Keywords: Antimicrobial, Garlic, Microbiota, Pig AGP, antibiotic growth-promoters, CFU, colony forming units, PTS, propyl propane thiosulfinate, PTS-O, propyl propane thiosulfonate

A number of in vitro experiments were designed to evaluate the effects of two different industrial products, namely PROALLIUM-S-DMC and PROALLIUM-SO-DMC (DMC Research Center, Granada, Spain), obtained from garlic (Allium sativum) on the faecal microbiota of pigs. The effects of three different concentrations (50, 200 and 400 ppm) of the active compounds (PTS and PTS-O, respectively) from both industrial products on the gastrointestinal microbiota of pigs were tested. Growth medium without any additive (0 ppm) was used as control. Predominant bacterial groups (total aerobes, total anaerobes, lactobacilli, bifidobacteria, coliforms, enterobacteria, bacteroides and clostridia) were studied. Results showed that both PTS and PTS-O have significant (P<0.01) antimicrobial activity against every group studied, although enterobacteria and coliforms were the most affected populations (P<0.01). Time kill curves for Escherichia coli and Salmonella Typhimurium, two common pathogens of pigs, showed that both compounds had a bactericidal effect against these strains. For the bacterial groups here studied, the antimicrobial effect of PTS-O was significantly (P<0.001) stronger than that of PTS.

Trials in vivo are in course to study the potential use of these products as alternatives to antibiotics in pig feeds.

 4) enhanced uptake and use of nutrients through the thinner intestinal wall associated with antibiotic-fed animals (Visek, 1978;[START_REF] Anderson | Gut microbiology and growth-promoting antibiotics in swine[END_REF][START_REF] Dibner | Antibiotic growth promoters in agriculture: history and mode of action[END_REF]. However, evidence shows that antibiotic resistance genes can be transmitted from animal to human microbiota [START_REF] Greko | Safety aspects on non-use of antimicrobials as growth promoters[END_REF], and the detection of pathogenic bacteria resistant to a number of antimicrobial agents [START_REF] Aarestrup | Effects of termination of AGP use on antimicrobial resistance in food animals[END_REF] have resulted in the full ban of these additives in animal nutrition in the European Union from January 2006 onwards [START_REF] Anderson | Gut microbiology and growth-promoting antibiotics in swine[END_REF][START_REF] Dibner | Antibiotic growth promoters in agriculture: history and mode of action[END_REF]. This new situation has promoted the investigation on alternative new feed additives with the benefits of AGP but without their observed drawbacks. Some plant extracts have been proposed as potential alternative candidates to the use of AGP. Among those, garlic (Allium sativum) has been traditionally used for its antimicrobial, antifungal and antioxidant properties, which have been known for centuries. Recently, some stable well characterized active compounds have been industrially obtained from garlic so that they can be used for different purposes. Due to their antimicrobial activity, these garlic derived compounds might represent a useful alternative to AGP.

Accordingly, the work here described was designed to evaluate in vitro the effects of two of these garlic-derived compounds (PTS and PTS-O) on predominant faecal microbial populations of swine, and to determine the concentrations active against some of the most relevant populations of swine intestinal microbiota. Additionally, activity against Escherichia coli and Salmonella Typhimurium, two common pathogens of pigs, was also tested. The final aim was to explore the potential effectiveness of these compounds before testing in vivo. Concentrations of active compounds (PTS and PTS-O) in the products were 10.7% and 11.3%

for PROALLIUM-S-DMC and PROALLIUM-SO-DMC respectively, as determined by HPLC according to the procedure described by [START_REF] Iberl | Quantitative determination of allicin and alliin from garlic by HPLC[END_REF].

PTS and PTS-O are organosulfurate compounds obtained by decomposition of initial compounds present in garlic bulbs as alliin and allicin (Figures 1c and1d). PTS is the acronym for propyl propane thiosulfinate, and PTS-O for propyl propane thiosulfonate. Both PTS and PTS-O were incorporated in an inert commercial alimentary support (cyclodextrin) to produce PROALLIUM-S-DMC and PROALLIUM-SO-DMC, respectively. The activity of these products was first tested in pure cultures in vitro. Three different final concentrations of PROALLIUM-S-DMC (0.47, 1.88 and 3.76 mg/ml) and PROALLIUM-SO-DMC (0.44, 1.77 and 3.54 mg/ml) corresponding to 50, 200 and 400 ppm of active ingredients (PTS and PTS-O, respectively) in the growth medium were tested. Growth medium without any additive (0 ppm) was used as control.

2.2.-In Vitro Fermentation Procedure

Fermentations were conducted in triplicate in 50 mL sterile polypropylene tubes. The composition of the semi-defined medium used for fermentations is presented in Table 1 ( [START_REF] Smiricky-Tjardes | Dietary galactooligosaccharides affect ileal and total-tract nutrient digestibility[END_REF]. All components except the vitamins solutions were mixed before autoclave sterilization of the medium. Filter-sterilized vitamin solutions were added immediately before dispensing the medium. Aliquots (26 mL) of the medium were aseptically transferred into fermentation tubes (which contained the appropriate amounts of additive) under a stream of N 2 .

A c c e p t e d M a n u s c r i p t

Tubes with no additive were used as controls. Tubes were placed in a 37ºC incubator 30 min before inoculation.

Faeces from 3 pigs of approx. 20 kg live weight, receiving a cereal-based diet [composition (g/kg): barley meal, 695.00, defatted soy, 113.80, fish meal, 35.00, maize starch, 109.04, vit. + min. mix, 30.10, β-glucanase, 1.00, supplemented amino acids, 5.86, cellulose, 7.39, vegetable oil, 2.81], which met the nutritional requirements of piglets at this age, and free of any antimicrobial agent, were collected immediately after deposition in plastic bags, which were sealed after expressing excess air and maintained at -80ºC until inoculum was prepared.

Equal amounts of faeces from the three animals were thawed at room temperature and immediately diluted 1:10 in 150 mM NaHCO 3 buffer adjusted to pH 7.4 (1g of faeces in 9 ml of buffer), and mixed in a stomacher for 2 min. Blended, diluted faeces were filtered through miracloth (Calbiochem, 475855) and sealed in serum bottles under a N 2 stream.

Appropriate sample (n= 3) and control (n= 3) tubes were aseptically inoculated with diluted faeces. Four millilitres of inoculum were added to 26 ml of medium (see above) in each tube under N 2 stream. Sealed tubes were incubated at 37ºC with periodic mixing for 24 h. After this time, tubes were placed into a -80ºC freezer until used.

2.3.-Microbial Analyses

Fermentation samples were thawed at room temperature and immediately serially diluted with sterile buffered peptone water (BPW) (Cultimed, 413795) and plated in triplicate to be used in colony-counting assays. The BPW dilutions were used for counts of bacteroides, clostridia, bifidobacteria, lactobacilli, coliforms, enterobacteria, total aerobes and total anaerobes, by using the following media and procedures:

Clostridia: Reinforced clostridial agar (Oxoid, CM0151B) with 20 mg/ml of polymixin B (Sigma, P4932) after incubation at 37ºC for 48 h. Lactobacilli and Bifidobacteria: Man, Rogosa and Sharpe (MRS) agar (Oxoid, CM 0361) for lactobacilli and the same media supplemented with 0.5 mg/l dicloxacillin sodium salt hydrate (Sigma, D9016), 1g/l LiCl (Sigma, L9650) and 0.5 g/l L-cysteine hydrochloride (Sigma, C1276) for bifidobacteria. Incubations were performed at 37ºC for 48 h under anaerobic conditions.

Enterobacteria and Coliforms: 3M Petrifilm Enterobacteriaceae Count (EB) plates and 3M Petrifilm Coliform Count (CC) plates respectively. Incubations were performed at 37ºC for 24 h.

Total aerobes: 3M Petrifilm Aerobic Count (AC) plates after incubation at 37ºC for 24 h.

Total anaerobes: Brain Heart Infusion agar (Oxoid, CM0375) after incubation at 37ºC for 72 h.

2.4.-Bacterial Strains

Escherichia coli ATCC 25922 and Salmonella Typhimurium ATCC13311 were donated by DMC Research Center SL. Mixed cultures of enterobacteria and coliforms were obtained by isolation of these bacterial groups from pig faeces in 3M Petrifilm Enterobacteriaceae Count (EB) plates and 3M Petrifilm Coliform Count (CC) plates respectively.

2.5.-Minimal Inhibitory (MIC) and Minimal Bactericidal Concentration (MBC) Determinations

MIC and MBC were performed as described by [START_REF] Bakri | Inhibitory effect of garlic extract on oral bacteria[END_REF]. PTS and PTS-O were serially diluted in sterile LB broth in microtitre wells and each inoculated with 10 µl of standardised cell suspension (2 x 10 6 CFU/ml). Wells were incubated at 37ºC overnight and the highest dilution where no growth was observed was recorded as the MIC. For MBC testing, aliquots (10 µl) of broth from wells containing no growth were plated onto LB agar and again incubated overnight at 37ºC. The highest dilution where no survivors were observed was recorded as the MBC. Controls were performed using sterile LB without PTS or PTS-O.

A c c e p t e d M a n u s c r i p t

2.6.-Time-Kill Curves

Two organisms, Escherichia coli and Salmonella Typhimurium (1x10 7 CFU/ml) were incubated in LB broth with PTS or PTS-O at a dilution equivalent to their MBC. Control suspensions without additives were incubated in parallel. At timed intervals, aliquots were removed, serially diluted and the number of survivors estimated on LB agar using a variation of the [START_REF] Miles | The estimation of the bactericidal power of the blood[END_REF] technique, which involves spotting 100 µl aliquots of serial dilutions onto agar plates and counting the colonies in each inoculated spot (n= 3).

2.7.-Statistical Analysis

Statistical analysis of data was performed by using SPSS software (SPSS 15.0 for Windows, SPSS Inc., 2006). The effects of the concentrations used [0 (control), 50 ppm, 200 ppm and 400 ppm] for both active ingredients (PTS, PTS-O) on microbial counts were analyzed by the leastsquares method using a model with one main effect (concentration). The effects of active ingredients (PTS, PTS-O) and concentrations (50, 200 and 400 ppm) on bacterial counts were analysed by the least-squares method using a model with two main effects (Additive and Concentration) and their interactions. An orthogonal polynomial procedure (Table 2) was used in concentrations (50, 200, 400 ppm) sum of squares into its linear and quadratic components. Two-way ANOVA analysis (Table 2) showed that PTS-O had stronger (P<0.001) antimicrobial activity than PTS for lactobacilli, bifidobacteria, bacteroides, clostridia and total anaerobes populations, while PTS was clearly more active (P<0.001) than PTS-O against enterobacteria, coliforms and total aerobes. The addition of 200 and 400 ppm of PTS and PTS-O caused similar effects in lactobacilli, bifidobacteria, coliforms, enterobacteria and total anaerobes populations. Bacteroides, clostridia and total aerobes showed a dose-dependent decrease in the presence of both compounds. Coliforms and enterobacteria were the groups most affected by the A c c e p t e d M a n u s c r i p t addition of PTS or PTS-O. A very significant decrease (P<0.001) in viable counts was found with the addition of 50 ppm of both additives in the media. Doses of 200 ppm or higher gave place to the disappearance of any viable cell of these genera in the conditions here used.

3.-Results

3.1.-Effects of PTS and PTS-O on Microbial

3.2.-Time Kill Curves in the Presence of PTS and PTS-O.

Time kill curves were performed for two common enteric pig pathogens, namely Escherichia coli and Salmonella Typhimurium (Figure 3). Both species showed identical MIC (100 ppm) and MBC (100 pm) values for PTS and PTS-O. For both E. coli and S. Typhimurium, PTS-O showed a higher (P<0.01) activity than PTS. Killing of E. coli and S. Typhimurium began almost immediately after incubation with PTS-O. After 5h of incubation, no viable cells were recovered.

E. coli was more sensitive (P<0.01) than S. Typhimurium to the presence of PTS, showing a drop greater than 5 log units in viability after 24 h of incubation. Control cultures without additives showed an exponential plus a stationary growth phase, with no drop in viability over the same period. Time kill curves were also performed for mixed cultures of enterobacteria and coliforms isolated from pig faeces. Results (Figure 4) showed that for both groups PTS-O showed a higher (P<0.01) activity than PTS. Coliforms were more sensitive (P<0.01) than enterobacteria to the action of both compounds. These results are in agreement with those obtained with pure cultures of E. coli and S. Typhimurium.

Cultures containing only the inert commercial alimentary support (where PTS and PTS-O were incorporated to produce the commercial preparations) as additive showed a behaviour similar to that of cultures without additives (data not shown).

4.-Discussion

Among the biologically active sulfur species in garlic, allicin has played the major role in garlic research. Allicin (Figure 1a) is formed from the chemically rather unreactive sulfoxide precursor A c c e p t e d M a n u s c r i p t alliin (Figure 1b) in a reaction catalysed by the C-S-lyase enzyme alliinase. Chemically speaking, allicin is a thiosulfinate, a reactive sulfur species which kills various bacteria, fungi, yeasts and even cancer cells. Decomposition and degradation of allicin results in a range of "second generation" compounds (diallylsufide, diallyldisulfide, diallyltrisulfide and diallyltetrasulfide) frequently found in garlic extracts, such as garlic oils. Other decomposition products of allicin include the dithiins 3-vinyl-3,4-dihydro-1,2-dithiin and 2-vinyl-2,4-dihydro-1,3-dithiin and more complex chemical structures, such as E-ajoene and Z-ajoene (Figure 1e). Follow-on reactions of allicin and polysulfides with intracellular thiols result in additional sulfur species, such as S-allylmercaptocystein. Each of these sulfur species exhibits its own chemical properties and biochemical activity. The central role of allicin in garlic chemistry is presently being challenged by a number of findings which have confirmed antibiotic and anticancer activities for diallylsulfides similar or even superior to the ones of allicin [START_REF] Münchberg | Polysulfides as biologically active ingredients of garlic[END_REF].

The interest on the beneficial properties of products derived from garlic has risen in the last decades due to its cardiovascular, antineoplastic and antimicrobial properties [START_REF] Harris | Antimicrobial properties of Allium sativum (garlic)[END_REF][START_REF] Ariga | Antithrombotic and anticancer effects of garlic-derived sulphur compounds: a review[END_REF]. In particular, a wide range of microorganisms including Grampositive and Gram-negative pathogenic bacteria, fungi, protozoa and viruses have been shown to be sensitive to crushed garlic preparations [START_REF] Ankri | Antimicrobial properties of allicin from garlic[END_REF][START_REF] Harris | Antimicrobial properties of Allium sativum (garlic)[END_REF][START_REF] Ruddock | Garlic natural health products exhibit variable constituent levels and antimicrobial activity against Neisseria gonorrhoeae, Staphylococcus aureus and Enterococcus faecalis[END_REF]. Antibacterial properties of garlic against some enteric bacteria have been previously described by [START_REF] Ross | Antimicrobial properties of garlic oil against human enteric bacteria: evaluation of methodologies and comparisons with garlic oil sulfides and garlic powder[END_REF]. These authors demonstrated that products such as garlic oil and garlic powder inhibited the growth of a range of gram-positive and gram-negative enteric bacteria, including both pathogenic and commensal bacteria.

Results showed that both PTS and PTS-O had a significant (P<0.05) antimicrobial effect in vitro against all populations of intestinal microbiota tested. The antibacterial activity of garlic is widely attributed to allicin, the major thiosulfinate in garlic [START_REF] Cavallito | Allicin, the antibacterial principle of Allium sativum. Isolation, physical properties and antibacterial action[END_REF].

A c c e p t e d M a n u s c r i p t

Thiosulfinates (as allicin and PTS) are organosulfur compounds originated by the transformation of S-alk(en)yl-L-cysteine sulfoxides (ACSOs) by the enzyme alliinase. ACSOs are cleaved by alliinase to sulfenic acids, ammonia, and pyruvate. Sulfenic acids are highly reactive and transient compounds. In fresh tissue, sulfenic acids with the same or different alk(en)yl substituents condense to form thiosulfinates. Thiosulfinates have been implicated as a principle source of the antiplatelet property of raw onion and garlic juice. In fact, allicin and PTS have a significantly greater antiplatelet potency than aspirin [START_REF] Briggs | Differential inhibition of human platelet aggregation by selected Allium thiosulfinates[END_REF].

More importantly in the present context, thiosulfinates derived from garlic have also shown antimicrobial activity against Gram-positive and Gram-negative bacteria (Yoshida et al., 1999). The main antimicrobial effect of thiosulfinates as allicin has been reported to be due to its chemical reaction with thiol groups of various enzymes such as the acetyl-CoA-forming system, consisting of acetate kinase and phosphotransacetyl-CoA synthetase [START_REF] Focke | Allicin, a naturally occurring antibiotic from garlic, specifically inhibits acetyl-CoA synthetase[END_REF]. RNA polymerase might also be a target for allicin. In Salmonella Typhimurium, allicin at bacteriostatic concentrations inhibits RNA synthesis [START_REF] Feldberg | In vitro mechanism of inhibition of bacterial cell growth by allicin[END_REF]. This same enzyme contains in E. coli a single sulfhydryl group which might react with allicin [START_REF] Ozolin | Specific modification of the alpha-subunit of Escherichia coli RNAS polymerase by monomercuric derivative of fluorescein mercuric acetate[END_REF].

The significance of allicin as a biological effector molecule is not only due to its high reactivity with low and high molecular weight thiols, but also to its accessibility resulting from high membrane permeability [START_REF] Miron | The mode of action of allicin: its ready permeability through phospholipid membranes may contribute to its biological activity[END_REF]. The condensation product of allicin, ajoene (Figure 1e), which has a similar oxygenated sulphur group also showed a broad-spectrum antimicrobial activity, inhibiting the growth of gram-positive and gram-negative bacteria. In this case, the disulfide bond in ajoene appears to be necessary for its antimicrobial activity [START_REF] Naganawa | Inhibition of microbial growth by ajoene, a sulphur-containing compound derived from garlic[END_REF].

Thiosulfonates (such as PTS-O) have only sporadically been studied to date. Oxidation of a disulfide or thiosulfinate are possible thiosulfonate formation pathways, although the reaction of certain C-S-lyase cleavage products might also result in this chemotype. Like thiosulfinates, A c c e p t e d M a n u s c r i p t thiosulfonates react readily with thiols, yet are less potent as oxidants. The antimicrobial activity of some thiosulfonates has also been previously reported. For example, pseudoallicin, a thiosulfonate found in garlic, possess about half the antibacterial activity of allicin.

Thiosulfonates, which possess a similar reactivity, are more stable than thiosulfinates [START_REF] Jacob | A scent of therapy: pharmacological implications of natural products containing redox-active sulfur atoms[END_REF].

Although the two compounds here assayed were active against all bacterial strains tested, either pathogenic or not, their activity was not the same. While PTS was more active against coliforms and enterobacteria, PTS-O showed higher activity against lactobacilli, bifidobacteria, bacteroides and clostridia (Figure 2). The groups most sensitive to the action of PTS and PTS-O were enterobacteria and coliforms, which were not detectable in the presence of the lower concentration of PTS here used (50 ppm). On the contrary, lactobacilli and bifidobacteria were the most resistant populations to the action of PTS and PTS-O. These results are in agreement with the antibacterial effects of garlic previously described in the literature. Thus, it has been shown that garlic products exert a broad-range of antimicrobial activity against gram-positive and gram-negative bacteria, including species of Escherichia, Salmonella, Klebsiella, Streptococcus, Lactobacillus, Bacillus, Bacteroides, Proteus [START_REF] Ross | Antimicrobial properties of garlic oil against human enteric bacteria: evaluation of methodologies and comparisons with garlic oil sulfides and garlic powder[END_REF], Staphylococcus, Micrococcus, and Clostridium [START_REF] Sharma | Antibacterial property of Allium sativum Linn. In vivo and in vitro studies[END_REF][START_REF] De Witt | Effect of garlic oil or onion oil on toxin production by Clostridium botulinum in meat slurry[END_REF][START_REF] Ruddock | Garlic natural health products exhibit variable constituent levels and antimicrobial activity against Neisseria gonorrhoeae, Staphylococcus aureus and Enterococcus faecalis[END_REF].

Even acid-fast bacteria such as Mycobacterium are sensitive to garlic (Uchida et al., 1975;[START_REF] Delaha | Inhibition of mycobacteria by garlic extract (Allium sativum)[END_REF]. Garlic extracts are also effective against Helicobacter pylori, the causal agent of gastric ulcers [START_REF] O'gara | Activities of garlic oil, garlic powder and their diallyl constituents against Helicobacter pylori[END_REF]. Also, [START_REF] Rees | A quantitative assessment of the anti-microbial activity of garlic (Allium sativum)[END_REF] documented that garlic exerts differential inhibition between beneficial intestinal microflora and potentially harmful enterobacteria, and lactic acid bacteria were the least sensitive microorganisms to the inhibitory effects of garlic. In mixed culture studies of Lactobacillus acidophilus and E. coli, garlic prevented the establishment of the last one.

A c c e p t e d M a n u s c r i p t

A differential effect of PTS and PTS-O was also reflected in the time kill curves performed in this study for E. coli and S. Typhimurium, two of the most important pathogens of pigs responsible for great economic losses due to mortality, morbidity, decreased growing rate, cost of medication, etc. [START_REF] Moxley | Comparative pathology of bacterial enteric diseases of swine[END_REF][START_REF] Fairbrother | Escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention strategies[END_REF]. Killing of both strains was much faster in the presence of PTS-O, and PTS was more effective against E. coli than it was against S. Typhimurium. Apart from reactivity and reaction stoichiometry, these differences are likely to be related with differences in the structure (Figure 1c and1d) or chemical stability of both compounds (see above). Also, the differential inhibition showed by PTS and PTS-O may be due to differing composition of bacterial membranes and their permeability to these compounds [START_REF] Miron | The mode of action of allicin: its ready permeability through phospholipid membranes may contribute to its biological activity[END_REF]. Anyway, the same results were obtained when time kill curves were performed for enterobacteria and coliforms isolated from pig faeces.

As the effects were not so strong as they were with identified strains, the bactericidal effect is probably not the same against all bacterial strains of these genera present in the pig intestine.

Based on the results found in the current study, further work is merited to establish the effectiveness and possible mechanism of action of both compounds in vivo. In fact, the results found in a preliminary in vivo trial carried out with a limited number of growing pigs (data not shown) were promising and comparable to those in vitro here described. Thus, the inclusion of PTS-O in an experimental pig diet free of antibiotics gave place to higher faecal counts of the lactic bacteria strains and lower of coliforms and enterobacteria than a commercial diet used as control which contained formic and propionic acids as antimicrobial agents.

  Garlic derivatives (PTS and PTS-O) differently affect the ecology of swine faecal microbiota in vitro Raquel Ruiz a , M. P. García b# , A. Lara b , L. A. Rubio a* a Dpto. de Fisiología y Bioquímica de la Nutrición Animal (IFNA, EEZ, CSIC), Profesor Albareda, 1, 18008 Granada, Spain. b Dpto. I+D/Area Biotecnología, DMC Research Center SL, Camino de Jayena s/n, 18620, Alhendín, Granada, Spain.

  Both therapeutic and growth-promoter antibiotics (AGP) have been regularly used in order to improve animal performance and to prevent gastrointestinal disorders. A number of different mechanisms have been proposed on the mode of action of AGP: (1) inhibition of sub-clinical infections, (2) reduction of growth-depressing microbial metabolites, (3) reduction of microbial use of nutrients and (

  -DMC and PROALLIUM-SO-DMC were the commercial preparations used in this study. Both products were provided by DMC Research Center SL, Granada, Spain.

  Bile aesculin agar (Oxoid, CM0888) after incubation at 37ºC for 24 h.

  Populations.Total counts of the microbial populations studied are shown in Figure2. The three different concentrations ofPTS (50, 200 and 400 ppm) tested resulted in a dose-dependent reduction (P<0.01) of all the microbial populations studied. Enterobacteria and coliforms populations were affected the most by the presence of PTS: both populations diminished below the detection limit even in the presence of the lower concentration of PTS used in this study (50 ppm). Bacteroides, A c c e p t e d M a n u s c r i p t clostridia, total aerobes and total anaerobes showed a dose-dependent reduction. Lactobacilli and bifidobacteria populations showed a similar pattern, i. e. both populations decreased in a dosedependent manner, but the effect of the addition of 400 ppm of PTS was not different (P= 0.0634) from the addition of 200 ppm. As shown in Fig 2, the effect of PTS-O was similar to the effect of PTS: bacterial populations showed a dose-dependent reduction with PTS-O addition (P<0.01). The populations most affected by the presence of PTS-O were enterobacteria and coliforms, and no detectable values were recorded with the addition of over 200 ppm of PTS-O. Lactobacilli and bifidobacteria showed a similar pattern to that found with PTS. Counts of both groups slightly decreased with the addition of 50 ppm of PTS-O, while the addition of 200 and 400 ppm of PTS-O gave place to the disappearance of one half of viable counts for both populations. Bacteroides and clostridia showed a dose-dependent reduction. These populations were more sensitive to the presence of PTS-O than they were to PTS, as species of these genera could not be detected in the presence of 200 ppm of PTS-O. Total aerobes population decreased significantly after addition of 50 ppm and 200 ppm of PTS-O, but the presence of 400 ppm of the additive caused the loss of more than a half of the group. Total anaerobes were more sensitive than total aerobes to the presence of PTS-O, this population showing a significant decrease in the presence of 200 ppm of PTS-O.

  In conclusion, the active ingredients of PROALLIUM-S-DMC and PROALLIUM-SO-DMC (PTS and PTS-O, respectively), two organosulphurate compounds industrially obtained by decomposition of initial compounds present in garlic such as alliin and allicin, have antimicrobial

  s c r i p t activity against every bacterial group of pig faces studied, although enterobacteria and coliforms were the most affected populations. Furthermore, both products had a bactericidal effect against Escherichia coli and Salmonella Typhimurium, two common pathogens of pigs.A c c e p t e d M a n u s c r i p t

  Figure 1.

  Figure 2.

Legend to Figure 4

 4 Figure captions

Table 1 .

 1 Composition of medium used for in vitro fermentation

	1

Table 2 .

 2 Effect of the type and concentration of additive on the bacterial counts [log 10 (CFU/g faeces)].

				Concentration (ppm) of active
		Additive		ingredient
									C 3
							Pooled
	Bacterial group 1	PTS	PTS-O	50	200	400	SEM	A x C 2 L	Q
	Lactobacilli	6.558 a	6.097 b	8.560 a	5.217 b	5.206 b	0.0369	*	*** ***
	Bifidobacteria	6.535 a	6.228 b	8.571 a	5.273 b	5.300 b	0.0326	***	*** ***
	Bacteroides	7.002 a	6.461 b	8.898 a	4.565 b	LOQ	0.0259	***	***	-
	Clostridia	6.402 a	8.697 b	8.706 a	4.089 b	LOQ	0.0277	***	***	-
	Coliforms	ND a	5.967 b	5.967	LOQ	LOQ	0.0063	-	-	-
	Enterobacteria	ND a	6.108 b	6.108	LOQ	LOQ	0.0230	-	-	-
	Total aerobes	6.529 a	7.337 b	8.989 a	7.815 b	3.995 c	0.0748	***	*** ***
	Total anaerobes	6.650 a	6.060 b	8.999 a	5.079 b	4.987 b	0.0491	NS	*** ***
	1 For each factor (additive, concentration), means in the same raw with different superscript letters differ (P<0.01). NS, not significant. LOQ,
	too low to count. 2 A x C: significance of interaction additive x concentration (*, P<0.05; ***, P<0.001). 3 Linear (L) and quadratic (Q) effects
	of concentrations (C). No quadratic affect could be analysed for variable A because there were only 2 additives (***, P<0.001).

Composition (mg/100mL): EDTA, 500; FeSO

.7H 2 O, 200; ZnSO 4 .7H 2 O, 10; MnCl 2 .4H 2 O, 3;

Acknowledgements

Authors are grateful to Drs. L. Lara and E. Rubio, for excellent technical assistance. R. Ruiz acknowledges receipt of an I3P contract.

Yoshida, H., Katsuzaki, H., Ohta, R., Ishikawa, K., Fukuda, H., Fujino, T., Suzuki, A., 1999.

Antimicrobial activity of the thiosulfinates isolated from oil-maceratedgarlic extract.

Biosci. Biotechnol. Biochem. 63 (3), 591-594.


Elena Castellanos

Beatriz Romero

Sabrina Rodríguez

Lucía De Juan

Javier Bezos

Ana Mateos

Lucas Domínguez

Alicia Aranaz
email: alaranaz@vet.ucm.es

Molecular characterization of Mycobacterium avium subspecies paratuberculosis Type II and III isolates by a combination of MIRU-VNTR loci

Keywords: MIRU, VNTR, Mycobacterium avium subspecies paratuberculosis, Type II, Type III

Mycobacterial interspersed repetitive units and variable number tandem repeats typing (MIRU-VNTR) is a useful technique that has been recently applied to characterize members of the Mycobacterium avium complex (MAC). The aim of this study was to examine the genetic variability among a collection of Spanish Mycobacterium avium subspecies paratuberculosis (M. a. paratuberculosis) isolates with a combination of MIRU-VNTR loci. For this purpose we tested six MIRU-VNTR loci (MIRU-2, MIRU-3, VNTR-25, VNTR-32, VNTR-292 and VNTR-259) in 70 M. a. paratuberculosis isolates of Types II and III that were recovered from 22 Spanish localities during a nineyear period (1998-2007). The combination of five loci (MIRU-2, MIRU-3, VNTR-25, VNTR-32 and VNTR-259) enabled the differentiation of 12 allelic profiles, with a resulting Hunter and Gaston discriminatory index (HGDI) of 0.84. Moreover, we obtained MIRU-VNTR patterns that were unique for each of the M. a. paratuberculosis

Types analyzed (II and III); other patterns were host-related or restricted to geographic areas. Therefore, this MIRU-VNTR approach could be a useful sub-typing molecular tool in order to get a better sense of the epidemiology of Johne's disease.

A c c e p t e d M a n u s c r i p t

Introduction

Mycobacterium avium subspecies paratuberculosis (M. a. paratuberculosis), the etiological agent of Johne's disease or paratuberculosis is difficult to isolate, often hindered by long-time incubation periods and culture growth requirements [START_REF] Cocito | [END_REF]de Juan et al., 2006).

Tandem repeats (TRs) are DNA elements dispersed throughout the genome and are divided into microsatellites (sequence repeats of 1-13 bp) and minisatellites (10-100 bp sequence repeats) [START_REF] Supply | Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome[END_REF]. Included within the minisatellites are the variable number tandem repeats (VNTR) and the mycobacterial interspersed repetitive units (MIRU). MIRUs were first described in the genome of Mycobacterium tuberculosis (M. tuberculosis) [START_REF] Supply | Identification of novel intergenic repetitive units in a mycobacterial two-component system operon[END_REF]. MIRUs are characterized by the presence of small open reading frames (ORFs) that overlap and are oriented in the same direction as the contiguous ORFs. In recent studies, these VNTR and MIRU have been used to sub-type the members of the Mycobacterium avium complex (MAC) [START_REF] Bull | Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex[END_REF][START_REF] Overduin | Use of multilocus variable-number tandem-repeat analysis for typing Mycobacterium avium subsp. paratuberculosis[END_REF][START_REF] Romano | Further analysis of VNTR and MIRU in the genome of Mycobacterium avium complex, and application to molecular epidemiology of isolates from South America[END_REF][START_REF] Thibault | New variable-number tandemrepeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing[END_REF][START_REF] Möbius | High genetic diversity among Mycobacterium avium subsp. paratuberculosis strains from German cattle herds shown by combination of IS900 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive unit-variable-number tandemrepeat typing[END_REF][START_REF] Thibault | Combined multilocus short-sequence-repeat and mycobacterial interspersed repetitive unit-variable-number tandem-repeat typing of Mycobacterium avium subsp. paratuberculosis isolates[END_REF][START_REF] Möbius | Unique genotypes of Mycobacterium avium subsp. paratuberculosis strains of Type III[END_REF].

M. a. paratuberculosis has been classified into Types I, II and III according to the differences observed at the genetic level [START_REF] Dohmann | Characterization of genetic differences between Mycobacterium avium subsp. paratuberculosis type I and type II isolates[END_REF][START_REF] De Juan | Genetic diversity of Mycobacterium avium subspecies paratuberculosis isolates from goats detected by pulsed-field gel electrophoresis[END_REF][START_REF] Castellanos | Use of Single Nucleotide Polymorphisms in inh-A gene to characterize Mycobacterium avium subspecies paratuberculosis into Types I, II and III[END_REF][START_REF] Griffiths | Sequence polymorphisms in a surface PPE protein distinguish types I, II, and III of Mycobacterium avium subsp. paratuberculosis[END_REF][START_REF] Turenne | Mycobacterium avium subsp. paratuberculosis and M. avium subsp. avium are independently evolved pathogenic clones of a much broader group of M. avium organisms[END_REF]Castellanos et al., 2009a;Castellanos et al., 2009b). Regarding MIRU-VNTR typing on M. a. paratuberculosis Type III, there is only one report where three Type III isolates were analyzed [START_REF] Möbius | Unique genotypes of Mycobacterium avium subsp. paratuberculosis strains of Type III[END_REF]. The main purpose of this study was to evaluate a set of MIRU-VNTR loci in a varied collection of M. a. paratuberculosis Types II and III isolates of Spanish origin to observe the differences present at the number of repeats and also at the nucleotide level.

A c c e p t e d M a n u s c r i p t 4

Materials and methods

Isolates

We selected a panel of M. a. paratuberculosis isolates (n=70) from goats, cattle, and wild animals collected from different Spanish locations over a period of nine years (1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007). Isolates from domestic animals representing 22 different herds [nine from cattle (Bos taurus) and 13 from goats (Capra aegagrus hircus)] and two isolates from wild animals included in the study [fallow deer (Dama dama) and mouflon (Ovis orientalis musimon)] were obtained from different locations in Spain (Table 1). Every isolate was processed, cultured and classified into M. a. paratuberculosis Type I, II or III as formerly described (de Juan et al., 2006;[START_REF] Castellanos | Use of Single Nucleotide Polymorphisms in inh-A gene to characterize Mycobacterium avium subspecies paratuberculosis into Types I, II and III[END_REF].

MIRU-VNTR typing

We analyzed five specific loci MIRU-2, MIRU-3 (alias X3 and VNTR-1658) [START_REF] Bull | Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex[END_REF][START_REF] Overduin | Use of multilocus variable-number tandem-repeat analysis for typing Mycobacterium avium subsp. paratuberculosis[END_REF][START_REF] Thibault | New variable-number tandemrepeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing[END_REF], VNTR-25, VNTR-32 and VNTR-292 [START_REF] Thibault | New variable-number tandemrepeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing[END_REF][START_REF] Möbius | High genetic diversity among Mycobacterium avium subsp. paratuberculosis strains from German cattle herds shown by combination of IS900 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive unit-variable-number tandemrepeat typing[END_REF], using the same specific primers that were reported. Moreover, we selected one additional locus (VNTR-259) that was identified by using in-silico analysis software, Germany). As suggested before [START_REF] Möbius | High genetic diversity among Mycobacterium avium subsp. paratuberculosis strains from German cattle herds shown by combination of IS900 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive unit-variable-number tandemrepeat typing[END_REF], primer concentrations for MIRU-3 were three times higher than for the rest of the loci.

PCR conditions for MIRU-2, VNTR-25, VNTR-32, VNTR-292 and VNTR-259 were denaturation at 95°C for 15 min, then 40 cycles of denaturation at 95°C for 30 sec, annealing at 58°C for 1 min, extension at 72°C for 2 min, and a final cycle of extension at 72°C for 10 min, as reported by Frothingham et al. [START_REF] Frothingham | Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats[END_REF] with slight modifications. For the particular case of MIRU-3 and VNTR-32 the recommended Tm (52-55°C) were used [START_REF] Thibault | New variable-number tandemrepeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing[END_REF][START_REF] Möbius | High genetic diversity among Mycobacterium avium subsp. paratuberculosis strains from German cattle herds shown by combination of IS900 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive unit-variable-number tandemrepeat typing[END_REF].

Afterwards, PCR products were analyzed in a 2.5% agarose gel stained SYBR safe in DMSO (Invitrogen, Eugene, Oregon, USA) at 45 V for 2 h, using a 100 bp molecular ladder (Biotools B&M Labs) to detect the size of the PCR amplicons and to determine the number of repeats for each locus.

To generate an allele calling [START_REF] Benson | Tandem repeats finder: a program to analyze DNA sequences[END_REF]). An allele calling table

was designed with the data obtained to assign the hypothetical size of each PCR product to their correspondent tandem repeat copy numbers for every locus analyzed (MIRU-2, MIRU-3, VNTR-25, VNTR-32, VNTR-292 and VNTR-259) (Table 2).

Discriminative power calculation

The index of discrimination (HGDI) [START_REF] Hunter | Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity[END_REF][START_REF] Hunter | Reproducibility and indices of discriminatory power of microbial typing methods[END_REF]) is based on the probability that two unrelated strains are typed and placed into different groups.

This index is based on the following equation:

) 1 ( ) 1 ( 1 1 1       j S j j x x N N D
, where N is the total number of isolates in the study, s is the total number of types described, and

x j the number of strains belonging to the jth type. This index was calculated by using in silico analysis software (http://insilico.ehu.es/mini_tools/discriminatory_power/).

A c c e p t e d M a n u s c r i p t

Results

Selection of a novel TR (VNTR-259)

We identified a novel locus (VNTR-259) by in-silico analysis of the genome of M. a. paratuberculosis K-10. This locus is located between genes MAP0259 and MAP0260, being the last gene part of the two-component regulatory system

AfsQ1/AfsQ2, according to the predicted function obtained with in-silico analysis software BacMap: genome atlas (http://wishart.biology.ualberta.ca/BacMap/index.html) [START_REF] Stothard | BacMap: an interactive picture atlas of annotated bacterial genomes[END_REF]. In the reference strain, this VNTR had a repetition pattern size of 54 bp, was easily distinguishable by gel electrophoresis, had a total copy number of 2.1 and showed 99% of nucleotide identity amongst different repeat units.

MIRU-2 and VNTR-292

MIRU-2 and VNTR-292 have been used as independent markers [START_REF] Thibault | New variable-number tandemrepeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing[END_REF][START_REF] Möbius | High genetic diversity among Mycobacterium avium subsp. paratuberculosis strains from German cattle herds shown by combination of IS900 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive unit-variable-number tandemrepeat typing[END_REF], although they are both located in the same region of the M.

a. paratuberculosis genome. As it was reported [START_REF] Bull | Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex[END_REF], MIRU-2 was structurally comprising a series of four motifs A (24 bp) and three motifs B (29 bp), while VNTR-292 [START_REF] Thibault | New variable-number tandemrepeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing[END_REF] was characterized as a TR with a MIRU structure with a 53 bp length and was described as present in three copies in the M. a.

paratuberculosis genome. Nevertheless, in the results obtained from the sequences and the in-silico analysis, we observed that motif A2a of MIRU-2 was comprehended within part of the MAP2191c gene (20 bp), whereas repeat units of VNTR-292 were just restricted to the intergenic region (Figure 1). As a result, there was a proportional and conserved ratio (i.e. 5-2, 7-3, etc) among TRs units of MIRU-2 and VNTR-292 in every isolate analyzed, although in previous findings some of the isolates did not follow this correlation [START_REF] Möbius | High genetic diversity among Mycobacterium avium subsp. paratuberculosis strains from German cattle herds shown by combination of IS900 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive unit-variable-number tandemrepeat typing[END_REF]. Therefore, locus VNTR-292 was excluded for the combined calculation of the HGDI.

A c c e p t e d M a n u s c r i p t

MIRU-VNTR typing

Locus-specific PCR primers were directed to six TRs in a panel of 70 M. a.

paratuberculosis field isolates from Spain. All loci presented allelic variation (Table 3), although MIRU-2 (Figure 2) gave the highest allelic variation, with eight variants. The majority of the isolates of Type III displayed a higher number of repeats compared to

Type II isolates at this locus.

M. a. paratuberculosis Type II was also distinguished from Type III by the number of repeats at MIRU-3 and VNTR-259, where fewer repeats at both loci were observed in Type III isolates. At MIRU-3 Type II isolates showed five (one exception of seven) and two repeats at VNTR-259, while Type III showed three and one, respectively. Finally, loci 25 and 32 yielded limited variability with only two allelic variants.

The analysis of MIRU-VNTR in our panel of field isolates revealed double alleles in five of them (Supplementary Figure 1). In these cases, the band representing each of the alleles was neat, did not appear as a ladder of lower intensity and did not correspond to the size of PCR fragments that lacked one or more repeat units as in the case of stutter peaks, which are related to strand slippage of the polymerase during PCR amplification [START_REF] Supply | Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units[END_REF].

The overall analysis yielded 12 different MIRU-VNTR genotypes (16 by including the double alleles). Besides the variability obtained combining all these loci, there were allelic profiles represented by one unique isolate (eight in total), whereas others seemed to be more predominant. For instance, MIRU-VNTR profile no. 1 (5-5-3-9-2), 9 (9-5-3-9-2) and 13 (13-3-3-9-1) represented 35.71%, 21.43% and 12.86% of the isolates tested respectively (Table 3). It is remarkable that every Type II isolate from goat origin (n=20) was comprehended into the most common allelic profile, no. 1 (5-5-

A c c e p t e d

M a n u s c r i p t 9 3-9-2), which was also present in five isolates from dairy cattle. This finding could show the re-circulation of the same clonal variant within these herds, which are geographically proximal and where animal movements among them have existed. On the other hand, it could indicate that these loci are not discriminative enough in these herds. We tested 19 Type III isolates, of which 63.16% belonged to allelic profiles no.

13 (13-3-3-9-1) (47.37%) and 14 (23-3-3-9-1) (15.79%). Isolates from bullfighting cattle showed unexpected diversity. Two M. a. paratuberculosis types were found in herd 15; Type II isolates were all clustered into allelic profile no. 9 (9-5-3-9-2) and

Type III isolates from this same herd were also grouped into one allelic profile no. 15

(27-3-3-9-1). Type III isolates from herds 16 and 17 (same epidemiological unit)

showed three allelic profiles, two of them are shared with some of the Type III isolates from goats. In addition, allelic profile no. 11 appears to be a mixed infection with the most common allelic profile for Type II isolates (allelic profile no.1) and one of the most common allelic profiles for Type III isolates (allelic profile no. 14). Regarding the isolates from wild animals (both Type II), one of them was included within one of the most common allelic profiles (no. 10), and the other isolate within allelic profile no. 2, which represented 5.71% of the isolates screened.

Index of discrimination

For the calculation of HGDI only strains not derived from an epidemiological setting were considered, thus identical isolates from a farm were counted as one regardless of the year of isolation. The isolates with double alleles at any of the loci studied were excluded from the analysis as was done in preceding studies [START_REF] Allix-Beguec | Three-year population-based evaluation of standardized mycobacterial interspersed repetitive-unit-variablenumber tandem-repeat typing of Mycobacterium tuberculosis[END_REF][START_REF] Romero | Persistence and molecular evolution of Mycobacterium bovis population from cattle and wildlife in Donana National Park revealed by genotype variation[END_REF]. The HGDI for individual locus calculated for all the isolates revealed broad differences among them, varying from 0.095 for VNTR-32 which divided our isolates into two allelic profiles to 0.783 in the case of MIRU-2 that 4). In addition, by further combining five MIRU-VNTR loci, we reached the greatest allelic diversity with a D value of 0.84. The second best combination of loci was obtained with MIRU-2 and MIRU-3 which gave a D value of 0.805, thus allowing the division of our isolates into 10 allelic profiles.

HGDI for Types II and III in separate for individual loci and combined loci was also calculated (Table 4). HGDI values for Type II isolates varied from 0 in the case of VNTR-259 to 0.561 for MIRU-2. For Type III isolates, HGDI values ranged from 0 for loci MIRU-3, VNTR-25, 32 and 259 and to 0.889 for MIRU-2. The combination of five MIRU-VNTR loci calculated independently for Types II and III gave a D value of 0.674 and 0.889 respectively.

Sequence analysis of MIRU-VNTR

We sequenced forward and reverse strands of a selection of each allelic variant for MIRU-2 (24 PCR amplicons) and MIRU-3 (19 PCR amplicons) to obtain the structure of motifs A and B. As it was reported [START_REF] Bull | Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex[END_REF], the motif structure in MIRU-2 and MIRU-3 was A-B-A (A motifs are 24 bp length and B motifs 29 bp).

However, both MIRUs presented unique sequences for each primary motif A(n)a, each B(n) motifs, for the internal A(n)i motifs and for the terminal motifs A(n)b (specified in Supplementary Tables 1 and2). Analysis of the sequences in MIRU-2 (Supplementary Table 1), revealed a Type-specific single nucleotide polymorphism (SNP) at Motif A2a in every Type III isolate sequenced, with a T instead of the A that appeared in every Type II isolate. All MIRU-3 sequenced showed a complete match amongst the isolates studied but presented a SNP with a C to A in position 14 of motif A3a with respect to the original description [START_REF] Bull | Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex[END_REF] (Supplementary Table 2).

Additionally, we sequenced a representation of PCR amplicons (n=16) for each of the number repeats obtained from the rest of the loci to confirm the results found in 2.

The analysis of the sequences disclosed the presence of SNPs in some of the TRs, hence confirming the existence of polymorphisms not only at the number of copies but also at the nucleotide level. From the results obtained, one SNP at VNTR-292 was type-specific at position 52 of TR no. 4 (except in MI07/02728 where the SNP was located in TR no. 3 and 464 in TR no. 2), thus differentiating between Type II (with an A) and Type III isolates (with a T).

In the case of VNTR-259, isolate 464 revealed a SNP between TR units (A to G at position 35 in TR no. 1 in comparison to TR no.2). For VNTR-25, every isolate presented a SNP (T to C) at position 11 of TR no.1 and a G to A at position 18 of the last TR, also TR no. 1 and 3 varied from TR no. 2 and 4 (in the case of isolates with a total of five repeat units and only variation from TR no. 1 to 2 in the rest) in an A to G (position 54), and CA to TG (positions 57-58). Conversely, in VNTR-32, we obtained a complete nucleotide match among the isolates sequenced. However, although the repeat unit in this locus is constituted by 18 nucleotides, according to in-silico analysis with Tandem Repeat Software, the percentage of matches between adjacent copies overall was only 72% with only seven nucleotides conserved among individual repeat units.

A c c e p t e d M a n u s c r i p t

Discussion

In this work, the use of a sub-typing PCR-based molecular approach in our collection of M. a. paratuberculosis isolates highlighted the presence of a multigenotype population. Beforehand, M. a. paratuberculosis isolates of Spanish origin were typified by PFGE, technique that yielded a final HGDI of 0.693 [START_REF] Sevilla | Pulsed-field gel electrophoresis profile homogeneity of Mycobacterium avium subsp. paratuberculosis isolates from cattle and heterogeneity of those from sheep and goats[END_REF]. This approach revealed a low level of variability in the collection of 'cattle'

isolates tested compared to the 'sheep' isolates included in this study (nomenclature Type I, II and III not provided). However, PFGE is time consuming and is not applicable for some of our M. a. paratuberculosis isolates, which failed to give enough growth to perform this technique. Due to this, the application of an efficient, reproducible sub-typing technique with low DNA template requirements is important.

Herein, by testing the loci selected (MIRU-2, MIRU-3, VNTR-25, VNTR-32 and VNTR-259) we have achieved a HGDI value of 0.84. The technique presents the advantage that it is easy to perform but sometimes the interpretation of TR numbers in an agarose gel is hindered by the presence of partial repeats. Therefore, the description of novel VNTR with an ETR (exact tandem repeats) structure as in M. tuberculosis [START_REF] Frothingham | Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats[END_REF] would be useful, or the use of an allele calling table similar to the one proposed in this study (Table 2), as the one published for M. tuberculosis typing [START_REF] Supply | Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis[END_REF].

Moreover, M. a. paratuberculosis Types II and III were subdivided into two groups by VNTR-259 (two and one TR respectively) and MIRU-3 (where all Type III presented three TR). This variant at MIRU-3 (three repeats) has been also described in two M. a. paratuberculosis Type I isolates [START_REF] Bull | Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex[END_REF], and in the vaccine strain 316F (Type II) too. In contrast to our results, the number of repeats at MIRU-3 locus in A c c e p t e d M a n u s c r i p t 13 three Type III isolates of German origin was five [START_REF] Möbius | Unique genotypes of Mycobacterium avium subsp. paratuberculosis strains of Type III[END_REF]. At MIRU-2 locus, the two Type I isolates presented 15 repeats, a number that has not been observed in any of the isolates we tested.

In the case of Type II isolates, we obtained the same allelic variants as in the M.

a. paratuberculosis isolates from Germany, France and United Kingdom [START_REF] Bull | Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex[END_REF][START_REF] Thibault | New variable-number tandemrepeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing[END_REF][START_REF] Möbius | High genetic diversity among Mycobacterium avium subsp. paratuberculosis strains from German cattle herds shown by combination of IS900 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive unit-variable-number tandemrepeat typing[END_REF], with the exception of some of the allelic variants at MIRU-2 (VNTR-292). The overall HGDI obtained in our study with the combination of five MIRU-VNTR loci was 0.84, which is in the range of values obtained in previous studies where they attained 0.751 using eight loci [START_REF] Thibault | New variable-number tandemrepeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing[END_REF] and 0.905 with nine loci [START_REF] Möbius | High genetic diversity among Mycobacterium avium subsp. paratuberculosis strains from German cattle herds shown by combination of IS900 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive unit-variable-number tandemrepeat typing[END_REF].

Beforehand, it was observed that phenotypic variation among different strains could be explained by polymorphisms in TR units due to modifications at the gene transcription level, although the precise mechanisms implicated still remain unknown [START_REF] Stragier | Genotyping Mycobacterium ulcerans and Mycobacterium marinum by using mycobacterial interspersed repetitive units[END_REF]. In our results, the variability observed in these particular VNTR loci is in contraposition to former findings where each M. a. paratuberculosis Type seemed to be a tightly homogeneous cluster and showed highly conserved genomic regions (Castellanos et al., 2009b). As a consequence, we could also suspect that these loci might act as a source of strain diversity and they are possibly playing a role in their different pathogenesis.

According to that of particular interest is MIRU-3, located between genes SenX3

and RegX3, components of a regulatory system maybe involved in the protective response to oxidative stress in M. tuberculosis and probably associated to its virulence [START_REF] Rickman | A two-component signal transduction system with a PAS domain-containing sensor is required for virulence of Mycobacterium tuberculosis in mice[END_REF]. Previously, it was found that Mycobacterium bovis (M. bovis)

BCG but not M. tuberculosis strains had a reduction of 53 bp in this locus [START_REF] Magdalena | Specific differentiation between Mycobacterium bovis BCG and virulent strains of the Mycobacterium tuberculosis complex[END_REF]. Interestingly in our study, every Type II isolate displayed five copies of (Supplementary Table 2), probably related to a lower degree of pathogenicity and a higher immunogenicity in comparison with Type II isolates. On the other hand, it is of special interest the isolate MI08/02728 (Type II) which had two extra motifs of this MIRU-3 (seven motifs), which represented an additional 53 bp with respect to the rest of the Type II isolates studied. Furthermore, two other isolates from the same herd showed two alleles in three of the loci analyzed. This result highlights another interesting feature in this particular herd, where the presence of paratuberculosis was accompanied by bovine tuberculosis (unpublished data) leading to a peculiar clinical status of the animals on these farms. The presence of doubles alleles in some of our isolates (n=4) could be the result of either the re-infection of the animal with another clonal variant present in the same herd, due to a mutation at these particular locus, or cross-contamination in the abattoir. The presence of mycobacterial mixed-infections within herds is not an unusual event, and several outbreaks have been reported in the literature [START_REF] Sevilla | Pulsed-field gel electrophoresis profile homogeneity of Mycobacterium avium subsp. paratuberculosis isolates from cattle and heterogeneity of those from sheep and goats[END_REF][START_REF] Michel | High Mycobacterium bovis genetic diversity in a low prevalence setting[END_REF][START_REF] Romero | Persistence and molecular evolution of Mycobacterium bovis population from cattle and wildlife in Donana National Park revealed by genotype variation[END_REF].

Furthermore, the novel locus VNTR-259 located within a two-component regulatory system, AfsQ1/AfsQ2, as in the case of MIRU-3. These two genes (AfsQ1/AfsQ2) in combination with sigQ have been widely studied for its implication in the regulation of antibiotic production in Streptomyces, and its role in the regulatory strategies of some other Actinomycetes [START_REF] Shu | AfsQ1-Q2-sigQ is a pleiotropic but conditionally required signal transduction system for both secondary metabolism and morphological development in Streptomyces coelicolor[END_REF] is already being studied.

Strikingly, every Type II isolate displayed two copies at this locus, whereas Type III isolates presented only one copy. Polymorphisms in number of TR units at these two loci could possibly lead to the differences in virulence mechanisms among M. a.

paratuberculosis types which have been previously described (Verna et al., 2007).

A c c e p t e d M a n u s c r i p t

Finally, from our observations it appeared to be genotypes specific to hosts and M. a. paratuberculosis types, and some others that dominate specific geographic regions, thus each molecular type appear to be responsible of a microepidemy.

Nonetheless, in recent studies based on MIRU-VNTR and short sequence repeats typing (SSR), did not observe these possible correlations in the M. a. paratuberculosis isolates that were tested [START_REF] Thibault | Combined multilocus short-sequence-repeat and mycobacterial interspersed repetitive unit-variable-number tandem-repeat typing of Mycobacterium avium subsp. paratuberculosis isolates[END_REF]. In addition, one study carried out in Spain based on SSR analysis identified 19 different SSR types in a collection of 268 isolates, reaching a D value of 0.691 [START_REF] Sevilla | Comparative analysis of Mycobacterium avium subsp. paratuberculosis isolates from cattle, sheep and goats by short sequence repeat and pulsed-field gel electrophoresis typing[END_REF]. Hereby, with the analysis of the selected combination of MIRU-VNTR within a panel of 29 epidemiological unrelated isolates, we obtained the 12 different genotypes, with a final HGDI of 0.84. For this reason, as it has been also suggested before [START_REF] Thibault | Combined multilocus short-sequence-repeat and mycobacterial interspersed repetitive unit-variable-number tandem-repeat typing of Mycobacterium avium subsp. paratuberculosis isolates[END_REF], the use of MIRU-VNTR typing of M. a. paratuberculosis isolates would be more accessible, feasible and useful in diagnostic laboratories than applying SSR analysis.

Furthermore, the molecular classification yielded by MIRU-VNTR analysis is consistent with the epidemiology of the screened isolates; therefore this data could be used to track the infections. Our results are in accordance with former findings where the majority of M. a. paratuberculosis isolates were grouped into specific genotypes, suggesting that these particular genotypes could be more transmissible or more likely to cause disease [START_REF] Bull | Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex[END_REF][START_REF] Harris | Short-sequence-repeat analysis of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium isolates collected from animals throughout the United States reveals both stability of loci and extensive diversity[END_REF][START_REF] Thibault | New variable-number tandemrepeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing[END_REF][START_REF] Sevilla | Comparative analysis of Mycobacterium avium subsp. paratuberculosis isolates from cattle, sheep and goats by short sequence repeat and pulsed-field gel electrophoresis typing[END_REF][START_REF] Möbius | High genetic diversity among Mycobacterium avium subsp. paratuberculosis strains from German cattle herds shown by combination of IS900 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive unit-variable-number tandemrepeat typing[END_REF].

However, further analysis of the correlation between each of the genetic variants obtained and their clinical importance is needed, with particular interest in those herds where multiple co-infections were suspected. It would be important to find out whether the polymorphisms found both in TRs units and at the nucleotide level in MIRU-3 and VNTR-259 had a relationship with the different pathogenicity of these strains.

A c c e p t e d M a n u s c r i p t

Conclusion

To sum up, the analysis with a combination of MIRU-VNTR loci revealed genetic variability among Spanish M. a. paratuberculosis isolates. Some MIRU-VNTR patterns were unique for each of the M. a. paratuberculosis Types analyzed (II and III); whereas other patterns were host-related. Therefore, this MIRU-VNTR approach could be a fast and useful molecular tool to understand the epidemiology of the infection.

It would be helpful to establish a consensus and to refine protocols for the MIRU-VNTR typing of M. a. paratuberculosis, thus allowing uniformity that would enable the comparison of the different outbreaks that occur world-wide. a Assigned according to PFGE data for the isolates with enough growth [START_REF] De Juan | Genetic diversity of Mycobacterium avium subspecies paratuberculosis isolates from goats detected by pulsed-field gel electrophoresis[END_REF], and PCR-REA of gyrB [START_REF] Castellanos | Polymorphisms in gyrA and gyrB genes among Mycobacterium avium subsp. paratuberculosis type I, II, and III isolates[END_REF] or PCR-REA of inhA [START_REF] Castellanos | Use of Single Nucleotide Polymorphisms in inh-A gene to characterize Mycobacterium avium subspecies paratuberculosis into Types I, II and III[END_REF].

b Wild animals did not belong to a particular herd.

A c c e p t e d M a n u s c r i p t [START_REF] Skuce | Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets[END_REF].

373

b Bold letters represent the alleles found in the panel of isolates included in this study.

374

c Amplicons sizes for the partial repeats at this locus: 2.7 TR (interpreted as 3): 352 bp and 4.7 TR (5): 457 bp.

375

d Amplicons sizes for partial repeats at this locus: 8.6 TR (interpreted as 9): 300 bp and 5.9 TR (6): 264 bp. a Sequenced isolates for specific VNTR locus are in bold.

b These isolates were sequenced only for VNTR-292.

c Partial repeats were considered as entire repeats if they consisted of a significant proportion of the entire repeat [START_REF] Skuce | Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets[END_REF].

d In these isolates with double alleles, two clear separate bands were observed. These isolates (n=4) were excluded for the HGDI calculation.

e Results at VNTR-292 have not been considered for the HGDI calculation because they are proportional to that of MIRU-2.. molecular ladder (100 bp); lane 2, isolate 733; lane 3, MI06/00285-2; lane 4, MI07/04010-2; lane 5, MI07/06579-2; lane 6, molecular ladder (100 bp); lane 7, 634; lane 8, MI06/00307-2; lane 9, D206; lane 10, 464; lane 11, negative PCR control; lane 12, molecular ladder (100 bp).

A c c e p t e d M a n u s c r i p

  ), 10% of DMSO (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) and 2.5 U of Hot-Start-Taq-Polymerase (Qiagen GmbH, Hilden,

  A c c e p t e d M a n u s c r i p t 10 differentiated eight allelic profiles (Table

  s c r i p t the gel. The complete sequence analysis highlighted the presence of partial repeats whose lengths differed among loci. In-silico analysis of the sequences of amplicons of VNTR-292 and VNTR-259 found partial repeats of three and two nucleotides respectively. However, these partial repeats constitute part of the genes flanking the tandem repeats and are not part of a repeat unit. On the other hand, we also found partial repeats in VNTR-25 (42 nucleotides) and VNTR-32 (nine or 15 nucleotides) in all studied isolates. These long partial repeats could hamper the interpretation of the alleles in the agarose gel; however, we have shown their correspondent molecular sizes in Table

  Type III isolates only three, as the vaccine strain 316F

  VNTR-292: 2.1 TR (interpreted as 2): 245 bp, 3.1 TR (3): 300 bp, 4.1 TR (4): 353 bp, 6.1 TR 377 (6): 460 bp, 8.1 TR (8): 566 bp, 11.1 TR (11): 720 bp.

f

  Wild animals did not belong to a particular herd.g Herds no. 16 and 17 have been considered the same epidemiological unit because animal movements between them have occurred in the past.

  .E., Garcia-Pariente, C., Muñoz, M., Moreno, O., García-Marín, J.F., Romano, M.I., Paolicchi, F., Pérez, V., 2007. Variation in the immuno-pathological responses of lambs after experimental infection with different strains of Mycobacterium avium subsp. paratuberculosis. Zoonoses. Public Health 54(6-7), 243-252.

Fig. 1 .

 1 Fig. 1. Diagram representing the structure of MIRU-2 and VNTR-292. MIRU-2 was

Fig. 2 .

 2 Fig. 2. Agarose gel representing the eight allelic variants observed at MIRU-2. Lane 1,

  Figure 1 Click here to download high resolution image

Figure 2

 2 Figure 2 Click here to download high resolution image

  Tandem Repeats Database,

	Laboratory	of	Biocomputing	and	Informatics,	Boston	University
	Specific primers for VNTR-259, F (5' GGGTGTGGAGCTACGACTTC 3') and R (5'
	GAGCTGCTTGACCAGGTGAT 3'), targeting flaking genes were designed using
	Primer 3 software (http://biotools.umassmed.edu/bioapps/primer3_www.cgi).
	PCR reactions were carried out in a final volume of 50 µl, containing 5 µl of

(https://tandem.bu.edu/cgi-bin/trdb/trdb.exe) on the genome of M. a. paratuberculosis K-10 (GenBank accession number. AE016958; reference sequence NC_002944).

DNA template, 1x standard reaction buffer containing 1.5 mM MgCl 2 , 200 µM deoxynucleoside triphosphate (Biotools B&M Labs, S.A, Madrid, Spain), upstream and

  Tandem Repeats Finder Software, Laboratory of Biocomputing and Informatics, Boston University (http://tandem.bu.edu/trf/trf.html)

	M a n u s c r i p t
	QIAquick PCR purification kit (Qiagen GmbH), and sequenced using an ABI Prism 3730 DNA Sequencer (Applied Biosystems) (CIB Sequencing Facilities, Madrid, Spain). Sequences of both strands were aligned with Biological Sequence Aligment A c c e p t e d
	Editor Software and then examined with ClustalW2 software, European Molecular
	Biology Laboratory (EMBL), (http://www.ebi.ac.uk/Tools/clustalw2/index.html) and
	Molecular Genetic Analysis Software (Mega) 3.1 to detect the degree of nucleotide
	identity amongst isolates. Finally, the number of total repeats were analyzed by using
	6

table (with PCR amplicons size and its correspondent number of repeats), a selection of 59 PCR amplicons representing the different sizes at the different TR locus from the different herds were purified with a

Table 1

 1 Panel of Mycobacterium avium subspecies paratuberculosis isolates (n=70) in the study

	Isolate	Map Type a	Host/Breed	Farm	Geographic distribution
	CAM 72			Herd 1
	MI05/03722-2, MI05/03723-2			Herd 2
	CAM 19			Herd 3
	CAM 63		Guadarrama goat	Herd 4	Madrid (Central)
	413, 415, 417, 464,
	MI05/00483-2, MI05/00484-2, MI05/00490-2, MI05/00494-2,			Herd 5
	MI05/00503-2
	CAM 20			Herd 6
	574			Herd 7	Ciudad Real (Central-South)
	MI05/04734, MI05/04735, MI05/04736, MI05/04737,		Murciano-Granadina goat	Herd 8	Toledo (Central-South)
	MI05/02938-2, MI05/02943-2,
	MI05/02945-2, MI05/02948-2,			Herd 9	Canary Islands
	MI05/02950	II
	25, 27			Herd 10	Cantabria (North)
	648, 682		Dairy cattle	Herd 11	Madrid (Central)
	MI07/02713, MI07/02721,
	MI07/02728, MI07/02730, MI07/02731			Herd 12	Valladolid (Central-North)
	MI07/03564			Herd 13
	MI06/06133-2, MI06/06134-2			Herd 14	Zamora (Central-North)
	D206		Fallow deer	-b	Huelva (South)
	1		Mouflon	-b	Soria (Central-North)
	740, 750, 752, 896, 940,
	MI05/02685-2, MI05/02686-2,
	MI05/02693-2, MI06/00304-2,		Bullfighting cattle	Herd 15	Salamanca (Central-North)
	MI06/00305-2, MI06/00306-2,
	MI06/00307-2
	733, MI05/02547-2			Herd 15	Salamanca (Central-North)
	619, 634, 841, MI06/00285-2, MI06/00286-2		Bullfighting cattle	Herd 16	Albacete (Central-South)
	MI07/06579-2, MI07/06582-2,			Herd 17	Ciudad Real (Central-South)
	MI07/01787-2	III		Herd 18
	CAM 38, CAM 40, CAM 42, CAM 86, CAM 87		Guadarrama goat	Herd 19	Madrid (Central)
	CAM 78			Herd 20
	MI07/04010-2			Herd 21
	793, MI05/03132		Murciano-Granadina goat	Herd 22	Toledo (Central-South)

Table 2 369

 2 Hypothetical correspondence between PCR amplicons (in bp) and numbers of whole If the partial repeat at VNTR-25 and VNTR-32 markers is a significant proportion of the entire repeat, it is 372 considered a whole repeat, as done previously in other mycobacteria

	370
	371	repeat units for each MIRU-VNTR locus
		No. of TR a	MIRU-2	MIRU-3	VNTR-25 c	VNTR-32 d	VNTR-292 e	VNTR-259 f
		0	116	168	192	146	137	271
		1	140	192	250	164	190	325
		2	169	221	308	182	243	379
		3	193	245	366	200	296	433
		4	222	274	424	218	349	487
		5	246 b	298	482	236	402	541
		6	275	327	540	254	455	595
		7	299	351	598	272	508	649
		8	328	380	656	290	561	703
		9	352	404	714	308	614	757
		10	381	433	772	326	667	811
		11	405	457	830	344	720	865
		12	434	486	888	362	773	919
		13	458	510	946	380	826	973
		14	487	539	1004	398	879	1027
		15	511	563	1062	416	932	1081
		16	540	592	1120	434	985	1135
		17	564	616	1178	452	1038	1189
		18	593	645	1236	470	1091	1243
		19	617	669	1294	488	1144	1297
		20	646	698	1352	506	1197	1351
		21	670	722	1410	524	1250	1405
		22	699	751	1468	542	1303	1459
		23	723	775	1526	560	1356	1513
		24	752	804	1584	578	1409	1567
		25	776	828	1642	596	1462	1621
		26	805	857	1700	614	1515	1675
		27	829	881	1758	632	1568	1729
		a

Table 3

 3 List of allelic variants obtained for the MIRU-VNTR loci selected from the panel of Mycobacterium avium subspecies paratuberculosis isolates

	Isolate a	2	MIRU-VNTR locus no. 3 25 32 292 e	259	Allelic profiles	Map Type	Host	Herd no. f
	CAM 19, CAM 20, CAM 63,
	CAM 72, 25, 27, 413, 415, 417,
	464, 574, 648, 682, MI05/00483-2,
	MI05/00484-2, MI05/00490-2, MI05/00494-2, MI05/00503-2, MI07/02713, MI05/04734,	5	5	3	9	2	2	1		Goat (n=20), Dairy cattle (n=5)	1-8, 10, 11, 12
	MI05/04735, MI05/04736,
	MI05/04737, MI05/03722-2,
	MI05/03723-2
	D206, MI05/02945-2, MI05/02948-2, MI05/02950	7	5	3	9	3	2	2		Fallow deer (n=1), Dairy cattle (n=3)	9, -e
	MI05/02938-2	5+9 d	5	3	9	2+4 d	2	3			9
	MI07/02721, MI07/02731	5+7 d	5	3+5 d	9	2+3 d	2	4	II
	MI07/02728	7	7	5	9	3	2	5		Dairy cattle	12
	MI07/02730	7	5	5	9	3	2	6
	MI06/06134-2	5+11 d	5	3+5 d 6+9 d 2+5 d	2	7			14
	MI06/06133-2	5	5	5	6	2	2	8
	1, 740, 750, 752, 896 b , 940,
	MI05/02685-2, MI05/02686-2,									Mouflon (n=1),
	MI05/02693-2, MI05/02943-2, MI06/00304-2, MI06/00305-2,	9	5	3	9	4	2	9		Bullfighting cattle (n=12), Dairy cattle	-e , 9, 13, 15
	MI06/00306-2, MI06/00307-2,									(n=2)
	MI07/03564
	CAM 38	7	3	3	9	3	1	10			19
	MI07/01787-2	5+23 d 3+5 d	3	9	2+11 d 1+2 d	11		Goat	18
	MI07/04010-2	21	3	3	9	10	1	12			21
	CAM 40 b , CAM 42, CAM 78, CAM 86, CAM 87, 619, 634, 841, MI07/06582-2	13	3	3	9	6	1	13	III	Goat (n=5), Bullfighting cattle (n=4)	16 g , 17 g , 19, 20
										Goat (n=2),
	793, MI05/03132, MI06/00285-2	23	3	3	9	11	1	14		Bullfighting cattle	16 g , 22
										(n=1)
	733, MI05/02547-2 MI06/00286-2, MI07/06579-2	27 17	3 3	3 3	9 9	13 8	1 1	15 16		Bullfighting cattle	15 16 g , 17 g

Table 4

 4 Individual and combined value of Hunter and Gaston discriminatory index (HGDI) for the MIRU-VNTR locus used Isolates with double alleles at any of the loci were excluded from the analysis.

	Locus	No. of allelic variants b Type II Type III Overall	Allelic diversity Type II Type III Overall
	MIRU-2 a	3	6	8	0.561	0.889	0.783
	MIRU-3	2	1	3	0.111	0	0.453
	25 a	2	1	2	0.209	0	0.173
	32 a	2	1	2	0.118	0	0.095
	259	1	1	2	0	0	0.403
	MIRU-2+MIRU-3	4	6	10	0.605	0.889	0.805
	MIRU-2+MIRU-3+ VNTR-25+32+259	6	6	12	0.674	0.889	0.837

a b Unrelated isolates are those that have been obtained from different herds.

f Amplicons sizes for VNTR-259: 2.1 TR (interpreted as 2): 381 bp ; 1.1 TR (1): 327 bp.

Acknowledgments

This research was funded by the EU Project ParaTBTools FP6-2004-FOOD-3B-023106, and by the Spanish Ministry of the Environment and Rural and Marine Affairs. E.C. is recipient of a grant (AP2005-0696 GAN) of the Science and Innovation Ministry. The group is a partner of EU Project VENoMYC SSPE- CT-2004-501903. We would like to thank the CIB Sequencing Facilities of Madrid, Spain. We are also grateful to Devin John Morey for careful revision of the manuscript.

Distribution and number of A and B motifs in MIRU-2 for the Mycobacterium avium subspecies paratuberculosis isolates sequenced

a SNP (A to T) in MIRU-2 Motif A (A2a). The structure of motifs was as described [START_REF] Bull | Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex[END_REF]): A2a: GGATGAGGAGCGCCCTAAGTGACA; B2:

CGCGCCGACGACGATGCAGAGCGCAGCGA; A2i: TGAGGAGGAGCGGCGCAGATGACT; A2b: TGAGGAGGAGCGGCGCAGATGACA b Number and distribution of motifs in M. a. paratuberculosis Vaccine strain 316F reported previously [START_REF] Bull | Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex[END_REF].

supplementary CGCGCGTATGACGATGCAGAGCGTAGCGA; A3b: TGAGGAGGAATCGCGCCGATGACC and A3i: TGAGGAGGAATCGCGCCGATGACT. In position 14 of motif A3a a C to A was observed in all the isolates sequenced.

b Number and distribution of motifs in M. a. paratuberculosis Vaccine strain 316F reported previously [START_REF] Bull | Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex[END_REF].

supplementary Table 2 A c c e p t e d M a n u s c r i p supplementary Figure 1 Click here to download high resolution image


Iza Marfisi-Schottman

Sébastien George
email: sebastien.george@insa-lyon.fr

Environnement Web multi-acteurs pour la conception de Serious Games

Les Serious Games (SG) sont des environnements éducatifs qui utilisent les ressorts du jeu pour impliquer les apprenants. Ils utilisent notamment des mécanismes de jeu et des interactions (gameplay) proches de ceux des jeux vidéo. Mais leur conception est souvent coûteuse : on estime que le coût de revient d'un SG varie habituellement entre 10 and 300 mille dollars 1 . Le coût de conception des SG à l'INSA de Lyon

Environnement multi-acteurs

La création d'un SG est un processus complexe de collaboration entre différents acteurs humains. Chacun doit, selon son rôle (expert pédagogique, cogniticien, codeur…), exécuter des tâches en fonction de ses compétences et savoir-faire. Le commanditaire (client) spécifie ses besoins et contraintes. Le chef de projet identifie et distribue les tâches aux équipiers tout en supervisant leurs 1 . Aldrich C. The Complete Guide to Simulations and Serious Games. Pfeiffer, October 12, 2009, pp. 576. 2 . http://liesp.insa-lyon.fr/nosSeriousGames 3 . http://www.wired.com/gamelife/2009/12/americas-army-budget 4 . Marfisi-Schottman, I. « Environnement informatique pour la conception, la production et le suivi de Serious Games »,Troisième rencontre Jeune chercheurs, Environnement informatique pour l'apprentissage Humain RJC-EIAH 2010, 6 et 7 mai 2010, pp.53-58.

IZA MARFISI-SCHOTTMAN ET SEBASTIEN GEORGE, « ENVIRONNEMENT WEB MULTI-ACTEURS POUR LA CONCEPTION DE SERIOUS GAMES » ACTES DE LA JOURNEE SCIENTIFIQUE « CONCEPTION DES EIAH A L'ERE DU WEB 2.0 ET A L'AUBE DU 3.0 », AMIENS, 2 JUILLET 2010.
bonnes exécutions. Le cogniticien travaille avec un ou plusieurs experts du domaine, souvent désignés par le client, pour formaliser les connaissances et savoir-faire à l'aide de techniques de maïeutique par exemple. Ensuite, l'expert pédagogique rassemble ces compétences de manière à identifier les connaissances principales et pour définir les objectifs pédagogiques du SG. Le scénariste doit ensuite structurer le scénario pédagogique et l'apparier à un scénario ludique. Cette étape, très délicate, est faite en étroite collaboration avec le directeur artistique qui propose différents ressorts de jeu pour porter l'apprentissage. Le directeur artistique spécifie également tous les aspects visuels, auditifs et sensoriels. Cette spécification doit être très précise afin d'éviter toute incompréhension avec les sous traitants concernés dans la phase de production des ressources (graphiste, acteur, cinéaste, sound manager…). Enfin, les codeurs reprennent les maquettes et les documents spécifiés en amont, dans la phase de conception, pour développer le SG.

La création d'un SG se fait donc par la collaboration d'acteurs ayant des compétences et des tâches très variées. Dans ce sens, notre environnement donne la possibilité au chef de projet de construire son équipe en attribuant les différents rôles et tâches à accomplir. Chaque équipier pourra ensuite se connecter à l'environnement et utiliser les interfaces et les outils proposés en fonction de l'avancement du projet.

Environnement de conception assistée

Notre environnement se concentre principalement autour d'outils pour assister les phases de conception. En effet, ce sont le manque d'organisation et les incompréhensions entre coéquipiers qui causent les pertes de temps les plus importantes. Des documents standardisés servent de références et de moyens de communication clairs. Parmi ces documents, les plus importants sont :

 le cahier des charges,  la liste des compétences métiers ciblés avec leurs descriptions,  le graphe du scénario pédagogique,  les ressorts de jeu utilisés (histoire avec quête, mécanisme de compétition, éléments d'hasard…),  les descriptions détaillées des scènes, des lieux du monde virtuel et des personnages, Nous proposons également des aides contextualisées pour assister les acteurs dans leurs tâches au fil de la conception.

Environnement Intuitif

Les auteurs qui interviennent dans la phase de conception n'ont pas de compétences informatiques. C'est pourquoi nous fournissons des interfaces ergonomiques et intuitives privilégiant des mécanismes de « drag & drop ». L'interface est constituée de palettes, ou widgets, interchangeables, modulables et adaptables... Les palettes correspondent à des informations complémentaires sur le SG. L'idée est qu'un acteur puisse voir la conception du jeu sous plusieurs angles (connaissances, scénario, gameplay, …). Pour le moment, notre interface se compose des palettes suivantes :

 palette Compétences qui montre les compétences en jeu dans le SG. Nous définissons une compétence par un ensemble organisé de connaissances et de comportements s'exerçant dans un contexte (Figure 1). Les informations définies à ce niveau sont ensuite reliées au scénario et ressources du jeu par exemple.

Un environnement dynamique suivant les approches actuelles du web

Perspectives

L'environnement de conception de SG est en cours de finalisation et l'objectif est de le tester avec des élèves-ingénieurs de l'INSA de Lyon qui sont amenés à concevoir des SG dans le cadre de projet pédagogique collectif. Nous pourrons ainsi comparer leur productivité par rapport à celle des groupes des années précédentes tout en évaluant l'utilisation de l'environnement.

Nous comptons également capitaliser et analyser les différents scénarios créés avec l'environnement. Il existe de nombreuses théories sur la modélisation des scénarios pédagogiques et ludiques issues, notamment, des recherches en didactique ou sur l'art du « story telling ». Les scénarios capitalisés peuvent être vus de différentes façon à travers ces modélisations. En jouant avec cette variété de vues, nous espérons être capables d'identifier des patterns récurrents qui mettent en relation les ressorts ludiques et les mécanismes d'apprentissage. Nous pourrons ainsi enrichir notre environnement et les assistances proposées.

Figure 1 .

 1 Figure 1. Palette compétences en vue "édition des compétences"

Figure 2 .

 2 Figure 2. Palette pour chercher des composants en fonction des besoins

  Toutes les palettes décrites ci-dessus sont mises à la disposition des acteurs de la conception d'un SG. Elles ne seront pas toutes disponibles au même moment et pour tout le monde. L'idée globale est bien d'avoir une recomposition dynamique de l'environnement en fonction du contexte particulier : rôle d'un acteur, phase du projet et avancées des autres acteurs. Cet environnement s'inscrit, d'une part, dans une approche web 2.0 de par sa malléabilité et ses interfaces et, d'autre part, dans une approche web 3.0 de par la gestion des compétences et des documents.

(pilotée par le LIESP), est évalué à au moins 15 000 € par heure de jeu d'apprentissage. Certains jeux peuvent se rapprocher de l'industrie cinématographique, avec par exemple plus de

million de dollars pour American Army 3 , un jeu en réseau massivement multijoueurs. Encore plus que pour les autres EIAH, les auteurs de SG ont donc besoins de méthodes de conception et de production efficaces pour réduire les coûts et les temps de production.Pour pallier ce problème, nous proposons un environnement web multi-acteurs avec un ensemble d'outils facilitant la conception et la production de SG. Cet environnement vise à guider les membres de l'équipe de conception à travers les étapes d'une méthode d'ingénierie globale, spécifique aux SG, issue de nos recherches précédentes 4 .