Neil Leblanc
email: neil.leblanc@sva.se

Mikael Leijon

Magnus Jobs

Jonas Blomberg

Sándor Belák

A novel combination of TaqMan RT-PCR and a suspension microarray assay for the detection and species identification of pestiviruses

Keywords: pestivirus, CSFV, BDV, BVDV1, BVDV2, TaqMan real-time PCR, suspension microarray

The genus Pestivirus contains four recognized species: classical swine fever virus, Border disease virus, bovine viral diarrhoea virus types 1 and 2. All are economically important and globally distributed but classical swine fever is the most serious, concerning losses and control measures. It affects both domestic pigs and wild boars.

Outbreaks of this disease in domestic pigs call for the most serious measures of disease control, including a stamping out policy in Europe. Since all the members of the Pestivirus genus can infect swine, differential diagnosis using traditional methods poses some problems. Antibody tests may lack specificity due to cross-reactions, antigen capture ELISAs may have low sensitivity, and virus isolation may take several days or even longer time to complete. PCR-based tests overcome these problems for the most part, but in general lack the multiplexing capability to detect and differentiate all the pestiviruses simultaneously. The assay platform described here addresses all of these issues by combining the advantages of real-time PCR with the multiplexing capability of microarray technology. The platform includes a TaqMan real-time PCR designed for the universal detection of pestiviruses and a microarray assay that can use the amplicons produced in the real-time PCR to identify the specific pestivirus.

A c c e p t e d M a n u s c r i p t 3

Introduction

The genus Pestivirus in the family Flaviviridae includes economically important, widespread pathogens causing postnatal and prenatal infections in a range of host animals, such as cattle, swine, and sheep (Moennig & Plagemann, 1992). The recognized species are classical swine fever virus (CSFV), Border disease virus (BDV), and bovine viral diarrhoea (BVD) virus types 1 and 2 (BVDV1, BVDV2). Whereas CSFV appears to be restricted to swine and wild boars, BDV, BVDV1 and BVDV2 can infect various domestic and wild ungulate species, including swine [START_REF] Becher | Genetic characterization of novel pestivirus genotypes: implications for classification[END_REF]. Cattle are susceptible for infections with both BVDV types and rarely with BDV, sheep can be infected with the two BVD viruses as well as BDV and pigs can be infected with CSFV, BDV, BVDV 1 and 2 [START_REF] Deregt | A multiplex DNS suspension microarray for simultaneous detection and differentiation of classical swine fever and other pestiviruses[END_REF].

Pestiviruses are enveloped, positive sense, single-stranded RNA viruses with a genome of approximately 12 kb, which consists of one large open reading frame flanked by highly conserved 5' and 3' non-translated regions. They have closely related genomes and are antigenically cross-reactive, which can cause diagnostic uncertainty and errors (Hoffman et al., 2005). This is particularly relevant to the diagnosis of classical swine fever (CSF), where a false positive result may have severe economic consequences. CSF is on the list of diseases notifiable to the World Organization for Animal Health (OIE). In 1997, an outbreak of CSF in the Netherlands resulted in approximately 11 million pigs being put down and caused direct losses (excluding loss of exports) of over two billion U.S. dollars [START_REF] Pluimers | Classical swine fever in The Netherlands 1997-1998: a description of organisation and measures to eradicate the disease[END_REF][START_REF] Terpstra | The 1997/1998 epizootic of swine fever in the Netherlands: control strategies under a non-vaccination regimen[END_REF]. As evident by inspection of the data given by the OIE World Animal Health Information Database (WAHID), BDV causes Border disease, a congenital disease affecting sheep and goats, although rare in goats. Clinical signs include abortions, barren ewes, stillbirths and small weak lambs, which can show tremors, abnormal body conformation and abnormal birthcoat [START_REF] Nettleton | Border disease of sheep and goats[END_REF].

BVDV is endemic in most cattle-raising countries globally, with 60-85% of adult cattle being antibody positive (Houe, 1999). The outcome of infection of susceptible females with BVDV includes embryonic death, abortion, foetal anomalies and/or the birth of persistently infected (PI) animals. After birth, BVDV may cause a range of disease syndromes and might also have immunosuppressive potential, which can result in increased susceptibility and more severe clinical effects of other infectious agents [START_REF] Potgieter | Immunology of bovine viral diarrhea virus[END_REF].

Recent progress in real-time PCR-based diagnostics has been reported employing a variety of novel molecular chemistries including molecular beacons (McKillen et al., 2006) and primer-probe energy transfer [START_REF] Hakhverdyan | Development of a real-time PCR assay based on primer-probe energy transfer for the detection of swine vesicular disease virus[END_REF] with the attempt to improve factors such as specificity, multiplexing and robustness towards mutation in the probe region. However, in real-time PCR the number of parallel detections are limited by the number of available fluorescence channels on the real-time fluorescence reader (to our knowledge at most six at the time of writing) and by difficulties caused by cross-talk between the fluorescence channels and interactions between simultaneously amplifying PCR and for these reasons rarely exceeds four or five (Vet et al., 1999;[START_REF] Molenkamp | Simultaneous detection of five different DNA targets by real-time TaqMan PCR using the Roche LightCycler480: Application in viral molecular diagnostics[END_REF]. In this paper, we describe an assay platform that combines general TaqMan real-A c c e p t e d M a n u s c r i p t 5 time PCR for optimized high sensitivity detection of pestiviruses with a low-density microarray for subsequent species identification. The assay is well suited for implementation in a routine diagnostic laboratory for the screening of clinical samples as well as confirming and identifying virus isolates in culture.

Materials and Methods

Sample preparations

The quantitated pestivirus RNA samples used in the present study were taken from a panel of pestivirus strains and isolates supplied by The Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Insel Riems, Germany (Table 1). This panel of pestiviruses represented a collection of CSFV, BDV, BVDV1 and BVDV2, which was pre-tested by the EPIZONE international consortium, thus, it provided a reliable basis for the estimation of pestivirus detection capacity. Samples were provided with a concentration of 10 4 copies/µl. All experiments, excluding sensitivity tests, were performed using samples that were diluted 100 times in RNase-free water unless otherwise mentioned and used in the PCR experiments without further purification. In addition, one isolate, representing a recently discovered atypical bovine pestivirus, was provided by our laboratories at the Swedish National Veterinary Institute (SVA). This isolate, originating from cattle in Thailand, was currently referred to as TKK virus [START_REF] Ståhl | Natural infection of cattle with an atypical 'HoBi'-like pestivirus -Implications for BVD control and for the safety of biological products[END_REF]Liu et al., 2008).

Sensitivity was estimated using a dilutions series of the Alfort 187 1.1 CSFV strain from the pestivirus panel. Specificity tests were performed using RNA extracted A c c e p t e d M a n u s c r i p t 6 from four swine influenza isolates propagated in eggs. Cultured samples of bluetongue virus subtypes 2 and 8, porcine herpesvirus type 1 and bovine herpesvirus type 1 were also used to test specificity. Extractions were performed by Trizol-chloroform method according to manufacturer's instructions (Invitrogen). Negative serum, intestine and semen samples from swine and negative blood samples from cattle were also tested. For these samples the RNA was extracted using the GenoM-48 workstation (GenoVision/Qiagen) and the MagAttract Virus Mini M48 kit (Qiagen). 400 µl of sample material was extracted according to MagAttract Virus Mini Protocol version 0.5.2 and eluted in 100µl RNase free water containing 0.04% sodium azide.

Reverse transcription and real-time PCR

Three sets of previously published primers, all from the conserved 5'-UTR region, were evaluated on the entire set of panel samples. The primers were those published by Vilcek et al (1994): forward primer, ATG CCC (T/A)TA GTA GGA CTA GCA ; reverse primer, TCA ACT CCA TGT GCC ATG TAC, by [START_REF] Elvander | An experimental study of a concurrent primary infection with bovine respiratory syncytial virus (BRSV) and bovine viral diarrhoea virus (BVDV) in calves[END_REF]:

forward primer, GCT AGC CAT GCC CTT AGT AGG A; reverse primer, ATC AAC TCC ATG TGC CAT GTA CAG C, and by [START_REF] Deregt | A multiplex DNS suspension microarray for simultaneous detection and differentiation of classical swine fever and other pestiviruses[END_REF]: forward primer, CAT GCC C(A/G)(C/T) AGT AGG ACT AGC; reverse primer, ATG TGC CAT GTA CAG CAG AG. In the followings, the primer pairs published in the above listed three articles will be referred to by Vil, Elv and Der, respectively. It has been shown that asymmetric PCR, which creates an excess of one of the strands, yields better signal-to-noise on Luminex equipment [START_REF] Deregt | A multiplex DNS suspension microarray for simultaneous detection and differentiation of classical swine fever and other pestiviruses[END_REF]. In the present study we take a different approach and use Lambda exonuclease to digest one of the amplicon strands. To facilitate A c c e p t e d M a n u s c r i p t 7 the digestion, the forward primers were 5´-phosphorylated. In addition the reverse primers were 5'-biotinylated to allow conjugation of streptavidin-phycoerythrin for later detection by the Luminex instrument. The real-time probe construct was as follows: 5´ 6-Fam-TGG GCA TGC CCT CGT CCA C-BHQ-1 3´. This probe was designed, validated and is currently in use at the routine diagnostic laboratory of the Swedish National Veterinary Institute (P. Thorén, SVA, personal communication). One-step RT-PCR reactions were employed using the SuperScript™ III One-Step RT-PCR System with Platinum® Taq from Invitrogen. The reaction volume was 12.5 µl and the primer and probe concentrations were 1.3 and 0.25 µM, respectively. The PCR cycling profile was as follows: a reverse transcription step at 50°C for 30 minutes, an enzyme activation step at 94°C for 2 minutes and, finally, 50 PCR cycles with 15 seconds denaturation at 95°C, 15 seconds primer annealing at 53°C and 40 seconds elongation at 72°C. Fluorescence was read during the annealing phase. PCR was performed on a Rotor-Gene 6000 (Corbett Life Science, Sydney, Australia). To carry out the nuclease digestion 0.15 µl Lambda exonuclease (Fermentas Life Sciences, Burlington, Canada) was added to the PCR mixture. The single strand product was generated by incubating the samples at 37°C for 30 minutes, followed by an enzyme inactivation step of 15 minutes at 80°C.

Suspension microarray

Eight amino-functionalised ssDNA oligonucleotides (Biomers) ranging from 18 to 45 bases were selected from a previous study [START_REF] Deregt | A multiplex DNS suspension microarray for simultaneous detection and differentiation of classical swine fever and other pestiviruses[END_REF] as capture probes for the array. Species specific probes included two universal CSFV probes (CSFV A and CSFV B) and one each for BVDV 1, BVDV 2, and BDV. Also, one universal BVDV The bead stocks (50,000 beads/µl) were diluted in 1.5× TMAC hybridization buffer such that 5,000 of each probe bead were present in each sample well.

Hybridization reactions were carried out in a total volume of 50 µl/well consisting of 5 µl of the PCR product, 10 µl TE buffer and 35 µl of the bead mix in duplicate in 96-well microtiter plates (Fisher Scientific, Waltham, MA, USA).. For a background control, a no template control from the PCR assay was used. The microtiter plates were incubated on a shaker plate at 50°C for 30 minutes at 600 rpm. A 12 µl volume of streptavidin-Rphycoerythrin (Caltag Laboratories, Burlingame, CA, USA) in 1× TMAC hybridization buffer, at a concentration of 0.01 mg/ml, was added and mixed with the reactants and incubation was continued at 50°C for 15 min. The median fluorescent intensity (MFI) of each reaction was measured in Luminex LX200 instrument at 50°C. A minimum of one hundred beads per probe was analyzed to calculate the MFI of each reaction.

Results and Discussion

Primer evaluation

All three primer pairs performed competently, successfully amplified the target viruses in the majority of samples, however, the Elv primers failed to amplify CSFV The Der primers proved, however, more robust over a broad range of primer concentrations during the optimization experiments and at optimized conditions never failed to amplify these strains while the Vil primers failed 44% of the time even at optimal primer concentration. A failure considered a low fluorescence (< 10:1 signal to noise with NTC) or a Ct > 35. Figure 1 shows results from one experiment using the Vil

Real-time detection followed by identification on suspension microarray

All 24 pestivirus strains and isolates (Table 1), except the TKK isolate, were run in duplicate in a single experiment on a RotorGene 6000 real-time PCR instrument. For The TKK amplification curve, likewise displayed as the average of a duplicate run, was obtained in a separate experiment but with the same temperature cycle. All strains were readily detected at the level of 100 genome copies per reaction with the Der primers (Figure 2, panels A, C & E); hence the detection rate in this study was 100%. In accordance with the fact that all samples were pre-quantified, all strains belonging to the same species (CSFV, BDV, BVDV1 or BVDV2) appeared at a similar cycle threshold (Ct) with the sole exception of the Congenital Tremor 3.1 strain that appeared 2-3 cycles later. This observation corroborates the generally observed greater difficulty to detect this sample as compared to the other strains investigated in this study. The late Ct value of the TKK isolate is not remarkable since the genome equivalents of this isolate were not precisely quantified in the present study. No amplification could be traced in the "no template" control samples (Figure 2A and2E).

In the microarray experiments, all nine probes generated a similar signal for no template control samples with an average MFI (median fluorescence intensity) of 38 and a standard deviation of 12. The threshold MFI signifying a pestivirus positive sample was set to 500 corresponding to ten times the sum of the average background and one standard deviation. The cross-species specificity was good as well with an average MFI of 43, only slightly higher than observed for the non-template control and a standard deviation of 33. The higher standard deviation originates from a slight interaction between the BDV probe with some other species isolates and a minor cross-reaction between the types 1 and type 2 BVDV specific probes. The strongest cross-species signal The PCR product of the atypical bovine pestivirus, TKK, did not cross-react with any of the other species specific and no cross-reactivity was observed between the new TKK probe and the recognized pestivirus strains (Figure 2).

Considering the positive signals in the microarray experiments it is clear that the common pestivirus probe A is less efficient than the B variant (Figure 2). The A probe frequently produces signals close to the threshold, e.g. the MFI for the CSFV strains: C-Stamm (1.1), Alfort 187 (1.1), Brescia (1.2), Schweiz II (1.2), D4886/82/Ro (2.2) the MFI's are 467,737,627,573 and 541,respectively,and for the CSFV Kanagawa (3.4) strain the probe even failed with an MFI of 217 (see Figure 2 and supplementary information). In addition, for the BDV strains, the common pestivirus probe A failed in three of four cases (Figure 2D). The common pestivirus probe B usually produce a stronger signal and detects all strains from the pestivirus panel, however, the novel atypical bovine pestivirus TKK was not detected by this probe (Figure 2F). The significance of the probe redundancy used by [START_REF] Deregt | A multiplex DNS suspension microarray for simultaneous detection and differentiation of classical swine fever and other pestiviruses[END_REF] is illustrated by this observation; the two common pestivirus probes together detect all 24 samples while A c c e p t e d M a n u s c r i p t 12 neither of the probes could do that alone. In common with [START_REF] Deregt | A multiplex DNS suspension microarray for simultaneous detection and differentiation of classical swine fever and other pestiviruses[END_REF] we observe (Figure 2B) that the species specific CSFV probe A are superior to the CSFV probe B that fails to detect five strains (Schweiz II (1.2), Paderborn (1.2), Bergen (2.2), D4886/82/Ro (2.2) and Uelzen (2.3)) and detects a forth strain weakly (Spante (2.3)).

However, it is interesting to note that, in our hands, the CSVF B probe is specific for genotype 1 and 3 while genotype 2 is not detected by this probe or detected weakly (Figure 2B). Hence, the combination of CSFV probes A and B provides some genotyping capacity. As mentioned above, the Luminex BDV probe detects three of four BDV strains. Finally, the common BVDV probe successfully detects all six strain of BVDV in the pestivirus panel and the type specific probes detect type 1 or 2 exclusively. However, none of the three BVDV probes produced a signal from the PCR product of the atypical bovine pestivirus TKK that only could be observed with the TKK specific probe (Figure 2F).

Sensitivity and specificity

In view of the similarity of the cycle threshold values of all pre-quantified samples from the pestivirus panel we only carried out sensitivity determinations on the CSFV strain Alfort 187 (1.1). Since the CSFV strain Congenital Tremor (3.1) (Figure 2A) and the BVDV 2 strains (Figure 2E) appeared later by 2-3 cycles than what was observed for the other strains it can be anticipated that the sensitivity estimate obtained with Alfort 187 (1.1) is an upper limit for these strains. In panel A of Figure 3 Specificity tests revealed that none of the heterologous viruses and clinical negative samples listed in section 2.1 were detected in real-time or on the microarray (data not shown).

Conclusions

In the present study we have demonstrated the utility of combining a TaqMan principle based real-time PCR detection with Luminex typing of pestiviruses. To maintain optimized condition in the TaqMan amplification, asymmetric PCR was not used to generate the single-strand target for subsequent Luminex experiments as previously reported [START_REF] Deregt | A multiplex DNS suspension microarray for simultaneous detection and differentiation of classical swine fever and other pestiviruses[END_REF]; Lambda exonuclease was used instead. The realtime PCR and the Luminex experiments had almost identical sensitivity, with the only difference being the appearance of an amplification curve for a BDV isolate that failed to reach the threshold level in the Luminex experiment. However, even in that instance the signal for BDV on the Luminex was more than 4 times higher than for the other specific pestivirus probes, which provided a good indication of a BDV positive sample. Given this correlation between the real-time PCR and Luminex results, the real-time PCR can A c c e p t e d M a n u s c r i p t 14 function as a gateway where samples can be deemed negative or passed on for typing by Luminex experiments. The TaqMan real-time PCR also gives a quantitative readout with a wide dynamic range, which can be useful for research purposes. We also readily extended the microarray probe set to include a novel atypical bovine pestivirus isolate, TKK [START_REF] Ståhl | Natural infection of cattle with an atypical 'HoBi'-like pestivirus -Implications for BVD control and for the safety of biological products[END_REF]. The usefulness of redundancy in the Luminex probe set was noted since none of the two common pestivirus probes detected the respective viruses in all 24 samples but results from the two probes combined managed to do so. These observations illustrate the strength of the low density suspension microarrays coupled with real-time single-plex detection as compared to conventional PCR-based multiplex techniques; redundancy can be introduced to make the assay more robust and the assay format is easily extensible to include novel or emerging pathogens. The strains and isolates used in this study were obtained from the European Network of Excellence, EPIZONE, 2007 pestivirus ring trial, confirming the validity of this platform for clinically important variants of the four pestiviruses.

  emerge and re-emerge on all continents and improved differential diagnosis is an important requirement internationally.

  A c c e p t e d M a n u s c r i p t 8 probe (BVDV Com) and two universal pestivirus probes (Pesti Com A and Pesti Com B) were used. A ninth probe with the sequence: 5´-CGA CGC ATC AAG GAA TGC CTC GAG was added for the detection of the atypical bovine pestivirus TKK. The oligonucleotides were coupled to carboxylated microspheres (Luminex Corporation, Austin, TX, USA) according to the protocol recommended by the manufacturer.

  3.1) and BDV 137/4 (1) at 10 000 copies, Congenital Tremor 3.1 amplified but poorly and 137/4 (1) failed six out of seven times, amplifying very poorly the one time it was successful (data not shown).In initial tests the Vil and Der primer combinations amplified all target viruses. Both primers sets were less effective compared with the other strains, for these three pestivirus strains: CSFV Congenital Tremor (3.1), BDV Gifhorn (3) and BDV 137/4 (1).

(

  empty symbols) and Der (filled symbols) primer pairs for amplification of 100 viral genome copies of the CSFV Congenital Tremor (3.1), BDV 137/4 (1) and BDV Gifhorn (3) strains. All amplifications were detected with the same TaqMan probe (see Materials and Methods). It is seen that the fluorescence signals appear 2-5 cycles earlier with the Der primers and yield higher signal than with the Vil primers. Similar results were obtained for other experiments with these strains (data not shown) and changes of primer ratios or concentrations could not alter this general pattern. For these reasons the Der primers where chosen for further experiments.

  the duplicates have been averaged and the amplification curves are shown in Figure 2 in three different panels: panel A (CSFV); panel C (BDV); panel E (BVDV1, BVDV2 and TKK).

  the threshold level at 281 MFI and observed for the BVDV 2 probe with the BVDV 1 NADL strain. Using the threshold at 500 MFI, indicated with a horizontal line in panels B, D and F of Figure2, all samples were correctly identified by their virus species except for the BDV Isard (2) that fell below the threshold level with a MFI value of 285. However, since the real-time PCR data clearly shows the presence of a pestivirus for this sample the weak signal could be taken as evidence for the presence of a border disease virus or warrant further investigations of the nature of the virus species. This highlights the strength of the combination of real-time PCR with the Luminex typing.

  the real-time PCR results of a serial dilution of the Alfort 187 (1.1) strain is shown and in panel B the subsequent Luminex suspension microarray data obtained with the common pestivirus A c c e p t e d M a n u s c r i p t 13 probes (circles) and specific CSFV probes (squares) are shown. The real-time data represent the average of duplicate or triplicate runs. The detection sensitivity as revealed by the PCR experiment is 6.4 viral genome copies/reaction. Correspondingly, the Luminex signals are above the threshold with all four probes down to the 6.4copies/reaction. The practical utility of this observation is that samples negative in the real-time PCR experiment do not need to be run in the suspension microarray experiment.

Figure 1 :

 1 Figure Legends

Figure 2 :

 2 Figure 2: The TaqMan real-time PCR data obtained for the (A) CSFV strains of

Figure 3 :

 3 Figure 3: (A) The real-time PCR data of a serial dilution of the CSFV strain Alfort 187

Acknowledgments

The authors would like to thank European FP6 project, LAB-ON-SITE (SSP3-513 645) for the primary financial support of this project as well as the European FP6 Network of Excellence for Epizootic Disease Diagnosis and Control (EPIZONE) for the contribution of samples used in this study. In particular, Drs. Martin Beer and Bernd Hoffmann (FLI, Institute of Diagnostic Virology, Germany), who prepared the ring trial under work package 4.1 "Real-time PCR diagnostics" and Dr. Ana Hurtado (Neiker-authors also would like to acknowledge the European FP6 projects, CSF&WILD-BOAR (SSP1-501599) and EPIZONE (FOOD-CT-2006-016236) for their scientific and financial contribution to this study.

Conflict of Interest Statement

There is no conflict of interest to declare in regard to this manuscript. Multiplex detection of four pathogenic retroviruses using molecular beacons. Proc. Natl. Acad. Sci. U.S. A. 96(11), 6394-6399.

Vilcek, S., Herring, A.J., Herring, J.A., Nettleton, P.F., Lowings, J.P., Paton, D.J., 1994.

Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch.

Virol. 136, 309-323.


K Ståhl

M Beer

H Schirrmeier

B Hoffmann

S Belák

S Alenius

Atypical 'HoBi'-like pestiviruses-recent findings and implications thereof

Keywords: Pestivirus, BVDV, HoBi, FCS, Contamination

. A few years later, a closely related virus (Th/04_KhonKaen) was detected in serum from a calf in Thailand, indicating that this group of atypical pestiviruses already is spread in cattle populations in various regions of the world. At the Friedrich-Loeffler-Institute, Insel Riems, Germany, FCS batches are regularly tested for pestivirus contamination, in general with positive PCR results, and in some cases the contaminants have been typed as 'HoBi'-like. At the National Veterinary Institute (SVA) in Uppsala, Sweden, a recent event with contaminated FCS ruined much of the ongoing cell culture work. From the FCS and the contaminated cells we were able to amplify and sequence nucleic acid from three different pestivirus strains, including BVDV-1,

A c c e p t e d M a n u s c r i p t 2 -2 and 'HoBi'-like; this in a commercial FCS that had been tested free from pestivirus by the manufacturer.

In this short communication we review the current status of atypical 'HoBi'-like pestiviruses, describe recent findings and discuss the implications thereof.

Introduction

Bovine Viral Diarrhoea Virus (BVDV) is the denomination of a heterogeneous group of viruses in the family Flaviviridae, genus Pestivirus, which are economically important pathogens that primarily infect ruminants. Infections with BVDV are endemic in cattle populations in most parts of the world. The prevalence of antibody positive cattle is in general high, and 1-2% of the cattle are persistently infected (PI) with the virus [START_REF] Houe | Epidemiological features and economical importance of bovine virus diarrhoea virus (BVDV) infections[END_REF].

Because of the high prevalence of PI cattle, BVDV is also a common contaminant of FCS, which is used to supplement growth medium for cell cultures for diagnostic or research purposes, or for the production of biological products such as vaccines [START_REF] Bolin | Prevalence of bovine viral diarrhea virus genotypes and antibody against those viral genotypes in fetal bovine serum[END_REF]. One possible consequence of such contamination became evident during a compulsory vaccination campaign against bovine herpesvirus 1 in Dutch cattle, when virulent BVDV type 2 (BVDV-2) was spread with a contaminated live vaccine, causing high morbidity and mortality (Barkema et al., 2001).

Currently there are two recognized genotypes of BVDV (BVDV-1 and -2) that together with Border Disease Virus (BDV) and Classical Swine Fever Virus (CSFV) constitute the four accepted species of the genus Pestivirus. In addition, a tentative fifth species is represented by a virus isolated from a giraffe [START_REF] Becher | Genetic and antigenic characterization of novel pestivirus genotypes: implications for classification[END_REF]. The pestivirus genome consists of single stranded positive-sense RNA of approximately 12.3 kb., with one open reading frame flanked by 5´ and 3´ untranslated regions (UTR). Classification of pestiviruses is based on the genetic relatedness between isolates, including genetic similarity with the type virus of the species A c c e p t e d M a n u s c r i p t 3 [START_REF] Heinz | Family Flaviviridae[END_REF]. Furthermore, because pestiviruses are serologically cross-reactive, species demarcation considers antigenic relationships based on binding assays with monoclonal antibodies or cross-neutralisation assays with polyclonal antisera [START_REF] Hamers | Diversity among bovine pestiviruses[END_REF][START_REF] Becher | Genetic and antigenic characterization of novel pestivirus genotypes: implications for classification[END_REF].

Atypical 'HoBi'-like pestiviruses

In 2004 an atypical pestivirus named D32/00_'HoBi' was isolated from FCS originating from Brazil [START_REF] Schirrmeier | Genetic and antigenic characterization of an atypical pestivirus isolate, a putative member of a novel pestivirus species[END_REF]. Due to the genetic and antigenic properties that markedly differed from previously described pestivirus species, this virus was proposed as a representative of a possible novel species. This was the first of several reports describing more or less closely related atypical pestiviruses: [START_REF] Stalder | Genetic heterogeneity of pestiviruses of ruminants in Switzerland[END_REF] described 'HoBi'-like viruses isolated from contaminated cell culture (probably originating from a batch of FCS produced in South America) and from a Brazilian buffalo; [START_REF] Cortez | Genetic characterization of Brazilian bovine viral diarrhea virus isolates by partial nucleotide sequencing of the 5'-UTR region[END_REF] detected 'HoBi'-like viruses in aborted Brazilian bovine fetuses; [START_REF] Ståhl | Natural infection of cattle with an atypical 'HoBi'-like pestivirus--implications for BVD control and for the safety of biological products[END_REF] detected a related virus in serum from a calf in Thailand, and [START_REF] Kampa | Bovine herpesvirus type 1 (BHV-1) and bovine viral diarrhoea virus (BVDV) infections in dairy herds: Self clearance and the detection of seroconversions against a new atypical pestivirus[END_REF] further described evidence of exposure to 'HoBi'-like viruses in additional cattle herds in Thailand. In sum, these reports suggested that 'HoBi'-like pestiviruses already were spread in cattle populations, particularly in Brazil, but also outside South America. Whereas the main concern caused by the detection of 'HoBi'-like viruses in FCS focused on issues related to the biosafety of biological products of bovine origin, the isolation of 'HoBi'-like pestiviruses from aborted foetuses also indicated direct clinical implications [START_REF] Cortez | Genetic characterization of Brazilian bovine viral diarrhea virus isolates by partial nucleotide sequencing of the 5'-UTR region[END_REF].

Recent findings at FLI and SVA

Since pestivirus contamination of FCS is a permanent problem in cell culture laboratories, the virological institutes are regularly screening serum batches for the presence of these viral A c c e p t e d M a n u s c r i p t 4 agents. The screening is mainly focusing on the presence of BVDV-1 and -2. However, recently the contamination with atypical pestiviruses is also a question of increasing importance. At the Friedrich-Loeffler-Institute a recent investigation of 25 batches was made using virus isolation on calf oesopharynx cells (KOP-R, L244, Collection of Cell Lines in Veterinary Medicine (CCLV), Insel Riems), and conventional and real-time RT-PCR protocols for the amplification of the 5' UTR [START_REF] Vilcek | Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis[END_REF][START_REF] Gaede | Detection and species-specific differentiation of pestiviruses using real-time RT-PCR[END_REF][START_REF] Hoffmann | A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses[END_REF], in some cases followed by typing of the contaminating viruses by sequencing of the amplicons. In all but one batch, traces of pestivirus nucleic acids were detected, demonstrating pestivirus contamination. In two of the batches, the contaminating virus was typed as 'HoBi'-like. These 'HoBi'-like viruses were only detected by the real-time RT-PCR assays and not by the conventional RT-PCR protocol based on the commonly used 324/326 primer pair, a finding that is in accordance with previous observations [START_REF] Schirrmeier | Genetic and antigenic characterization of an atypical pestivirus isolate, a putative member of a novel pestivirus species[END_REF]. Furthermore, BVDV type 1 was isolated from three of the contaminated batches; the other virus isolation attempts remained negative also after three passages.

At SVA bovine turbinate (BT) cells are used for virus isolation within the Swedish BVD control and eradication programme. For almost 20 years pestivirus-free FCS was produced inhouse, but since the last few years, however, commercial FCS is being used for the cell cultures. Although all commercial FCS is being tested for pestivirus contamination, a recent event demonstrated the vulnerability of the system. A new batch of FCS of Brazilian origin had been used for the BT cells between passages 4 and 9 when suddenly all virus isolations turned positive for BVDV. Further investigations demonstrated contamination also in other cell cultures where the FCS had been used, and the FCS was considered the most likely source. From the FCS and the contaminated cells, the 5' UTR from three different pestivirus strains, including BVDV-1, -2 and 'HoBi'-like, could be amplified and sequenced (Figure 1).

Genetic diversity

To date at least nine atypical 'HoBi'-like pestiviruses have been described; eight of known or probable Brazilian origin, and one of Thai origin. Phylogenetic analysis based on the longest available overlapping segment of the 5'UTR (approximately 140 nucleotides) demonstrates genetic diversity within the group and suggests that two subgroups can be defined: one represented by D32/00_'HoBi' and the seven other 'HoBi'-like viruses of Brazilian origin, and the other by Th04_KhonKaen (Figure 1). The sequence identity within the partial 5'UTR used for the analysis ranges between 90 and 93% between the subgroups, and between 96 and 99% within the 'HoBi' subgroup.

Discussion

The growing number of reports on 'HoBi'-like pestiviruses, including the recent findings reported herein, raises two main concerns. The first is related to the general problem of pestivirus contamination of FCS with implications for the biosafety of biological products that contain FCS, such as vaccines, diagnostic reagents and kits, stem cells and other products of the bioindustry. The second is related to the emergence and spread of atypical pestiviruses, possibly via biological products (such as vaccines), with possible implications for animal health and disease control. Both issues have to be seriously considered at a global perspective.

The first issue is well known and has been addressed in several recent publications (Audet et al., 2000;[START_REF] Studer | Detection and characterization of pestivirus contaminations in human live viral vaccines[END_REF][START_REF] Makoschey | Bovine viral diarrhoea virus antigen in foetal calf serum batches and consequences of such contamination for vaccine production[END_REF], and was also the main topic during an international workshop organized by the EDQM in Paris 2001 [START_REF] Edqm | Pestivirus contamination of bovine sera and other bovine virus contamination[END_REF]. The European Committee for Medicinal Products for Veterinary Use (CVMP) prescribes inactivation, such as gamma irradiation, of FCS used in the production immunological medicinal products such as vaccines, and testing for BVDV before and after the inactivation process using virus isolation on bovine cells sensitive to Pestiviruses (three passages recommended; see EMEA/CVMP/743/00-Rev-2, http://www.emea.europa.eu/pdfs/vet/iwp/074300en.pdf). Given that the inactivation process is properly validated and properly carried out, this should ensure a safe end product. However, it is not always evident that the inactivation process is efficiently applied, as shown by the contaminated herpesvirus vaccine in the Netherlands (Barkema et al., 2001), and even very low levels of contamination may have severe consequences. And although testing is carried out after the inactivation in accordance with the A c c e p t e d M a n u s c r i p t 7 CVMP guidelines, a low-level pestivirus contamination may be missed, as shown by the recent experience at SVA where the contaminating virus was not detected until after five passages. Moreover, the legislation as well as the commonly used diagnostic methods are optimized to ensure freedom from BVDV-1 and -2, and might not be optimal for the atypical bovine pestiviruses, and as already mentioned this is particularly true for commonly applied RT-PCR protocols.

Since the first description of D32/00_'HoBi', eight other related atypical pestivirus strains have been detected and/or isolated seemingly independently of one another. Together with the phylogenetic data presented herein that, albeit based on a short fragment of the genome, demonstrate genetic diversity and indicate subgrouping, this suggests that atypical 'HoBi'like pestiviruses are not recently emerged viruses of cattle, but rather that they have existed long enough in the cattle population to spread and evolve. The observation that the only virus detected outside of the South American continent, i.e. Th/04_KhonKaen, is the most divergent member of the group and forms a possible subgroup, is interesting. However, whether this subgrouping correlates with geographical origin, or not, cannot be concluded at this stage, but requires further studies.

Conflicts of interest

Figure 1 .

 1 Figure 1. Unrooted Neighbor-joining phylogram generated from a 140 nucleotides fragment of the 5´ UTR sequences of pestivirus contaminants detected in FCS at FLI and SVA (in bold), and representatives of each known species within the genus Pestivirus, including the tentative pestivirus of giraffe and the atypical 'HoBi'like pestiviruses. The sequences of the reference strains NADL, Osloss, US890, Giraffe H138, Alfort187, BDV137/4, Moredun, D32/00_'HoBi', the previously described Th/04_KhonKaen and the Brazilian strains

  , EF683558 and EF683557 were obtained from the GenBank (accession nos. NC001461, M96687, Z79772, AB040131, X87939, U65052, U65022, AY489116, DQ897641, AY379547, AF410789, EF683558 and EF683557).

  Audet, S.A., Crim, R.L., Beeler, J., 2000, Evaluation of vaccines, interferons and cell substrates for pestivirus contamination. Biologicals 28, 41-46. Barkema, H.W., Bartels, C.J., van Wuijckhuise, L., Hesselink, J.W., Holzhauer, M., Weber, M.F., Franken, P., Kock, P.A., Bruschke, C.J., Zimmer, G.M., 2001, [Outbreak of bovine virus diarrhea on Dutch dairy farms induced by a bovine herpesvirus 1 marker vaccine contaminated with bovine virus diarrhea virus type 2.]. Tijdschr Diergeneeskd 126, 158-165.


Hana Van Campen

Title: Epidemiology and Control of BVD in the U.S

Keywords: Bovine viral diarrhea, epidemiology, U.S

The apparent prevalence of bovine viral diarrhea virus (BVDV) persistently infected cattle has been found to be low in U.S. dairies, beef herds and feedlots. Current management practices within U.S. cattle industries that impact the epidemiology of BVDV infections include purchasing untested cattle, lack of biosecurity procedures, large herd sizes, mixing cattle from multiple sources, high cattle densities in dairy and feedlot operations, synchronous breeding of beef herds, communal grazing and widespread vaccination. Evidence for BVDV infection has been found in farmed and free-ranging wildlife in North America; however the risk of BVDV transmission from wildlife to cattle is not known. The perception of a low prevalence of BVDV herd infections, the unrestricted sale of PI cattle, lack of economic data, intensive marketing of vaccines, reluctance to accept federal regulations, and a "gambler's" attitude among producers are impediments to implementation of a national systematic BVD control program. Since 2004, voluntary BVDV control programs have been organized in nine states reflecting the recognition of BVD as an important and preventable problem in the U.S.

The Epidemiology of Bovine Viral Diarrhea Virus in the U.S.: There are 104.3 million cattle in the U.S. distributed over 1 million farms, ranches, feedlots and other premises covering a broad spectrum of environments and diverse cattle production systems (NASS, 2008). While bovine viral diarrhea virus (BVDV) transmission and associated diseases are the same as found elsewhere, differences in cattle management practices in the U.S. result in clinical manifestations at indicate that the prevalence of persistently infected (PI) animals in the U.S. cattle population is low, ≤0.3% in surveys of beef herds (Wittum et al., 2001;O'Connor et al., 2007), feedlots (Loneragan et al., 2005, Fulton et al., 2006), and dairies (Houe et al., 1995;Munoz-Zanzi et al., 2003). The prevalence of BVDV-infected beef herds, as defined by PI animal detection within herds, is 4% for beef cattle (Wittum et al., 2001;O'Connor et al., 2007). The prevalence of BVDV-infected dairy herds is 15% based on PI cattle detected in Michigan herds (Houe et al., 1995), and 1.7% in a national survey based on detecting BVDV by RT-PCR in bulk milk samples (USDA 2007). In the latter report, the percent of BVDV RT PCR positive dairy herds ranged from 0% in herds with less than 100 cows to 12.8% in herds with ≥500 cows.

The apparently low prevalence of BVDV-infected beef herds based on PI animal detection is likely to be an underestimate of true herd prevalence. Some BVDV-infected beef herds may be misclassified as uninfected herds if there is no PI animal alive at the time of testing. Test methods that are currently available do not detect PI fetuses, and the infection at the herd level continues when these calves are born. Surveying dairy herds A c c e p t e d M a n u s c r i p t 4 by reverse transcriptase-polymerase chain reaction (RT-PCR) assays of bulk milk samples may result in the misclassification of herds as uninfected if there are no PI animals in the lactating cow herd. This testing strategy underestimates the prevalence of infected herds as the young stock, which is not surveyed by this method, is more likely to include PI animals than older cattle.

Accurately determining the prevalence of BVDV infection in individual cattle or herds in the U.S. is problematic for a number of reasons. Herd sizes are relatively large and testing every individual animal is neither logistically or economically feasible.

Although testing strategies for pooled samples have been developed, they do not replace the need to obtain samples from each animal (Kennedy et al., 2006). An estimated 80% of cattle in the U.S. are vaccinated with either inactivated or modified live viral vaccines containing BVDV (USDA 1995; USDA 2007); consequently, BVDV seroprevalence is high, and the serologic tests are unable to distinguish between vaccinated and naturally infected cattle [START_REF] Paisley | Prevalence of bovine viral diarrhea antibodies in 256 U.S. cow-calf operations: a survey[END_REF]. The testing strategies used in Europe to identify BVDV-infected herds based on serology are not useful in this situation as they would misclassify vaccinated-uninfected herds as being BVDV-infected. Thus, testing U.S. herds is a challenge for any rigorous BVD control program.

If the prevalence of BVDV-infected herds in the U.S. is truly low, then one could surmise that control of BVDV-associated diseases by eradication could be easily accomplished. The low prevalence, however, also means that a large proportion of U.S. herds are susceptible to the introduction of BVDV infections. As the sale of PI animals is BVDV-associated diseases are not reportable and the diseases may be misdiagnosed; therefore, there is no data enumerating BVDV-associated cases. Occurrences of diseases are only captured in a few case reports.

Cattle management in the U.S. that impact BVDV infections:

The trend in U.S. dairies has been towards fewer farms and larger herd sizes. The rapid growth of large dairies has increased the demand for bred replacement heifers.

Risk factors for herds with positive BVDV RT-PCR bulk milk tank samples include large herd size (>500 cows) and purchasing animals (USDA 2007). To meet the demand, heifers are raised and bred in commercial facilities where thousands of cattle from multiple origins are co-mingled. Contact with PI animals in these facilities during the breeding period generates PI fetuses. When these heifers return to dairies, they give birth to PI calves which then are a source of BVDV infection for the rest of the herd. Dairy herd expansion may occur more rapidly than optimal for the facilities and personnel resulting in crowding and insufficient attention to biosecurity and sanitation measures.

Inadequate separation of pregnant cows from young stock including PI calves leads to additional fetal infections. In registered herds where the genetic value of individual animals is emphasized, PI cattle may be retained in the herd despite poor health and phenotype. [START_REF] Rauff | Evaluation of the results of a survey of dairy producers on dairy herd biosecurity and vaccination against bovine viral diarrhea[END_REF][START_REF] Rush | Descriptive epidemiology of postnatal bovine viral diarrhea virus infection in intensively managed dairy heifers[END_REF]. Infertility as judged by the percent of cows >150 days open is higher (18.2%) for BVDV-infected operations compared to noninfected dairies (USDA 2007), and supports the finding that infertility in heifers has A c c e p t e d M a n u s c r i p t 6 been linked to congenital BVDV infection (Munoz-Zanzi et al., 2004). As infertility is an important factor in culling decisions made on dairies, a cycle of culling infertile cows necessitating increased purchases of at-risk heifers. BVDV RT-PCR positive dairies are more likely to use BVDV-containing vaccines than non-infected operations suggesting that vaccines are being used in response to problems perceived to be due to BVDV infections (USDA 2007). An important difference between dairy and beef herds with respect to the epidemiology of BVD is that breeding in dairy herds occurs over the entire year. Exposure of the cows to a PI animal results in the infection of fetuses whose ages cover the entire gestational period. Calves with congenital defects resulting from BVDV infection are more likely to be born into dairy herds which aids in recognition of herd infection.

Beef herds engage in similar high risk practices as dairy herds including the purchase of untested cattle. The purchase of pregnant heifers and utilization of heifer development feedlots has become common in the beef industry and like the dairies, generate PI fetuses which then serve as the source of infection for the cow herd when the heifers return and calve. In addition, ranchers share summer pastures where cows in their first trimester of pregnancy may contact PI calves from other herds [START_REF] Sanderson | Biosecurity practices of beef cowcalf producers[END_REF]. The practice of synchronous, seasonal breeding in beef cattle has a marked impact on the manner in which BVDV-associated reproductive losses present. Contact with a single PI calf can infect a large proportion of cows during the first trimester of pregnancy.

In this scenario, the first observation is a distinct rise in infertility cases determined at pregnancy examination. Typically, this is followed by explosive abortion "storm" A c c e p t e d M a n u s c r i p t 7 beginning one month prior to the planned calving interval and followed by increased stillbirths, the birth of stunted calves and weak, non-viable calves. In some cases, BVDV epidemics result in losses of 50% of the calf crop. In contrast to diary herds, classic congenital defects such as cerebellar hypoplasia are infrequently observed in beef herds due to the early gestational age of fetuses at the time of exposure of cow-calf herds to PI calves.

There are two possible outcomes following the epidemic year of BVDV infection.

If a PI calf is not born or does not survive into the breeding season, then BVDV infection of the herd will not be sustained and the herd will be uninfected. Detection of BVDV antibodies in the cow herd at this point would falsely classify the herd as infected (Van Campen et al., 1998). Secondly, the herd can enter an endemic state of infection if a PI calf survives into the breeding season. In endemically infected beef herds, approximately 50% of the calves will have become infected by the time they are weaned due to contact with PI calves present in the same cohort [START_REF] Cleveland | Removal of bovine viral diarrhea virus persistently infected animals from an endemically infected beef herd: effect on PI animal prevalence and BVDV seroprevalence[END_REF]. Calfhood infections may be clinically inapparent during the period that the calf is protected by maternal antibodies, but manifest as diarrhea and pneumonia when maternal antibodies wane. The rest of the calf crop may be impacted by BVDV in more subtle ways such as lower weaning weights and rate of gain (Waldner and Kennedy, 2008). In either scenario, cows rendered immune by the initial infection will protect subsequent pregnancies from antigenically similar BVD viruses for the rest of their life. Their offspring will not be PI or suffer other consequences of congenital infection; however, their offspring will be susceptible to infection. If susceptible heifers are retained in the herd and remain A c c e p t e d M a n u s c r i p t 8 unexposed, e.g., by virtue of being raised separately from the cow herd, they serve as the source of additional PI animals following exposure to PI calves when they enter the cow herd and are bred.

On average, 10,000,000 head of cattle are on feed in the U.S. in just under 1,000,000 premises. Approximately one-third of the inventory is fed in feedlots with over 1,000 head of cattle (NAAS 2008). By necessity, maintaining this inventory requires purchasing cattle from multiple sources and co-mingling animals at high animal densities. In addition, calves pass through several sales facilities prior to entry into the yard which represents another opportunity for exposure to pathogens. The percentage of PI cattle entering feedlots is low; however, cattle in adjacent pens as well as with the home pen are subject to infection. In some cases, the immunosuppressive effects of BVDV likely play a role in increased risk of morbidity and mortality due to respiratory disease (Loneragan et al. 2005). However, other surveys of beef cattle failed to find any significant effect of PI calves on herd health (O'Connor et al., 2007). In terms of transmission, the feedlot is an endpoint for the viruses, and would not impact their maintenance in the breeding cattle population. The economic impact on feeder calves is more easily apparent which explains the interest in BVDV control from this segment of the cattle industry.

Wildlife and other sources of BVDV:

The U.S. enjoys ample populations of wild species of ruminants which share range and forage with domestic cattle. Serologic surveys have indicated that these A c c e p t e d M a n u s c r i p t 9 animals become infected with BVDV and other pestiviruses (reviewed by Van Campen et al., 2001a). Evidence for PI white-tailed and mule deer have been found in several states [START_REF] Chase | Bovine viral diarrhea virus multiorgan infection in two white-tailed deer in southeastern South Dakota[END_REF]Duncan et al., 2008b;[START_REF] Pogranichniy | Prevalence and characterization of bovine viral diarrhea virus in the white-tailed deer population in Indiana[END_REF]Van Campen et al., 2001b), and PI white-tailed fawns have been generated experimentally by the inoculation of pregnant does (Duncan et al., 2008a). Clearly, free-ranging wildlife species are a potential source of infection for cattle; however, transmission from wildlife to cattle has not been demonstrated to have occurred in nature and the risks of infection from this source have not been examined. Recently, the persistent infection of alpacas with a noncytopathic type 1b BVDV was reported [START_REF] Carman | Bovine viral diarrhea virus in alpaca: abortion and persistent infection[END_REF]. The monetary value placed on individual alpacas, the aggressiveness with which breed associations pursued PI testing and the small number of PI crias identified makes this species an unlikely source of infection for cattle. Few surveys of Pestivirus infections have been conducted in domestic sheep and goats in the U.S. that might indicate the extent of these infections or whether these populations pose a risk to cattle.

BVDV Control and Eradication Programs in the U.S.:

Prior to 2004, BVDV control largely rested on the use of inactivated and modified live BVDV vaccines. For decades, BVDV vaccines included either the Singer or NADL cytopathic type 1a BVD viruses. These vaccines were used to prevent both fetal infections with accompanying reproductive losses as well as to prevent diseases due to acute infections. The recognition of type 2 BVDV associated with severe disease and occurrence of fetal losses in vaccinated cows (Van Campen et al., 2000) led to the inclusion of a cytopathic type 2 BVDV in many vaccines. Since then experimental vaccines on health remain a factor in their use [START_REF] Ellsworth | Fetal protection following exposure to calves A c c e p t e d M a n u s c r i p t 16[END_REF]Grooms et al., 2007;[START_REF] Schnackel | Modified-live bovine viral diarrhea virus (BVDV) type 1a vaccine provides protection against fetal infection after challenge with either type 1b or type 2 BVDV[END_REF]. A significant consequence of the widespread use of BVDV vaccines is the inability to use serologic techniques for the diagnosis of BVD herd infections and for surveillance purposes in U.S. cattle. As important is the perception by producers that BVDV vaccines will prevent the introduction of BVDV into their herds.

Given this mindset, biosecurity measures to prevent the introduction of BVDV and other pathogens are often ignored.

In 2003, the Academy of Veterinary Consultants published a position statement promoting the control and eventual eradication of BVD from the U.S. (http://www.avcbeef.org/links/BVDLinks.asp). This announcement was followed in Jan. 2004 Currently mandatory, systematic BVD control programs similar those in several European countries do not exist in the U.S. (Lindberg et al., 2006). The concept of BVD control by eradication has been slow to find acceptance in the U.S. Impediments include reluctance to institute a government-regulated control program. Available data indicating a low prevalence of BVDV infection in beef and dairy herds and uncertainty on the part of individual producers about the economic benefits of BVDV control. The lack of a clear danger is compounded by the "Gambler's" mentality among individual cattle producers. The low herd prevalence validates the general belief that the application of BVDV vaccines is a "cure-all" rather than an aid in prevention of BVDV infections.

However, producers and veterinarians who have experienced losses due to BVDVassociated diseases are more motivated to adopt preventive measures. Producer and veterinarians' awareness of the potential impact is the impetus behind the BVDV testing recently adopted by bull sales, purebred stock sales, livestock shows and specific feedlots. The proliferation of BVD control and eradication programs in the U.S. is encouraging and highlights recognition of BVDV's importance to cattle health.

  that are different from those observed in Europe, and which present challenges to controlling the viral infection. Prevalence of BVDV infections in U.S. cattle. An important difference between European cattle prior to the institution of bovine viral diarrhea (BVD) eradication programs, and the U.S. is the low prevalence of BVDV persistent infection in U.S. cattle. Data collected from recent surveys of beef cattle

  epidemics of BVDV-associated diseases occur each year as the viruses are introduced into these herds through the purchase of PI cattle or cows bearing PI fetuses.

  cows has established the superiority of MLV BVDV vaccines in providing fetal protections; however, concerns about safety and subtle effects of live viral

  through their extension service that is tailored to dairy farms (http://nyschap.vet.cornell.edu/module/bvd/bvd.asp). An exciting addition to this list is the BVD eradication program (http://www.msue.msu.edu/workspaces/one.cfm?workspace_id=28413&object_id=45518 1) offered to beef and dairy herds in the Upper Peninsula of Michigan through the extension service of Michigan State University.All programs are voluntary, associated with a university and organized in conjunction with other beef or dairy quality assurance programs. Program elements include: 1) education about BVDV transmission and diseases, 2) required testing procedures, 3) documentation of biosecurity practices to prevent the re-introduction of BVDV, and 4) verified use of a vaccination schedule. A wide range of resources are available to veterinarians, diagnosticians and producers seeking information about the control and prevention of BVDV infections. These subjects have continued to receive extensive coverage in magazines and the websites of veterinary medical and producer organizations, by university extension personnel and vaccine companies. Consultation on BVD control is provided at no cost to the producer by extension personnel, clinical faculty at veterinary teaching hospitals and by diagnostic laboratories. Two BVD control programs have funding for to partially cover the costs of testing and limited funding for indemnification of owner's of PI animals. The biosecurity requirement in some programs means the development of a written biosecurity plan by each producer, and the systematic element of follow-up testing in subsequent years is encouraged.Funding the existing programs is a constant challenge. To date, BVD control programs A c c e p t e d M a n u s c r i p t 12 have relied on grants from producer organizations, state departments of agriculture, vaccine and pharmaceutical companies, and university resources.

  ., Brownlie J, Gunn GJ, Houe H, Moennig V, Saatkamp HW, Sandvik T, Valle PS. 2006. The control of bovine viral diarrhoea virus in Europe: today and in the future. Rev Sci Tech. 25, 961-79. Loneragan, G. H., Thomson, D. U., Montgomery, D.L., Mason, G. L., Larson, R.L. 2005. Prevalence, outcome, and health consequences associated with persistent infection with bovine viral diarrhea virus in feedlot cattle. JAVMA 226(4), 595-601. Munoz-Zanzi, C. A., Hietala, S. K., Thurmond, M. C., Johnson, W.O. 2003. Quantification, risk factors and health impact of natural congenital infection with bovine viral diarrhea virus in dairy calves. Am J Vet Res 64(3), 358-365. Munoz-Zanzi, C. A., Hietala, S. K., Thurmond, M. C., Johnson, W.O. 2004. Effect of bovine viral diarrhea virus infection on fertility of dairy heifers. Theriogenology 61(6), 1085-1099. National Agricultural Statistics Service, Agricultures Statistics Board, USDA. http://www.nass.usda.gov/index.asp O'Connor, A.M., Reed, M. C., Denagamage, T. N., Yoon, K.-J., Sorden, S.D. 2007. Prevalence of calves persistently infected with bovine viral diarrhea virus in beef cowcalf herds enrolled in a voluntary screening project. JAVMA 230(11), 1691-1696.

  //www.vetmed.wsu.edu/bvdcep/) with a focus on beef herds. New York has a BVD

	by
	development of a voluntary BVD control program offered in conjunction with Colorado
	State University
	(http://www.dlab.colostate.edu/BVDControlProgram/bvdcontrolprog_main.cfm). Since
	then, the number of BVD prevention and control programs has expanded to include
	Alabama (http://www.aces.edu/counties/Marion/files/bvd.pdf), Georgia, Mississippi
	(http://www.mbah.state.ms.us/disease_programs/bvd/MS_PI_BVD_Program.pdf),
	Montana (http://www.mtbqa.org/news/2007%20Montana%20BVD-
	PI%20Herd%20Screening%20Project.doc), Oregon
	(http://ans.oregonstate.edu/bvd/index.html) and Washington
	(http:

Acknowledgements: The author would like to thank the organizers of the 7 th ESVV Pestivirus Symposium for their invitation and encouragement.


Le Potier
email: sophie.rossi@oncfs.gouv.fr

Preventive vaccination contributes to control classical swine fever in wild boar (Sus scrofa sp.)

). This has generally led to an enhanced seroprevalence and a decreased viroprevalence at the scale of the whole vaccinated populations, but no quantitative analysis has demonstrated the protective effect of preventive vaccination or intensive baiting. In the present paper we conducted an retrospective analysis at the scale of the municipality, taking into account the local dynamics and possible covariates of infection to test the effect of preventive vaccination and of the baiting effort. To be efficient, vaccination was expected to increase seroprevalence above the level considered as suitable for preventing disease invasion (40-60%) independently of infection, to protect free areas from disease invasion or contribute to control subsequent disease intensity and duration. We also hypothesized that a better baiting effort would be correlated with an improvement of immunisation and disease control.

In uninfected municipalities, seroprevalence increased up to 40% after one year, i.e. three vaccination campaigns. We observed a significant protective effect of preventive vaccination, especially within municipalities that had been vaccinated at least one year before disease emergence and where virus detection did not last more than one quarter. On the other hand, we did not detect a significant effect of the baiting effort on local seroprevalence or disease dynamics, suggesting that the baiting system could be improved. We discuss these results regarding the improvement of management measures and further research perspective.

Key-words : wild boar / classical swine fever / preventive vaccination 1. INTRODUCTION

The control of classical swine fever (CSF) in the Wild Boar (Sus scrofa sp.) is required in the European Union, since this species may act as a wild reservoir of the virus [START_REF] Fritzmeier | Epidemiology of classical swine fever in Germany in the 1990's[END_REF], Anon 2001[START_REF] Artois | Classical swine fever (hog cholera) in wild boar in Europe[END_REF].

Mathematical models of this host-virus system have been developed and suggested that preventive immunisation (40 to 60%) of susceptible populations may prevent disease emergence [START_REF] Hone | Evolution of the dynamics and rate of transmission of classical swine fever (hog cholera) in wild pigs[END_REF][START_REF] Guberti | Estimate the threshold abundance for the persistence of classical swine fever in the wild boar population of the eastern Sardinia[END_REF].

In the field, oral vaccination has been implemented using a Riems C-strain vaccine and baits attractive to wild boars [START_REF] Kaden | Oral immunisation of wild boar against classical swine fever: evaluation of the first field study in Germany[END_REF]. This process has been improved in Germany during the 2000's to maximize individual antibody titre and seroprevalence [START_REF] Kaden | Oral immunisation of wild boar against classical swine fever: concluding analysis of the recent field trials in Germany[END_REF][START_REF] Kaden | Evaluation of the oral immunisation of wild boar against classical swine fever in Baden-Wurttemberg[END_REF][START_REF] Kaden | Does multiple vaccination of wild boar against classical swine fever (CSF) have a positive influence on the immunity?[END_REF]. Retrospective analysis showed that high seroprevalence (>60%) and low viroprevalence (<0.1%) were maintained in vaccinated areas [START_REF] Kaden | Oral immunisation of wild boar against classical swine fever: concluding analysis of the recent field trials in Germany[END_REF][START_REF] Kaden | Evaluation of the oral immunisation of wild boar against classical swine fever in Baden-Wurttemberg[END_REF][START_REF] Louguet | Mesures de gestion de la peste porcine classique sur la faune sauvage : stratégie vaccinale[END_REF], Kaden et al., 2006, Rossi et al., 2006a[START_REF] Von Rüden | Retrospective analysis of the oral immunisation of wild boar populations against classical swine fever virus (CSFV) in region Eifel of Rhineland-Palatinate[END_REF].

However, assessing the effect of vaccination on disease dynamics has been a difficult issue because antibodies generated by the Riems C-strain vaccine and by the natural infection are similar [START_REF] Kaden | Oral immunisation of wild boar against classical swine fever: evaluation of the first field study in Germany[END_REF]. Furthermore, retrospective studies have often considered data from a large infected area as a whole and have thus not taken into account the complex spatiotemporal evolution of outbreaks. During the epizootic phase, the virus spreads over space and locally decreases over time, moreover many other covariates interfere with disease dynamics such as population size and landscape structure (Rossi et al., 2005a, Rossi et al., 2005b[START_REF] Kramer-Schadt | A review of potential ecological and epidemiological factors affecting the persistence of Classical Swine Fever in wild boar populations[END_REF]. According to the principle of pseudo-mass action and critical community size developed by modellers, the number of hosts is supposed to favour disease A c c e p t e d M a n u s c r i p t 4 invasion and persistence at both local and global level [START_REF] Hone | Evolution of the dynamics and rate of transmission of classical swine fever (hog cholera) in wild pigs[END_REF][START_REF] Begon | A clarification of transmission terms in host-microparasite models: numbers, densities and areas[END_REF], Rossi et al., 2005b[START_REF] Kramer-Schadt | A review of potential ecological and epidemiological factors affecting the persistence of Classical Swine Fever in wild boar populations[END_REF]. Forest continuity is known to influence the probability of contacts between neighbouring wild boars and the resulting probability of disease spreading and persistence at the scale of the whole area [START_REF] Grenfell | Metapopulation dynamics of infectious diseases[END_REF], Rossi et al., 2005b, Kramer-Schadt et al., 2007, Kramer-Schadt in press). Lastly, even though the baiting effort, i.e., the number of baits delivered per wild boar, is strongly related to the cost of vaccination, its effect has not been tested so far.

In the present paper we assess the effectiveness of preventive vaccination by considering data at the municipality level, i.e., at a spatial scale where wild boars experienced the same epidemiological situation, where we could differentiate preventive from post-active vaccination and avoid confounding effects of other covariates. We considered the data collected in the Vosges mountains from 2003 to 2007. We targeted the following questions:

1. in uninfected areas, did vaccination increase seroprevalence above the level considered as suitable for preventing disease invasion(40-60%)?

2. did preventive vaccination protect from disease invasion or contribute to control subsequent disease intensity and duration? 3. was baiting effort correlated with a better immunisation and control of the outbreak?

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

MATERIALS AND METHODS

Study area, epidemiological context and vaccination process

The study area is located in France in the Vosges Mountains (48° 50' N and 7° 30' E); it covers 3,030 km2 including 1,180 km2 of forest land (Figure 1). Motorways, canals, rivers or towns constitute relative barriers to wildlife movements towards the west, east and south.

Towards the north there is no physical barrier: the forest is uninterrupted between the Vosges and the Palatinate Forest (Germany) which constitute a large metapopulation (Rossi et al., 2005a).

In this area a previous outbreak of CSF occurred during the 90's and disappeared by 2000 (Rossi et al., 2005a). However, since April 2003 CSF has re-invaded the whole study area [START_REF] Louguet | Mesures de gestion de la peste porcine classique sur la faune sauvage : stratégie vaccinale[END_REF][START_REF] Pol | Two outbreaks of classical swine fever in wild boar in France[END_REF]. Oral vaccination has been implemented since August 2004 [START_REF] Louguet | Mesures de gestion de la peste porcine classique sur la faune sauvage : stratégie vaccinale[END_REF] using a modified live vaccine (Riems C-strain) encapsulated into maize baits [START_REF] Kaden | Oral immunisation of wild boar against classical swine fever: evaluation of the first field study in Germany[END_REF]. A double vaccination scheme has been adopted as recommended by [START_REF] Kaden | Does multiple vaccination of wild boar against classical swine fever (CSF) have a positive influence on the immunity?[END_REF], vaccination campaigns comprising two baits distributions have been performed three time a year: by February/March, June/July and August/September (2005)(2006) or October/November (2007). On average, 40 baits were delivered per vaccination place each time, the area comprising on average 1.8 vaccination places per square kilometre [START_REF] Kaden | Oral immunisation of wild boar against classical swine fever: concluding analysis of the recent field trials in Germany[END_REF][START_REF] Kaden | Does multiple vaccination of wild boar against classical swine fever (CSF) have a positive influence on the immunity?[END_REF], Kaden et al., 2006). Over the whole forested area, a total of 500 000 baits have been delivered each year; hunters buried each bait on feeding grounds where wild boars were attracted with maize and where hunting was temporarily interrupted to minimize the disturbance of animals [START_REF] Louguet | Mesures de gestion de la peste porcine classique sur la faune sauvage : stratégie vaccinale[END_REF].

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

Sampling and diagnosis designs

Over the whole study area, blood and spleen were compulsorily sampled in all hunted wild boars for serological and virological examinations [START_REF] Louguet | Mesures de gestion de la peste porcine classique sur la faune sauvage : stratégie vaccinale[END_REF]. Antibody detection was performed using commercial ELISA kits according to the manufacturer's instructions (Herdcheck CSFV Antibody test kit or CHEKIT CSF SERO Antibody, both distributed by IDEXX® and of similar sensitivity). The genome of CSFV was amplified by real-time polymerase chain reaction (r-RT-PCR) using a commercial r-RT-PCR kit according to the manufacturer's instructions (TAQVET PPC® or ADIAVET CSF®). When positive or doubtful r-RT-PCR results were observed, subsequent virus isolation was performed by the French National Reference Laboratory for CSF (AFSSA) according to the EU-Diagnostic Manual for CSF (Anon 2002). An animal was considered to be viropositive when either CSFV could be isolated or the PCR threshold cycle was below 40 [START_REF] Dewulf | Analytical performance of several classical swine fever laboratory diagnostic techniques on live animals for detection of infection[END_REF], Le Potier et al., 2006[START_REF] Depner | Diagnostic evaluation of a real-time RT-PCR assay for routine diagnosis of classical swine fever in wild boar[END_REF][START_REF] Le Dimna | Validation of two commercial real-time RT-PCR kits for rapid and specific diagnosis of classical swine fever virus[END_REF].

Covariates

• Vaccination

We considered the number of baits per shot wild boar as an indicator of the baiting effort (Bait) that varied from 0.3 to 7 with an average of 4.4 baits delivered each time per shot wild boar. The number of vaccination campaigns (T V ) conducted in each municipality was used as an indicator of the vaccination effort over time.

Among the infected municipalities, we took into account the number of vaccination campaigns since the first viropositive result (Vacc); we defined three different treatments: For the municipalities that were not yet infected at the beginning of vaccination, we estimated the Euclidean distance (in Km) to the centroids of the closer infected municipalities (wave front) (Dist). We considered that the municipalities located far from the wave front had been preventively vaccinated as opposed to the others.

• Other covariates

For the infected municipalities, we defined a relative time scale (T R ) that was the sum of We distinguished two ages classes according to the body mass (Age). The dressed carcasses under 30kg were classified as juveniles, i.e., less than one year old, whereas the others were classified as more than one year old (Rossi et al., 2005a).

The hunting bag in 2006 was used as an indicator of the number of wild boars inhabiting the municipality (Num). We took into account the proportion of forested areas (Forest) as an index of connectivity between neighbouring municipalities. The number of roads separating A c c e p t e d M a n u s c r i p t 8 one municipality from its neighbours was calculated to measure a potential barrier effect of roads (Road).

Dependent variables and tested hypotheses

We used data collected from April 2003 to December 2007 and defined four dependent variables at the municipality level: the proportion of immune animals among unvaccinated municipalities, the infectious status of municipalities, the proportion of viropositive animals and the duration of infection.

• Proportion of immune animals

We considered that the municipalities exhibiting no viropositive result had not been infected.

In these municipalities, we studied the proportion of immune animals hypothesizing that seropositive animals had been vaccinated and not infected.

The proportion of immune animals was likely to increase during the first vaccination campaigns and no more subsequently due to a likely saturation of the vaccination process [START_REF] Louguet | Mesures de gestion de la peste porcine classique sur la faune sauvage : stratégie vaccinale[END_REF][START_REF] Von Rüden | Retrospective analysis of the oral immunisation of wild boar populations against classical swine fever virus (CSFV) in region Eifel of Rhineland-Palatinate[END_REF], we thus tested the effect of the number of vaccination campaigns performed in each municipality (T V ). Given that wild boars less than 4.5 months old are poorly attracted by baits (Brauer et al., 2006, Rossi et al., in prep.) we expected a lower immunity in juvenile wild boars compared to animals more than one year old (Age). We also expected a difference between seasons (Season) (autumn/winter versus spring/summer) since young wild boars grow during the year and since piglets born in spring or summer enter the hunting bag each autumn, i.e., when hunters start to hunt them.

We expected that to delay vaccination would increase the consumption of vaccine in autumn because most of the wild boars would then be older than 4.5 months (Season). We finally • Infectious status of municipalities

The municipalities exhibiting at least one viropositive result were considered as infected whereas the others were considered as uninfected. This status was examined in the municipalities that were not yet infected when the vaccination started (August 2004). This information was summarized as a binomial variable (0 for not infected / 1 for infected) to study the factors influencing the probability of having been infected during the vaccination period.

The probability of being infected was supposed to be influenced by the proportion of forest (Forest), the number of roads (Road) and the number of wild boars (Num) since we assumed that both forest continuity and a sufficient number of hosts were required for new areas to be infected [START_REF] Hone | Evolution of the dynamics and rate of transmission of classical swine fever (hog cholera) in wild pigs[END_REF], Rossi et al., 2005b).

We expected a significant effect of the distance (Dist) from the wave front and of the baiting effort (Bait) because we assumed preventive vaccination and intensive baiting could prevent virus spread.

• Proportion of viropositive animals

At the level of each infected municipality, we analysed the virological results collected since the first viropositive result. We considered the proportion of viropositive individuals as an indicator of infection intensity.

The proportion of viropositive individuals was expected to be higher in juvenile wild boars than in older individuals (Age) [START_REF] Kern | Incidence of classical swine fever (CSF) in wild boar in a densely populated area indicating CSF virus persistence as a mechanism for virus perpetuation[END_REF], Rossi et al. 2005b[START_REF] Von Rüden | Retrospective analysis of the oral immunisation of wild boar populations against classical swine fever virus (CSFV) in region Eifel of Rhineland-Palatinate[END_REF].

The proportion of viropositive individuals was also supposed to decrease over time (T R )

A c c e p t e d M a n u s c r i p t 10 ( [START_REF] Kern | Incidence of classical swine fever (CSF) in wild boar in a densely populated area indicating CSF virus persistence as a mechanism for virus perpetuation[END_REF], Rossi et al., 2005b[START_REF] Von Rüden | Retrospective analysis of the oral immunisation of wild boar populations against classical swine fever virus (CSFV) in region Eifel of Rhineland-Palatinate[END_REF]. We expected that the number of wild boars (Num), the proportion of forest (Forest) and the number of roads between neighbouring municipalities (Road) would significantly influence the proportion of viropositive animals because the number of hosts and the green corridors are supposed to increase the probability of contacts between infected and susceptible animals [START_REF] Begon | A clarification of transmission terms in host-microparasite models: numbers, densities and areas[END_REF][START_REF] Hone | Evolution of the dynamics and rate of transmission of classical swine fever (hog cholera) in wild pigs[END_REF][START_REF] Guberti | Estimate the threshold abundance for the persistence of classical swine fever in the wild boar population of the eastern Sardinia[END_REF], Rossi et al., 2005b).

Lastly, we expected that vaccination timing (Vacc) and baiting effort (Bait) would influence the proportion of viropositive animals because we assumed that to apply preventive vaccination and to intensify baiting could at least decrease the probability of virus transmission [START_REF] Hone | Evolution of the dynamics and rate of transmission of classical swine fever (hog cholera) in wild pigs[END_REF][START_REF] Guberti | Estimate the threshold abundance for the persistence of classical swine fever in the wild boar population of the eastern Sardinia[END_REF][START_REF] Kaden | Oral immunisation of wild boar against classical swine fever: concluding analysis of the recent field trials in Germany[END_REF].

• Duration of infection

We considered the sum of quarter(s) with viropositive results as an indicator of the local duration of infection in the infected municipalities.

We expected that the number of wild boars (Num), the proportion of forest (Forest) and the number of roads between neighbouring municipalities (Road) would significantly influence the duration of infection. Indeed, at the level of the municipality, disease persistence is more likely above a threshold population size and because at the level of a network of neighbouring municipalities virus circulation is more likely if the forested areas are uninterrupted between them [START_REF] Grenfell | Metapopulation dynamics of infectious diseases[END_REF]HarForest 1997, Rossi et al., 2005b, Kramer-Schadt in press).

We finally expected the duration of infection to be influenced by the vaccination timing (Vacc) and the baiting effort (Bait) because we assumed that preventive vaccination and intensive baiting would decrease the probability of a continuous chain of transmission over time.

A c c e p t e d M a n u s c r i p t 11

Statistical analyses

We used Poisson models to study the duration of infection and logistic models for the other dependent variables [START_REF] Dohoo | Veterinary epidemiologic research[END_REF]. The sum of quarters with viropositive results (Poisson model) was weighted by the sum of quarters with both viropositive and vironegative results observed at the level of each infected municipality.

We adopted a step by step descendant process to test for the effect of each expected covariable. We used the Akaike Information Criterion (AIC) to select the best model. When the difference in the AIC was less than 2, we selected the most parsimonious model, i.e., the model with the fewest parameters [START_REF] Burnham | Model selection and inference: a practical information-theoretic approach[END_REF]. We tested for a significant difference between specific parameters using Wald tests [START_REF] Burnham | Model selection and inference: a practical information-theoretic approach[END_REF]. All statistical analyses were performed using R 2.7.2 (the R foundation for statistical computing 2008).

A c c e p t e d M a n u s c r i p t 12 3. RESULTS

Sampled animals and disease evolution in the whole study area

Within the 266 municipalities comprising the study area, 31,892 wild boars were sampled, of which 30,097 exhibited a conclusive (positive/negative) virological result and 27,501 exhibited a conclusive serological result. At the level of the whole area, the proportion of seropositive wild boars increased to 40% in juveniles and to 70% in older animals (Figure 2).

From 2003 to 2005 infection spread over the study area, and infection subsequently persisted in a more enzootic way, the proportion of viropositive being less than 1% since 2006 (Figure 2).

Proportion of immune animals

Within the 168 municipalities with no viropositive result, 5,893 wild boars were sampled and exhibited a conclusive serological result. The number of vaccination campaigns (T v ), age (Age), season (Season) and an interaction between age and season (Age*Season) were significantly correlated with the proportion of seropositive animals (table I). Contrary to our assumptions, the number of baits delivered per wild boar (Bait) had no effect on seroprevalence (table I). As expected, seroprevalence increased over the first three vaccination campaigns and no more subsequently (Figure 3). As expected, seroprevalence was lower in juveniles compared to older animals and this difference was even more important during autumn/winter than in spring/summer (Figure 2). The proportion of juvenile seropositive animals was slightly higher in autumn/winter 2007 (delayed vaccination) than in I).

3.3.Infectious status of municipalities

Within the 232 municipalities that were not yet infected when vaccination began, 5,908 wild boars were sampled and exhibited a conclusive virological result. 91 municipalities among 232 exhibited at least one virological result and were considered as infected. The number of wild boars (Num), the proportion of forest (Forest) and the distance from the wave front (Dist) were significantly correlated with the status of the municipality (infected/not infected) (table II). Contrary to our expectations, the number of roads (Road) and the number of baits (Bait) had no effect on the status of the municipality (table II). As expected, the probability of being infected was positively correlated with the number of wild boars (OR 1 shot wild boar =1.05, P-Wald=<0.001) and the proportion of forest (OR 1% of Forest =6.03, P-Wald=0.038) and negatively correlated with the distance from the wave front (OR 1km =0.88, P-Wald=<0.001).

Proportion of viropositive animals

Within 91 infected municipalities, 2,065 wild boars exhibited a conclusive virological result and were sampled after the observation of the first viropositive result. The number of wild boars (Num), age (Age), time (T R ), the number of roads (Road), the vaccination treatment (Vacc) and an interaction between time and vaccination treatment were significantly correlated with the proportion of infected animals (table III). Contrary to our assumptions the proportion of forest (Forest) and the number of baits (Bait) had no significant effect on the proportion of infected animals (table III). As expected, the proportion of infected individuals A c c e p t e d M a n u s c r i p t 14 was higher in juveniles than in older animals (OR young/old =1.70, P-Wald<0.001). As expected, the proportion of infected animals was positively correlated with the number of wild boars (OR 100 shot wild boar =1.35, P-Wald<0.001) and slightly negatively correlated with the number of roads separating neighbouring municipalities (OR 1 road =0.94, P-Wald=0.016). The proportion of infected animals decreased faster in the municipalities that had been vaccinated contemporaneously with disease emergence compared to late vaccinated municipalities (Figure 4). In municipalities vaccinated early, infection lasted no more than one quarter (Figure 4).

Duration of infection

The number of wild boars (Num), the number of roads (Road), the vaccination treatment (Vacc) and number of baits (Bait) had a significant effect on the duration of infection (table IV). As expected, the duration of infection was positively correlated with the number of wild boars (OR 100 shot wild boars =1.41, P-Wald<0.001) and was slightly negatively correlated with the number of roads between neighbouring municipalities (OR 1 road =0.94, P-Wald<0.001). As expected, the duration of infection was shorter in the municipalities vaccinated contemporaneously with disease emergence compared to the municipalities vaccinated late (OR contemporaneous/late =1.39, P-Wald<0.001) and even shorter in the municipalities vaccinated early (OR early/contemporaneous =1.54, P-Wald<0.001). As assumed, the duration of infection and the number of baits were slightly negatively correlated (OR 1 bait/shot wild boar =0.98, P-Wald=0.016).

A c c e p t e d M a n u s c r i p t 15

DISCUSSION

As expected, the proportion of infected wild boars varied over time according to the municipality; we thus confirm that in a study area comprising municipalities that were not infected at the same time it is more appropriate to explore the effect of vaccination at the municipality level than at the level of the whole study area. As expected wild boar abundance and forest continuity had an important impact on infection emergence, intensity and persistence, which is consistent with previous results in the same area (Rossi et al. 2005b) and justifies the inclusion of these variables in the analysis.

As observed in previous field or experimental studies, juveniles were less efficiently vaccinated and more frequently infected than older wild boars [START_REF] Kern | Incidence of classical swine fever (CSF) in wild boar in a densely populated area indicating CSF virus persistence as a mechanism for virus perpetuation[END_REF], Rossi et al. 2005b[START_REF] Brauer | Oral immunisation of wild boar against classical swine fever: uptake studies of new baits and investigations on the stability of lyophilised Cstrain vaccine[END_REF], VonRüden et al. 2008). The delay of vaccination until October/November slightly increased the proportion of juvenile seropositive animals in comparison with August/September, possibly because a larger part of them were old enough to be attracted by baits [START_REF] Brauer | Oral immunisation of wild boar against classical swine fever: uptake studies of new baits and investigations on the stability of lyophilised Cstrain vaccine[END_REF], Rossi et al., 2006a); one may hypothesize that to delay vaccination until winter could be even more effective. Obviously, duplicating the experiment several times is required to test the effect of the different vaccination time-tables; moreover, other factors must be considered: baits may be less attractive in October/November owing to the seasonal oak mast production and competition may arise between juveniles and adults even though baits are attractive to young wild boars (Rossi et al., 2006b[START_REF] Von Rüden | Retrospective analysis of the oral immunisation of wild boar populations against classical swine fever virus (CSFV) in region Eifel of Rhineland-Palatinate[END_REF]). Our study does not properly address the problem of young animals but our results confirm the interest of specific studies aiming to evaluate the role of young animals (Kaden et al. 2006, Kramer-Schadt et al. in press, Rossi et al. in prep.) and to improve their vaccination et al. 2006, Faust et al. 2007, Ballesteros et al. in press).

In the municipalities where no viropositive result was detected, i.e., where we assumed that the population was initially susceptible, the proportion of immune animals increased during the first three vaccination campaigns before reaching the seroprevalence level required by mathematical models (40-60%) for preventing disease emergence in a susceptible population [START_REF] Hone | Evolution of the dynamics and rate of transmission of classical swine fever (hog cholera) in wild pigs[END_REF][START_REF] Guberti | Estimate the threshold abundance for the persistence of classical swine fever in the wild boar population of the eastern Sardinia[END_REF]. Accordingly, even though preventively vaccinated municipalities became temporarily infected, we observed a lower intensity and duration of infection in the municipalities where one to three vaccination campaigns had been performed before disease emergence. These findings confirm the protective effect of preventive or fenced motorways, preventive vaccination helped CSF control at a distance of 24km from the wave front. Our results suggest that to vaccinate only part of a green corridor is not a suitable option because CSF is likely to spread to free areas without any protective effect of vaccination.

The number of baits delivered per wild boar neither affected the proportion of immune animals nor the intensity of infection, which suggests that increasing the baiting effort did not increase vaccination effectiveness. The slight negative correlation we observed between the baiting effort and disease duration, although statistically significant, is difficult to interpret.

These surprising findings suggest that the baiting process was saturated; this possibly arises A c c e p t e d M a n u s c r i p t 17 because dominant wild boars had exclusive access to feeding grounds [START_REF] Hebeisen | Population size, density and dynamics, and social organization of wild boar (Sus scrofa) in the Basin of Geneva[END_REF]) and consumed most of the baits delivered by hunters. Consequently, many baits might be wasted and the cost-effectiveness of the system could be improved in the future by delivering fewer baits or the same number of baits in a different way. To optimise the baiting effort will require one to develop alternative delivery systems and to study the uptake pattern in detail. In this respect, the use of biomarkers such as the iophenoxic-acid recently tested by [START_REF] Massei | Iophenoxic Acid as a Long-Term Marker for Wild Boar[END_REF] in this species would be helpful for exploring the bait uptake according to different delivery systems.

Our retrospective analysis confirmed that preventive vaccination contributed to better control CSF. However, to assess vaccination effectiveness in achieving disease eradication in the areas still infected is a much more complex issue. [START_REF] Kaden | Oral immunisation of wild boar against classical swine fever: concluding analysis of the recent field trials in Germany[END_REF][START_REF] Kaden | Evaluation of the oral immunisation of wild boar against classical swine fever in Baden-Wurttemberg[END_REF]. These different outcomes may possibly arise because the virulence of recently observed strains was lower than in the past, owing to larger wild boar numbers, or owing to the enhanced sampling effort and sensitivity of diagnostic techniques (r-RT-PCR).

Exploring this question requires specific theoretical approaches simulating different vaccination scenarios and specifying the required conditions for CSF eradication (Kramer-Schadt et al., 2007, Kramer-Schadt et al., in press).

  twenty years, oral vaccination implementing a live attenuated vaccine has been experimented in Europe in order to control classical swine fever (CSF) in Wild Boar (Sus scrofa sp.

  i.e., when at least three campaigns had been performed before the first viropositive result, ii)Contemporaneous vaccination, i.e., when one or two vaccination campaign(s) had been performed before the first viropositive result, iii) Late vaccination, i.e., when the 1 st PCR positive result had occurred before the first vaccination campaign.

  quarters since the first viropositive result. We defined two seasons (Season): autumn/winter from the first of October to the end of March and spring/summer from the first of April to the end of September. Autumn/winter 2007 was considered as a third season type given that vaccination was delayed to October-November in 2007 instead ofAugust-September in 2005 and 2006.

  number of baits delivered per wild boar (Bait) would be positively correlated with the proportion of immune animals.

  years (figure3) but this difference was not significant (OR 2007/other =1.09, P-Wald=0.520) (table

  vaccination and encourage the implementation of preventive rather than post-active vaccination. During recent CSF outbreaks the wave front spread ate has been about 8km per quarter over forested areas (von Rüden et al 2008, Rossi unpublished data); in such conditions, i.e., an epizootic occurring in the absence of physical barriers such as open fields

  of logistic models dealing with the proportion of immune animals observed in uninfected municipalities (the selected model is in bold and italic).

Figure 1 :

 1 Figure 1: The study area is located in north-eastern France at the border of the

Figure 2 :

 2 Figure 2: Observed evolution of the proportion of immune and infected animals over the

Figure 3 :

 3 Figure 3: Observed evolution of the proportion of immune animals over time (Seasons)

Figure 4 :

 4 Figure 4: Predicted evolution of the proportion of infected animals over time according

Table II : Comparison of logistic models dealing with the infectious status of municipalities (the selected model is in bold and italic).

 II

	Covariates		Number of	AIC
	Age*Season + Bait + Ln(T v +1) Age + Season + Bait + Ln(T v +1) Age*Season + Ln(T v +1) Age"*"season + Bait Age + Season+ Ln(T v +1) Age*Season Covariates Forest + Road + Bait + Dist + Num Forest + Road + Bait + Dist Forest + Road + Bait + Num Forest + Road + Dist + Num Forest + Bait + Dist + "Num Road + Bait + Dist + Num Forest + Road + Dist	parameters a n u s c r i p t 8 7014.2 6 7022.0 7 7012.3 7 7641.1 4 7020.0 5 7645.7 M Number of parameters AIC 6 185.1 5 205.6 5 199.0 5 183.1 5 186.6 5 189.1 4 205.1 M a n u s c r i p t
	Forest + Road + Num		4	197.2
	A c c e p t e d Forest + Dist + Num Road + Dist + Num A c c e p t e d	4 4	184.8 187.1
		25 27		Page 27 of 33

Table IV : Comparison of Poisson models dealing with the duration of infection in 1 infected municipalities (the selected model is in bold and italic).

 IV

	2
	3
	Covariates	Number of	AIC
		parameters
	Num + Forest + Road + Bait + Vacc	7	411.7
	Num + Forest + Road + Bait	5	480.5
	Num + Forest + Road + Vacc	6	417.4
	Num + Forest + Bait + Vacc	6	483.0
	Num + Road + Bait + Vacc	6	411.3
	Forest + Road + Bait + Vacc	6	684.4
	Num + Road + Bait	4	478.4
	Num + Road + Vacc	5	415.4
	Num + Bait + Vacc	5	487.3
	Road + Bait + Vacc	5	785.3

Acknowledgements

The authors would like to thank the hunters, game keepers, local veterinary services and laboratories who performed a huge effort to maintain the quality of vaccination and CSF monitoring. Particular thanks go to Mrs. C. Carter who revised the English language and Pr.

G. Gunn who reviewed the oral presentation of this paper during the 7 th Pestivirus congress (Uppsala 2008).