Chen Wang

Isabelle Bourven

Abdel Lakel

Michel Baudu
email: michel.baudu@unilim.fr

Comparison of wastewater treatment efficiencies in packed bed bioreactors according to the nature of materials

Keywords: packed bed bioreactor, filtration, purification, river sand, crushed aggregate, biomass

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

Introduction

On-Site Wastewater Treatment (OWTS) plants typically comprise a septic tank for primary wastewater treatment and infiltration/filtration systems (featuring soil infiltration or sand filtration) for secondary treatment. The septic tank enables solid/grease/liquid separation and settling, in addition to the hydrolysis processes of solids, digestion of certain organic matter and transformation of organic nitrogen into ammonium [START_REF] Jr | Fate and transport of biological and inorganic contaminants from on-site disposal of domestic wastewater[END_REF]. Pre-treated effluent, known as septic effluent, which is considered a source of groundwater contamination and water resources eutrophication in the presence of phosphorus and nitrogenous pollutants and microorganisms [START_REF] Yates | Septic Tank Density and Ground-Water Contamination[END_REF][START_REF] Withers | Quantifying the impact of septic tank systems on eutrophication risk in rural headwaters[END_REF]. This septic effluent is spread over soil absorption fields or sand filtration systems flowing under unsaturated conditions. Packed bed bioreactors are used for very different water treatment objectives like domestic sewage or synthetic industrial wastewater [START_REF] Chen | Immobilized cell fixed-bed bioreactor for wastewater decolorization[END_REF][START_REF] Oliveira Netto | Treatment of domestic sewage in an anaerobic-aerobic fixed-bed reactor with recirculation of the liquid phase[END_REF][START_REF] Anjaneya | Decolourization of Amaranth dye by bacterial biofilm in batch and continuous packed bed bioreactor[END_REF]. For OWTS, unsaturated filtration treatments are often applied in conjunction with on-site soil absorption fields. Soil conditions however are sometimes unsuitable for wastewater treatment, hence the soil reconstruction with other packing materials is needed; the most widely used filter medium is natural sands from alluvial deposits, e.g. river sands. Grain size distribution is the most important characteristic of a porous medium. French and American authorities have imposed particle size distribution of a filter medium. Liénard et al. [START_REF] Liénard | Choice of the sand for sand filters used for secondary treatment of wastewater[END_REF] have also suggested a narrower range for D 10 (i.e. 0.2 mm < D 10 < 0.4 mm) so as to ensure an effective purification process. The discontinuous feed maintains the filter medium unsaturated and aerated, in addition to facilitating aerobic bacterial growth; consequently, sand filters can be considered as aerobic fixed-biomass reactors [START_REF] Kristiansen | Sand-Filter Trenches for purification of septic tank effluent: I. The clogging mechanism and soil physical environment[END_REF]. Rodgers M. [START_REF] Rodgers | Different depth sand filters for laboratory treatment of synthetic wastewater with concentrations close to measured septic tank effluent[END_REF] considered that biomass developed inside the filter medium contributes to purification performance via the degradation of organic matter consumed by heterotrophic bacteria. The purification process is influenced by both environmental and operational conditions, such as: temperature, packing material characteristics, hydraulic loading, organic loading, and dosing frequencies [START_REF] Kristiansen | Sand-Filter Trenches for purification of septic tank effluent: I. The clogging mechanism and soil physical environment[END_REF][START_REF] Rodgers | Different depth sand filters for laboratory treatment of synthetic wastewater with concentrations close to measured septic tank effluent[END_REF][START_REF] Kristiansen | Sand-Filter Trenches for purification of septic tank effluent: I. The clogging mechanism and soil physical environment[END_REF][START_REF] Rodgers | Different depth sand filters for laboratory treatment of synthetic wastewater with concentrations close to measured septic tank effluent[END_REF][START_REF] Rout | Role of Bacillus cereus GS-5 strain on simultaneous nitrogen and phosphorous removal from domestic wastewater in an inventive single unit multi-layer packed bed bioreactor[END_REF].

Due to over-exploitation, river sands are less and less available as packing bed materials.

Crushed aggregate resulting from the quarrying of massive rocks is considered a potentially suitable material. Some studies have shown that crushed aggregate offers a very broad size distribution with angular, irregular particle shapes [START_REF] Withers | Quantifying the impact of septic tank systems on eutrophication risk in rural headwaters[END_REF][START_REF] Cho | Particle shape effects on packing density, Stiffness, and Strength: Natural and Crushed Sands[END_REF]. The lack of scientific base of filter conception don't permit a good potential information on the long-term functioning. Research works have been made with the purpose of finding the correlation between filter sizing and long term functioning (clogging). Authors tented to propose the operating conditions and sizing criteria provide a balance between necessary nutrients loads and clogging produced by organic and particulate matters accumulation [START_REF] Healy | Site evaluation and design of seepage fields[END_REF]. Later, design criteria have been improved since more full-scale soil infiltration beds and sand filters have been investigated by Jenssen & Siegrist [START_REF] Jenssen | Technology assessment of wastewater treatment by soil infiltration systems[END_REF].

Investigations of clogging lead to three major mechanisms: pore blocking caused by suspended solids [START_REF] Vries | Soil filtration of wastewater effluent and the mechanism of pore clogging[END_REF]; over developed biomass and accumulation of extracellular polymers secreted, such as proteins and polysaccharides [START_REF] Torrens | Impact of design and operation variables on the performance of vertical-flow constructed wetlands and intermittent sand filters treating pond effluent[END_REF] and precipitation of certain metals such as FeS and CaCO 3 [START_REF] Cooke | Modeling biochemically driven mineral precipitation in anaerobic biofilms[END_REF].

Fewer studies have based on the clogging process and packing materials characteristics. Thus, the real impact of solid particles shape and surface proprieties are never investigated and how the materials of different nature impact the development of clogging or the purification efficiency remains unclear. Only one study on small scale pilot unit and only during the first step of colonization (1 month) [START_REF] Wanko | Biological Processing Capacities and Biomass Growth in Waste Water Treatment by Infiltration On two Kinds of Sand[END_REF] seems to show similar efficiencies for organic and suspended solids removal with river sand and crushed aggregate. For a better knowledge on the influence of packing bed material and a more complete information about the potential substitution of sand in OWTS, we have conducted this study using real septic effluent with various filter materials and the purification efficiency was monitored throughout the year following process startup.

Materials and methods

Packed bed materials

Two natural materials (river sands RS1 and RS2) and two crushed aggregates (CA1 and CA2) were tested. The packing material characteristics are displayed in Table 1. The influence of effective size (D 10 ), uniformity coefficient and packing material chemical composition (divalent cations Ca 2+ and Mg 2+ ) on the treatment efficiencies were studied and taken into account for purposes of statistical analysis. Statistical analysis was performed with the SYSTAT 12 program (Systat Software, Inc.). A p value of 0.05 was established as the parameter level of significance on treatment efficiency in the steady state. For p values of less than 0.05, the parameter was considered to exert influence on the treatment efficiency.

Pictures of the sand samples were processed by ImagJ Software. After an 8-bit binary conversion, the picture scale was set and the particle analyses performed with a dedicated plugin.

The filter was introduced with limitations to both its size (0.1 -4 mm) and circularity (> 0.75) so as to ensure effective particle isolation. Two parameters were determined: Circularity = 4π × (Area) / (Perimeter) 2 ; Roundness = 4 × (Area)/π × (Major axis) 2 .

Pilot batch experiment

The reactor configurations and feeding conditions are described in Table 2. The experiment was carried out on a laboratory-scale pilot equipped with 12 columns 30 cm in diameter featuring 3 different heights: 15, 30, and 70 cm. French standards recommended the material layer of 70cm as real scale sand filtration bed practice. The feed water was stored in a mixed storage tank and replenished roughly every 7 to 10 days. Figure 1 provides an overview of the reactor set-up. The feed water was spread by the distribution disk perforated with 15 holes. These experiments were conducted in a controlled environment at 20 ± 2°C. The columns were discontinuously supplied with 10 batches of septic effluent in order to maintain the unsaturated conditions. The hydraulic loading was set at 12 cm/day for all reactors, except columns 10 and 12, which were set at 20 cm/day. Hydraulic conductivities were estimated by infiltration tests using the columns with an 8-cm diameter and 20-cm height [START_REF] Liénard | Choice of the sand for sand filters used for secondary treatment of wastewater[END_REF]. The hydraulic residence time (HRT) was characterized in the 30and 70-cm reactors by instantly injecting inert tracer lithium chloride (LiCl, 1 g Li/l) and monitoring Li concentration in the treated effluent. Li was analyzed using Atomic Absorption Spectroscopy (SpectrAA 220, VARIAN).

In an initial step, the depollution performance was compared by running filtration reactors with a 30-cm diameter and 30-cm thickness on four different packing materials (river sands RS1 and RS2, and crushed aggregates CA1 and CA2) because the upper 30-cm filter medium is the more active part in a vertical filtration bed [START_REF] Cooke | Modeling biochemically driven mineral precipitation in anaerobic biofilms[END_REF]. Operational parameters were modified on two selected materials for subsequent comparisons; these parameters were bed thickness and hydraulic loading. The depollution efficiencies with four different filter materials were monitored in terms of: particulate matter (TSS), organic matter with a Chemical Oxygen Demand (COD) determination, nitrogenous pollutants (ammonium, nitrates and total nitrogen), and phosphate pollutants.

2.3.Characterization of feed water and treated effluent

The feed water was collected from the septic effluent tank. Its main characteristics were monitored throughout the operating period. The average values of each characteristic are listed in Table 3. The feed water and treated effluent from 12 columns were characterized by physicochemical analysis conducted with Spectroquant ® photometric testing (Merck). The chemical oxygen demand (COD) was determined according to the rapid-digestion method (EN ISO 15705); total nitrogen (TN) was derived after decomposition with potassium peroxodisulfate with the finer materials (RS1 and CA1) confirmed that the reactors of 70cm remained oxygenated at the beginning of the operation (see Table 2). The microbiological parameters were only monitored at the end of the process on the 70-cm columns with viable aerobic flora (EN ISO 6222:1999). All feed water characteristics are summarized in Table 3.

Material characterization during the process

The total organic matter attached to the various materials was quantified in terms of volatile dry weight (VDW), with the weight loss of sampled materials being measured by incineration between 105° and 550°C [START_REF] Wanko | Biological Processing Capacities and Biomass Growth in Waste Water Treatment by Infiltration On two Kinds of Sand[END_REF]. The total organic distribution across the depths of the four materials was evaluated at the end of the second study period.

The bacterial flora was extracted from the three different layers in river sand 1 and crushed aggregates 1 and 2 at the end of the first period. A 20-g mass of colonized materials was sampled from 0~5 cm, 10~15 cm and 30 cm. These materials were then washed with 100 mL of a 9%

NaCl solution, and 10 g of washed sample were extracted using a 20-mL phosphate buffer (0.

Results

Impact of materials on treatment efficiencies

The first part of this study investigated the efficiency of 4 reactors with varied materials through several parameters: TSS, nitrogen forms and phosphates. The filtration reactors with a 30-cm active layer were fed with inlet water at a rate of 12 cm/day. The results obtained over 360 days of operations are presented in Figure 2. The TSS removals (Fig. 2a) shows similar and satisfactory efficiency rates with the finer river sand (RS1) and both crushed aggregates (CA1 and CA2) after 60 days of operations. RS1 demonstrated quick and effective removals at the beginning of the process. In contrast, the coarser river sand (RS2) yielded lower and less stable TSS removals throughout the operations, i.e. typically below 80%. COD removals behaved similarly to TSS. The coarser river sand (RS2) was rated at the lowest efficiency (< 70%, > 125 mgO 2 /L) throughout the operating period, while the finer river sand (RS1) and crushed aggregate 1 (CA1) exhibited similar removal rates (85%, 60 mg O 2 /L) after running for 120 days (Fig. 2b).

Compared to RS1, the two crushed aggregates took longer (100 days for CA1, 180 days for CA2)

to reach a similar efficiency level. The effective size of materials has shown a most significant correlation (treatment efficiency increased with a decrease in effective size) in both TSS and COD removals (significant at p = 0.001 < 0.05 for TSS and p = 0.007 < 0.05 for COD). The material uniformity coefficient also indicated a slight correlation with COD removal (p = 0.04 < 0.05).

Even though an increase in removal rate can be noticed in RS2, this material still produced the lowest and least stable organic removals.

The nitrogenous pollutants in feed water were dominated by organic and ammonium nitrogen; the nitrate traces were negligible. By relying on sample averaging, the nitrite level in treated effluent also dipped below the detection limit (< 0.01 mg N-NO 2 -/L). As indicated in the TSS and organic removals, RS1 and CA1 showed similar satisfactory efficiencies in ammonium removals after 100 days (> 95%), with RS1 displaying early stabilization (at around 45 days) (Fig. 2c). The removals in CA2 indicated slightly less stability (60~120 days). The highly permeable material (RS2) of coarser size did not improve ammonium elimination (< 75%) due to its poor HRT (Table 2), which led to incomplete nitrification. Similar to TSS and COD, the effective size and uniformity of materials exhibited significant correlations with ammonium removals (p = 0 < 0.05 for D10 and p = 0.0003 < 0.05 for UC). Aside from this exceptionally coarse sand, RS1 and the two crushed aggregates revealed no significant difference in ammonium removal after 100 days of operations.

Phosphorus pollutants in the feed water were dominated by phosphates; their removal rates were typically below 60% (2~9 mg P/L). Figure 2f shows that phosphate removals via the filtration reactors were inconsistent and less effective. At the beginning of the process, two CA specimens exhibited higher phosphate removals than the river sands (around 70% for CA1 and CA2 vs. approx. 50% for RS1). Although the phosphate sorption tests showed no significant phosphate adsorption by all four materials.

Degraded conditions

For RS1 and CA1-packed reactors, reducing bed thickness (from 30 to 15 cm) led to lower purification efficiency, especially with respect to organic and ammonium removals. In contrast, the thicker beds improve efficiency and provide for both satisfactory removals and better stability due to the longer HRT (Table 2) [START_REF] Torrens | Impact of design and operation variables on the performance of vertical-flow constructed wetlands and intermittent sand filters treating pond effluent[END_REF]. With 70-cm filtration reactors, RS1 yielded slightly better efficiencies in organic and ammonium removals, but also significantly higher total nitrogen removal rates (39% [21%, 58%] for RS1 and 22% [4%, 36%] for CA1).

Another comparison was drawn between an HLR of 12 cm/day vs. 20 cm/day using two selected filter media. In sum, increasing the HLR led to a decrease in the HRT value, especially for RS1 (Table 2), along with a slight decrease in the efficiency of several parameters (Table 5):

TSS, organic matter, ammonium and total nitrogen, specifically at the beginning of operations when the biomass is still immature. Removal rates gradually rose with operating time, and the bed thickness also compensated for the resistance to hydraulic loads; a higher hydraulic loading significantly reduces organic removal efficiency in shallower beds (30cm) [START_REF] Van Cuyk | Hydraulic and purification behaviors and their interactions during wastewater treatment in soil infiltration systems[END_REF].

Ammonium removal efficiencies presented fluctuations under higher hydraulic loading, yet both materials seemed to be sufficiently resistant and removals typically remained above 90% (Figs. 3 and4). Stabilization was established early under the lower hydraulic loading, notably in the fine river sand (RS1), whereas a longer operating time was needed for both materials at a higher loading. At the beginning of operations, both adsorption and incomplete nitrification were present in the immature system [START_REF] Healy | Site evaluation and design of seepage fields[END_REF]. The higher water flow reduced ammonium adsorption and limited the nitrification process [START_REF] Pell | Infiltration of Wastewater in a Newly Started Pilot Sand-Filter System: III. Transformation of nitrogen[END_REF][START_REF] Pell | Microbial numbers and activity during infiltration of septic-tank effluent in subsurface sand filter[END_REF]. This impact of hydraulic loading has been confirmed by nitrate contents as well as by total nitrogen removals in the higher loading reactors, where denitrification has considerably limited the reduction in HRT values.

The differences observed between materials can be discussed from the implantation of the biomass in the reactors.

Filter materials and biomass distribution

Given the observations recorded for the various filter media at the end of the study, total organic contents (expressed as volatile dry weight, i.e. mg VDW/g of material) could be assessed by core sampling from 3 layers in the filtration reactors (0~5 cm, 10~15 cm and 30 cm) following process stabilization (Table 6). Due to poor colonization onto coarse sand (RS2 with a VDW of 3.69 mg/g), cell extractions were not carried out with this material. Total organic contents decreased from the top layer throughout the bed in all materials studied. The fine river sand (RS1)

showed higher accumulations at the surface and less organic matter in the deeper part. The total VDW is highest for RS1, in accordance with grain size (see Table 1, average grain diameter of 0.82 mm for RS1, vs. 2.66 mm for RS2). The organic matter accumulation results matched the correlation between the TSS, COD removals and the effective sizes shown in Section 3.1 (p < 0.05).

The results of total viable aerobic heterotrophic flora and autotrophic nitrifying flora are listed in Table 7. The distribution of heterotrophic bacteria in the fine river sand (RS1) appears to be higher in the top 15 cm than the deeper layer, which follows organic matters accumulation pattern especially at the top regarding the VDW values. The distribution of autotrophic nitrifying bacteria is less prevalent at the surface of the three filter media since the presence of higher substrate concentrations favors the growth of heterotrophic flora. The results showed that, for the fine river sand (RS1), nitrifying bacteria are mainly concentrated in the 10~15-cm layer but not in the deeper part (30 cm) , where as they were embedded relatively deeper in the crushed aggregate (CA1 and CA2).

Discussion

A medium composed of finer material enabled earlier stabilization in both TSS (particulates)

and organic matter (particulates and dissolved forms) removals under the action of mechanical retention at first by the reactor surface layer [START_REF] Wanko | Biological Processing Capacities and Biomass Growth in Waste Water Treatment by Infiltration On two Kinds of Sand[END_REF], and then uptaken by the increased biomass (Table 4) [START_REF] Healy | Treatment of dairy wastewater using constructed wetlands and intermittent sand filters[END_REF][START_REF] Rodgers | Organic carbon removal and nitrification of high strength wastewaters using stratified sand filters[END_REF]. The cumulative particulate pollutants and heterotrophic biomass growth on material over time are expected to contribute to increased TSS removals [START_REF] Bandick | Field management effects on soil enzyme activities[END_REF], and the hydrolysis and assimilation by heterotrophic biomass also played a key role in organic pollutant removals [START_REF] Anjaneya | Decolourization of Amaranth dye by bacterial biofilm in batch and continuous packed bed bioreactor[END_REF][START_REF] Hedström | Adsorption and desorption of ammonium by clinoptilolite adsorbent in municipal wastewater treatment systems[END_REF][START_REF] Siegrist | Wastewater-induced soil clogging development[END_REF]. CA1 and CA2 are relatively coarser media but a lower circularity and roundness coefficients (table 1) leads to a lower organization of the stack and the presence of fine particles reducing pore spaces and increasing surface area. This particularity of crushed aggregate increase efficiency with an accumulation of retained particles. Crushed aggregates exerted no significant impact on either TSS or organic removals, provided an adequate stabilization time has been allocated (i.e. more than 60 days of operations). Consequently, the physical characteristics of materials may influence COD and TSS removals either directly or indirectly (particulate or dissolved organic carbon catabolism affected by the velocity of microbial implantation on the material). The small size could explain the higher TSS retention as well as the higher bacterial implantation due to a greater specific surface area. Results from this study indicated that for a filter medium with an average grain size of less than 1.6 mm (i.e. D 10 < 0.44 mm, CA2), 30-cm media were sufficiently effective for TSS removals [START_REF] Rodgers | Different depth sand filters for laboratory treatment of synthetic wastewater with concentrations close to measured septic tank effluent[END_REF][START_REF] Schwager | Transport phenomena in intermittent filters[END_REF]. A narrower range for D10 (i.e. 0.2 mm < D10 < 0.4 mm) so as to ensure an effective purification process have also suggested [START_REF] Liénard | Choice of the sand for sand filters used for secondary treatment of wastewater[END_REF].

As for the COD removal, the material's physical characteristics could therefore impact ammonium removals both directly (portion of ammonium linked to TSS) and indirectly (ammonium oxidation affected by the microbial implantation on the material). An earlier and faster ammonium removal by RS1 (over the first 60 days) was due to assimilation by both bacterial growth (heterotrophic and autotrophic flora) and TSS retention, which takes place higher in the top layer of river sand. On the other hand, TN removal is limited (to around 20%) for all materials (even lower for RS2) with no improvement in incubation time (Fig. 2e). Rodgers et al. [START_REF] Healy | Treatment of dairy wastewater using constructed wetlands and intermittent sand filters[END_REF] have demonstrated that unsaturated sand filters often yield low total nitrogen removals due to a slight denitrification under aerobic conditions. Relatively higher TN removals have been observed on a real scale constructed of wetland and soil trenches (Table 4) [START_REF] Kristiansen | Sand-Filter Trenches for purification of septic tank effluent: I. The clogging mechanism and soil physical environment[END_REF][START_REF] Ardakani | Oxidation of ammonium to nitrate in a soil column[END_REF]: the presence of local suitable conditions for denitrification was facilitated by the long-term accumulation of organic matter (particulates and biomass). In the four reactors studied, results indicate that the denitrifying bacteria activities never take place inside the reactor due to overly-saturated conditions. However, the relatively higher phosphate removals by four materials during the earlier operation (Day 0 to Day 360) might be related to the biological assimilation. No adsorption capacity was shown with any of the materials, due to their nature of material surface, which contains mainly the oxide of silica and results in slightly negative surface charge [START_REF] Jacobs | Kinetic adhesion of bacterial cells to sand: Cell surface properties and adhesion rate[END_REF]. Authors suggested that a stratified packed bed reactor using dolochar as filter material is capable for phosphate removal due to its composition, which contains mainly the carbonates of calcium and magnesium; the prediction breakthrough and exhaustion time can be relayed by increasing bed thickness and initial phosphate concentration or by decreasing the flow rate using several bed column models [START_REF] Rout | Evaluation of kinetic and statistical models for predicting breakthrough curves of phosphate removal using dolochar-packed columns[END_REF].By drawing another comparison between RS1 and CA1 with a 70-cm medium, the material characteristics revealed a major influence on total nitrogen removal efficiency. Using the 70-cm reactor packed with RS1, the improved removal rate (39% [21%, 58%] for RS1, 22% [4%, 36%] for CA1) was due to the more extensive denitrification within the fine medium under low hydraulic loading. Relatively saturated conditions ( [START_REF] Rodgers | Different depth sand filters for laboratory treatment of synthetic wastewater with concentrations close to measured septic tank effluent[END_REF][START_REF] Pell | Infiltration of Wastewater in a Newly Started Pilot Sand-Filter System: III. Transformation of nitrogen[END_REF]%, oxygen gas variation, see Table 2), when the effluent inlet arrives and stagnates at the surface of the fine uniform medium (Table 1), were temporarily created. Moreover, the pore seal caused by an accumulation of particulates and biomass further improved the appearance of local anoxic microsites and favoured denitrification [START_REF] Kristiansen | Sand-Filter Trenches for purification of septic tank effluent: I. The clogging mechanism and soil physical environment[END_REF]. The denitrification condition was sensitive to a point that increasing HLR led to limiting denitrification by reducing the corresponding HRT value (23% for RS1 at 20 cm/day and 39% for RS1 at 12 cm/day).

In terms of biomass distribution which plays an important role in purification and pore sealing, both the retention of particulate matters and the straining of bacterial cells suggested that RS1, mechanical filtration is an active surface phenomenon over the top part of the filter (organic matter is included in TSS) thanks to its fineness, and soluble substrate is a limited factor to colonization along the column depth where substrates, bacterial cells and oxygen are relatively concentrated compared to the deeper part [START_REF] Rodgers | Different depth sand filters for laboratory treatment of synthetic wastewater with concentrations close to measured septic tank effluent[END_REF][START_REF] Bahgat | Sand-filters used for wastewater treatment: buildup and distribution of microorganisms[END_REF]. In contrast, this surface filtration phenomenon does not exist with crushed aggregate (CA1 and CA2). Both revealed a slightly higher penetration of particulates and cells into the beds. Their heterogeneous grain sizes (UC = 5~10, while UC = 2 for RS1) yields larger pores and a smaller specific surface area, allowing the bacteria to penetrate and also be retained in the deeper part of the filters: the distribution of heterotrophic bacteria appear to be homogeneous in the top 15 cm to the deeper layer even if VDW is higher at the top.

More, the colonization of heterotrophic bacteria at the top is lower in CA1 and 2 than in RS1 due to smaller specific surface area. This could impact the clogging velocity at long term at the top layer the more thickness is the biofilter the more efficient it is (table4 COD%). On the other hand, the concentration of autotrophic nitrifying bacteria in RS1 suggests that their distribution is also limited by another factor: oxygen content. The lower oxygen gas content (Table 2) in RS1 during batch operations (varies from 20% to 11% during the rest-batch sequence) indicates a relatively saturated environment and the lack of access to oxygen in the deeper layer. Otherwise, the relatively deeper implantation of nitrifying bacteria in crushed aggregates might be explained by :i) the size heterogeneity and angularity of crushed aggregate, which allow for bacterial migration; ii) the competition with heterotrophic bacteria for NH 4 + is lower (less viable heterotrophic bacteria after 0-5 cm); and iii) the porosities are relatively higher and the media less saturated (e.g. CA1, see Table 2). Some authors have pointed out that nitrifying bacteria are most active in the 10-12 cm below the infiltrative surface [START_REF] Jenssen | Technology assessment of wastewater treatment by soil infiltration systems[END_REF][START_REF] Hedström | Adsorption and desorption of ammonium by clinoptilolite adsorbent in municipal wastewater treatment systems[END_REF]. However, with a more permeable material, nitrification also occurs between depths of 0.3 and 1 m in the medium [START_REF] Bahgat | Sand-filters used for wastewater treatment: buildup and distribution of microorganisms[END_REF].

So, soluble substrate (organic) are metabolized with similarly efficacy from the top to the deeper part in CA biofilter. This is confirmed by the FDA activity upon the biofilter level at day 360 in CA1 and 2 (figure 5). FDA (fluorescein-diacetate (3'-6' diacetyl-fluorescein)) is used for determining total heterotrophic microbial activity. Nevertheless, colonization of the filter by heterotrophic bacteria is not so homogenous: at day 60 and especially day 210, the implantation of bacteria starts and follow the accumulation of TSS: distribution of heterotrophic bacteria appears to be higher in the top 15 cm than the deeper layer (figure 5). At the first step of colonization in the CA biofilter distribution of heterotrophic bacteria is similar to the stationary step of RS1.

Conclusion

From the above, bacteria influence the treatment efficiencies (through the final removal value and stabilization time, as described above) of soluble pollutants, especially given that heterotrophic metabolisms remove organic carbon and may cause denitrification in an anoxic environment, while the nitrification metabolism removes ammonium ions. Bioreactor with RS1 material should be more favourable to the denitrification (more heterotrophic bacteria, more anoxic) whereas bioreactor with CA material should be suitable to nitrification. The crushed aggregate, as a potential substitution for river sands whose deposits are becoming depleted, may provide similar treatment functionality, namely suitable pollutant removals and biomass support.

References TABLES

( 3 -)NO 2 -,

 32 EN ISO 11905-1); ammonium (NH 4 + ) and nitrates (NO were found by applying the indophenol blue method and dimethylphenol method (EN ISO 7890-1), respectively; and lastly orthophosphates (PO 4 3-) were determined by the phosphomolybdenum blue method (EN ISO 6878). Suspended solids were measured by weight loss after heating to 105°C for 24 h. The feed water and treated effluent were stored in open systems; hence, the dissolved oxygen (O 2 ) and nitrite (DIN EN 26777) values were not recorded. An oxygen gas evolution test over 24 h

1 M

 1 at pH = 8.0) by sonication for 15 min. The suspended slurry was then diluted and inoculated with nutrient agar for aerobic heterotrophic flora (n=2). The nitrifying bacteria were also assessed with a liquid medium(ISO 4831:2006). 200 µL of diluted extracted sample were added to 1.8 mL of liquid ammonium medium (N-NH 4 , 0.6 g/L) that also contained bicarbonate anions (NaHCO 3 , 1 g/L) at pH 8.2. After 72 hours of incubation at 30°C, the presence of nitrites was revealed by adding sulfuric acid and alpha-naphtylamine (reagent Nit 1 and Nit 2 of analytical profile index: API20E) into each tube. In the absence of nitrites, zinc powder was added to recover the nitrates (n=5). FDA (fluorescein-diacetate (3'-6' diacetyl-fluorescein)) hydrolytic activity was determined by the enzymatic assay procedures[START_REF] Pell | Infiltration of Wastewater in a Newly Started Pilot Sand-Filter System: III. Transformation of nitrogen[END_REF]. A 0.5-g mass of colonized CA materials was sampled from 0~5 cm, 10~15 cm and 30 cm.0.5g. Material was placed into a 50 mL Erlenmeyer flask. 20 mL of 60 mM sodium phosphate buffer (pH 7.6) and 100 µL of 4.8 mM FDA were added into the flask. Samples were incubated for 60 min on a shaker at room temperature. After 60 min, 10 mL of acetone were added to terminate the hydrolysis reaction. Samples then were centrifuged for 5 min and Fluorescein (the product of FDA hydrolysis) concentration was determined spectrophotometrically at 490 nm.The phosphate sorption kinetic tests were also performed on all four materials. 200mL of phosphate solution of 20mg P-PO 4 3-/L (prepared from Na 2 HPO 4 ) at pH=7 was added to 20g of each material. The batches were mixed at 110 rpm during 24 hours. The mixture were sampled at 0, 30min, 1h30, 5h, 10h and 24h, and the P-PO 4 3-concentrations were measured with 0.45µm filtrates.

[ 3 .FIGURESFigure 1 :

 31 FIGURES

Figure 4 :

 4 Figure 4: Total nitrogen removal efficiencies with 70-cm reactors for two materials under different hydraulic loadings

Table 1 :

 1 Packing material characteristics

	Filter material	River sand 1 (RS1)	River sand 2 (RS2)	Crushed aggregate 1 (CA1)	Crushed aggregate 2 (CA2)
	Picture
	Effective size D 10	0.38	1.60	0.17	0.44
	(mm)
	Average diameter D m	0.82	2.26	1.36	1.6
	(mm)
	Uniformity coefficient	2.78	1.75	10	5
	(UC) (D 60 /D 10 )
	Circularity	0.849 (±0.060)	0.819 (±0.037)	0.824 (±0.088)	0.800 (±0.109)
	Roundness	0.74 (±0.11)	0.76 (±0.10)	0.73 (±0.15)	0.67 (±0.15)
	Fine particles (%)	0.4%	0.5%	5%	2.4%
	Porosity [min, max]	[30%, 33%]	[37%, 41%]	[38%, 41%]	[38%, 44%]
	Specific surface area
	(m 2 /kg)	4.04	1.18	2.78	2.88
	Estimated hydraulic	[8.25~9.53]×10 -4	-	[2.79~2.88]×10 -4 [7.49~9.73]×10 -4
	conductivity (m/s)

Table 2 :

 2 Reactors and feeding conditions354

Table 4 :

 4 Historic data of similar processes aimed at the treatment efficiencies of key

	363
	364	pollutants
	365
						Removal efficiencies (%)
		Reference	Location	Process type	Operating conditions	COD or BOD 5	SS	NH 4 TN + or
				Vertical
		Molle et al. (2003)	France	flow constructed	D 10 =0.22 mm, UC=5.1	84 on COD	95	85 on TN
				wetland
		Torrens et al. (2009)	France	Sand filter	D 10 =0.25 mm, UC=4.7, depth=25 cm of RS	42 on COD	63	82 on NH 4 +
				Planted
		Neralla et al. (2000)	USA	horizontal flow constructed	30 cm of 0.95-cm gravel media	84 on BOD 5	70	-
				wetland
		Van Cuyk et al. (2000)	USA	Soil lysimeter	D 10 =0.22 mm, UC=2.7, depth=60 cm of aggregate free soil	97 on COD / 83 on BOD 5	84	3 on TN / 100 on NH 4 +
		Elbana et al. (2012)	USA	Sand filter	D 10 =0.32 mm, of RS UC=3.2, depth=50 cm	-	60	-
		Gill et al. (2009)	Ireland	Soil trench	depth=30 cm of sandy soil	77 on COD	-	59-89 on TN
		Rodgers et al. (2011)	Ireland	Sand filter	D 10 =1 mm, UC=2.95, depth=30 cm of RS	82 on COD	74	92 on NH 4 +
		Pramanik et al. (2015)		Sand filter	Depth = 50 cm of RS	45 on DOC	-	89 on NH 4 +
	366
	367

Table 5 :

 5 Treatment efficiencies (% removals) of filtration reactors with two selected materials (RS1 and CA1) under different operating conditions

			Removal (%) and NO 3 values (average value [min, max], n = 18-22)
	Operating conditions	TSS%	COD%	N-NH 4	+ %	-(mg N/L) NO 3	Tot-N%
	HLR	Thickness
	(cm/day)	(cm)

Table 6 :

 6 Distribution of total organic matter in four materials at 360 days

	Layer: VDW
	(mg/g of	RS1	RS2	CA1	CA2
	aggregate)
	0~5 cm	12.49	3.69	10.03	8.01
	10~15 cm	4.10	2.19	4.81	5.21
	30 cm	2.98	1.63	3.39	3.56
	VDW decreasing from 0~30 cm (%)	76%	56%	66%	56%

Table 7 :

 7 Distributions of heterotrophic and nitrifying bacteria throughout the depth of three

	materials at 360 days
	Heterotrophic flora (CFU*/g of material)	RS1	CA1	CA2
	0~5 cm	4.7×10 7 [2.5×10 7 ; 6.9×10 7 ]	4.28×10 6 [2.2×10 6 ; 6.3×10 6 ]	2.30×10 6 [1.2×10 6 ; 3.4×10 6 ]
	10~15 cm	4.98×10 7 [2.6×10 7 ; 7.3×10 7 ]	2.98×10 6 [1.6×10 6 ; 4.4×10 6 ]	1.25×10 6 [6.6×10 5 ; 1.9×10 6 ]
	30 cm	5.05×10 5 [2.7×10 5 ; 7.5×10 5 ]	2.93×10 6 [1.5×10 6 ; 4.3×10 6 ]	1.66×10 6 [8.7×10 5 ; 2.4×10 6 ]
	Nitrifying flora (MPN*/g of material)	RS1	CA1	CA2
	0~5 cm	20 [12; 48]	130 [78; 312]	50 [30; 120]
	10~15 cm	6.00×10 3 [3.6×10 3 ; 1.4×10 4 ]	1.80×10 3 [1.1×10 3 ; 4.3×10 3 ]	3.50×10 3 [2.1×10 3 ; 8.4×10 3 ]
	30 cm	N/A	[5.4×10 2 ; 2.2×10 3 ] 9.00×10 2	6.00×10 3

Reactor number in Fig. Abs at 490 nm AU/g material Abs at 490 nm AU/g material A B


María Cevallos-Almeida

Laure Martin

Catherine Houdayer

Valerie Rose

Jean-Marie Guionnet

Frédéric Paboeuf

Martine Denis

Annaëlle Kerouanton
email: annaelle.kerouanton@anses.fr

Experimental infection of pigs by Salmonella Derby, S. Typhimurium and monophasic variant of S. Typhimurium: Comparison of colonization and serology

Keywords: Salmonella, Typhimurium, Derby, monophasic, Pig, colonization, antibody

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

Introduction

Salmonella (or S. enterica, subspecies enterica) is responsible for human and/or animal salmonellosis and is one of the most broadly-distributed foodborne pathogens worldwide. In Europe, it is the second-ranked cause of zoonosis after Campylobacter sp., with a notification rate of 21.2 cases per 100,000 population [START_REF] Efsa | The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2015[END_REF]. In France, Campylobacter spp., Salmonella spp., and norovirus were responsible for 73% of all foodborne illnesses and 76% of all associated hospitalizations [START_REF] Van Cauteren | Estimated Annual Numbers of Foodborne Pathogen-Associated Illnesses, Hospitalizations, and Deaths, France, 2008-2013[END_REF]. S.

Typhimurium is recognized as the predominant serovar isolated from humans in Europe and S. Derby was one of the ten serovars most frequently isolated from humans in different countries [START_REF] Boyen | Non-typhoidal Salmonella infections in pigs: a closer look at epidemiology, pathogenesis and control[END_REF][START_REF] Efsa | The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2015[END_REF]. The monophasic variant of Salmonella Typhimurium is an emerging serovar in human infections around the world [START_REF] Switt | Emergence, distribution, and molecular and phenotypic characteristics of Salmonella enterica serotype 4,5,12:i[END_REF][START_REF] Weaver | Longitudinal study of Salmonella 1,4,[5],12:i:-shedding in five Australian pig herds[END_REF]. Source attribution studies have established that pork has a key role in foodborne outbreaks of human salmonellosis, because in several investigations many isolates detected in pigs were responsible for these human cases [START_REF] Bonardi | Salmonella in the pork production chain and its impact on human health in the European Union[END_REF]. Pigs can be infected by several Salmonella serovars and the occurrence of these serovars is also partly geographically-determined [START_REF] Boyen | Non-typhoidal Salmonella infections in pigs: a closer look at epidemiology, pathogenesis and control[END_REF][START_REF] Xu | Phenotypic Characteristics and Genetic Diversity of Salmonella enterica Serotype Derby Isolated from Human Patients and Foods of Animal Origin[END_REF]. S. Typhimurium, S.

Derby, and the monophasic variant of S. Typhimurium are nowadays the most frequently isolated serovars from swine and pork products in the European Union [START_REF] Bonardi | Salmonella in the pork production chain and its impact on human health in the European Union[END_REF][START_REF] Efsa | The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2015[END_REF][START_REF] Gosling | Observations on the distribution and persistence of monophasic Salmonella Typhimurium on infected pig and cattle farms[END_REF]. In France, S. Derby and S. Typhimurium are also the most frequently isolated serovars in pig production [START_REF] Denis | Distribution of serotypes and genotypes of Salmonella enterica species in French pig production[END_REF] and, in the last ten years, the French Salmonella Network has observed an increase in the diagnosis of monophasic serovars in animal feed and environmental sources (Bugarel et al., 2012b).

Differences in the excretion and immune response in pigs have been reported between Salmonella serovars [START_REF] Ivanek | Salmonella fecal shedding and immune responses are dose-and serotype-dependent in pigs[END_REF][START_REF] Osterberg | Direct and indirect transmission of four Salmonella enterica serotypes in pigs[END_REF], but these studies have never included the monophasic variant of S. Typhimurium.

While all three serovars are present in pork products prior to ingestion, serovar Typhimurium and its monophasic variant are more widely implicated in human salmonellosis. In addition, the infection produced by S. Derby seems to be less severe than that produced by S. Typhimurium [START_REF] Ivanek | Salmonella fecal shedding and immune responses are dose-and serotype-dependent in pigs[END_REF][START_REF] Matiasovic | Immune response of pigs to Salmonella enterica serovar Derby and Typhimurium infections[END_REF][START_REF] Osterberg | Patterns of excretion and antibody responses of pigs inoculated with Salmonella Derby and Salmonella Cubana[END_REF]. To better understand the duration and dynamics of fecal shedding and seroconversion after infection with these three pig-associated serovars, we carried out an experimental trial, which was conducted with the same parameters as those previously used for an experimental trial on monophasic S. Typhimurium [START_REF] Cevallos-Almeida | Colonization of Pigs Experimentally Infected with a Monophasic Variant of Salmonella Typhimurium[END_REF].

Materials and methods

Strains

Three strains of Salmonella from the ANSES laboratory's private collection were used to inoculate the piglets. The strains had been isolated in pigs' fecal samples at farm level or in their mesenteric lymph nodes at the slaughterhouse (Table 1). To ensure that strains could be easily enumerated during the trial, they were made resistant to Rifampicin (Rif) after several passages on XLD agar (Biokar, France) supplemented with Rifampicin (100 mg/lt, Sigma, France). Rif strains were checked to confirm similarity to parental strains using conventional serotyping, PFGE typing [START_REF] Ribot | Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet[END_REF] and antimicrobial susceptibility testing (data not shown).

Experimental design

This in vivo trial protocol was approved by the Ethics Committee on Animal Research No. 16, of the French Ministry of National Education, Higher Education and Research (APAFiS license no. 7689-2016112311346069). The trial was conducted on 28 secondary specificpathogen-free (SPF) Large White piglets born at ANSES Ploufragan's protected animal facilities. The secondary SPF piglets are naturally born from SPF sows controlled for presence of several pathogens (Le [START_REF] Devendec | Development of a pig infection model with colistin-resistant Escherichia coli[END_REF].

At 4 weeks of age, three weeks before inoculation, the piglets were weaned and placed in separate hermetic experimental animal rooms of biosecurity level 3. The 24 piglets intended to be inoculated were placed in three separate rooms, with two pens per room (eight piglets per room, four per pen). Each room was considered a group. In Groups SDb, ST, and mST, piglets were inoculated by S. Derby, S. Typhimurium and the monophasic variant of S.

Typhimurium respectively.

Staff at the animal facility complied with all the necessary biosecurity measures. The piglets were monitored daily; their rectal temperature was taken and any clinical manifestations were recorded. Food consumption and body weight were reported weekly.

Experimental Inoculation

Seven days before inoculation, the 28 piglets were tested and confirmed to be free of Salmonella. Fecal samples from each piglet were analyzed in accordance with the NF U47-102 method [START_REF] Afnor | Animal health analysis methods -Isolation and identification of any Salmonella serotype or of specified Salmonella serotypes among mammals[END_REF]. On day 0, the 24 piglets of seven weeks of age were inoculated as previously described for an earlier experimental study [START_REF] Cevallos-Almeida | Colonization of Pigs Experimentally Infected with a Monophasic Variant of Salmonella Typhimurium[END_REF]. The inoculum were prepared after an overnight culture of the three strains at 37°C. Cultures were adjusted in Tryptone Salt Broth (SLT) (bioMérieux, France), after a DO measurement, in order to obtain an inoculum of about 10 8 CFU/ml (8Log10CFU/ml). After numeration, inoculum concentrations were 8. 23, 8.40, 8.19 Log10CFU/ml respectively for SDb, STM and vmST. Then, piglets were inoculated orally, using a cannula connected to a screw syringe, with 10 ml of the inoculum. The four control piglets were given 10 ml of SLT orally.

Sampling and necropsy

Fecal samples (at least 30 g) were taken directly from the animal rectum on 1, 2 and 3 DPI in the first week, and then twice a week until 49 DPI.

To determine seroconversion, blood samples were taken on the same dates as fecal samples.

The blood was then centrifuged at 3500 rpm for 5 minutes to recover the serum in order to determine anti-Salmonella antibody levels. These sera were stored at -20°C until they were analyzed.

The three groups were necropsied at different ages: 21 DPI for 14 piglets (four in each group) and 49 DPI for the remaining piglets. The four control piglets were kept in a separate room and also slaughtered at different ages: two piglets on 21 DPI and two piglets on 49 DPI.

Piglets were euthanized through intravenous injection with an overdose of tiletamine and zolazepam (Zoletil 100, Virbac, France). The subsequent necropsies primarily examined organs and tissues in the abdominal and thoracic cavities. The tonsils, mesenteric lymph nodes (MLN) and intestinal contents of the duodenum, jejunum, ileum and cecum were collected.

Enumeration and detection of Salmonella

All the analyses were performed as previously described [START_REF] Cevallos-Almeida | Colonization of Pigs Experimentally Infected with a Monophasic Variant of Salmonella Typhimurium[END_REF].

Briefly, fecal and necropsy samples were diluted 1:10 in Buffered Peptone Water (BPW, bioMérieux, France). Serial dilution was then performed in the SLT until a dilution of 10 -4 was obtained. One milliliter of the 10 -1 dilution was seeded on three plates of XLD supplemented with rifampicin (XLD-Rif), and 100 µl of each of the subsequent dilutions was plated on separate plates of XLD-Rif. After incubation at 37°C for 24 hours, typical black colonies of Salmonella were counted to enumerate Salmonella in terms of CFU/g for each sample. If enumerations for Salmonella sp. were negative due to being below the enumeration limit (10 CFU/g), the NF U47-102 method (AFNOR, 2008) was used to enrich the sample so as to confirm the presence or absence of Salmonella in the sample. This method was also applied to the samples collected from control piglets throughout the assay in order to confirm the sustained absence of Salmonella.

Antibody response to Salmonella

For antibody screening, we used the IDEXX Swine Salmonella Ab Test® (IDEXX, France). This indirect ELISA is based on detecting the Salmonella lipopolysaccharide (LPS) antigen.

In accordance with the manufacturer's recommendations, samples with an optical density (OD) equal to or greater than 15%were regarded as positive.

Data Management and statistical analysis

Statistical analysis was performed using R software (R version 3.2.4). The bacterial excretion level was compared between groups for each sampling point post-inoculation with a Tukey test (p<0.05). The CFU/g in feces from each sampling point was normalized logarithmically and plotted in order to calculate the cumulative area under the log curve (AULC) to determine total Salmonella shedding for each piglet over the course of the study. A Kruskal-Wallis test was used to compare the AULC between groups (p<0.05). For the data obtained from necropsy, we compared the contamination levels of each group at the two dates of necropsy with a Kruskal-Wallis Test (p<0.05).

Results

Clinical monitoring

No clinical signs were found in the control piglets. The clinical signs displayed by the inoculated piglets differed depending on the strain inoculated. Transitory diarrhea was found in all groups at least once: for Group SDb on 14 DPI, for Group ST on 7 DPI and 10 DPI, and for Group mST on 21 DPI. For Group mST, no fever was observed at any point during the trial. Furthermore, we found no statistical difference (p>0.05) between inoculated piglets and the control group in terms of weight or feed consumption.

Excretion in fecal samples

The control piglets remained negative for Salmonella in their feces throughout the trial. In Group mST, all the piglets were positive on all sampling days, and fecal excretion was continuous. For Group SDb, one piglet was negative on the day 21 after inoculation (Fig 2 .).

In Group ST, two piglets were negative; for one on the day 14 after inoculation (Fig 2 ) and, another one, on the day 45 after inoculation.

Mean excretion and standard deviation data for all groups are presented in Table 2.

We found a statistical difference by the Holm multiple comparison test (p<0.05) between the average excretion levels obtained in the groups on 14, 17 and 21 DPI (Figure 1). On 14 DPI, Group SDb differed from Group ST; on 17 DPI, Group SDb differed from Group mST; and on 21 DPI, Group mST differed from Groups SDb and ST.

The inoculated piglets in Groups ST and mST euthanized on 21 DPI showed an excretion peak on 1 DPI, with a variation in levels during the trial. Group SDb also showed a high excretion level on 1 DPI but the peak was on 14 DPI, linked to a case of diarrhea the same day (Figure 2). By comparing AULC measurements from the excretion curves of the 24 piglets over 21 DPI using the Kruskal-Wallis test, a significant difference (p=0.008) was evident between Group ST and Group mST (Figure 3).

Salmonella colonization in organs and intestinal contents

At each necropsy date, 100% of the cecum content and tonsil samples were colonized in every group. Between 25% and 75% of mesenteric lymph nodes were colonized depending on the serovar and the necropsy date. For all serovars, contamination levels in intestinal contents increased through the intestinal passage up until the ileum, then began to decrease in the cecum and fecal samples. For all serovars, the lowest levels of contamination were found in the duodenum (0.7 to 1.3 Log10CFU/g) and mesenteric lymph nodes (0.7 to 2.3 Log10CFU/g), while the tonsils were the most significantly contaminated (p<0.01) on both necropsy days, 21 and 49 DPI (4.7 to 5.7 Log10CFU/g) (Table 3). On the basis of the analysis performed at necropsy, we found no statistical difference between serovars, regardless of the type of sample, either on day 21 or on day 49 (p>0.05).

Antibody responses

The OD% means per day are shown in Table 1. Piglets produced detectable antibodies to the Salmonella LPS antigen, indicating detectable seroconversion, on 7 DPI for piglets in Groups SDb and mST, and 10 DPI for piglets in Group ST. The seroconversion of 100% of piglets was completed by different dates depending on the serovar: all piglets in Group ST had seroconverted by 28 DPI, piglets in Group mST by 31 DPI, and piglets in Group SDb by 38 DPI.

On 1 and 3 DPI, the bacterial excretion frequencies and levels were high in each group (Figure 4) and the frequency of seroconverting piglets started to increase in all groups from day 7. On day 49, 100% of piglets shedding Salmonella were shown to have seroconverted through an ELISA.

Discussion

Salmonella serovars commonly associated with swine are a major human health problem. In our study we experimentally inoculated three groups of piglets with strains of three major swine serovars: S. Derby, S. Typhimurium and monophasic S. Typhimurium. We compared excretion patterns, seroconversion and the colonization of several organs in the three groups of animals. We found differences in some clinical signs, such as diarrhea and fever in piglets. While all groups had transient diarrhea at least once, no fever was ever recorded in the mST group. In other studies comparing either infection by S. Typhimurium versus S. Yoruba, or S.

Derby versus S. Cubana, no clinical signs or diarrhea were observed [START_REF] Osterberg | Patterns of excretion and antibody responses of pigs inoculated with Salmonella Derby and Salmonella Cubana[END_REF][START_REF] Osterberg | Effects of a challenge dose of Salmonella Typhimurium or Salmonella Yoruba on the patterns of excretion and antibody responses of pigs[END_REF]. However, for S. Typhimurium, several clinical signs such as fever, diarrhea and anorexia were reported in animals inoculated with a dose of 10 9 CFU [START_REF] Boyen | Porcine in vitro and in vivo models to assess the virulence of Salmonella enterica serovar Typhimurium for pigs[END_REF].

The excretion dynamics for all groups showed that the highest level of excretion was reached on 1 DPI, except for the group inoculated with S. Derby. In this group, the excretion was also very high on day 1 DPI, but the maximum amount of Salmonella excreted was observed on 14 DPI. In the days following inoculation, Salmonella excretion varied depending on the serovar inoculated. Only piglets in the mST group were shown to shed Salmonella continuously throughout the experimental trial. In the SDb group, one piglet was negative for Salmonella on one day of sampling 21 DPI, but he was then necropsied. At necropsy, their tonsils, ileum and caecum were Salmonella positive. For ST group, two pigs had feces salmonella-free, one on 14 DPI and the other on 45 DPI. The two pigs became again salmonella shedders at the following sampling dates, indicating intermittent shedding.

By comparing total shedding (AULC), we found statistical differences between piglets inoculated with serovars S. Typhimurium and those inoculated with the monophasic variant of S. Typhimurium. The monophasic variant strain caused a higher excretion level than the S. Typhimurium strain. This study is the first comparison of colonization by S. Typhimurium versus that by monophasic S. Typhimurium. Even though the monophasic variant is genetically related to S. Typhimurium (Bugarel et al., 2012a;[START_REF] Soyer | Salmonella enterica serotype 4,5,12:i:-, an emerging Salmonella serotype that represents multiple distinct clones[END_REF][START_REF] Vieira-Pinto | Virulence Characterization of Salmonella Typhimurium 1,4,[5],12:i:-, the New Pandemic Strain[END_REF], further research appears necessary to establish the differences between these two serovars in their colonization of pigs.

On the other hand, no difference was observed between Sdb and ST or between Sdb and mST.

In 2012, Ivanek noted that pigs inoculated with the pig-associated serovars S. Typhimurium and S. Derby tend to shed Salmonella in shorter periods of time (both as part of continuous and intermittent shedding) than pigs inoculated with the feed-associated serovars S. Yoruba and S. Cubana detected in Sweden [START_REF] Ivanek | Salmonella fecal shedding and immune responses are dose-and serotype-dependent in pigs[END_REF]. However, pigs infected with S.

Typhimurium and S. Derby were more likely to (re-)enter an intermittent non-shedding state.

Consequently, they showed a tendency towards longer-lasting host infection overall, with a similar or longer total duration of fecal shedding.

A recent longitudinal study in pigs naturally infected with Salmonella Typhimurium indicates that this serovar may also cause high shedding rates among colonized pigs in natural conditions, particularly in younger animals [START_REF] Weaver | Longitudinal study of Salmonella 1,4,[5],12:i:-shedding in five Australian pig herds[END_REF].

At necropsy, on 21 and 49 DPI, the tonsils and cecum of all the inoculated piglets were systematically found to be contaminated by one of the three Salmonella serovars. A previous study demonstrated that several serovars can contaminate the tonsils and cecum 3 hours after inoculation [START_REF] Loynachan | Acute infection of swine by various Salmonella serovars[END_REF]. In 2004, Cote et al. also reported 100% colonization for the tonsils, cecum and MLN on 14 DPI [START_REF] Cote | Distribution of Salmonella in tissues following natural and experimental infection in pigs[END_REF]. On average, the occurrence of positive samples was the lowest in MLN, then in the contents of the duodenum. The jejunum and ileum were more highly colonized, with 87.5% and 95.8% respectively. These observed trends are the same regardless of the serovar used for inoculation. [START_REF] Matiasovic | Immune response of pigs to Salmonella enterica serovar Derby and Typhimurium infections[END_REF] showed that after S. Typhimurium inoculation, only the tonsils and lymph nodes remained culture-positive up until 28 DPI and only the tonsils and cecum remained culture-positive following inoculation with S. Derby. In 2010, Osterberg et al. compared direct and indirect transmission of S. Derby and S. Typhimurium. For indirect transmission, pigs were necropsied after 14 days in a contaminated environment. For S. Derby, the cecum content, colon tissue and ileocecal lymph node were found to be contaminated in only one pig out of six (16%). For S. Typhimurium, four out of six of the pigs (66%) were culture-positive for cecum and/or ileocecal lymph node samples. In terms of data for the monophasic variant, in a previous study we found similar results for the colonization of tonsils and intestinal contents, but MLNs were more highly contaminated, with 100% and 62.5% of the samples positive on 21 and 49 DPI respectively [START_REF] Cevallos-Almeida | Colonization of Pigs Experimentally Infected with a Monophasic Variant of Salmonella Typhimurium[END_REF]. In 2004, Loynachan detected that 75% of tonsils were culture-positive 3 hours after inoculation with the monophasic variant [START_REF] Loynachan | Acute infection of swine by various Salmonella serovars[END_REF]. Tonsils are also a common site of contamination at slaughter, with about 10% contaminated with Salmonella [START_REF] Van Damme | Quantification of hygiene indicators and Salmonella in the tonsils, oral cavity and rectal content samples of pigs during slaughter[END_REF][START_REF] Vieira-Pinto | Occurrence of salmonella in the ileum, ileocolic lymph nodes, tonsils, mandibular lymph nodes and carcasses of pigs slaughtered for consumption[END_REF], and are a major source of carcass contamination, along with the contents of the intestinal tract [START_REF] Berends | Identification and quantification of risk factors regarding Salmonella spp. on pork carcasses[END_REF].

For all serovars, the highest level of contamination in organs and intestinal contents was found in the tonsils, cecum and jejunum. The least contaminated sites were the lymph nodes and the duodenum. In 2008, Scherer et al. found similar amounts of Salmonella to those we discovered in the tonsils (4.4 Log10CFU/g) and ileum (2.7 Log10CFU/g) after inoculation with S. Typhimurium [START_REF] Scherer | Time course of infection with Salmonella typhimurium and its influence on fecal shedding, distribution in inner organs, and antibody response in fattening pigs[END_REF]. In a recent study in which pigs were inoculated with 1x10 9 CFU of a multidrug-resistant monophasic S. Typhimurium strain, Shippy et al. made contrasting observations after 7 DPI. Specifically, tissues including the ileocecal lymph nodes were more highly contaminated than cecal content, and the tonsils were the least contaminated organ, with a mean of 1 Log10CFU/g [START_REF] Shippy | Porcine Response to a Multidrug-Resistant Salmonella enterica serovar I 4,[5],12:i:-Outbreak Isolate[END_REF].

In our study, piglets seroconverted at different times depending on the serovar inoculated. We found 100% of seroconversion in group mST piglets by 31 DPI. Our results are similar to those of Lynch et al., who found that 85% of pigs had seroconverted by 28 DPI [START_REF] Lynch | Evaluation of an Alternative Experimental Infection Method, Which Closely Mimics the Natural Route of Transmission of Monophasic Salmonella Typhimurium in Pigs[END_REF]. For piglets in groups ST and SDb, seroconversion was 100% completed by 28 and 38 DPI. [START_REF] Matiasovic | Immune response of pigs to Salmonella enterica serovar Derby and Typhimurium infections[END_REF] previously described a lower immune response after infection with S. Derby than with S. Typhimurium. Another study revealed a different result, with seroconversion of 100% of pigs within two weeks of infection [START_REF] Osterberg | Effects of a challenge dose of Salmonella Typhimurium or Salmonella Yoruba on the patterns of excretion and antibody responses of pigs[END_REF]. For S. Derby our results are different to those found by [START_REF] Osterberg | Patterns of excretion and antibody responses of pigs inoculated with Salmonella Derby and Salmonella Cubana[END_REF], with 100% seroconversion after 21 days.

Conclusion

This study revealed similar profiles of Salmonella excretion and colonization regardless of the serovar used for experimental inoculation. The amount of excretion, however, was different between groups inoculated with S. Typhimurium 07CR095 and monophasic S. Typhimurium S12AK50. Seroconversion had occurred by different days depending on the serovar used for inoculation. Further research is necessary to establish the virulence gene patterns of the serovars in order to understand the differences in colonization and pathogenicity among serovars colonizing pigs.

Figure 1 :

 1 Figure 1: Average fecal excretion in Log10 UFC/g over 21 days; calculations based on the

Figure 2 :

 2 Figure 2: Kinetics of Salmonella excretion in Log10 UFC/g for the piglets in each group (8

Figure 3 :

 3 Figure 3: Comparison of AULC obtained for each group (21 days). SDb: S. Derby, ST:

Figure 4 :

 4 Figure 4: Comparison between the percentage (%) of Salmonella-seroconverted pigs (in

Acknowledgments

We gratefully acknowledge funding from SENESCYT-ECUADOR (Secretaría de Educación Superior Ciencia y Tecnología Del Ecuador; Programa de Becas para Docentes Universitarios http://www.educacionsuperior.gob.ec), and would also like to thank Christelle Fablet, from the ANSES Ploufragan Unit of Swine Epidemiology and Welfare Research, for her help in the statistical analysis.

1 MLN : mesenteric lymph node; 2 Minimal inhibitory concentrations (MIC) of antibiotics were determined for all strains using the broth dilution method with a customing Sensititre® plates (Trek diagnostic, Biocentric, France); 3 For MLVA, the 5-loci MLVA allele profiles were determined according to the method described by Larsson et al. [START_REF] Larsson | Development of a new nomenclature for Salmonella Typhimurium multilocus variable number of tandem repeats analysis (MLVA)[END_REF].