Winy Messens
email: winy.messens@ilvo.vlaanderen.be

Johan Goris

Noël Dierick

Lieve Herman

Marc Heyndrickx

Inhibition of Salmonella Typhimurium by medium chain fatty acids in an in vitro simulation of the porcine cecum

Salmonella Typhimurium was responsible for more than half of the reported cases of human salmonellosis in Belgium in 2007 and was the predominant serovar isolated from slaughter pig carcasses. To lower the Salmonella contamination of pork meat, measures can be taken at the primary production level, e.g. by reducing the shedding of Salmonella through the use of feed additives such as medium chain fatty acids (MCFA's). An in vitro continuous culture system, simulating the porcine cecum, was developed for investigating the effect of MCFA's (sodium caproate, sodium caprylate and sodium caprinate) on the pig intestinal microbial community. The system was monitored by plating on selective media, PCR-DGGE and HPLC analysis of fermentation products. An inoculated Salmonella Typhimurium strain could be maintained by the system at a population size of about 5 log 10 cfu/mL. By addition of 15 mM caprylate, significant reductions of coliforms and Salmonella counts by 4.69 log 10 units (95% confidence interval: 4.19-5.18) could be achieved, while other bacterial populations were clearly less affected. This concentration seems economically feasible in pig feed, provided that the substance can reach the cecum without being absorbed. Thus, caprylate, for example in the form of encapsulated beads or as triacylglycerol oil, might have potential as a Salmonella-reducing additive in pig feed.

Introduction

In 2007, salmonellosis was the second most commonly reported zoonotic disease in the European Union [START_REF] Efsa | The community summary report on trends and sources of zoonoses and zoonotic agents in the European Union in 2007[END_REF]. A quantitative microbial risk assessment (QMRA) from farmto-fork that was developed to assess the risk for human salmonellosis through consumption of fresh minced pork meat in Belgium estimated the mean number of annual cases as 20,513 [START_REF] Bollaerts | Development of a quantitative microbial risk assessment for human salmonellosis through household consumption of fresh minced pork meat in Belgium[END_REF]. In Belgium, Salmonella Typhimurium was responsible for 56.2% of the reported cases of human salmonellosis in 2007 [START_REF] Anonymous | Salmonella en Shigella stammen afgezonderd in België in 2007[END_REF] and was the predominant serovar (63%) isolated from slaughter pig carcasses [START_REF] Efsa | The community summary report on trends and sources of zoonoses and zoonotic agents in the European Union in 2007[END_REF]. Several risk management strategies aiming to reduce the Salmonella contamination in the pork production have been suggested [START_REF] Goldbach | A cost-benefit analysis of Salmonella-control strategies in Danish pork production[END_REF][START_REF] Goldbach | A cost-benefit analysis of Salmonella-control strategies in Danish pork production[END_REF][START_REF] Ojha | Approaches for reducing Salmonella in pork production[END_REF][START_REF] Ojha | Approaches for reducing Salmonella in pork production[END_REF] such as the use of acidified feed for slaughter pigs. If the gastrointestinal tract (GIT) of the pig can be made an unfavourable environment for Salmonella growth, e.g. through the use of feed additives, decreased pathogen shedding will result leading to a reduced Salmonella load of the pigs and carcasses. Medium-chain fatty acids (MCFA) are proposed as a valuable alternative to in-feed antibiotics, used for growth promotion, and even for the preventive and curative treatment of gastrointestinal diseases [START_REF] Decuypere | The combined use of triacylglycerols containing medium-chain fatty acids and exogenous lipolytic enzymes as an alternative to in-feed antibiotics in piglets: concept, possibilities and limitations. An overview[END_REF]. For caprylic acid (= octanoic acid), an inhibitory effect on Salmonella was obtained in standard bacteriological media [START_REF] Skrivanova | In vitro effect of C2-C18 fatty acids on Salmonellas[END_REF]. Although MCFA preparations are commercially available and used in pig farming, their effect on the gut bacterial ecosystem remains largely unknown, especially in the hindgut.

In this study, the effect of selected MCFA's on the GIT microbial community was evaluated using an in vitro model mimicking the conditions in the porcine cecum, with special emphasis on their inhibitory activity against S. Typhimurium.

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

Materials and methods

Bacterial strain

S. Typhimurium MB 2184 was originally isolated from pig colon and belongs to phage type DT104. The strain was stored at -80 °C in brain-heart infusion medium (BHI; Oxoid, Basingstoke, UK) supplemented with 15% glycerol as a cryoprotectant (w/v) [START_REF] Botteldoorn | Salmonella on pig carcasses: positive pigs and cross contamination in the slaughterhouse[END_REF]. The strain was cultivated for 24 h on Trypton Soy Agar or on Brain Heart Infusion agar (BHI; Oxoid). Then one colony was picked and transferred to 10 mL of Mueller-Hinton broth (Oxoid) which was incubated overnight at 37 °C. The culture vessel was inoculated with this overnight-grown culture.

Cecal culture conditions

Cecal growth conditions were mimicked in an in vitro system, which operated as a continuous culture system. The fibre and mucin containing incubation medium for porcine cecum simulation was as described by [START_REF] Dierick | The combined use of triacylglycerols (TAGs) containing medium chain fatty acids (MCFAs) and exogenous lipolytic enzymes as an alternative for nutritional antibiotics in piglet nutrition. I. In vitro screening of the release of MCFAs from selected fat sources by selected exogenous lipolytic enzymes under simulated pig gastric conditions and their effects on the gut flora of piglets[END_REF], with some modifications. The fibre content was halved to 3.8% and the concentration of the phosphate buffer was lowered to 0.01 M. The medium was acidified to pH 2.0 and kept in autoclaved 13 LPyrex bottles at 4 °C under constant agitation to prevent fibre precipitation. Equipment for continuous culture consisted of a BioFlo110 unit (New Brunswick Scientific, Edison, New Jersey, USA) with a 1.3 Lfermentor vessel. Fresh medium was added via a peristaltic pump at a constant rate of about 1.8 mL/min and spent culture liquid was wasted at the same rate to maintain a constant working volume of 500 mL (corresponding to 4.6 h retention time). The pH was kept constant A c c e p t e d M a n u s c r i p t 5 at 6.2 with 5 N NaOH and the temperature was maintained at 37 °C. Temperature and pH control were performed on-line and moderate agitation at 200 rpm was performed. Anaerobic conditions were maintained by flushing the headspace of the vessel with a nitrogen-carbon dioxide mixture (80 and 20%, respectively) at a flow rate of 20 mL/min. The fermentation was started with pooled cecal content as inoculum. Directly after commercial slaughter, cecal content from twelve pigs at slaughter age (originating from seven different farms) was pooled, homogenized with a kitchen blender, divided in aliquots of 15 mL supplemented with 15% (w/v) glycerol and frozen at -80 °C. Salmonella was not detected in the pooled cecum content as black colonies were not observed upon plating on Xylose-Lysine-Desoxycholate agar (XLD). After inoculation with an aliquot of pooled cecum content, the fermentor was operated in batch mode for 24 h. Continuous culture was then started by switching on the peristaltic pumps. Unless otherwise stated, ten mL of S. Typhimurium MB 2184 grown overnight in Mueller-Hinton broth (Oxoid) was added after the switch to continuous culture.

Addition of medium-chain fatty acids

After S. Typhimurium reached steady-state conditions, the blank fresh medium stock was replaced by a stock containing 15 mM of one of the following MCFA's (sodium caproate, sodium caprylate, sodium caprinate; all of Sigma-Aldrich (St Louis, MO, USA). From that moment on, fermentor liquid was gradually replaced by medium containing one of the MCFA's. Sodium caprylate was also evaluated at a concentration of 5 and 30 mM. Separate fermentation experiments were performed for each condition.

Analyses

A c c e p t e d M a n u s c r i p t 6 Bacterial populations were monitored by plating on selective media: Reinforced Clostridial Medium (RCM) for total anaerobes, Trypton Soya Agar for total aerobes, de Man, Rogosa & Sharpe medium (MRS) for lactic acid bacteria, Slanetz and Bartley medium for streptococci, MacConkey agar for coliforms, and XLD for Salmonella. All media were from Oxoid. For Bifidobacteria, modified Trypticase-phytone-yeast extract (MTPY) was used [START_REF] Rada | A new selective medium for the isolation of glucose non-fermenting bifidobacteria from hen caeca[END_REF]. For some experiments, a theoretical dilution curve in case of no Salmonella growth was calculated based on the hydraulic retention time and Salmonella count for the sample taken prior to MCFA addition. Cultivation-independent community analysis was done by 16S rDNA PCR denaturing gradient gel electrophoresis (PCR-DGGE), as described by [START_REF] Boon | Bioaugmentation of activated sludge by an indigenous 3-chloroaniline-degrading Comamonas testosteroni strain, I2gfp[END_REF]. PCR-DGGE patterns were numerically compared using the BioNumerics software package version 4.6 (Applied Maths, St.-Martens-Latem, Belgium). Organic acids formed during fermentation (formic, acetic, propionic, butyric and lactic acid) were quantified by HPLC analysis using an Aminex HPX-87H column (BioRad Laboratories, Hercules, CA, USA), as described before [START_REF] Van Coillie | Identification of lactobacilli isolated from the cloaca and vagina of laying hens and characterization for potential use as probiotics to control Salmonella Enteritidis[END_REF].

Statistical analysis

A two-sample two-sided t-test was performed to assess whether the bacterial counts (in log 10 cfu/mL) differed before and after the replacement of the blank medium by medium containing one of the MCFA's. Counts were used after reaching an equilibrium (approximately 3 days after replacement of the medium). The t-test was performed assuming equality of variances. The significance level α was set at 0.05. All analyses were done in R version 2.7.2 (2008-08-25).

Results

In a first experiment, simulating the porcine cecum without any MCFA treatment (blank), the microbial community was left to stabilize for nine days in continuous culture before S. Typhimurium MB 2184 was added at a final concentration of 4.3 log 10 cfu/mL. From 42 h after inoculation onwards, a stable population size of this strain was reached, averaging 5.2 ± 0.2 (standard deviation) log 10 cfu/mL for at least four operating days (data not shown). In a second blank experiment, S. Typhimurium MB 2184 was added immediately after switching to continuous culture (data not shown). A mean population density of 4.8 ± 0.8 log 10 cfu/mL for at least 8 operating days was obtained. Other mean population sizes were 8.7 ± 0.3, 8.5 ± 0.4, 5.5 ± 0.6, 7.2 ± 0.8 log 10 cfu/mL for total anaerobes, lactic acid bacteria, coliforms and streptococci, respectively. Organic acid concentrations were 0.7 ± 0.5, 11 ± 2, 94 ± 13, 14 ± 3 and 26 ± 4 mM for lactic, formic, acetic, propionic and butyric acid, respectively. In both experiments, a stable PCR-DGGE pattern (data not shown) was obtained after day three of continuous culture.

The effect of different MCFA's was evaluated in the in vitro cecum model upon achieving steady state conditions in the microbial population. Mean changes in bacterial populations by addition of MCFAs are given in Table 1. In Fig. 1, the bacterial populations and organic acid production are depicted for addition of 15 mM sodium caprylate (= sodium octanoate). After four days of blank operation, gradual replacement of the medium in the fermentor vessel by 15 mM sodium caprylate-containing medium resulted in a rapid and significant decrease of the Salmonella population by on average 4.69 log 10 units (from 6.80 to a stable value of 2.11 ± 0.10 log 10 cfu/mL after 3.5 days) and the coliform population by on average 3.62 log 10 units (from 6.63 to 3.01 ± 0.37 log 10 cfu/mL) populations. Small but significant changes were noticed for total anaerobes and lactic acid bacteria (decreases by respectively on average 0.75 A c c e p t e d M a n u s c r i p t 8 and 0.39 log 10 units). The fermentation pattern changed after the addition of sodium caprylate: propionic, formic and butyric acid production decreased, while lactic acid production increased. Acetic acid concentrations gradually increased in the course of the experiment.

When the feed was switched back to blank medium (after 11 days of operation), no concomitant increase of the Salmonella or coliform population was observed while propionic, butyric and lactic acid production returned to their initial levels. Cultivation-independent microbial community analysis via PCR-DGGE for this experiment is shown in Fig. 2. At day 1 (directly after the start of continuous culture) equilibrium was obviously not reached yet, as shown by a community pattern quite distinct from all later samples. From day 3 on, DGGE profiles were all fairly similar, indicating a stable ecosystem. During sodium caprylate treatment, one band (indicated by the arrow in Fig. 2) seemed to be more prominent in the DGGE pattern. On day 15, i.e. 4 days after adding blank medium again, the pattern became as before the switch to sodium caprylate containing medium.

The fermentation patterns for 15 mM sodium caproate (= sodium hexanoate) are shown in Fig. 3. For the first seven days, the cecum simulation was run under blank medium conditions, after which the fermentor liquid was gradually replaced by 15 mM sodium caproatecontaining medium. The treatment was maintained until the end of the experiment at day 14.

The high counts of Salmonella and coliforms on day 3 were due to the large inoculum size of strain MB 2184 and the fact that no equilibrium was reached yet at the time of sampling.

From day six on and before addition of 15 mM sodium caproate, bacterial counts and organic acid production remained rather steady. The mean Salmonella count was 4.62 ± 0.22 log 10 cfu/mL. After addition of 15 mM sodium caproate, no relevant effects on total microbial counts (Table 1) or produced organic acids were observed. Salmonella counts were reduced significantly by 0.70 log 10 units and averaged 3.92 ± 0.17 log 10 cfu/mL upon reaching stable The effect of 15 mM sodium caprinate (= sodium decanoate) addition on the bacterial populations was not very pronounced: streptococci, Salmonella and coliform counts decreased during 2-3 days after addition but then stabilised, while total anaerobes and lactic acid bacteria remained steady. Propionic acid production decreased rapidly after sodium caprinate addition, while concentrations of lactic, formic, acetic and butyric acid remained at the same level (Fig. 4). Using PCR-DGGE, it was clear that bacterial community patterns differed in several bands after addition from patterns before addition (data not shown).

Since sodium caprylate was the most effective MCFA in reducing Salmonella at a concentration of 15 mM, a higher (30 mM) and lower (5 mM) concentration were evaluated.

In comparison with 15 mM sodium caprylate, very similar observations could be made using 30 mM sodium caprylate (Table 1). Mean Salmonella counts decreased significantly by 3.81 log units (from 5.72 to 1.91 ± 0.01 log 10 cfu/mL) and mean coliform counts by 3.93 log units (from 5.88 to 1.95 ± 0.07 log 10 cfu/mL). Likewise, propionic and butyric acid production decreased rapidly after sodium caprylate addition while lactic concentration increased. The effect of 5 mM sodium caprylate was evaluated in duplicate. In the first experiment, Salmonella counts significantly decreased by 2.37 log units (from 6.03 to 3.66 ± 0.19 log 10 cfu/mL) and coliform counts by 2.50 log units (from 6.30 to 3.80 ± 0.17 log 10 cfu/mL) upon addition of 5 mM sodium caprylate. In the second experiment, Salmonella counts significantly decreased by 1.95 log units (from 4.99 ± 0.06 to 3.04 ± 0.09 log 10 cfu/mL) and coliform counts by 1.1 log unit (from 5.3 ± 0.2 to 4.2 ± 0.1 log 10 cfu/mL) upon addition of 5 mM sodium caprylate. Counts for other bacterial groups remained relatively constant in both experiments (Table 1). The shift in fermentation profile, as observed with higher sodium Considering the data of all experiments, the original pooled cecal content inoculum, derived from pigs at slaughter age, evolved in the in vitro model within 3-4 days after the onset of continuous culture into a relatively stable microbial ecosystem with stable fermentation characteristics, as observed from plating results, PCR-DGGE analysis and organic acid production. When operated under blank conditions, population densities of total anaerobic bacteria, lactic acid bacteria, Salmonella, and coliforms stabilized to a mean value of 8.7 ± 0.5, 8.1 ± 0.7, 5.3 ± 0.9, 5.6 ± 0.8 (mean of 8 fermentations) log 10 cfu/mL, respectively. For total aerobes, streptococci and bifidobacteria stabilisation to a mean value of 7.9 ± 1.1 (mean of 6 fermentations), 7.1 ± 0.7 (mean of 5 fermentations), and 8.90± 0.05 (mean of 3 fermentations) log 10 cfu/mL, respectively was obtained. Mean organic acid concentrations of 8 fermentations were 2.2 ± 2.8, 11 ± 11, 84 ± 21, 17 ± 16 and 26.5 ± 3.2 mM for lactic, formic, acetic, propionic and butyric acid, respectively.

Discussion

The use of an in vitro model facilitates the study of the GIT microbial community and circumvents animal-to-animal variation encountered in animal trials. In vitro techniques can play an important role as fast screening methods for potential antimicrobial feed additives and feed ingredients before in vivo trials are planned. A review is given in [START_REF] Williams | In vitro assessment of gastrointestinal tract (GIT) fermentation in pigs: fermentable substrates and microbial activity[END_REF].

Existing continuous culture GIT models are often operated on particle free medium with a chemical composition that is quite distinct from actual chymus. In our model, we mimicked the physiological conditions in the porcine cecum as closely as possible through the use of a cellulose (non-digestible fibre) and mucin (polysaccharides isolated from the porcine A c c e p t e d M a n u s c r i p t 11 stomach)-containing incubation medium. The cecum was specifically selected, because the large intestine (comprising the colon and cecum) is the most heavily colonized region of the GIT [START_REF] Williams | Fermentation in the large intestine of single-stomached animals and its relationship to animal health[END_REF] and also because the cecum is the site where Salmonella is predominantly colonizing and invading.

The original pooled cecal content inoculum, derived from pigs at slaughter age, evolved in the in vitro model within 3-4 days after the onset of continuous culture into a relatively stable microbial ecosystem with stable fermentation characteristics, as observed from plating results, PCR-DGGE analysis and organic acid production. Bacterial counts as well as organic acid concentrations in our model corresponded well with literature data available for the porcine cecum [START_REF] Mikkelsen | Effects of physical properties of feed on microbial ecology and survival of Salmonella enterica serovar Typhimurium in the pig gastrointestinal tract[END_REF]. It should be remarked, however, that the same steady-state conditions were not always reached. Concentrations of formic (from 1 to 34 mM) and propionic acid (from 0 to 37 mM) varied most widely among different experiments, while within a single experiment typically much less variation was found. For repetitions of experiments, different PCR-DGGE profiles were obtained that typically showed similarity to other profiles in only a few (1-3) bands. It was not our aim to identify the PCR-DGGE bands but PCR was used to demonstrate the change in the fermentation pattern after the addition of MCFA. It has already been observed in continuous-flow bioreactor cultures simulating the chicken cecal bacterial community, that there are significant differences in bacterial concentrations and variations in bacterial diversity in replicate bioreactors initiated from the same stock, but no significant differences in bacterial concentrations of repetitive samples from the same bioreactor occurred [START_REF] Crippen | Bacterial concentration and diversity within repetitive aliquots collected from replicate continuous-flow bioreactor cultures[END_REF]. Since Salmonella was not detected prior to the deliberate inoculation with S. Typhimurium MB 2184, the in vitro simulation can be used to specifically monitor the behaviour of the inoculated strain via enumeration on XLD agar plates.

A c c e p t e d M a n u s c r i p t 12

The effect of MCFA on Salmonella and other microbial populations was investigated in the in vitro cecum model. At a dose of 15 mM (corresponding to a practically and economically feasible concentration in pig feed), caproate only had a small effect, while a clear Salmonella inhibiting effect was found for caprylate. Earlier, [START_REF] Skrivanova | In vitro effect of C2-C18 fatty acids on Salmonellas[END_REF] found caprylic acid to be the only Salmonella-inhibiting compound among the 15 fatty acids tested in liquid cultures. Inhibition (measured as a 50% decrease in glucose utilisation) by caprylic acid occurred at a dose of 6 mM, while caproic and capric acid did not show any effect at 30 mM for three Salmonella strains (S. Enteritidis, S. Infantis and S. Typhimurium).

On the contrary, Van Immerseel et al. (2004a) found significant bacteriostatic effects on a S. Enteritidis strain at concentrations as low as 5 mM (for caproic and capric acid) or 10 mM (for caprylic acid), as assessed by performing growth curves in Luria-Bertani broth. Our results seem to be more in line with those of [START_REF] Skrivanova | In vitro effect of C2-C18 fatty acids on Salmonellas[END_REF], although strain-specific or methodological differences should not be ruled out. Also in our experiments, cecal flora was present while pure Salmonella cultures were used in both cited studies. In our experiments, doubling the caprylate dose from 15 to 30 mM did not result in an improved Salmonella inhibition. However, when comparing the actual decrease of Salmonella counts with the theoretical dilution curve, i.e. considering Salmonella as a non-growing particle in the continuous culture, for the different caprylate concentrations evaluated, a slower decrease is noticed when lower caprylate concentrations were applied. This could be explained by a certain lag time before an effective caprylate concentration is reached in the fermentor (with lower concentrations in the fresh medium stock, this process takes longer) and by partial inhibition of Salmonella, i.e. decreased growth rate as opposed to zero growth. At a concentration of 5 mM, the inhibitory effect of sodium caprylate is clearly lower and more delayed, but nevertheless a significant reduction of Salmonella with about two log 10 units was achieved. With none of the evaluated caprylate concentrations, Salmonella counts dropped A c c e p t e d M a n u s c r i p t 13 below the detection level. This means that S. Typhimurium MB 2184 is still able to grow in the system, albeit at a much reduced growth rate. The observed effect does not seem to be Salmonella-specific, since coliform counts showed a similar inhibition in response to caprylate (Fig. 1, Table 1). Other bacterial groups, such as lactic acid bacteria, were only marginally affected by the addition of MCFA's. This is advantageous because there is a delicate balance between beneficial and pathogenic bacteria in the GIT, and many symbiotic and competitive interactions occur between them [START_REF] Williams | Fermentation in the large intestine of single-stomached animals and its relationship to animal health[END_REF]. MCFAs may increase the lactic acid bacteria/E. coli ratio, which is considered as one of the best gut health criteria for the pig gut. The susceptibility of bacteria to MCFAs is most probably related to their membranes. It is suggested that at low pH the outer lipopolysaccharide membrane of the Gram-bacteria become less dense and allow the MCFAs to penetrate into the cell membrane.

Recent studies show that minimal inhibitory concentration (MIC) values depend on chain length, decrease with decreasing pH and are lower for Gram-than Gram+ bacteria [START_REF] Sun | The antimicrobial properties of milkfat after partial hydrolysis by calf pregastric lipase[END_REF][START_REF] Thormar | The role of microcidal lipids in host defense against pathogens and their potential as therapeutic agents[END_REF].

As judged from PCR-DGGE patterns, caprylate addition in concentrations of 5, 15 or 30 mM had a visual effect on the composition of the microbial community in the in vitro caecum simulation. However, PCR-DGGE might be a too coarse method to detect minor shifts in abundance, since the decline of the Salmonella population escaped detection as well with this molecular cultivation-independent method. The observed shift in fermentation pattern as induced by caprylate (lower propionic and butyric acid production and higher lactic acid production) and caprinate (decreased propionic acid production) addition might therefore be due to minor changes in abundance of certain bacterial groups that escaped detection by PCR-DGGE or -perhaps more likely-to metabolic shifts occurring in an unaltered microbial community. It has been shown that butyrate decreases Salmonella invasion in epithelial cell lines [START_REF] Van Immerseel | Invasion of Salmonella enteritidis in avian intestinal epithelial cells in vitro is influenced by short-chain fatty acids[END_REF] and lowers Salmonella colonisation in chickens (Van In conclusion, an in vitro continuous culture model for the microbial community in the porcine cecum was developed and validated. The model can therefore be used in future studies on the composition of cecal flora in response to several parameters such as feed composition and -additives, pH, presence of probiotic strains, etc. In the present study, caprylate was found to selectively inhibit Salmonella and coliforms at cecal concentrations that seem economically feasible, provided that the substance can reach the cecum without being absorbed. Therefore, caprylate, for example in the form of encapsulated beads (Van Immerseel et al., 2004b) or as triacylglycerol oil [START_REF] Dierick | The combined use of triacylglycerols (TAGs) containing medium chain fatty acids (MCFAs) and exogenous lipolytic enzymes as an alternative for nutritional antibiotics in piglet nutrition. I. In vitro screening of the release of MCFAs from selected fat sources by selected exogenous lipolytic enzymes under simulated pig gastric conditions and their effects on the gut flora of piglets[END_REF], might have potential as a Salmonella-reducing additive in pig feed. Although MCFA oils are absorbed in the proximal regions of the digestive tract (stomach 25%, ileum remainder), significant effects on the flora and metabolism at the distal regions (cecum, colon) are possible [START_REF] Dierick | Effect of medium chain fatty acids and benzoic acid, as alternatives for antibiotics, on growth and some gut parameters in piglets[END_REF]. The use of MCFA oils with lipases has additional advantages over the use of MCFA oils solely [START_REF] Dierick | The combined use of triacylglycerols (TAGs) containing medium chain fatty acids (MCFAs) and exogenous lipolytic enzymes as an alternative for nutritional antibiotics in piglet nutrition. I. In vitro screening of the release of MCFAs from selected fat sources by selected exogenous lipolytic enzymes under simulated pig gastric conditions and their effects on the gut flora of piglets[END_REF].

  A c c e p t e d M a n u s c r i p t 9 conditions after 3-7 days. Also changes in PCR-DGGE patterns were not introduced (data not shown).

  was less pronounced or even absent when a 5 mM dose was used (data not shown).

  ., 2004b). It would be interesting to evaluate in in vivo pig trials whether the beneficial Salmonella-inhibiting effect of caprylate would not be counterbalanced by an increased colonization due to a lower cecal butyric acid concentration resulting from caprylate application.

Fig. 1 :

 1 Fig. 1: Effect of 15 mM sodium caprylate on the in vitro porcine cecum simulation. Bacterial

Fig. 2 :

 2 Fig. 2: Denaturing gradient gel electrophoresis patterns of the microbial populations in the

Fig. 3 :

 3 Fig. 3: Effect of 15 mM sodium caproate on the in vitro porcine cecum simulation. Bacterial

  Fig. 1

  Figure 2

Table

Acknowledgements

This study was financed by the Institute for the Promotion of Innovation by Science and Technology in Flanders. Jessy Claeys is acknowledged for excellent technical assistance.


Belén Rodríguez-Sánchez

Pedro J Sánchez-Cordón

Verónica Molina

María A Risalde

Ana Cristina Pérez De Diego

José C Gómez-Villamandos

José M Sánchez-Vizcaíno

DETECTION OF BLUETONGUE SEROTYPE 4 IN MOUFLONS (Ovis aries musimon) FROM SPAIN

Keywords: bluetongue, serotype-4, mouflons, wild ruminants, RT-PCR, paraffinembedded tissues

Bluetongue serotype 4 (BTV4) has been detected for the first time in tissue samples from 2 mouflons (Ovis aries musimon) from the South of Spain, in a retrospective study. The samples included in this study had been fixed and paraffinembedded for over a year prior to their analysis using a BTV group-specific and a BTV4 specific RT-PCR test. Lung and lymphatic nodes were found positive in both specimens. The amplified DNA was confirmed to be BTV4 by sequencing the RT-PCR products and comparing them with other sequences from GenBank. The combination of RNA extraction from paraffin-embedded samples and serotype-specific real-time RT-PCR assays provides the tools for the detection of BTV from samples stored for a long time. The results shown in this study set out the basis for a greater survey with fixed samples from different species of wild ruminants that the veterinary services have been collecting for years.

Introduction

Bluetongue (BT) is a noncontagious, vector-borne disease of domestic and wild ruminants caused by an Orbivirus (Family Reoviridae) that includes at least 24 different serotypes [START_REF] Mathews | Classification and nomenclature of viruses[END_REF][START_REF] Mertens | The bluetongue virus core: a nano-scale transcription machine[END_REF]. Traditionally, sheep have been considered the most severely affected livestock species, whereas cattle and goats usually appeared as asymptomatic reservoir hosts [START_REF] Maclachlan | The pathogenesis and immunology of bluetongue virus infection of ruminants[END_REF]. However, the recent outbreaks of BT occurred in both southern and northern Europe caused by different A c c e p t e d M a n u s c r i p t 3 serotypes, some of them never described previously in these countries, displayed severe clinical signs in cattle, goats and different species of wild ruminants characterized by fever, depression, nasal discharge, respiratory distress, oral ulceration and haemorrhages, subcutaneous oedema and cyanotic tongue [START_REF] Maclachlan | Experimental reproduction of severe bluetongue in sheep[END_REF][START_REF] Saegerman | Bluetongue epidemiology in the European Union[END_REF][START_REF] Backx | Clinical signs of bluetongue virus serotype 8 infection in sheep and goats[END_REF][START_REF] Dercksen | First outbreak of bluetongue in goats in The Netherlands[END_REF]. Wild ruminants have been considered for a long time as asymptomatic hosts. However, recent studies have reported BT characteristic symptoms [START_REF] Mauroy | Bluetongue in captive yaks[END_REF] and the presence of the virus [START_REF] Fernandez-Pacheco | Bluetongue virus serotype 1 in wild mouflons in Spain[END_REF] or BT specific antibodies [START_REF] Linden | Bluetongue virus antibodies in wild red deer in southern Belgium[END_REF][START_REF] Ruiz-Fons | Spatial and temporal evolution of bluetongue virus in wild ruminants, Spain[END_REF] in wild species like yaks (Bos grunniens grunniens), mouflons (Ovis aries musimon) or wild red deer (Cervus elaphus), respectively. The role of wildlife in the prevalence and spreading of BT is beginning to be elucidated [START_REF] Ruiz-Fons | Spatial and temporal evolution of bluetongue virus in wild ruminants, Spain[END_REF].

The analysis of samples from wild ruminants is becoming increasingly important in order to develop control programs and to elucidate the role of wildlife in the spreading and persistence of the disease. Recently, a study on the presence of BTV antibodies in wild ruminants in Spain [START_REF] Ruiz-Fons | Spatial and temporal evolution of bluetongue virus in wild ruminants, Spain[END_REF] and the characterization of BTV1 in mouflons (Ovis aries musimon) [START_REF] Fernandez-Pacheco | Bluetongue virus serotype 1 in wild mouflons in Spain[END_REF] have been published. The adaptation of rapid molecular techniques, such as real-time PCR, to these samples can provide valuable information about the BTV serotype present in it, just by sequencing the PCR product. The advantages provided by the PCR techniquerapidity, high through-put, robustness-could replace or accompany seroneutralization as a method to characterize BTV serotypes in wildlife species.

In October 2007, two mouflons (Ovis aries musimon) -one male and one femalewere found dead in the North of the Córdoba province (Spain). The animals were referred to the Diagnostic Services of the Veterinary Faculty (Córdoba University) for A c c e p t e d M a n u s c r i p t 4 routine necropsy. Tissue samples from these two animals have been analyzed in a retrospective study in order to detect the serotype of BTV that caused their death.

Materials and Methods

The samples analyzed in this study belong to two mouflons found dead in the field and transported to the Diagnostic Services of the Veterinary Faculty (Córdoba University, Spain) in order to determine the cause of their death. Macroscopical examination revealed the presence of subcutaneous oedema of the head and neck, hydrothorax and hydropericardium. Lungs appeared pale and non-collapsed, showing alveolar oedema. Subintimal petechial hemorrhages at the base of the pulmonary artery were also observed, together with the presence of diffuse hemorrhages in subendocardial and subepicardial locations of the heart. During necropsy, tissue samples from lung, heart, pulmonary artery, trachea, lymph nodes (submandibular and tracheobronchial), tongue, spleen, liver, small intestine, rumen, kidney, urinary bladder, adrenal gland, ovary and uterus were taken and fixed in 10% buffered formalin solution during 24h. After that, samples were routinely processed and embedded in paraffin wax. 4 μm sections were cut and stained with haematoxylin and eosin (H&E) for histopathological study. Microscopical examination (Table 1) confirmed the lesions observed at necropsy. Both, histopathological and macroscopical studies were consistent with BTV infection.

Paraffin-embedded samples that remained after the study carried out by the Diagnostic Services of the Veterinary Faculty (Córdoba University, Spain) in order to determine the cause of death of the mouflons, were kept at RT and further analyzed in this study. One year after they were processed, the slides were included in a retrospective study that tentatively tried to identify the BTV serotype that caused the A c c e p t e d M a n u s c r i p t 5 death of the mouflons. Paraffin-embedded tissues from sheep experimentally infected with BTV1 and BTV4 were included in this study as positive control. These two serotypes were circulating in Spain at the time the mouflons were found dead, while BTV 8 was not detected in the country until January 2008, i. e. three months later (http://www.oie.int/wahis/public.php?page=single_report&pop=1&reportid=668). As negative controls As negative controls, paraffin-embedded tissue samples from BTVfree sheep and from a PRRS-infected pig were included in this study.

In order to avoid RNA contamination, disposable razors and the microtome components in contact with the samples were treated with RNaseZap (Ambion) prior to cutting each paraffin block. Furthermore, 10 μm sections were obtained by cutting a paraffin block from pig tissues inoculated with PRRS after each paraffin block from mouflons. Pig tissues were included as RNA extraction controls and negative controls for BTV. After trimming the excess of paraffin, tissues were scrapped off from the slides and their RNA was extracted using RNeasy ® FFPE kit (Qiagen) following the manufacturer's instructions. Briefly, tissues were incubated for 10 sec at RT with xylene (Sigma-Aldrich) and ethanol 100% (Panreac). After centrifuging for 2 min at full speed, the supernantants were removed by pipetting and the tubes remained with the lids open until all residual ethanol evaporated. Then, the samples were incubated for 15 min at 55ºC and then 15 min at 80ºC with Proteinase K and a buffer that reverses formaldehyde modification of nucleic acids. The mixture was transferred to a silicon column where the genomic DNA present in the sample was bound. After a short centrifugation, the flow-through was collected and mixed with 100% Ethanol in order to precipitate RNA. From this step on, the protocol proceeded as a regular RNA extraction protocol using spin columns. In the final step, RNA was resuspended in 20μl of PCRquality water and frozen at -80ºC. 3μl of the RNA obtained were amplified by RT-PCR.

A c c e p t e d M a n u s c r i p t 6 Three RT-PCR assays were applied to the RNA samples: 1) a gel-based, group-specific RT-PCR that amplifies a common sequence -274bp-within BTV segment 5 [START_REF] Agüero | Molecular differentiation between NS1 gene of a field strain Bluetongue virus serotype 2 (BTV-2) and NS1 gene of an attenuated BTV-2 vaccine[END_REF], 2) a gel-based, serotype specific RT-PCR using a set of primers that flank a 765bp sequence within the segment 2 of BTV1 [START_REF] Mertens | Design of primers and use of RT-PCR assays for typing European bluetongue virus isolates: differentiation of field and vaccine strains[END_REF] and3

Results

Tissue samples from BTV-free sheep and PRRS infected pigs were negative to the three BTV RT-PCR assays.

Fixed and fresh tissues from sheep experimentally infected with BTV1 and BTV4 were also included in the RT-PCR assays. Tissues from sheep infected with BTV1 -mainly lung-were positive to the group-specific RT-PCR and to the BTV1 specific RT-PCR, but all the tissues from the mouflons were negative to this assay (data not shown). Lung and tracheobronchial nodes were the samples that gave positive results for the group-specific RT-PCR and for BTV4-specific RT-PCR assay, while BTV-free tissues were always negative for these two tests (Fig 1). Two samples from lung tissue and lymph node, one from each mouflon, gave a positive plot for BTV4 real- 1) the presence of BTV4 in wild ruminants, as it has been described in this study, may be due to the arrival of infected Culicoides in Spain -in this case, the vaccination campaign would have proved to be highly effective-or 2) the persistence of infected vectors in the area due to milder temperatures. In the latter case, wild ruminants could have a role as a reservoir for BT. Thus, efforts should be focussed on controlling and surveying the wildlife, as well as improving the methods to detect and eliminate the vector.

The fact that the samples were not taken right after the death of the animals and, moreover, they were fixed and paraffin-embedded prior to RNA extraction and amplification by RT-PCR, shows that this method preserves the tissues and allows the detection of BTV by RT-PCR [START_REF] Parsonson | Retrospective diagnosis of bluetongue virus in stored frozen and fixed tissue samples using PCR[END_REF]. Thus, the implementation of specific RT-PCR tests to fixed samples facilitates the development of retrospective studies. This methodology provides an efficient and rapid tool for surveillance and epidemiological studies.

Conclusions

To the authors' knowledge, this is the first identification of BTV4 in wild ruminants. The fact that the analysed samples from which BTV4 was detected had been fixed and paraffin-embedded for over one year prior to the isolation of BTV RNA proves that this method preserves enough nucleic acids to allow the detection of putative pathogens present in tissue samples.

  PCR (Fig 1). The same samples were also positive to the group-specific RT-PCR assay (data not shown).BTV4-specific RT-PCR products were purified and sequenced. When these sequences were compared with other sequences from GenBank using BLAST software, the homology with other BTV4 segment 2 sequences ranged between 90% and 100% (Fig2). The homology variation was due to the length of the RT-PCR product and to the genome variability in BTV segment 2. The sequences from the dead mouflons were 100% homologous to BTV4 circulating in Portugal -EF434176-, Corsica -AY839945and Turkey -DQ825670-or with BTV4 reference strains -AJ585125-and 90% homologous to other BTV4 strains -DQ191281, DQ191280-. No homology was found with other BTV serotypes.DiscussionIdentification of BTV serotypes has been previously carried out in mouflons[START_REF] Fernandez-Pacheco | Bluetongue virus serotype 1 in wild mouflons in Spain[END_REF]. In their study, the authors identified BTV1 in blood samples from animals that had been affected by BTV during the outbreak that took place in the south of Spain in 2007. In October 2004, a BTV4 outbreak occurred in southern Spain. The virus was detected for the last time one year later, in October 2005 (http://www.oie.int/wahis/public.php?page=event_summary&this_country_code=ESP& reportid=5615). Domestic ruminants were vaccinated with the appropriate BTV vaccine -a live attenuated BTV4 vaccine in 2005 and an inactivatedBTV4 from 2006 to 2008 (Savine et al., 2008)-. This control measure avoided the subsequent presence of both serotypes in the locations where they were first detected. However, the results of this study show the presence of BTV4 in wild ruminants from the same geographical area, at BTV4 was not detected in livestock anymore. Two hypotheses are possible:

Figure legends Table 1 .

 1 Figure legends

Figure 1 .

 1 Figure 1. Amplification plots obtained from the specific BTV4 real time RT-PCR on

Figure 2 .

 2 Figure 2. DNA sequence from the RT-PCR product. The sequence from the amplified

  Figure 1

Acknowledgements

This work was supported by grants from the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (P05-AGR-00132). P. J. Sánchez-Cordón holds a contract of the "Ramón y Cajal programme" (Ministry of Education and Science, Spain). The authors want to thank the Spanish Ministry of Natural, Rural and Marine Environment for funding this project (RASVE 274/2007), Dr. Marisa Arias, Technical Director of the CISA-INIA laboratory (Madrid, Spain) for kindly proving us with BTV1 and BTV4 positive controls, and the lab technicians from the Animal Health Department -Belen Rivera & Rocio Sanchez-for their enthusiasm and dedication to this study.


Sabrina Rodríguez

Beatriz Romero

Javier Bezos

Lucía De Juan

Julio Álvarez

Elena Castellanos

Nuria Moya

Francisco Lozano

Sergio González

José Luis Sáez-Llorente

Ana Mateos

Lucas Domínguez

Alicia Aranaz
email: alaranaz@vet.ucm.es

High spoligotype diversity within a population: clues to understanding the demography

Keywords: Mycobacterium bovis, tuberculosis, spoligotyping, epidemiology

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

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

Introduction

Mycobacterium bovis, the main causative agent of bovine tuberculosis, affects a wide range of domestic animals and wildlife. This infection entails important environmental, economic and public health risks [START_REF] Briones | Bovine tuberculosis and the endangered Iberian lynx[END_REF][START_REF] Corner | The role of wild animal populations in the epidemiology of tuberculosis in domestic animals: how to assess the risk[END_REF][START_REF] Michel | Wildlife tuberculosis in South African conservation areas: implications and challenges[END_REF].

In Spain all regions are subjected to a national eradication programme based on intradermal tuberculin testing of cattle and the slaughter of reactor animals. The annual herd period prevalence rate has been reduced to 1.63% in 2007. However, large differences exist between geographical areas (0.19-9.51%) (Spanish Ministry of Environment and Rural and Marine Affairs, http://www.mapa.es/ganaderia/pags/sanidad_ganadera/zoonosis/Informe2007.pdf) Nowadays we count on a set of molecular typing techniques in order to investigate epidemiological backgrounds. Direct variable repeat spacer oligonucleotide typing (DVR-spoligotyping) [START_REF] Kamerbeek | Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology[END_REF] detects polymorphism within the genomic Direct Repeat (DR) locus. The DR locus consists of multiple sequences interspersed with non-repetitive spacer sequences (spacers). Strains vary in the presence and absence of these spacers and can therefore be clustered in so-called spoligotypes. Since the implementation of spoligotyping for M. bovis [START_REF] Aranaz | Restriction fragment length polymorphism and spacer oligonucleotide typing: a comparative analysis of fingerprinting strategies for Mycobacterium bovis[END_REF][START_REF] Roring | Spacer oligotyping of Mycobacterium bovis isolates compared to typing by restriction fragment length polymorphism using PGRS, DR and IS6110 probes[END_REF], the technique has been considered useful as a fast and cost-effective method for firstline typing [START_REF] Haddad | Molecular differentiation of Mycobacterium bovis isolates. Review of main techniques and applications[END_REF][START_REF] Milian-Suazo | Molecular epidemiology of Mycobacterium bovis: Usefulness in international trade[END_REF]. The applicability of the technique can vary between countries or even regions, because of the A c c e p t e d M a n u s c r i p t 4 differences between strain diversity and the discriminatory power of spoligotyping. However, the reasons underlying these differences in diversity remain largely unknown.

This paper provides a survey of the M. bovis population in Spain by resuming the spoligotyping results for 6215 isolates obtained during the last 15 years. We describe the spoligotype diversity of M. bovis and offer insight into the usefulness of spoligotyping for epidemiological studies when a high degree of strain diversity is present.

A c c e p t e d M a n u s c r i p t 5

Materials and methods

Mycobacterial isolates and strains

This study comprises 6215 M. bovis isolates from Spain, collected between January 1992 and December 2007. Most of the isolates were cultured in the laboratory VISAVET (Facultad de Veterinaria, Universidad Complutense Madrid), but about 40% of the isolates were submitted by Regional Laboratories within the national bovine tuberculosis eradication program. The isolates were obtained from cattle (Bos taurus, n=5585), domestic goats (Capra aegagrus hircus, n=33), domestic pigs (Sus scrofa domestica, n=7), wild boars (Sus scrofa, n=204), red deer (Cervus elaphus, n=141), fallow deer (Dama dama, n=229), Iberian lynxes (Lynx pardinus, n=6), foxes (Vulpes vulpes, n=2), chamois (Rupicapra rupicapra, n=2), a badger (Meles meles, n=1), cats (Felis silvestris catus, n=3), a dog (Canis lupus familiaris, n=1) and a mouflon (Ovis musimon, n=1, zoo animal). The geographical distribution is represented in the supplementary figure according to the number of isolates.

The tissue samples were decontaminated and cultured onto Coletsos and 0.2% (w/v) pyruvate-enriched Löwenstein-Jensen media (bioMérieux España and Biomedics, Madrid, Spain) and incubated at 37 o C. The DNA was prepared from colonies by suspending them in 200 µl of distilled water and boiling for 10 min at 100ºC. The isolates were confirmed as members of the M. tuberculosis complex by acid-alcohol-fast staining and PCR amplification of Mycobacterium genusspecific 16S rRNA fragment and MPB70 sequences [START_REF] Wilton | Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube[END_REF].

A c c e p t e d M a n u s c r i p t 6

DVR-spoligotyping

The isolates were spoligotyped following the protocol described by [START_REF] Kamerbeek | Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology[END_REF]. The DR region was amplified using the primers DRa (GGTTTTGGGTCTGACGAC, 5' biotinylated) and DRb (CCGAGAGGGGACGGAAAC) (Roche Molecular Biochemical, Berlin, Germany) and the amplified product was hybridised onto a spoligotyping membrane (Isogen Bioscience BV, Maarssen, The Netherlands). The DNA was detected with the streptavidin-peroxidase conjugate (Boehringer-Mannheim, Germany) and the ECL system (GE Healthcare, Barcelona, Spain), followed by exposure of an X-ray film to the membrane. We included purified sterile water as a negative control and a clinical isolate of M. tuberculosis [START_REF] Aranaz | Use of polymerase chain reaction in the diagnosis of tuberculosis in cats and dogs[END_REF] as a positive control in every PCR-batch and hybridisation assay. Authoritative names (prefix SB followed by four digits) for spoligotype patterns were obtained from the Mycobacterium bovis Spoligotype Database website (http://www.Mbovis.org). Part of the results have been previously published [5% [START_REF] Aranaz | Bovine tuberculosis (Mycobacterium bovis) in wildlife in Spain[END_REF], 1.6% [START_REF] Romero | Persistence and molecular evolution of Mycobacterium bovis population from cattle and wildlife in Doñana National Park revealed by genotype variation[END_REF]].

The spoligotyping results were enlisted in a Microsoft® Office Access database along with the epidemiological data (isolation date, animal species and geographical origin).

Discriminatory power calculation

The index of discrimination (D) described by [START_REF] Hunter | Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity[END_REF] was calculated in order to determine the discriminatory power

Outline

The 6215 M. bovis isolates included in this study originated from sampling [START_REF] Aranaz | Use of polymerase chain reaction in the diagnosis of tuberculosis in cats and dogs[END_REF]] that clustered 27.89% of the isolates, followed by SB0134 (spb-13) and SB0339 (spb-16) which accounted for 11.19% and 8.05% of the isolates (Table 1).

The 237 other spoligotyping patterns comprised 153 patterns (1319 isolates)

with very low frequency (28.1% of them had been found infecting only one herd each), and 84 singular patterns, so-called orphans, which were only isolated once (Table 1).

Altogether we identified 148 spoligotypes that have not been previously

Discriminatory power and diversity ratio

We calculated the discriminatory power (D) for the spoligotyping of the Spanish M. bovis isolates, which means the average probability that the technique will assign a different type to two isolates randomly sampled in the population of M. bovis, using the equation by [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]. We achieved a discriminatory index D=0.87 at national level.

We also calculated the discriminatory index of spoligotyping in Spanish provinces from which more than 60 isolates were obtained, and D oscillated between 0.74 and 0.93. In addition, the strain/pattern ratio ranged from 1.5 to 7.1 (supplementary Figure ). We compared these results with the respective number of isolates and the annual herd period prevalence of bovine tuberculosis (MARM, 2007). In general, provinces with lower herd prevalence (<0.8%) showed a slightly higher D value (average D=0.90) compared to national average and lower strain/pattern ratio (average ratio=2.7), whereas provinces with extreme herd prevalence (>4%) showed lower D values (average D=0.82) and higher strain/pattern ratio (average ratio=4).

Geographical distribution

The M. bovis strains were unequally distributed all over Spain (supplementary out in those areas.

Animal species

The vast majority of the types were isolated from cattle, in fact 207 of our spoligotypes were found only in this species (

Frequency of spacers

We compared the frequencies of the 43 spacers throughout the 252 spoligotypes (Figure 1) and could confirm the absence of spacers 3, 9, 16, and 39 to 43. Though none of the spacers were present in all the patterns, we found a rather homogenous distribution. Each spacer was present in about 79.96% (69.5-89.2%) of the strains, except for spacer 21 which could be found in only 36% of our strains. The patterns which lack this spacer account for 67.13% of the typed isolates, but neither belonged to a determined animal species, nor geographical area.

A c c e p t e d M a n u s c r i p t 12

Discussion

In this study we report the characterisation of a representative collection of M. bovis isolates from the different animal host species described in Spain. This collection resulted from the involvement of numerous contributors which reflects the commitment at national level on the study of the epidemiology of bovine tuberculosis. The DVR-spoligotyping clustered the isolates in 252 spoligotypes with a discriminatory index of D=0.87. To our knowledge this study reveals the highest diversity within a M. bovis population that has been described in scientific literature.

While most spoligotypes were distributed throughout the country, a small number of patterns were restricted to determined regions. Consequently, extension of the analysis to Spanish regions not studied before yields patterns not previously described. In general, we found a higher discriminatory index for spoligotyping in the Spanish northern regions which started eradication of bovine tuberculosis first and present lower annual herd period prevalence. However, some exceptions exist, likely reflecting animal species, cattle breeds and herd management. This fact suggests that test-and-slaughter policy has not affected the local strain diversity.

Spoligotyping results show common traits among western continental EU countries and to a limited extent also to the British Isles. As previously described in other reports, a frequent type clusters a high percentage of isolates. Our most often identified spoligotype, SB0121, is also the most frequent strain in Portugal, A c c e p t e d M a n u s c r i p t 13 the second most predominant type in France after SB0120 [START_REF] Haddad | Spoligotype diversity of Mycobacterium bovis strains isolated in France from 1979 to 2000[END_REF] and one of the five most frequent Italian types [START_REF] Boniotti | Molecular Typing of Mycobacterium Genotyping[END_REF]. The similarities between Spain, France, Portugal and Italy can be due to the geographical nearness and trade relationship. SB0121 has also been found with a very low frequency (<1%) in mainland Great Britain [START_REF] Hewinson | Recent advances in our knowledge of Mycobacterium bovis: a feeling for the organism[END_REF].

Surprisingly, SB0120 represents 3.95% of the isolates in Spain but is infrequent in Portugal [START_REF] Duarte | Spoligotype diversity of Mycobacterium bovis and Mycobacterium caprae animal isolates[END_REF]. Spoligotypes SB0130 and SB0134, which make up 3.39 % and 11.19% of our isolates respectively, were isolated frequently in Britain [START_REF] Smith | Bottlenecks and broomsticks: the molecular evolution of Mycobacterium bovis[END_REF] and Ireland (only SB0130) [START_REF] Costello | Study of restriction fragment length polymorphism analysis and spoligotyping for epidemiological investigation of Mycobacterium bovis infection[END_REF][START_REF] Skuce | Discrimination of isolates of Mycobacterium bovis in Northern Ireland on the basis of variable numbers of tandem repeats (VNTRs)[END_REF]. SB0134 was also reported from French cattle and wildlife [START_REF] Zanella | Mycobacterium bovis in wildlife in France[END_REF]. Furthermore, we have repeatedly isolated SB0140, the most common spoligotype of the British Isles Advanced studies using additional techniques such as Variable Number Tandem Repeat analysis would be needed to reveal further epidemiological relationships.

The analysis of diversity within each species offered insight into the relative contribution of livestock and wildlife to the epidemiology of bovine tuberculosis in Spain. Wildlife, especially wild boar has been suggested as a reservoir [START_REF] Aranaz | Bovine tuberculosis (Mycobacterium bovis) in wildlife in Spain[END_REF][START_REF] Hermoso | Bovine tuberculosis in wild boar (Sus scrofa), red deer (Cervus elaphus) and cattle (Bos taurus) in a Mediterranean ecosystem (1992-2004)[END_REF][START_REF] Naranjo | Evidence of the role of European wild boar as a reservoir of Mycobacterium tuberculosis complex[END_REF]. The bovis strains which have a maximum number of DVR spacers. This ancestor would derive from a M. tuberculosis-like organism [START_REF] Brosch | A new evolutionary scenario for the Mycobacterium tuberculosis complex[END_REF][START_REF] Mostowy | Revisiting the evolution of Mycobacterium bovis[END_REF][START_REF] Smith | Bottlenecks and broomsticks: the molecular evolution of Mycobacterium bovis[END_REF]. We assume that original cattle populations remained with human settlements, giving rise to locally adapted strains and further diversification. This evolutionary scenario is congruent with those described for the demography of the M. tuberculosis complex and its association with the human host [START_REF] Wirth | Origin, spread and demography of the Mycobacterium tuberculosis complex[END_REF][START_REF] Hershberg | High functional diversity in Mycobacterium tuberculosis driven by genetic drift and human demography[END_REF]. Cattle arrived into Spain through the Pyrenees and along the Mediterranean cost during the Neolithic period. Likely, the cattle population increased in the region providing an by this dual entrance of cattle from land and maritime routes. Nevertheless, we could not distinguish two specific spoligotyping signatures suggesting two major lineages that had evolved independently.

The evolution of the DR region is probably unidirectional, occurring by single spacer deletions or loss of contiguous spacer sequences [START_REF] Fang | IS6110 transposition and evolutionary scenario of the direct repeat locus in a group of closely related Mycobacterium tuberculosis strains[END_REF][START_REF] Van Embden | Genetic variation and evolutionary origin of the direct repeat locus of Mycobacterium tuberculosis complex bacteria[END_REF]. Among our strains, SB0120 (BCG-like) is the most likely common ancestor, from which SB0121 may have emerged by loss of spacer 21. The role of spacer 21 and the possible implications of its deletion have to be elucidated, as it has also been described for Portuguese isolates. . The SB numbers and the binary code of the 252 spoligotypes described in this study are represented, as well as the total number of isolates and the prevalence in Spain of each spoligotype at national and regional level.

Conclusion

DVR

  technique at the national level and for provinces (Spanish administrative subdivision). We used the in-silico website of the University of the Basque Country (http://www.insilico.ehu.es), filling in the number of unrelated strains for each spoligotype. For this purpose we only counted one spoligotype when isolates of the same herd or a precise geographical area (i.e. National Parks, hunting estates) shared identical patterns.Additionally, we calculated the quotient of the number of unrelated strains (with unknown epidemiological link) and the number of different spoligotyping patterns observed for that region in order to describe the degree of diversity in each province.

  .Mbovis.org. The 252 spoligotypes are attached as supplementary table.

  both regions intensive sampling for typing purposes has been carried out in 2006 and 2007.

  finding of 207 spoligotypes exclusive to cattle gives evidence that cattle-specific spoligotypes are responsible for much of the diversity of the Spanish M. bovis population. The involvement of wildlife in the epidemiology of the infection is highlighted by the fact that 12 out of the 15 most frequent types are present both in cattle and at least in one wild Artiodactyla species. The special farming system A c c e p t e d M a n u s c r i p t 14 in Spain may have promoted the transmission from cattle to sympatric wildlife and the broad host range of these strains reflects its success as a pathogen. The results of this study also contribute to the knowledge of the demography of M. bovis in Spain within the European context. A comparison of the spoligotypes and D values reported in publications could explain the spread of M. bovis infection. Modern genetic and archeological evidence suggests that the domestication centre of European cattle (B. taurus) was the Near East at the very beginning of the Neolithic period[START_REF] Beja-Pereira | The origin of European cattle: evidence from modern and ancient DNA[END_REF][START_REF] Edwards | Mitochondrial DNA analysis shows a Near Eastern Neolithic origin for domestic cattle and no indication of domestication of European aurochs[END_REF].Recent articles of cattle phylogeny based on mitochondrial DNA showed the origin of European cattle in the Fertile Crescent and the subsequent expansion from this ancestral population[START_REF] Götherström | Cattle domestication in the Near East was followed by hybridization with aurochs bulls in Europe[END_REF][START_REF] Beja-Pereira | The origin of European cattle: evidence from modern and ancient DNA[END_REF]. They also highlight the strong influence of cattle of North African origin introduced by maritime routes into the Mediterranean countries. These findings are coherent with the hypothesis of ancient cattle being infected with ancestral M.

  clonal expansion of the founder strains. Due to the orography and history of the Iberian Peninsula, the exchange of cattle between certain regions has been impeded for a long time and therefore favoured an independent evolution of M. bovis strains. The high diversity in Spain may have been caused

  -spoligotyping revealed a high degree of strain diversity among Spanish M. bovis isolates (D=0.87) and thus can be considered a useful tool for the study of epidemiology of the infection in our country. The distribution of spoligotypes was unequal in Spain both in terms of geographical presence and frequency, and even in infection in the different host species. The implementation of the international database (www.Mbovis.org) has simplified the comparison between countries. A comparison of spoligotypes available in the scientific A c c e p t e d M a n u s c r i p t 16 literature reveals a hypothesis about the spread of bovine tuberculosis that has shaped the M. bovis population structure in Europe. Our next target is the completion of a national database which integrates conventional epidemiology and GIS in order to support the national eradication campaign by exploiting the molecular epidemiology data. J. Morey for careful revision of the manuscript.The group is a partner of the coordination action "Veterinary European Network on Mycobacteria (VENoMYC)" funded by the European Union.

Figure 1 .

 1 Figure 1. Frequencies of the 43 spacers of the DR locus that are included in the

  org. b Spoligotype represented as binary code. 1: presence of spacer; 0: absence of spacer. c Total number of isolates. d Prevalence of each spoligotype within the 6215 M. bovis isolates from Spain. e Spanish Autonomous regions from which the M. bovis isolates were obtained: AR: Aragon; AS: Asturias; BC: Basque Country; CA: Cantabria; CT: Catalonia; GA: Galicia; LR: La Rioja; NA: Navarra; CL: Castile and Leon; CM: Castile-La Mancha; EX: Extremadura; MA: Madrid; BI: Balearic Islands; CI: Canary Islands. f Geographic location in Spain. N: North; C: Centre; S: South.

Table ) .

 ) The predominant patterns were repeatedly found in all

	regions and in different animal species. We observed accumulation of some
	spoligotypes in certain regions. For instance, SB0339 was isolated from animals
	from central and northern regions, whereas SB1230 originated from isolates from
	the South. The analysis of the geographical distribution of 137 types (excluding
	M the orphan and epidemiologically-linked isolates) showed that the majority (75.9%) is disseminated while 33 types appear localized. The strains that appeared in delimited areas and presented a high prevalence, especially SB0339 (Monte El Pardo Nature Reserve) and SB1232 (Doñana National Park), must not be a n u s c r i p t overestimated, given that intensive sampling for study purposes had been carried
	A c c e p t e d
	10

Table 2

 2

	). Six patterns were

animal species, as shown in Table

1

. There are only two exceptions: SB1305, isolated from cattle from the north of Spain, and SB1337 isolated from cattle from

TABLE 1 .

 1 Distribution of the spoligotypes of the Spanish Mycobacterium bovis

	isolates according to prevalence [frequent types (>1%), infrequent types and
	orphans (singular types)] by animal species.
						No. of isolates from
	Spoligotype	Cattle	Goat	Pig	Deer	Wild boar	Iberian	lynx	Fox	Dog	Cat	Mouflon	Badger	Chamois	Prevalence
	SB0140	62				1									1.01
	SB1305	65													1.05
	SB1258	66					1		1	1				1.11
	SB1337	90													1.45
	SB0135	104			1	1									1.71
	SB0152	106				1									1.72
	SB0119	112			4	13									2.08
	SB1232	37			49	54	3	1						2.32
	SB0130	204			2	4					1				3.40
	SB0120	241			2	3									3.96
	SB0295	240	1	1	3	8	2							4.10
	SB0265	377		6	2	7									6.31
	SB0339	235			254 12									8.06
	SB0134	686			2	9									11.21
	SB0121	1653 31		16	37									27.94
	Orphans (84)	76	1		3	2					1	1			1.35
	Others (153)	1231			32	52			1				1	2	21.22

Table 2 .

 2 Distribution of the Mycobacterium bovis isolates according to animal species, and number of spoligotypes which were found only in a determined species.

	Animal species Isolates	Types	Exclusive types
	Cattle	5585	239	207
	Wild boar	204	26	6
	Red deer	141	22	2
	Goat	33	3	1
	Cat	3	3	1
	Mouflon	1	1	1
	Fallow deer	229	13	1
	Pig	7	2	0
	Iberian Lynx	6	3	0
	Chamois	2	1	0
	Fox	2	2	0
	Badger	1	1	0
	Dog	1	1	0

Acknowledgements

This research was funded by EU project TB-STEP (KBBE-2007-1-3-04, no. 212414) and the Ministry of Environment and Rural and Marine Affairs. S.

Rodríguez is recipient of a PhD studentship AP2006-01630 of the Spanish Ministry of Science and Innovation.

We would like to thank the National and Regional Animal Health authorities, in especial L. Carbajo and J.L Paramio for their continuous encouragement. We are grateful to T. Alende and A. Gutiérrez for technical help. Contributing members to the Spanish Network on Surveillance and Monitoring of Animal Tuberculosis are F. Garrido (Laboratorio Central de Sanidad Animal de Santa Fé, Granada, MARM), staff of Government and Regional and Research Laboratories of Autonomous Communities [J. Tellez, C. Fornell, A. Jiménez, J.M. Gómez, E.J. Villalba and I. Muñoz (Andalucía), J. Gracia, I. Belanche, S. Izquierdo, N. Abacens, J.M. Malo (Aragón), E. Fernandez, M.F. Copano, I. Merediz, J.M. Prieto and A. Espí (Asturias), V. Vigo (Canarias), F.M. Fernández, E. Sola and C. Fernández (Cantabria), P. García, M.R. Bermúdez, V. Alcaide, C. Rojas, M.L. Rando, A. Sánchez, J. Alonso, F. Plaza, C. Fernández, J.A. Viñuelas, J. Alia and E. Grande (Castilla La Mancha), O. Mínguez, F. Fernández, C. Domínguez, J.A. Anguiano, F. Moreno, I. Romero, C. Martínez, I. Burón, A. Grau and O. Martín (Castilla y León), J. Gou (Cataluña), J.R. Puy (Euskadi), C. Sanz and E. Dorado (Extremadura), J.E. Mourelo, D. Fernández and C. Calvo (Galicia), M.J. Portau, C. Aguilo and C. Vidal (Islas Baleares), J. Carpintero, E.


I M G A Santman-Berends

L Van Wuijckhuise

P Vellema

P A Van Rijn

Vertical transmission of bluetongue virus serotype 8 virus in Dutch dairy herds in 2007

Keywords: bluetongue, BTV-8, calves, vertical transmission

In February 2008, evidence was found for transplacental infection of bluetongue virus serotype 8 (BTV-8) in PCR negative, seropositive heifers in Northern Ireland originating from the Netherlands. The relevance of this route of transmission was studied in Dutch cow-calf combinations in the Netherlands of which the calves were born in the same time period of the year as the calves from the exported heifers, the first quarter of 2008. Blood samples were tested Manuscript A c c e p t e d M a n u s c r i p t 2 from 385 cows and their calves, housed in 43 dairy farms that became naturally infected with BTV-8 for the first time in 2007. All calves were at least ten days old at the moment of first testing. In total 229 cows tested seropositive for BTV-8. Eight of these cows were still PCR positive. Out of the 229 seropositive cows, 37 calves (16.2%; 95% CI: 11.4-21.0) were tested PCR positive in the first sample taken in April 2008. In the first week of June, 34 out of the 37

PCR positive calves were still available for resampling. Three calves were still PCR positive; one was five months old, the other two were three months old. One month later, in the first week of July, all initially PCR positive calves, including the three still tested positive one month earlier, were PCR negative. We showed that BTV-8 can be vertically transmitted from cow to calf and can result in healthy looking viraemic calves remaining PCR positive for up to five months.

These PCR positive calves could play a role in the epidemiology, and in particular in overwintering of BT. However, further investigations are needed to evaluate the importance of this route of transmission.

Introduction

In 2006, bluetongue virus serotype 8 (BTV-8) emerged in the Netherlands for the first time (BTV8/net2006 in [START_REF] Maan | Sequence analysis of bluetongue virus serotype 8 from the Netherlands 2006 and comparisons to other European BTV strains[END_REF]. The infection started in August and spread until November. In that year BTV-8 only spread in the southern part of the Netherlands and approximately 200 cattle herds notified clinical signs to the Food and Consumer Safety Authority (VWA) [START_REF] Van Wuijckhuise | Bluetongue in the Netherlands; description of the first clinical cases and differential diagnosis. Common symptoms just a little different and in too many herds[END_REF][START_REF] Efsa | Scientific Report of the scientific Panel on Animal Health and Welfare on request from the Commission (EFSA-Q-2006-311) and EFSA self mandate (EFSA-Q-2007-063) on bluetongue[END_REF][START_REF] Elbers | Field observations during the bluetongue serotype 8 epidemic in 2006. I. Detection of first outbreaks and clinical signs in sheep and cattle in Belgium, France and the Netherlands[END_REF]. Overall, BTV-8 caused A c c e p t e d M a n u s c r i p t 3 only limited morbidity and mortality during this first episode in 2006 [START_REF] Elbers | Field observations during the bluetongue serotype 8 epidemic in 2006. I. Detection of first outbreaks and clinical signs in sheep and cattle in Belgium, France and the Netherlands[END_REF][START_REF] Van Schaik | Seroprevalence of bluetongue serotype 8 in cattle in the Netherlands in spring 2007 and its consequence[END_REF].

In the spring of 2007, after the first BTV-8 episode in the Netherlands, a small scale study was performed to determine the possibility of overwintering of BTV-8 by vertical transmission. In this study 50 suckling cows and their newborn calves, originating from 14 BTV-8 infected herds, were tested for BTV-8. From the suckling cows 36 (72%; 95% CI: 59.6-84.4) tested seropositive and PCR negative in blood and colostrum. All 31 newborn calves were tested PCR negative, before and after colostrum intake (van Wuijckhuise and Vellema, 2008). Based on these results, it was concluded that there was no or a small risk on vertical transmission to newborn calves.

However, in the third week of February 2008, one heifer and three calves from a group of 21 pregnant heifers imported from the Netherlands were tested BTV-8 PCR positive in Northern Ireland. Eight of these 21 heifers were BTV-8 seropositive, and all were PCR negative at the time of departure from the Netherlands and also at arrival in Northern Ireland. More than a month after arrival, one of the originally seronegative heifers tested PCR positive and three of four newborn calves from seropositive but PCR negative heifers were also tested PCR positive [START_REF] Menzies | Evidence for transplacental and contact transmission of bluetongue virus in cattle[END_REF]. Based on these results and taken the Culicoides-free period into account, it was likely that BTV-8 was transmitted to the fetus after infection of the pregnant cow.

Recently, it was shown that experimental infection with BTV-8 of a cow in the final stage of pregnancy could result in the birth of a viraemic calf. In the same study, it was demonstrated that infection by BTV-8 could occur after uptake of BTV-8 contaminated colostrum [START_REF] Backx | Transplacental and oral transmission of wild-type bluetongue virus serotype 8 in cattle after experimental infection[END_REF]. However, the relevance of transplacental and oral transmission for the epidemiology, the undesired movement of a possible BTV-8 reservoir, and in particularly the overwintering of BTV-8 was unknown.

Materials and Methods

Study population and data collection

In the spring of 2008, dairy herds were selected from a region in the eastern part of the Netherlands in which massive spread of BTV-8 had occurred for the first time in September and October 2007 [START_REF] Santman-Berends | The monthly increase of the BTV-8 within-herd prevalence and associated risk factors in Dutch dairy herds, during the second episode in[END_REF] (Figure 1). Based on an unknown expected prevalence of 50%, an accepted error of 5% and a confidence level of 95%, 400 cows and their newborn calves which were born in the first quarter of 2008, were blood sampled and BTV-8 PCR and serologically tested.

The inclusion criteria for a cow-calf combination were that both the cow and her calf remained on the farm that was affected by BTV-8 in 2007. Furthermore, the calf had to be at least 10 days old and had to be born in the first quarter of 2008.

(Figure 1) Dairy herds should have at least eight calves that fulfilled the criteria to be finally selected (Figure 2). Fifty-five herds met these criteria and were contacted. Eventually 43 farmers were willing to cooperate in this study. From these 43 dairy herds, 388 cows and their calves were sampled for serum and EDTA-blood between March 27 th and April 8 th (Figure 2). Three combinations were excluded from the study because of improper samples. Eventually, 385 cow-calf combinations remained for analysis. Sera from cows and calves were tested for antibodies to BTV-8 with a competitive VP7 ELISA (Institut Pourquier, Montpellier, France).

EDTA-blood samples were tested for BTV-8 with an in-house developed real time reverse transcriptase polymerase chain reaction assay (PCR test) [START_REF] Van Rijn | High throughput RRT-PCR diagnosis for Bluetongue base don genome segment 10[END_REF]. If possible, PCR positive calves in the first sampling (Figure 2) were monthly resampled for serum and EDTAblood, starting in June (Figure 2).

Statistical analysis

Proportion tests were used to determine the significance in difference between the number of cows and calves positive in ELISA or PCR test.

Results

Results of the first sampling

From the 385 available combinations, 229 (8+221) cows (59.5%) and 234 (37+197) calves (60.8%) were seropositive (Table 1). Of these 229 seropositive cows, eight cows were PCR positive (3.5%), of which three had a relatively weak PCR-signal. Of the 234 seropositive calves, 37 calves were PCR positive (15.8%).

There was no significant difference between the number of seropositive calves and cows at first testing (proportion test; P-value: 0.77). However, the number of PCR positive calves was All 156 seronegative cows (40.5%; 95% CI: 35.6-45.4%) also were PCR negative. Fourteen calves from these seronegative cows (9.0%; 95% CI: 4.5-13.5%) were seropositive (Table 1).

(Table 1)

From 229 seropositive cows (59.5%; 95% CI: 54.6-64.4%), 37 (3+34) calves (16.2%; 95% CI: 11.4-21.0) were both PCR positive and seropositive. Eight of these seropositive cows were PCR positive. Their respective calves were all seropositive and three out of eight were also PCR positive (Table 1). The remaining 221 seropositive cows (57.4%; 95% CI: 52.5-62.3%) were PCR negative. From their 221 respective calves, 178 calves (80.5%; 95% CI: 75.3-85.8%) were seropositive and PCR negative, however, 34 (15.4%; 95% CI: 10.6-20.1%) calves were positive in both tests and nine (4.1%; 95% CI: (1.5-6.8%) calves were negative in both tests (Table 1).

Results of the follow-up period

At first sampling, 37 out of 385 calves (9.6%; 95% CI: 6.6-12.6) were PCR positive (Table 1).

These PCR positive calves were resampled monthly from June 2008 until the end of the study period in September 2008.

At second sampling in the first week of June, 34 calves were available for resampling; two had died for unknown reasons and one was no longer present on the farm. Three out of 34 initially PCR positive calves were still PCR positive (8.8%; 95% CI: 1.9-23.7) (Figure 3). In the first week of July, all 34 initially PCR positive calves were PCR negative and remained PCR negative in following resamples (Figure 3).

All 34 initially PCR positive calves available for resampling were also seropositive in April. Of these 34 calves, 32 calves (94.1% 95% CI: 80.3-99.3) remained seropositive during the whole study period which was until September. Two initially PCR positive calves originating from different herds had no detectable levels of antibodies against BTV as determined by ELISA in September 2008 (Figure 3).

Discussion

In February 2008, BTV-8 seropositive but PCR negative heifers imported into Northern Ireland gave birth to PCR positive healthy looking calves during the Culicoides-free period, providing evidence of transplacental transmission of the virus [START_REF] Menzies | Evidence for transplacental and contact transmission of bluetongue virus in cattle[END_REF]. Before this event, it was generally assumed that infection in utero by field strains of BTV could not result in healthy looking calves. In utero infection resulting in healthy looking calves seemed only possible for laboratory-adapted or vaccine strains [START_REF] Osburn | Experimental viral-induced congenital encephalopathies. I. Pathology of hydranencephaly and porencephaly caused by bluetongue vaccine virus[END_REF][START_REF] Jochim | Bluetongue in cattle: immunogenic and clinical responses in calves inoculated in utero and after birth[END_REF]Waldvogel et al, 1992), whereas infection with field strains of BTV would result in abortion, malformed or weak calves, or dummy calves [START_REF] De Clercq | Transplacental bluetongue infection in cattle[END_REF][START_REF] Maclachlan | Induced brain lesions in calves infected with bluetongue virus[END_REF]Wouda et al., 2008;[START_REF] Maclachlan | Bluetongue virus-induced hydranencephaly in cattle[END_REF].

A small scale study performed in the Netherlands in the spring of 2007 (van Wuijckhuise and Vellema, 2008), demonstrated that there was no or a small risk on transplacental transmission.

Hence, it was unclear whether the findings of [START_REF] Menzies | Evidence for transplacental and contact transmission of bluetongue virus in cattle[END_REF] were a common or rare A c c e p t e d M a n u s c r i p t 8 observation and therefore a larger study was performed. From an area in the Netherlands which was heavily affected for the first time in 2007, 385 cow-calf combinations were selected in order to find cows which were likely to have been infected during gestation. In parallel to the observation in Northern Ireland, calves in this study were born in the first quarter 2008. From the 385 combinations selected for this study, 229 cows were seropositive at the end of March. This indicated that they were infected before calving, because the Culicoides-free period, which was based on outdoor temperatures and Culicoides activity, started the 13 th of December 2007 and lasted until the 21 st of April 2008. Furthermore, these cows probably were infected during gestation, as the area was first affected in 2007. Eight cows were still PCR positive at the end of March which is in agreement with previous observations that cattle can be PCR positive for more than 200 days post infection. The calves from these eight PCR positive cows were all seropositive and three out of eight were also PCR positive. This latter finding indicated that BTV-8 was vertically transmitted from cow to calf, since birth and sampling occurred in the period without Culicoides activity. In other words, it is most unlikely that these newborn calves were infected by midges. From the remaining 221 calves, originating from BTV-8 infected cows, 178 were seropositive, but PCR negative, and 34 calves were PCR positive and seropositive. The first group of 178 seropositive calves can be explained by the uptake of colostral antibodies, or by seroconversion in combination with clearance of BTV-8.

In April 2008, the number of PCR positive calves (37) was significantly larger than the number of PCR positive dams (8). Apparently, the PCR-signal remained longer in the newborn calves than in the cows, the fetal infection was significantly delayed or the fetal infection occurred later with respect to that of the cow. Alternatively, these calves could be infected by uptake of colostrum contaminated with BTV-8 like this has been shown experimentally [START_REF] Backx | Transplacental and oral transmission of wild-type bluetongue virus serotype 8 in cattle after experimental infection[END_REF]. There is no evidence that colostrum was contaminated by BTV-8, although colostrum can A c c e p t e d M a n u s c r i p t 9 easily contain blood due to collapsed veins in the udder of freshly calved cows. Furthermore, nine calves from 229 seropositive cows were negative in both tests. Probably, these calves did not receive antibodies by insufficient uptake of colostrum with antibodies or by uptake of colostrum from a seronegative cow.

In total, 37 calves out of the 229 seropositive cows tested PCR positive in our study, indicating that the transplacental transmission rate in our study was 16.2% (95% CI:11.4-21.0). This rate is lower than the transplacental transmission rates found in the study of [START_REF] Galleau | Can vaccination prevent transplacental transmission of BTV-8? In proceedings of Epizone[END_REF] and [START_REF] Batten | Transplacental transmission of bluetongue serotype 8 (BTV-8) in the United Kingdom (2007-2008)[END_REF]. [START_REF] Galleau | Can vaccination prevent transplacental transmission of BTV-8? In proceedings of Epizone[END_REF] tested 10 out of 24 calves (41.7%), born from seropositive dams, PCR positive within the first four days after birth. [START_REF] Batten | Transplacental transmission of bluetongue serotype 8 (BTV-8) in the United Kingdom (2007-2008)[END_REF] found 21 out of 61 calves (34.4%), born from seropositive dams, PCR positive within the first three days after birth. The difference between our study and theirs was that [START_REF] Batten | Transplacental transmission of bluetongue serotype 8 (BTV-8) in the United Kingdom (2007-2008)[END_REF] and [START_REF] Galleau | Can vaccination prevent transplacental transmission of BTV-8? In proceedings of Epizone[END_REF] tested the calves within three or four days after birth, whereas the calves in our study could be up to three months old at the moment of sampling. Our study and the study of [START_REF] Batten | Transplacental transmission of bluetongue serotype 8 (BTV-8) in the United Kingdom (2007-2008)[END_REF] showed that the PCR positive calves became PCR negative during the time period of the study. Thus the 16.2% PCR positive calves we found in April 2008 is probably an underestimation of the real transplacental transmission rate, because calves born PCR positive, could have become PCR negative at the moment of first testing.

From the 156 PCR-and seronegative cows, 14 calves tested seropositive. The most logical explanation is that these calves were fed colostrum of a seropositive cow.

In our study, no acute infections were detected in the first sampling at the end of March, as there were neither cows nor calves PCR positive and seronegative. This is in agreement with the absence of Culicoides activity in the Culicoides-free period.

A c c e p t e d M a n u s c r i p t 10 Fetal infection in the first three months of gestation has been assumed to result in abortion [START_REF] De Clercq | Transplacental bluetongue infection in cattle[END_REF][START_REF] Maclachlan | Induced brain lesions in calves infected with bluetongue virus[END_REF]Wouda et al., 2008;[START_REF] Maclachlan | Bluetongue virus-induced hydranencephaly in cattle[END_REF]. Fetal infection later in gestation could result in the birth of PCR positive calves up to six months after infection of the cow. At the time of birth, a new generation of Culicoides could be infected by feeding on these newborn viraemic calves and subsequently start spread of BTV-8 again after a Culicoides-free period. However, we were not able to cultivate virus from the samples of these calves with standard procedures, probably due to the presence of maternal neutralizing antibodies (data not shown). Nevertheless, the blood from the calves may be infectious to Culicoides. [START_REF] Mertens | Enhanced infectivity of modified bluetongue virus particles for two insect cell lines and for two Culicoides vector species[END_REF] showed that pretreatment of BTV-containing blood samples with serum or protease, increased the infectivity for Culicoides cells. We speculate that partial digestion in the gut of Culicoides could result in increased infectivity and thus in BTVreplication. In addition, in Dutch dairy herds about 10% to 25% of the newborn calves do not drink (sufficient) colostrum from their dams. In the absence of maternal antibodies, it was shown that BTV-8 could be easily cultivated from a viraemic newborn calf [START_REF] Backx | Transplacental and oral transmission of wild-type bluetongue virus serotype 8 in cattle after experimental infection[END_REF].

Our study also showed that calves could remain PCR positive up to five months of age. Although it is unknown whether PCR-positivity is related to infectivity of the midge and although the majority of the viraemic calves are PCR positive for only a short period of time, we emphasize that one or a few viraemic calves could induce the (re)start of BTV-8 circulation. Studies with species of Culicoides are needed to proof whether Culicoides can be infected by bites on these PCR positive calves. If so, BTV-8 could easily overcome a Culicoides-free period of 4-5 months by transmission to the fetus.

In seasons and/or in areas with a high level of Culicoides activity, vertical transmission will be of less or no importance for spread of BTV-8. However, in periods and/or areas without or with a Here we have shown that vertical transmission of BTV-8 in cattle is not rare and that, in the period without Culicoides activity, this mechanism could play an important role for the vectorborne disease bluetongue.

  a study to determine the percentage of PCR positive calves born in the first quarter of 2008 from cows infected with BTV-8 in 2007. Subsequently, PCR positive newborn calves were resampled to determine the length of PCR-and ELISA-positivity.

  to the number of PCR positive cows (proportion test; Pvalue:<0.001).

  From these three, one calf was born in January 2008 and the other two calves were born in March 2008.

  of Culicoides activity, vertical transmission could become relatively important by lack of other routes of transmission. Further research will be needed to proof the significance of potential routes of transmission of BTV-8 in order to survive the winter in north-western Europe.

  L., Vellema, P., 2008. The role of pregnant cows in the overwintering of bluetongue (in Dutch). Report number 2080005 GD Deventer, the netherlands Waldvogel, A.S., Anderson, G.A., Phillips, D.L., Osburn, B.I., 1992. Association of virulent and avirulent strains of bluetongue virus serotype 11 with premature births of late-term bovine fetuses. J. Comp. Pat. 106, 333-340. Wouda, W., Roumen, M.P.H.M., Peperkamp, N.H.M.T., Vos, J.H., Van Garderen, E., Muskens, J., 2008. Hydranencephaly in calves following the bluetongue serotype 8 epidemic in the Netherlands. Vet. Rec. 162, 422-423.

  Figure 1. The Netherlands has been divided in 20 compartments. The within-herd prevalence per

Figure 2 .Figure 3 .

 23 Figure 2. The timeline of the study in 43 Dutch dairy herds from January until September 2008.

Table 1 .

 1 Results of the PCR test and ELISA on 385 cow-calf combinations tested between March 27 th and April 8 th , 2008.

	Cows

Table 1

 1

Acknowledgements

This project was financially supported by the Dutch Ministry of Agriculture, Nature and Food Quality (LNV). We thank farmers and veterinarians for participation in this study.