMw: -266), KPR2 (ΔMw: -216) and KPR3 (ΔMw: -223). On the other hand, the of free procyanidins after interaction with pectins was lower than the of initial procyanidin sample, whatever pHs and species (Table 4), confirming that pectins preferentially interacted with highly polymerized procyanidins.

In comparison with the initial pectin solutions, the main peaks of free APs, BPs, KPRs and KPOs in the solutions slightly shifted to higher elution volumes indicating a decrease in hydrodynamic volume (Fig. 1). Moreover, the shoulder at a lower elution time (corresponding to the high molar mass fraction) in the main peak of AP6 and KPR/Os decreased after interaction due to complexation with procyanidins. The molecules that remained in supernatants after pectin-procyanidin interactions were smaller procyanidins DP9 and smaller pectins. The large-sized pectins and the larger procyanidins were co-aggregated, or the aggregates contained procyanidins of higher and pectins with larger hydrodynamic volume. Nevertheless, it was challenging to use the α value obtained from Mark-Houwink-Sakurada equation to determine which pectin conformation was more conducive to interaction as this value did not show clear trend probably because it only provided some general structural information on these pectins (Table 2).

Interactions with procyanidins of DP79

Isothermal titration calorimetry

Titration of all pectins by procyanidins DP79 showed complex curves characterized by strong exothermic peaks. Thermodynamic parameters are shown in Table 3B. Stoichiometry (n) results, suggested that approximately 1 (-)-epicatechin constitutive unit bound to 5 units of galacturonic acid for high linearity KPO2. For AP3, KPR3/6 and KPO3/6 with medium linearity, approximately 1 (-)-epicatechin constitutive unit bound to 10 units of galacturonic acid. While, the stoichiometry for AP2 with high molar mass and size, and BP2/3 with high arabinans side chains were ca. 0.5.

The association constant Ka between pectins and procyanidins DP79 ranged from 0.3 × 10 2 M -1 to 1.2 × 10 4 M -1 in the order BP2 ≈ BP3 ≈ BP6 <<KPR3 <KPR6 <AP6 <AP3 <<AP2 <KPO3 < KPO6 << KPR2 < KPO2. In particular, the BP2, AP2, KPR2 and KPO2 pectins were the most different from our sample set (Supplementary Fig. 1) and cover a wide range of affinity. Regarding the interactions between all pectins and procyanidins DP79, contribution of entropy (-TΔS from -12 to -19 kJ/mol) related to the exothermic reactions, indicated that the interactions were mostly driven by entropy indicating hydrophobic interactions and water released. Moreover, an enthalpy contribution especially for KPR2 (ΔH = -6.0 kJ/mol) and KPO2 (ΔH = -8.0 kJ/mol) was also observed indicating that hydrogen bonds were also involved.

Phase diagram

More precipitation was obtained with procyanidins of DP79 (Fig 3 . A,B) than of DP 9, regardless of pectin type. Turbidity increased dramatically with the increase of concentration of galacturonic acid and procyanidins for AP2, BP2, KPR2 and KPO2, indicating strong interactions, which confirmed the result of ITC. The turbidity for AP2, BP2, KPR2 and KPO2 with procyanidins DP79 at 15 mM galacturonic acid equivalent or 30 mM (-)-epicatechin equivalent increased in the following the order:

KPO6 < KPR6 <AP6 < KPR3 ≈ BP2 < AP2 < BP3<BP6 <KPO3<KPR2 <AP3 <KPO2.

Characterization of unbound pectin and procyanidins

After interactions between pectins (30 mM galacturonic acid equivalent) and procyanidin DP79 (60 mM epicatechin equivalent), 4% -11% of the pectins remained in the supernatants (Table 4). Similarly, more pectins remained in solution for AP6, BP6 and KPO/R6 than for AP2/3, BP2/3 and KPO/R2/3, respectively. w of all pectins and of procyanidin DP79 obviously decreased after interactions (Table 4). The ΔMw obtained between APs, BPs, KPR/Os and procyanidins DP79 were higher than the ones obtained with procyanidin DP9, especially for AP2. Procyanidin DP79 gave a higher ΔDPn for all pectins ranging from -56 to -38. This high decrease of the degree of polymerization and molar mass indicated that highly polymerized procyanidins of fraction DP79 associated selectively with pectins of higher molar mass. HPSEC-MALLS chromatograms of the pectins also support this conclusion.

Like with procyanidin DP9, the main population of free APs, BPs, KPRs and KPOs was slightly shifted to lower sizes after complexation/aggregation and the largest population disappeared (Fig. 3). This suggested that the large-sized pectin aggregates from the largest population preferentially interact with large-sized procyanidins DP79 and produce precipitates [START_REF] Alba | Pectin Conformation in Solution[END_REF][START_REF] Carn | Structural properties of colloidal complexes between condensed tannins and polysaccharide hyaluronan[END_REF]. However, this specificity can not be distinguished in different conformations of pectins, because α values of large populations are all in the range of 0 -0.41 representing large-sized folded structures or branched aggregates did not show clear trend (Table 2).

Discussion

Comparison of calorimetry and turbidity methods

Using both isothermal titration calorimetry and turbidity, pectins with different linearity (or side-chain abundance) and macromolecular characteristics were shown to interact with procyanidins (DP9 and DP79). These interactions were mainly driven by entropy, which may be caused by hydrophobic interactions, or changes in solvation and conformation [START_REF] Leavitt | Direct measurement of protein binding energetics by isothermal titration calorimetry[END_REF][START_REF] Poncet-Legrand | Interactions between flavan-3-ols and poly(L-proline) studied by isothermal titration calorimetry: Effect of the tannin structure[END_REF]. Enthalpy contributions were also observed indicating that interactions also involved some hydrogen bonds. Haze formation was observed for all pectins with procyanidins DP9, while for some pectin fractions, AP6, BP3/6, and KPO3/6, the interactions were below the detection limit for nano calorimetry. The reason may be that the ITC signal generated by these pectin-procyanidin interactions (exothermic) is masked by the concurrent endothermic signal from chain-chain interaction during pectin aggregation.

In addition, the pectins for which no titration could be detected were generally characterized by higher arabinose contents: although procyanidins do interact with arabinans [START_REF] Fernandes | Interactions of arabinan-rich pectic polysaccharides with polyphenols[END_REF], this polymer appeared to lead to less intense binding. This was not observed with procyanidins DP79, which had higher energies of interactions and generally higher affinities than DP9. Turbidity appeared to be more sensitive than ITC for detection of interactions. Haze formation is a complex process which involves the modification of intra-molecular and molecule-solvent interactions.

A limit to measuring turbidity however is that it must be observed under static conditions, and the lack of stirring during the complete measurement may result in uneven cloudiness / aggregate formation [START_REF] Watrelot | Comparison of microcalorimetry and haze formation to quantify the association of B-type procyanidins to poly-l-proline and bovine serum albumin[END_REF].

Moreover, procyanidins, and notably the larger procyanidins, can auto-aggregate [START_REF] Carn | Structural properties of colloidal complexes between condensed tannins and polysaccharide hyaluronan[END_REF] and their sedimentation increased with . The turbidity measurement provided information on the formation of insoluble complexes, but it cannot provide information on the mechanism and binding sites. These two methods are complementary, allowing higher sensitivity for detection of the interactions (haze formation) on the one hand and access to stoichiometric ratio and binding enthalpy side-chains may also have contributed to the higher affinity of KPR/Os for procyanidins DP79. Interactions between BPs and procyanidin DP79 showed association constants of the order of 10 2 M -1 . The differences in affinity constants between the different pH fractions were very limited, ranging between 0.3 × 10 2 M -1 (BP2) and 0.4 × 10 2 M -1 (BP3/6). The low affinity performance of BPs may be not only due to their complex arabinan side chain structures, but also to the presence of ferulic acid covalently linked to arabinans, and to their acetylation. Ferulic acid cross-linking, by further rigidifying the arabinan side-chains, might lower interactions with procyanidins due to steric hindrance. This is consistent with Fernandes et al.

(2020) who reported that the branched arabinan side chains in pectin limit their interactions with polyphenols.

Impact of pectins molar mass on interactions

AP2, with higher molar mass, intrinsic viscosity, hydrodynamic radius and lower density, had higher affinity for procyanidin DP9/79 than AP3 and AP6; this was also true when comparing KPR2 or KPO2 to KPR3/6 and KPO3/6. This was the same relation as with corn silk polysaccharides, for which the binding capacity to flavonoids increases with molar mass [START_REF] Guo | Studies on the binding characteristics of three polysaccharides with different molecular weight and flavonoids from corn silk (Maydis stigma)[END_REF]). However, this relationship only appeared true within a series with otherwise similar structural features, as KPR2 and KPO2 had lower molar masses but higher affinities than AP2.

This suggests that increasing the molar mass of pectins alone may not increase their adsorption capacity. The linear structure of pectins was more important than their molar mass for binding to procyanidins.

Higher molar mass pectins have more glycosidic bonds, therefore more potential binding sites, which contribute to the adsorption of polyphenols. Moreover, a larger hydrodynamic radius and lower density may also mean that there was more space for procyanidins to interact with the polysaccharide molecule. Meanwhile, a larger space for bending and turning, a larger effective volume, a larger flow resistance, and a higher frequency of collisions between segments produce high intrinsic viscosity [START_REF] Bemiller | Carbohydrates[END_REF]. Pectins with higher intrinsic viscosity, that is existing as expanded chains in solution, might better interact with procyanidins (Fig. 4A). In addition, most carboxyl groups in pectins are ionized when the pH is higher than their pKa, which was the case in the experiments (pH 3.8 > pKa 3.5). This ionization leads to coulomb repulsion between the consecutive free carboxyls on a pectin molecule, increasing its stiffness [START_REF] Stoddart | Solution properties of polygalacturonic acid[END_REF], and preventing spatial proximity between segments of two pectin molecules (Fig. 4B). This causes the chain to stretch, which may result in more space to adsorption.

Substitution of the galacturonic acids

The DM of pectin has already been demonstrated to be a very important factor for interactions. However, all the pectins used here had high degrees of methylation (> 70), allowing strong interactions with procyanidins [START_REF] Watrelot | Interactions between pectic compounds and procyanidins are influenced by methylation degree and chain length[END_REF]. Although KPR/Os were less methylated than APs and BPs, they still had a high DM (> 73), which did not limit their high affinity for procyanidins. Moreover, the intensity of the interaction seems to decrease with increasing acetylation. KPR/Os have the lowest degree of acetylation with a high affinity for procyanidins, while BPs have the highest degree of acetylation with the lowest affinity. However, a simple comparison may be misleading as there are confounding factors which should be considered, e.g., pectin linearity.

We noted that the overall enthalpy of interactions with KPR/Os were significantly higher than with APs and BPs, especially KPR/O2 (Table 3), suggesting more hydrogen bonds. Most previous studies focused on commercial pectin (usually not acetylated and sometimes demethylated), purified HG or RG. [START_REF] Watrelot | Interactions between pectic compounds and procyanidins are influenced by methylation degree and chain length[END_REF] reported that interactions between commercial apple and citrus pectins and procyanidins is also mostly driven by entropy. However, the interactions between rhamnogalacturonans and hairy regions of pectins and procyanidins could be driven by either enthalpy or entropy or both [START_REF] Watrelot | Neutral sugar side chains of pectins limit interactions with procyanidins[END_REF]. Similarly, the interactions between apple pectins extracted at pH 3.8 (from 'Ariane' cultivar) and procyanidins (DP9) are entropy-driven (Le Bourvellec et al., 2012). Most pectin-procyanidin interactions are driven by both entropy and enthalpy, but their proportions may be regulated by the degree of esterification of pectins.

Degree of polymerization of procyanidins

As expected, the interactions were higher with the procyanidins of the highest degree of polymerization. With procyanidins DP79, marked aggregation appeared for all pectin fractions (absorbance > 0.9) at the maximum concentration of both, while with procyanidins DP9 formation of cloud or aggregate were relatively lower.

However, two trends were observed for ITC results. APs, BP3/6, KPR2 and KPRs had a higher affinity with DP79 than DP9 by ITC, but BP2 and KPR3/6 had a lower affinity with DP79 than DP9, especially BP2, the affinity of which was lower by one order of magnitude. This may be due to the conformational constraints of the two molecules that limit their interactions with each other. BP2 had low pectin linearity and HG content, together with the highest neutral sugar side-chains content of the twelve pectins, especially arabinans, and high ferulic acid content. On the one hand, these structures might cross-link the chains and limit the available binding sites for complexation with procyanidins. On the other hand, procyanidins DP79 may exhibit longer length and larger size, and are less uniform than procyanidins DP30 and DP9.

Using the Fisher-Burford model to fit the SAXS spectrum, [START_REF] Vernhet | Condensed tannin changes induced by autoxidation: Effect of the initial degree of polymerization and concentration[END_REF] reported that the apple proanthocyanidins of DP69 and DP80

have a more dense shape with branches. [START_REF] Zanchi | Rigidity, conformation, and solvation of native and oxidized tannin macromolecules in water-ethanol solution[END_REF] described polymeric proanthocyanidins containing about 6% ramifications. Therefore, high DP procyanidins may induce intramolecular hydrophobic interactions and hydrogen bonds resulting in more aggregated and less extended structure. [START_REF] Watrelot | Interactions between pectic compounds and procyanidins are influenced by methylation degree and chain length[END_REF][START_REF] Watrelot | Neutral sugar side chains of pectins limit interactions with procyanidins[END_REF] showed that pectins interact preferentially with highly polymerized procyanidins DP30, and that structure and size of procyanidins DP30 facilitates aggregates formation. Using larger procyanidins facilitated detection of the selectivity of various pectins, DP79 being less favorable with some specific structures due to its conformation.

Each polysaccharide has its own unique chemical composition, structure, molecular architecture or conformation, and these factors interact with each other to influence their adsorption to procyanidins. The interaction between these factors remains complex, and more work will be needed to clarify their internal relationship.

Conclusions

Procyanidins showed different binding selectivity to apple, beet and kiwifruit pectins depending on the compositions and macromolecular features of the pectins and degree of polymerization of procyanidins. High molar mass and low density contributed to procyanidin adsorption. Pectins with high linearity and HG content, and low arabinan branching had highest interaction with procyanidins. On the contrary, high RG-I branching and ferulic acid content limited the affinity to procyanidins. Higher number average DP of procyanidins might hinder the adsorption of pectins with high side chain branching. The importance of factors affecting pectin selectivity were linearity (proportion of side-chains) > molar mass > density ≈ hydrodynamic radius, with high branching and density being detrimental to interaction while high molar mass was favorable. Combining different factors, capacity of association between procyanidin DP79 and pectins could be ranked: KPO2 > KPR2 > AP2 > BP2 (Fig. 5).

Consequently, a deep understanding of various processing-structures-binding capacity of pectins to procyanidins aids food workers customize the functional characteristics of plant-derived products and provide effective guidance for processing.

Systematic variation of pectin structural features thus allows to better understand polyphenol affinity and may pave the way to anticipate the variability of retention of polyphenols in different fruits and vegetables. (-)-epicatechin units, EC: (-)-epicatechin as flavan-3-ol monomer, DHC: dihydrochalcones, CQA: 5′-caffeoylquinic acid, PCQ, p-coumaroylquinic acid, FLV: flavonols.

Ratios are calculated using the yields of neutral sugar expressed in mol%. The ratio between different sugars can contribute to understand information on polymer levels. The ratios of Gal A / Rha, (Ara+Gal) / Rha and Ara / Gal are characteristic for pectin backbone homogalacturonan / rhamnogalacturonan contribution, the branching of RG and the proportion of arabinans / galactans, respectively. Xyl and FA are indicators for the presence of xylogalacturonans and ferulic acids, respectively. DM and DAc are the degree of methylation and acetylation, respectively. w, weight-average molar mass.

[η] z, z-average intrinsic viscosity. R Hz, z-average hydrodynamic radius.

̅ = (4 3) * R ⁄ ⁄
, average apparent molecular density. AP: pectins from apple cell wall, BP: pectins from beet cell wall, KP: pectins from kiwifruit cell wall, Gal A: galacturonic acid, Rha: rhamnose, Ara: arabinose, Gal: galactose, pH values-: 2: pH 2.0, 3: pH 3.5, 6: pH 6.0. Maturity-: R: -Ripe, O: -Overripe. * data adapted from [START_REF] Liu | Modification of apple, beet and kiwifruit cell walls by boiling in acid conditions: common and specific responses[END_REF]. Pooled SD: pooled standard deviation. normalized chain concentration of free pectins after interaction with DP79. Orange thick solid line : molar mass of pectins before interaction; Green thick solid line : molar mass of free pectins after interaction with DP9; Blue thick solid line : molar mass of free pectins after interaction with DP79.

Samples

  procyanidin solution was loaded into the injection syringe and titrated into the sample cell by 50 injections of 5 μL. Each injection lasted 5 s, with separating delay of 20 min. The content of the sample cell was stirred throughout the experiment at 90 rev/min. The raw ITC data as a plot of heat flow (microjoules per second) against time (minutes) were then integrated peak-by peak and normalized to obtain a plot of observed enthalpy change per mole of injectant (ΔH, kJ/mol) against the molar ratio (epicatechin/galacturonic acid). Peak integration was performed and the experimental data were fitted to a theoretical titration curve using the instrument software (NanoAnalyze 3.10.0). Control experiments include the titration of procyanidin fractions into buffer and are subtracted from titration experiments. The thermodynamic parameters including binding stoichiometry (n), binding constant (Ka), enthalpy (ΔH) and entropy (ΔS), the 'S' shape curve as adjustable parameters.

Fig. 1 .

 1 Fig. 1. HPSEC-MALLS chromatograms and molar mass vs elution volume of the pectin samples. A, B and C: AP2, AP3 and AP6, respectively; D, E and F: BP2, BP3 and BP6, respectively; G, H and I: KPR2, KPR3 and KPR6, respectively; J, K and L: KPO2, KPO3 and KPO6, respectively. Thin orange solid line : normalized chain concentration of pectins before interaction; Thin green solid line : normalized chain concentration of free pectins after interaction with DP9; Thin blue solid line :

  898

Fig. 4 .

 4 Fig. 4. (A) High and low intrinsic viscosity pectin chains, (B) Conformations of pectin chain extended (pH > pKa) and compact (pH < pKa).

  : high molar mass component. α: hydrodynamic coefficient for a given polymer calculated from 895 the Mark-Houwink -Sakurada equation ([η] = K & M ' ) for pectin samples in citrate/phosphate buffer 896 at pH 3.8, 0.1 M ionic strength. α = 0: Spheres, 0.5-0.8: Random coils, 1.0: Stiff coils, 2.0: Rods. NA:

		Main peak		HMM region
		Mi range (g/mol)	α	Mi range (g/mol)	α
	AP2	2×10 5 -2×10 6	0.55	NA	NA
	AP3	2×10 4 -2×10 5	1.17	2×10 5 -2×10 6	0.34
	AP6	2×10 4 -2×10 5	1.07	5×10 5 -5×10 6	0
	BP2	5×10 4 -2×10 5	1.04	2×10 5 -2×10 6	0.12
	BP3	5×10 4 -2×10 5	1.16	10 5 -10 6	0.41
	BP6	10 4 -10 5	0.96	10 5 -10 6	0.17
	KPR2	10 4 -2×10 5	NA	2×10 5 -2×10 6	0
	KPR3	10 4 -2×10 5	NA	2×10 5 -2×10 6	0
	KPR6	2×10 4 -8×10 4	1.16	10 5 -2×10 6	0
	KPO2	10 4 -7×10 4	1.02	10 5 -10 6	0.41
	KPO3	2×10 4 -7×10 4	1.32	10 5 -10 6	0.21
	KPO6	10 4 -6×10 4	0.94	6×10 4 -10 6	0.17
	897
	Not applicable.

HMM

Acknowledgements

LIU Xuwei would like to acknowledge China Scholarship Council (CSC) and Institut National de Recherche pour l'Agriculture, l'Alimentation, et l'Environnement (INRAE) for financial support to his PhD study. The authors thank the various CPER Platform 3A funders for the support to the HPSEC-MALLS measures as well as the related financial support from the European Union with the European Regional Development Fund, the State, the Provence-Alpes-Côte d'Azur Region, the Departmental Council of Vaucluse and the Urban Community of Greater Avignon.

(ITC) on the other hand.

Pectin linearity

The highest affinities for procyanidins DP9/79 were obtained for kiwifruit pectins KPO2 and KPR2, with both the highest Ka and the most marked aggregate formation, and the lowest affinities were observed for beet pectins. KPR/Os exhibited the highest linearity, HG and galactans contents, and lower RG-I content (Table 1 and Supplementary Fig. 1). Ripening involves a decrease in arabinose and a loss of pectic side chains [START_REF] Liu | Modification of apple, beet and kiwifruit cell walls by boiling in acid conditions: common and specific responses[END_REF] resulting in even higher linearity and higher HG ratio in KPO2, which strengthen the binding to procyanidins. [START_REF] Brahem | Pear ripeness and tissue type impact procyanidin-cell wall interactions[END_REF] also reported that cell walls from pear at an overripe stage have a higher affinity for procyanidins than those from the ripe stage, due to removal of arabinan and galactan pectin side chains during ripening, allowing better access to galacturonic acid-rich molecules. Moreover, the adsorption of anthocyanins on blueberry linear chelator-soluble pectins is four times higher than on the highly branched water-soluble pectin [START_REF] Koh | Binding kinetics of blueberry pectin-anthocyanins and stabilization by non-covalent interactions[END_REF]. On the other hand, [START_REF] Watrelot | Neutral sugar side chains of pectins limit interactions with procyanidins[END_REF] described that the affinity of procyanidin DP30 to pectic compounds increases in the following order: arabinans < arabinans + galactans II < galactan I . This may be due to the length and the flexibility of galactan side chains, while arabino-galactan side chains are short, more branched and stiff (M'sakni et al., 2006). Highly branched arabinans have more globular structures which limit their interactions with polyphenols [START_REF] Fernandes | Interactions of arabinan-rich pectic polysaccharides with polyphenols[END_REF]. KPR/Os contained more galactan side chains, while BPs contained more arabinans, followed by APs. Therefore, structure of the Table 4. Changes in the molecular mass and concentrations of pectins and the degree of polymerization of procyanidins before and after interactions between pectic fractions and procyanidins DP9/79 (30/60 mM (-)-epicatechin equivalent). = Unbound pectin concentration / Initial pectin concentration; b ΔMw: difference of weight-average molar mass between pectic fractions unbound to procyanidin solutions after interaction with procyanidins and initial pectic fractions in buffer; c ΔDPn: difference of degree of polymerization between procyanidins unbound to pectic fractions after interaction with pectins and initial procyanidins in buffer.

Sample


Ali Bouattour
email: ali.bouattour@pasteur.tn

Ahmed Chabchoub
email: chabchoubahmed2009@yahoo.fr

Iheb Hajjaji
email: ihebhajjaji@hotmail.fr

Youmna M'ghirbi
email: youmna.mghirbi@pasteur.tn

Hepatozoon canis and Babesia vogeli infections of dogs in Tunisia

Keywords: Canine Babesiosis, Babesia, Hepatozoon, Dogs, Tunisia, Rhipicephalus sanguineus

A paucity of studies is available on haemoparasites in dogs in Tunisia. In this study, we used molecular techniques (PCR/sequencing) to detect and characterize haemoprotozoa in sick dogs from Tunisia. A total of 99 dogs displaying such clinical symptoms as fever, anorexia, and depression were presented for treatment to the hospital of the Veterinary School of Sidi Thabet (Tunisia). Among dogs screened by PCR, five (5%) proved to be infected with a hemoprotozoa species.

An analysis of all the sequences that were obtained enabled us to identify two species of Protozoa: Babesia vogeli (in three dogs) and Hepatozoon canis (in two other dogs). This is the first time that an infection of dogs by Hepatozoon canis in Tunisia has been

Introduction

Species of Babesia and Hepatozoon infect a wide variety of mammals, including dogs [START_REF] Smith | The genus Hepatozoon (Apicomplexa: Adeleina)[END_REF][START_REF] Solano-Gallego | Babesiosis in dogs and cats-Expanding parasitological and clinical spectra[END_REF]. Some of the pathogens transmitted to dogs by ticks, such as hemoprotozoa, cause severe disease and serious illness. Several Babesia species, including B. canis, B. rossi, B. vogeli and B. gibsoni, which are the primary causes of babesiosis, have been reported in dogs [START_REF] Carret | Babesia canis canis, Babesia canis vogeli, Babesia canis rossi: differentiation of the three subspecies by a restriction fragment length polymorphism analysis on amplified small subunit ribosomal RNA genes[END_REF][START_REF] Irwin | Canine babesiosis: From molecular taxonomy to control[END_REF][START_REF] Solano-Gallego | A review of canine babesiosis: the European perspective[END_REF][START_REF] Baneth | Establishment of Babesia vulpes n. sp. (Apicomplexa: Babesiidae), a piroplasmid species pathogenic for domestic dogs[END_REF] and can cause infections whose clinical and pathological signs range from subclinical to severe (fever, haemolytic anemia, splenomegaly, thrombocytopenia…) and even death (Solano-Gallego and Baneth, 2011).

Small animal practitioners in Tunisia have frequently reported cases of canine babesiosis, which is most commonly caused by Babesia vogeli and transmitted by the dog brown tick Rhipicephalus sanguineus (M'ghirbi and [START_REF] Bouattour | Detection and molecular characterization of Babesia canis vogeli from naturally infected dogs and Rhipicephalus sanguineus ticks in Tunisia[END_REF]. Considered to be the least pathogenic species [START_REF] Köster | Canine babesiosis: a perspective on clinical complications, biomarkers, and treatment[END_REF], Babesia vogeli has also been reported in other African countries [START_REF] Collett | Survey of canine babesiosis in South Africa[END_REF][START_REF] Oyamada | Detection of Babesia canis rossi, B. canis vogeli, and Hepatozoon canis in dogs in a village of eastern Sudan by using a screening PCR and sequencing methodologies[END_REF], in southern Europe [START_REF] Caccio | Molecular characterisation of Babesia canis canis and Babesia canis vogeli from naturally infected European dogs[END_REF][START_REF] Solano-Gallego | A review of canine babesiosis: the European perspective[END_REF][START_REF] Solano-Gallego | Babesiosis in dogs and cats-Expanding parasitological and clinical spectra[END_REF], in North and South America [START_REF] Birkenheuer | Geographic distribution of babesiosis among dogs in the United States and association with dog bites: 150 cases[END_REF][START_REF] Passos | First molecular detection of Babesia vogeli in dogs from Brazil[END_REF], Asia [START_REF] Abd Rani | A survey of canine tick-borne diseases in India[END_REF] and in Australia [START_REF] Jefferies | Two species of canine Babesia in Australia: detection and characterization by PCR[END_REF].

Active during the warm season in Tunisia, R. sanguineus heavily infests dogs and can be a vector for several pathogens. In addition to B. vogeli, this tick species was reported to be infected with Anapalsma platys, Rickettsia massiliae Bar 29 and Rickettsia conorii [START_REF] Bouattour | Distribution and ecology of ticks (Acari: Ixodidae) infesting livestock in Tunisia: an overview of eight years of field collection[END_REF][START_REF] Sarih | Detection and identification of Ehrlichia spp. in Ticks collected in Tunisia and Morocco[END_REF][START_REF] Bouattour | Detection and molecular characterization of Babesia canis vogeli from naturally infected dogs and Rhipicephalus sanguineus ticks in Tunisia[END_REF][START_REF] Khrouf | Detection of Rickettsia in Rhipicephalus sanguineus ticks and Ctenocephalides felis fleas from southeastern Tunisia by reverse line blot assay[END_REF]. However, haemoparasites of the genera Hepatozoon have never been reported in dogs in Tunisia. Canine infections with H. canis have been reported in many parts of Africa [START_REF] Ibrahim | Neutrophilmyeloperoxidase deficiency associated with canine hepatozoonosis[END_REF][START_REF] Oyamada | Detection of Babesia canis rossi, B. canis vogeli, and Hepatozoon canis in dogs in a village of eastern Sudan by using a screening PCR and sequencing methodologies[END_REF][START_REF] Medkour | Canine vector-borne Protozoa: Molecular and serological investigation for Leishmania spp., Trypanosoma spp., Babesia spp., and Hepatozoon spp. In dogs from Northern Algeria[END_REF], Europe, America, Аsia, and Australia [START_REF] Eiras | First molecular characterization of canine hepatozoonosis in Argentina: evaluation of asymptomatic Hepatozoon canis infection in dogs from Buenos Aires[END_REF][START_REF] Li | Diagnosis of canine Hepatozoon spp. infection by quantitative PCR[END_REF][START_REF] Abd Rani | A survey of canine tick-borne diseases in India[END_REF]2011;[START_REF] Barker | Haemoparasites of Free-Roaming Dogs Associated With Several Remote Aboriginal Communities in Australia[END_REF]. The cosmopolite brown dog tick R. sanguineus was ascertained to be the primary invertebrate host for this Protozoa [START_REF] Christophers | The sexual life cycle of Leucocytozoon canis in the tick[END_REF].

Babesia and Hepatozoon are known to occur in endemic areas, which could partially explain the variations in clinical presentation and pathogenicity in infected dogs [START_REF] Shaw | Tick-borne infectious diseases of dogs[END_REF]. When examining tick-infested dogs that show clinical signs suggestive of hemoprotozoosis, veterinary practitioners often presume the cause to be babesiosis and omit hepatozoonosis as a possible cause without seeking laboratory confirmation.

Infection with Hepatozoon canis (Family Hepatozoidae) is also part of the differential diagnosis of hemoprotozoosis affecting dogs as intermediate hosts; the brown dog tick R. sanguineus is the definitive host and vector [START_REF] Baneth | Transmission of Hepatozoon canis to dogs by naturally-fed or percutaneously injected Rhipicephalus sanguineus ticks[END_REF]. Dogs with canine hepatozoonosis are often asymptomatic with low levels of parasitaemia whereas dogs with high levels of parasitaemia can present severe disease and show fever, lethargy, anaemia, and cachexia [START_REF] Ivanov | Hepatozoon canis and hepatozoonosis in the dog[END_REF] [START_REF] Baneth | Genetic and antigenic evidence supports the separation of Hepatozoon canis and Hepatozoon americanum at the species level[END_REF][START_REF] Caccio | Molecular characterisation of Babesia canis canis and Babesia canis vogeli from naturally infected European dogs[END_REF][START_REF] Inokuma | Molecular survey of Babesia infection in dogs in Okinawa, Japan[END_REF] and active Hepatozoon [START_REF] Oyamada | Detection of Babesia canis rossi, B. canis vogeli, and Hepatozoon canis in dogs in a village of eastern Sudan by using a screening PCR and sequencing methodologies[END_REF][START_REF] Medkour | Canine vector-borne Protozoa: Molecular and serological investigation for Leishmania spp., Trypanosoma spp., Babesia spp., and Hepatozoon spp. In dogs from Northern Algeria[END_REF] infections in dogs. This study therefore aimed to diagnose the causal agents of infections in dogs presenting suspected haemoparasitosis using PCR and sequencing methodologies.

Materials and methods

Dogs and blood samples

Our study was carried out during the warm season (April-July 2018) during which ticks are active. A total of 99 dogs of various breeds with clinical manifestations compatible with tick-borne diseases including apathy, dehydration, fever, anorexia, weight loss, and pale mucous membranes, were presented to Veterinary School of Sidi Thabet Hospital (Tunisia) for treatment. The dogs underwent clinical examinations and blood samples were collected from the radial vein in 5 mL tubes containing ethylenediaminetetraacetic acid (EDTA)-anticoagulated. Whole blood was collected for DNA extraction and hematological analysis.

DNA extraction

We used the QIAmp®DNA Mini kit (Qiagen GmbH, Hilden, Germany) for DNA extraction as per the manufacturer's instructions, adjusted to 100 μL with the elution buffer (AE) provided with the kit, and stored at -20°C for further use. To verify contamination, a control tube (distilled water) for each of the ten samples was included in the extraction protocol. The DNA yield was determined with a NanoDrop_ND-1000

Spectrophotometer (NanoDrop Technologies, DE, USA).

PCR

PCR was performed using a set of primers that amplified a 460 to 520 bp fragment in the V4 variable region of the 18S rRNA of Babesia and Theileria species [START_REF] Georges | Detection of haemoparasites in cattle y reverse line blot hybridisation with a note on the distribution of ticks in Sicily[END_REF]. Sequences of genus-specific primers were used to amplify the V4 region of the 18S rDNA were RLB-F2 5'-GACACAGGGAGGTAGTGACAAG-3' and RLB-R2

-5'-CTAAGAATTTCACCTCTGACAGT-3'. In our experiments, sequencing showed that these primers, previously reported as being able to amplify the Babesia and Theileria genus, also amplified Hepatozoon. The PCR products were separated by electrophoresis using 2% agarose gel containing ethidium bromide (10 mg/mL) before it was analyzed by sequencing.

DNA sequencing and data analysis

To confirm the PCR results, positive Babesia/Theileria amplified DNA (n=5) was analyzed by sequencing. The PCR products were purified using the ExoSAP clean-up procedure (Amersham Biosciences, Germany). All nucleotide sequences were obtained using the Big Dye Terminatorv.3.1 Cycle Sequencing Kit (Applied Biosystems) and the 3130 automated sequencer (Applied Biosystems).

The sequences generated in this study were edited and aligned using BioEdit [START_REF] Hall | BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF] and ClustalW software programs [START_REF] Thompson | improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF]. The BLAST program (http://www.ncbi.nlm.nih.gov/BLAST) was used to compare and analyze the data sequences.

Phylogenetic analysis

A phylogenetic tree was inferred using neighbor-joining analysis with MEGA5.2 software with a bootstrap of 1000 replications and evolutionary distances adjusted through Kimura-2 parameter nucleotide substitution [START_REF] Tamura | Mega4, Molecular Evolutionary Analysis (MEGA) software, version 4.0[END_REF]. The sequence of Plasmodium falciparum (M19172.1) was used as distant phylogenetic organisms to root the tree.

Nucleotide sequences data

Nucleotide sequences data reported in this paper is available in the GenBank™, EMBL and DDBJ databases under the accession numbers MT588813-MT588817.

Hematological analysis

White blood cell counts, Hematocrit, Hemoglobin, and platelet counts were performed by a fully automatic hematology analyzer --the BC-2800 Vet (Mindray, China).

Results

Of the 99 dogs examined by PCR, five (5%) showed a specific band of 460 bp for Protozoa. The sequences obtained were analyzed using Bioedit® software and then compared with the reference sequences in the GenBank (www.ncbi.nlm.nih.gov/BLAST). This enabled us to identify two species of Protozoa:

Babesia vogeli and Hepatozoon canis. Three sequences (354 sequenced nucleotides)

were found to be 100% identical to the partial sequence of B. vogeli (MK910150) reported in a dog from Colombia. In addition, two sequences (339 sequenced nucleotides) were 100% identical to the partial sequence of H. canis (MN628318) isolated in India (Fig. 1).

All positive dogs had a fever (39.4-41.3 °C) and were anorexic (Table1). Of these, only dogs 51 and 93, which were infected by B. vogeli and H. canis respectively, developed a mild non-regenerative anaemia. The hematocrit showed low values in dogs 32, 93 and 51. Dog 51 also presented a leukopenia (WBC = 5.2 ×10 3 /µL). Dogs 15, 18 and 51 suffered from thrombocytopenia (Table1). All dogs had at least two specimens of Rhipicephalus sanguineus ticks on their ears.

Discussion

Infections with Babesia or Hepatozoon provoked a wide range of clinical manifestations in dogs from asymptomatic to severe disease. Acute clinical and hematological signs include fever, lethargy, hemolytic anemia, and thrombocytopenia, [START_REF] Criado-Fornelio | New advances in molecular epizootiology of canine hematic Protozoa from Venezuela, Thailand and Spain[END_REF][START_REF] Irwin | Canine babesiosis: From molecular taxonomy to control[END_REF][START_REF] Solano-Gallego | Babesiosis in dogs and cats-Expanding parasitological and clinical spectra[END_REF][START_REF] Baneth | Canine hepatozoonosis: two disease syndromes caused by separate Hepatozoon spp[END_REF][START_REF] Kamani | An Annotated Checklist of Tick-Borne Pathogens of Dogs in Nigeria[END_REF]. However, clinical manifestations caused by haemoprotozoa are not so clearly defined that veterinary practitioners can make a diagnosis and adapt their treatments.

Molecular methods must be used to identify the causative species. Several techniques have been described, and conventional PCR used on blood proved more sensitive and specific than classical methods for diagnosing haematozoa [START_REF] Oyamada | Detection of Babesia canis rossi, B. canis vogeli, and Hepatozoon canis in dogs in a village of eastern Sudan by using a screening PCR and sequencing methodologies[END_REF][START_REF] Wahlang | Comparative analysis of conventional and real time PCR for detection of haemoparasites in dogs[END_REF]. In addition, sequencing facilitated the identification of hemoparasite species (Solano-Gallego and Baneth, 2011).

In our study, the DNA of 99 dogs presenting various clinical manifestations to the hospital of the Veterinary School of Sidi Thabet (Tunisia) was screened by PCR using a set of a genus-specific primers amplifying Babesia and Theileria species [START_REF] Georges | Detection of haemoparasites in cattle y reverse line blot hybridisation with a note on the distribution of ticks in Sicily[END_REF]. After sequencing the positive amplicons, we discovered that these same primers can also amplify Hepatozoon. [START_REF] Oyamada | Detection of Babesia canis rossi, B. canis vogeli, and Hepatozoon canis in dogs in a village of eastern Sudan by using a screening PCR and sequencing methodologies[END_REF] have reported similar results using Babesia spp. primers. Sequencing analysis of the positive amplicons that were obtained revealed that Babesia vogeli was present in three dogs (15, 18 and 51) and Hepatozoon canis was present in two dogs (32 and 93).

Our results support and confirm the presence of B. vogeli in dogs in Tunisia as we have previously reported. However, in the investigated sick dogs, the prevalence of the infection was only 3%, which is lower than that reported in our previous investigation (6.6%) on randomly studied dogs (M'ghirbi and [START_REF] Bouattour | Detection and molecular characterization of Babesia canis vogeli from naturally infected dogs and Rhipicephalus sanguineus ticks in Tunisia[END_REF]. In fact, infections in dogs with this large Babesia have been reported with prevalence range values that vary widely by locality: B. vogeli prevalence was 13.2% among 227 dogs screened by PCR in Algeria a neighboring country [START_REF] Medkour | Canine vector-borne Protozoa: Molecular and serological investigation for Leishmania spp., Trypanosoma spp., Babesia spp., and Hepatozoon spp. In dogs from Northern Algeria[END_REF]; 2% in 78 randomly chosen dogs from eastern Sudan [START_REF] Oyamada | Detection of Babesia canis rossi, B. canis vogeli, and Hepatozoon canis in dogs in a village of eastern Sudan by using a screening PCR and sequencing methodologies[END_REF]; 0.6% in dogs with clinical manifestations in Nigeria, [START_REF] Kamani | Molecular detection and characterization of tick-borne pathogens in dogs and ticks from Nigeria[END_REF]; in southern France, the prevalence was 13.6 % in 140 dogs tested by nested PCR [START_REF] René-Martellet | Update on epidemiology of canine babesiosis in Southern France[END_REF]; in southeastern Rio de Janeiro (Brazil), the prevalence was 15.66 % in 390 dogs tested by qPCR [START_REF] Paulino | Molecular epidemiology of Babesia vogeli in dogs from the southeastern region of Rio de Janeiro, Brazil[END_REF]. The variation in prevalence might be attributable to several factors including the DNA extraction methodology, the primers that were used and the targeted gene, which can have different sensitivities [START_REF] Valkiunas | A comparative analysis of microscopy and PCR-based detection methods for blood parasites[END_REF]. In addition, host-related variables such as age or breed and mainly tick infestations should also be considered when comparing varying levels of regional prevalence.

In addition to B. vogeli, we detected H. canis in the group of investigated dogs. This study is the first molecular report of H. canis in Tunisia. This parasite has been reported recently in dogs in Algeria with a prevalence of 41% [START_REF] Medkour | Canine vector-borne Protozoa: Molecular and serological investigation for Leishmania spp., Trypanosoma spp., Babesia spp., and Hepatozoon spp. In dogs from Northern Algeria[END_REF]. In addition, this Hepatozoon species was recorded in several countries of Africa: Sudan, Nigeria and Zambia with prevalence rates of 42.3%, 41.4%, and 13% respectively [START_REF] Oyamada | Detection of Babesia canis rossi, B. canis vogeli, and Hepatozoon canis in dogs in a village of eastern Sudan by using a screening PCR and sequencing methodologies[END_REF][START_REF] Kamani | Molecular detection and characterization of tick-borne pathogens in dogs and ticks from Nigeria[END_REF][START_REF] Kamani | An Annotated Checklist of Tick-Borne Pathogens of Dogs in Nigeria[END_REF][START_REF] Williams | Prevalence and diversity of Babesia, Hepatozoon, Ehrlichia, and[END_REF]. Studies in several European countries have reported different prevalence rates from low (1% and 3% in France and Spain respectively; [START_REF] Criado-Fornelio | Hemoprotozoa of domestic animals in France: prevalence and molecular characterization[END_REF][START_REF] Tabar | PCR survey of vector borne pathogens in dogs living in and around Barcelona, an area endemic for leishmaniasis[END_REF] to high (50.6% in southern Italy; [START_REF] Otranto | Diagnosis of Hepatozoon canis in young dogs by cytology and PCR[END_REF]. The prevalence of H. canis similarly varies by country in America: 53.3% in Brazil [START_REF] Rubini | Molecular and parasitological survey of Hepatozoon canis (Apicomplexa: Hepatozoidae) in dogs from rural area of São Paulo state, Brazil[END_REF] to 2.5% in the USA, where H. canis occurs together with H. americanum [START_REF] Li | Diversity of Hepatozoon species in naturally infected dogs in the southern United States[END_REF]. Dogs in several counties in Asia were also reported to show prevalence: in India where the hemoparasite was identified for the first time, the rate of prevalence was 30% [START_REF] Abd Rani | A survey of canine tick-borne diseases in India[END_REF].

Babesia vogeli and Hepatozoon canis are canine hematic Protozoa that are widespread due to the cosmopolitan nature of their main vector, Rhipicephalus sanguineus [START_REF] Shaw | Tick-borne infectious diseases of dogs[END_REF][START_REF] Bouattour | Detection and molecular characterization of Babesia canis vogeli from naturally infected dogs and Rhipicephalus sanguineus ticks in Tunisia[END_REF][START_REF] Irwin | Canine babesiosis: From molecular taxonomy to control[END_REF][START_REF] Solano-Gallego | Babesiosis in dogs and cats-Expanding parasitological and clinical spectra[END_REF], the most prevalent tick species infesting dogs worldwide. However, while

Babesia is transmitted to a dog by the tick's salivary gland during the blood meal [START_REF] Solano-Gallego | A review of canine babesiosis: the European perspective[END_REF], H. canis is transmitted by the ingestion of ticks containing mature oocysts [START_REF] Baneth | Transmission of Hepatozoon canis to dogs by naturally-fed or percutaneously injected Rhipicephalus sanguineus ticks[END_REF][START_REF] Baneth | Canine hepatozoonosis: two disease syndromes caused by separate Hepatozoon spp[END_REF].

In our study, infections with B. vogeli, considered to be the least virulent Babesia species, caused clinical diseases in three infected dogs that displayed fever, anorexia, dehydration depression, lethargy and weakness. All three dogs also suffered from thrombocytopenia whereas only one dog (number 51) had mild regenerative anaemia since it was infected with B. vogeli (30% hematocrit; 9g/dl hemoglobin). Similar clinical signs (fever, anorexia and weakness) were observed in the two dogs infected with H. canis; hematological abnormalities (anemia and low hematocrit) were also noted. Similar clinical and hematological manifestations caused by these two parasites were reported [START_REF] Sasanelli | Atypical clinical presentation of coinfection with Ehrlichia, Babesia and Hepatozoon species in a dog[END_REF][START_REF] Solano-Gallego | Babesiosis in dogs and cats-Expanding parasitological and clinical spectra[END_REF][START_REF] Solano-Gallego | A review of canine babesiosis: the European perspective[END_REF]. H. canis also causes subclinical infections in apparently healthy dogs.

Indeed, infection with this protozoon can induce severe illness and can be fatal; dogs with high levels of H. canis parasitaemia that often approaches 100% of the peripheral blood neutrophils can display lethargy, cachexia and anaemia [START_REF] Baneth | Canine hepatozoonosis: two disease syndromes caused by separate Hepatozoon spp[END_REF]. Two dogs presented to the clinic showed mild disease, which is the most common presentation of infection that is usually associated with low levels of H. canis parasitaemia (1-5% of neutrophils are infected) [START_REF] Baneth | Canine hepatozoonosis: two disease syndromes caused by separate Hepatozoon spp[END_REF][START_REF] Ivanov | Hepatozoon canis and hepatozoonosis in the dog[END_REF].

Conclusion

In Tunisia, warm weather from March to October prolongs the active season of Rhipicephalus sanguineus ticks that heavily infest dogs. This tick is a vector of several pathogens such as species of Ehrlichia and Babesia, previously reported [START_REF] Sarih | Detection and identification of Ehrlichia spp. in Ticks collected in Tunisia and Morocco[END_REF][START_REF] Bouattour | Detection and molecular characterization of Babesia canis vogeli from naturally infected dogs and Rhipicephalus sanguineus ticks in Tunisia[END_REF], and Hepatozoon canis that is described in this work. It should be noted that co-infections can occur in dogs, which might partially explain the variations in often unspecific clinical presentations [START_REF] Shaw | Tick-borne infectious diseases of dogs[END_REF][START_REF] Sasanelli | Atypical clinical presentation of coinfection with Ehrlichia, Babesia and Hepatozoon species in a dog[END_REF][START_REF] Kamani | Molecular detection and characterization of tick-borne pathogens in dogs and ticks from Nigeria[END_REF]. It is therefore clinically important that these diseases be diagnosed with a sensitive, specific, simple method and that the veterinary practitioner be aware of the existence of these pathogens in Tunisia and select a treatment adequate to each one.

911-918.

  The PCR reactions were performed in a final volume of 25 μL containing 75 ng of genomic DNA, 200 nM of each primer, 1× PCR buffer, 1.5 mM MgCl2, 200 µM of each dNTPs and 1 U of Taq Platinum Polymerase (Invitrogen, CA, USA).The reactions were performed on an automated DNA thermal cycler (Perkin-Elmer 2720, CA) as follows: starting with 15 min at 95° C and followed by 45 cycles of 94° C for 35 sec; 51° C for 35 sec and 72° C for 35 sec and a final elongation, 72° C for 10 min. Extraction controls and PCR negative (water) controls were included in each PCR reaction. Plasmid including the amplicon of the V4 region of the 18S rRNA gene of Babesia canis species was used as a positive control.

Table 1 .

 1 Results of sequencing PCR products and hematological values in dogs thatshowed positive for the first PCR Fig.1: A phylogenetic tree obtained using a small ribosomal subunit sequence Theileria annulata, 10 Babesia species/isolates and 6 Hepatozoon canis isolates. The Kimura-2 method was used to calculate distances, and tree topology was inferred by neighbour joining (MEGA 5.2 software). A total of 354 informative positions were used in the analysis. Numbers at the nodes represent the bootstrap support in 1000 replications.Scale bar indicates an evolutionary distance of 0.02 nucleotides per position in the sequence. New sequences are marked in bold.Table1 . Results of sequencing PCR products, clinical signs, and hematological values in dogs revealed positive by the PCR

	Dog	Age	Babesia	H.	Accession	Red blood	White	Hematocrit	Hemoglobin	Platelet		Clinical signs
	no.	/	vogeli	canis	number	cells	blood cells	(%)	(g/dl)	Count	T °C
		year				(10 6 /µl)	(10 3 /µl)			(10 3 /µl)
	Reference range*			5.50 -8.50 6.00 -17.0 37.0 -55.0 12.0 -18.0	200.0 -	38-39
										500.0
	15	2	+		MT588815	6.40	11.1	44	13	66	40.3	Anorexia, dehydrated,
												depression
	18	1	+		MT588814	7.09	7	54.4	15.8	120	41.3	Anorexia, dehydrated,
												depression
	32	3		+	MT588816	5.11	15.4	28	11.4	569	39.5	Anorexia, lethargy
	51	1.5	+		MT588813	5.20	5.2	30	9	140	39.4	Anorexia, lethargy,
												weakness
	93	1		+	MT588817	5.54	9.9	25	8.63	292	39.7	Anorexia, Weakness
	*From Sasanelli et al. (2009)
	+ = sequencing PCR products result

Acknowledgments

We thank Ministry of High Education and Research of Tunisia (Direction Generale de la Recherche) for financial support. We thank also Dr. Deborah Glassman, Washington D.C., for English corrections.

Ethical statement

All dogs were examined and sampled by the veterinary clinicians according to the procedures and the ethics committee of the Veterinary School of Sidi Thabet, and after the consent of the owner. Owners presented their dogs to the Veterinary School Hospital for diagnosis and treatment.

Conflict of interest

The authors declare that there is no conflict of interest.

Authors' contribution:

A.B (MDV, PhD) conceived, designed and wrote the manuscript. A. Ch (MDV, Professor) and I. H. (MDV) were the veterinary clinicians who examined the dogs and performed the hematological analysis.

Y.M (PhD) performed the molecular analysis and contributed to the manuscript.

All authors read and approved the final manuscript.