B J J Mccormick

A W Stott

F Brülisauer

B V Ahmadi

G J Gunn

An integrated approach to assessing the viability of eradicating BVD in Scottish beef suckler herds

Keywords: bovine viral diarrhoea (BVD), stochastic model, marginal benefits

The viability of eradicating bovine viral diarrhoea (BVD) in Scottish suckler herds is dependent on the continued compliance with eradication schemes. At the farm level, the costs of BVD have been identified in previous studies and show a substantial financial imperative to avoid infection. At a regional level the incentives of BVD eradication to individuals are unclear, for example the requirement for vaccination strategies despite achieving disease-free status. Ensuring farmer compliance with an eradication scheme is therefore difficult. Experience of eradicating BVD from beef-dominated areas is limited

and theoretical models have tended to focus on the dairy sector. Here we present a stochastic epidemiological model of a typical beef suckler herd to explore the interaction of a farm with a regional pool of replacements, utilising information from a BVD virus seroprevalence survey of Scottish beef suckler herds. Our epidemiological model is then used to assess the relative costs to individuals assuming different regional endemic prevalences, which are used to represent the likelihood of BVD re-introduction. We explore the relative cost of BVD, taken as likelihood and consequence, at an endemic steady state in contrast to previous models that have assumed the introduction or control of BVD in an epidemic state (e.g. a closed and mostly susceptible population). Where endemic, BVD is unlikely to affect all farms evenly and will cost most farmers very little due to herd immunity or self-clearance of the virus. Compliance is likely to be boosted by pump-priming to initiate and complete eradication schemes with cost-sharing.

Introduction

Bovine viral diarrhoea (BVD) is an endemic livestock disease causing substantial economic losses in dairy and beef cattle herds worldwide (Bennett and Ijpelaar 2005).

The biggest losses occur through reduced fertility and the generation of persistently infected (PI) animals that are a source of continued infection and can suffer high mortality from the development of mucosal disease [START_REF] Brownlie | Clinical aspects of the bovine virus diarrhoea/ mucosal disease complex in cattle[END_REF]Roeder and Drew 1984). BVD virus (BVDV) causes immunosuppression [START_REF] Chase | The immune response to bovine viral diarrhea virus: a constantly changing picture[END_REF], predisposing animals to a wide range of disease and resulting losses will often be attributed to secondary causes. As a consequence, economic as well as animal health impacts of BVD are often underestimated in herds with endemic infection.

A comparative study of the economic challenge of BVD in several European countries or regions [START_REF] Gunn | Assessing economic and social pressure for the control of bovine viral diarrhoea virus[END_REF], suggests that the UK may be at a relative disadvantage both in terms of the current challenge from BVD and in the costs to individual farmers of unilateral attempts to establish and maintain disease freedom. This highlights the need for a more detailed study of the economics of BVD at regional rather than just at individual farm level. Regional control has tended to focus on areas dominated by the dairy industry. This is not the case in Scotland, which has a much larger beef sector and where there is limited practical experience of eradicating BVDV from beef herds, although some success has been had on the Shetland and Orkney islands [START_REF] Synge | The control of bovine virus diarrhoea virus in Shetland[END_REF].

Based on the guidance of the OIE (2008), we use a risk analysis framework to integrate studies from complimentary disciplines to explore the feasibility of a regional eradication A c c e p t e d M a n u s c r i p t 4 scheme in the context of Scottish beef suckler herds. [START_REF] Mcinerney | Old economics for new problems -Livestock disease: Presidential address[END_REF] has suggested that the marginal costs of an eradication scheme might increase considerably towards the end as prevalence decreases and it becomes more difficult to find and eliminate the last and most intransigent infected herds. The problems experienced on the Shetland islands during the eradication of BVD [START_REF] Synge | The control of bovine virus diarrhoea virus in Shetland[END_REF]) lend weight to this argument. Marginal costs at the start of an eradication programme may also be higher as maintaining diseasefree herds will be more expensive while regional prevalence, and thereby challenge, of BVDV remains high [START_REF] Gunn | Assessing economic and social pressure for the control of bovine viral diarrhoea virus[END_REF]. At both extremes of the scheme farms are, therefore, less likely to either embark on or comply with the protocol because the balance of 'risks' is less favourable than mid-way through the programme. Identification of the relative cost, or their inverse benefits, to individuals and to the public (i.e. a wider farming community) may enable appropriate 'beneficiary pays' cost sharing to pumpprime an eradication scheme at its start and end. However, estimates of the potential extent of this effect are needed to inform policy.

We explore the financial incentives to control BVD, as an individual farm or a region and the identification of the 'beneficiary' who ought to pay based on the level of endemic disease. Endemic disease represents both the status quo, that which is accepted as the norm, and then by reducing the prevalence it represents stages of an eradication scheme which aims to reduce prevalence of disease to zero. For this purpose, we present a stochastic model of a typical Scottish beef suckler herd that interacts with a regional pool of replacement animals of known BVDV prevalence. Previous models [START_REF] Gunn | Modelling and costing BVD outbreaks in beef herds[END_REF][START_REF] Innocent | A computer simulation of the transmission dynamics and the effects of duration of immunity and survival of persistently infected animals on the spread of bovine viral diarrhoea virus in dairy cattle[END_REF][START_REF] Viet | A model of the spread of the bovine viral-diarrhoea virus within a dairy herd[END_REF]) have dealt with the incentives to control BVDV on Here we present a model of a farm that introduces animals from an external source to explore the longer term consequences of different likelihoods of reintroducing BVDV. Our model also explores the endemic state of BVDV, at which point there is an approximately stable prevalence of infected herds even though for any given farm the incidence of BVDV is very low.

Materials and Methods

Data

BVD seroprevalence in Scotland

The starting point for an eradication programme is to identify the prevalence level in order to assess the scale of the challenge and to establish a benchmark from which to estimate the changing marginal benefits of eradication postulated above. This study is based on the results of a survey estimating the national prevalence of active BVDV herd infection in Scottish beef suckler herds [START_REF] Brülisauer | Prevalence of bovine viral diarrhoea in Scottish beef suckler herds[END_REF].

A survey comprising 301 beef suckler herds, stratified by geographic region and herd size, was carried out in 2006-2007. Blood samples were taken from a randomised sample of between seven and ten youngstock (6 -16 months old) from each management group within the enterprise. Samples were tested by BVDV antibody ELISA and information gathered on farm characteristics and herd management factors using a standardised questionnaire.

A c c e p t e d M a n u s c r i p t 6 One third of beef suckler herds showed evidence of recent BVDV exposure, with at least one animal testing BVDV seropositive. Sixteen percent (95CI: 11.6, 19.7%) of herds had youngstock with a seroprevalence of more than 90%. This group therefore showed evidence for recent active BVDV infection and presently or in the past harbouring a PI animal.

Modelling

Epidemiological modelling

Following [START_REF] Gunn | Modelling and costing BVD outbreaks in beef herds[END_REF], we modelled a typical Scottish 100 cow beef suckler herd, using a state transition approach for the BVDV dynamics in an age stratified population.

Our stochastic simulation model was designed to account for the small finite population of beef cattle and featured a daily, rather than annual, time step to allow greater emphasis on the timing of (re-)introduction of BVDV via the purchase of animals. These features allowed us to better explore the importance of variability in the timing and likelihood of introduction of infectious animals from off-farm relative to rebreeding and other significant management activities on-farm. We modelled BVDV dynamics on a single typical farm using no BVDV control measures, which interacted with a regional pool of herds that were the source of replacement animals. The proportion of animals in each of the mutually exclusive disease states (susceptible, S; transiently and persistently infected/infectious, TI and PI respectively; and immune animals, R) on the farm was dynamic based on the assumptions in Table 1.

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

[Table 1]

Regional pool properties

The regional pool was designed as a collection of replacement animals and modelled as a separate set of disease-state classes. Both the number of animals and the prevalence of BVDV in the regional pool were assumed to be static during a given model scenario. The rationale was to mimic the risk of BVDV infection/re-infection to which an individual farm is exposed when buying replacements from a large pool of other farms (the region) assuming an endemic (i.e. steady) prevalence of BVDV equal to, or below, the current level. The regional herd in this model represented any large collection of available animals from a local cooperative to a national herd that can be assumed to have relatively stable, endemic, BVDV prevalence. The assumed stability of disease is supported by the apparent consistency of BVDV prevalence across different countries [START_REF] Houe | Epidemiological features and economical importance of bovine virus diarrhoea virus (BVDV) infections[END_REF], suggesting that in spite of the management, spatial and temporal heterogeneity, BVDV is relatively stable at a regional level.

We modelled different scenarios, each assuming a different prevalence of PIs in the regional pool, thus the probability of acquiring a PI was a function of the regional prevalence of PIs. The rest of the animals in the regional pool were assumed to be susceptible. This assumption served two functions; first it represents the worst-case scenario of buying either highly infectious PI animals, which are considered the primary source of BVDV on farms [START_REF] Houe | Epidemiological features and economical importance of bovine virus diarrhoea virus (BVDV) infections[END_REF], or susceptible animals that can then become A c c e p t e d M a n u s c r i p t 8 infected and do not contribute to the herd immunity. The presence of antibodies are not necessarily an indicator of immunity as they can arise from vaccination and they are not a reliable indication of the disease status of offspring which can be infected as foetuses before the cow recovers and develops an immune response. Second, complications associated with the changing risks of buying-in susceptible, immune, transiently infected or PI animals as an eradication programme progresses are eliminated. Although this level of complexity is involved in practice, we felt that the key driver would be the likelihood of purchasing PIs in an endemic situation.

Farm herd properties

Individual cows were modelled to utilise the higher temporal resolution of this model compared to [START_REF] Gunn | Modelling and costing BVD outbreaks in beef herds[END_REF], which modelled age cohorts. Cohorts were still retained for heifers and calves, but the individual level was deemed more appropriate for breeding cows since this allowed a more detailed exploration of the specific timing of transient infection during pregnancy and thus the probability of different outcomes of foetal infection (see the supplementary information for further details). We assume that the population is managed to maintain an approximately constant number of breeding cows (100) [START_REF] Gunn | Modelling and costing BVD outbreaks in beef herds[END_REF]), and stochastic fluctuations in the population size simulate additional births, deaths or management decisions to replace individual animals. We have assumed that deaths not directly attributable to BVD are accounted for in proportion to the population replaced during management activities (i.e. to maintain a constant population size). These additional deaths may include both mortality from A c c e p t e d M a n u s c r i p t 9 unrelated causes and secondary infection resulting from BVDV induced immunosuppression [START_REF] Brownlie | Pathogenesis and Epidemiology of Bovine Virus Diarrhea Virus-Infection of Cattle[END_REF].

Additional mortality resulting directly from persistent infection with BVDV is explicitly modelled. We have assumed an exponential probability of death capturing both those that are born weak and thereby predisposed to secondary pathogens and the mortality resulting from mucosal disease in older PIs (Duffell and Harkness 1985). Mortality was calculated according to the age of PIs reported by [START_REF] Rüfenacht | Prevalence of cattle infected with bovine viral diarrhoea virus in Switzerland[END_REF] using the best fitting exponential distribution (mean age of 342.3 days). This approximation produces similar results to those observed by Duffell and Harkeness (1985) with 65% of PIs dying in their first year and 20% surviving to 18 months [START_REF] Houe | Age Distribution of Animals Persistently Infected With Bovine Virus Diarrhea Virus in 22 Danish Dairy Herds[END_REF]. It is worth commenting that [START_REF] Rüfenacht | Prevalence of cattle infected with bovine viral diarrhoea virus in Switzerland[END_REF] reported the age of animals observed rather than their age of death, therefore if incorrect, the longevity of PI animals is underestimated (see the supplementary information).

In our model, cows were put to the bull for a fixed period of 85 days approximately following the upper limit of the recommend window for maximal productivity given by [START_REF] Caldow | Veterinary Intervention in the Reproductive Management of Beef Cow Herds[END_REF], and the probability of becoming pregnant was uniform within this period.

A proportion, 15%, of adult cows was replaced each year assuming an average herd life of seven years [START_REF] Sac | Farm Management Handbook 28th Edition[END_REF]. Replacements were home-bred in the first instance. The availability of home-bred replacements was dependent on the number of calves (and A c c e p t e d M a n u s c r i p t 10 thereby heifers) retained at the point when calves were sold and replacements potentially bought. If there were insufficient calves to meet the needs of the replacements, as resulted from stochastic fluctuations around the mean population size at the point of weaning, an appropriate number of animals were bought from the regional herd. In accordance with the prevalence survey, it was assumed that 80% of replacements were in-calf as this strategy (and cows with calf-at-foot) represents a major risk of (re-)introducing BVD onto the farm and thus are the most conservative worst-case estimates of marginal benefits. Replacement animals were assumed to be heifers, because it was deemed a more common strategy to replace older animals with younger ones where possible since younger animals would be worth more over time.

BVDV transmission

Transmission was based on [START_REF] Viet | A model of the spread of the bovine viral-diarrhoea virus within a dairy herd[END_REF] who modelled a dairy herd using a proportionate mixing term to describe the probability of encountering an infectious animal as a proportion of the herd. We assumed that in a suckler herd all animals ran together [START_REF] Gunn | Modelling and costing BVD outbreaks in beef herds[END_REF]) and therefore the probability of interacting with PIs from different age cohorts were equally likely. The daily mean probability of BVDV transmission was the proportion of PIs in the age cohort multiplied by the force of infection (i.e. the rate at which susceptible animals get infected), 0.5 [START_REF] Viet | A model of the spread of the bovine viral-diarrhoea virus within a dairy herd[END_REF]).

Similarly, virus transmission by transiently infected animals was assumed to be the proportion of TI animals and a force of infection of 0.03 [START_REF] Viet | A model of the spread of the bovine viral-diarrhoea virus within a dairy herd[END_REF]. As used by [START_REF] Gunn | Modelling and costing BVD outbreaks in beef herds[END_REF], the initial conditions of the model was a population with a single PI cow, ten TI cows and eight TI heifers along with a further 89 susceptible cows and 18 susceptible heifers.

Models were run for different regional PI prevalence scenarios (5-0% in 0.05 decrements) in order to cover a range of possible stages of BVDV prevalence from an endemic state and through to eradication. The simulation was run for thirty years, which allowed the first 15 years to be disregarded as the burn-in period and reach a representative steady state (i.e. endemic BVDV). Each scenario was repeated 1000 times to account for stochastic variation.

Economic modelling

The cost of disease health and labour consequences were derived from Stott and Gunn (2008). The cost of BVD was partitioned into five components, namely: (1) the cost of an abortion from a transiently infected cow, (2) the cost of a weak calf from a transiently infected cow, (3) the cost of a PI calf (from a TI or PI cow), (4) the cost of transient infection in calves and (5) the cost of a PI cow. The cost of an embryonic re-absorption A c c e p t e d M a n u s c r i p t 12 return to service, which constituted the sixth cost in the Stott and Gunn (2008) model was not calculated in this model. This was because the daily time step used in this model meant it was possible to account for the delay in rebreeding and hence in calving via the costs attributed to the calf rather than the cow. Therefore, including the costs of delayed conception would then constitute double counting. More detailed exploration of the interaction between BVD and infertility in suckler cows is undertaken by Varo Barbudo et al. (2008). We also excluded the fixed cost of immunosuppression in calves included by Stott and Gunn (2008) on the grounds that there is insufficient information to establish how this might vary with regional prevalence of BVDV. Our objective was to establish how the marginal cost (rather than absolute cost) of BVD changes as regional prevalence alters at different stages of an eradication programme. The costs of BVD in each run of the simulation model were summarised as the sum of all costs given above in years 15 to 30 of the simulated epidemic, discounted in the normal way (Boehlje and Eidman 1984) so that the total discounted costs (TDC) were expressed in present value terms. The discount factor used was 0.05 as in previous economic impact assessments of BVD in Scottish beef suckler herds [START_REF] Gunn | Modelling and costing BVD outbreaks in beef herds[END_REF]Stott and Gunn 2008). As cashflows were summed across years 15 to 30 of a simulated epidemic to represent the endemic disease situation and then discounted to present values for comparative purposes they were not comparable with published costs and benefits that are concerned with the more immediate costs of BVD in the early phases of an epidemic. Furthermore, our interest was in the relative incentive to deal with the disease under different regional epidemiological circumstances. Absolute costs are, in any case, dependent on price A c c e p t e d M a n u s c r i p t 13 assumptions made, which are likely to date quickly. For these reasons, we expressed all our financial results relative to the lowest TDC obtained.

For simplicity, any costs were incurred when the animal/event occurred. Whilst the cost of replacing a PI cow that is suffering mucosal disease (i.e. the loss of the cow, the cost of replacing an animal and additional labour and veterinary costs) may occur some years after the PI cow comes onto the farm, in this model the charge was noted at the point of occurrence to avoid double counting BVD related costs.

Results

The initial conditions of the model had a dramatic impact on the first 5 years of each iteration, however, these effects were negligible after 10 years. This outcome was in accordance with the observation of previous simulation models that observed selfclearance of BVDV after 7 years [START_REF] Humphry | Modelling BVD at herd level compared with individual animal level[END_REF]. We only, therefore, consider the last 15 years of the simulation (i.e. the first 15 years are discarded) such that we only explore the endemic (steady) state of BVDV infection in the modelled herd.

The median incidence of PIs, that is the average number of PIs occurring on the farm per year, was a linearly function of the regional prevalence of PIs (p <0.001) (Figure 1).

[Figure 1] A c c e p t e d M a n u s c r i p t 14 The median proportion of simulation time (years 15 to 30) for which BVDV was active (i.e. a non-zero count of PIs or TIs in the herd) was linearly related to the regional prevalence of PIs (p <0.001). There was a strong correlation between the proportion of time BVDV was active and the incidence of PIs in the herd (r 0.972), although the correlation decreased as the regional prevalence of PIs increased and the variability of time active BVDV increased, ranging from an inter-quartile range of 0.20 of simulation time with no PI re-introductions, to 0.58 with a PI prevalence of 5%.

[Figure 2] Assuming that each iteration of the simulation could be treated as a uniquely realised farm, comparison was made between the regional PI prevalence scenarios and the result of the seroprevalence survey for validation of the model results (Figure 2). The seroprevalence survey identified three herd cohorts: seronegative herds, herds with low seroprevalence (1-8 animals seropositive and a median within herd prevalence of 0.3) and high seroprevalence (9 or 10 animals seropositive and a median within herd prevalence of 0.9). Each of 1000 iterations of each of the regional prevalence scenarios were categorised into similar cohorts based on a single spot sample of youngstock taken in year 15, effectively replicating the seroprevalence spot sample with the model data. The simulated spot sample identified the proportion of youngstock (calves) in the immune (R) class just as the serology identified the immune status of real livestock. The distribution of herd seroprevalence for each scenario (columns 2-12 on Figure 2) was then compared to the distribution of farm seroprevalence results from the survey (first column on Figure ). The best-fitting (i.e. least significant difference, p = 0.0396) regional PI prevalence was 2.5%. This is within the range of previous estimates for PI prevalence from other studies (in other countries, e.g. [START_REF] Rüfenacht | Prevalence of cattle infected with bovine viral diarrhoea virus in Switzerland[END_REF]).

[Figure 3] The regional prevalence of PIs, rather than the herd seroprevalence (as defined by the simulated seroprevalence sampling), dominated the relative TDC due to BVDV (Figure 3). For clarity, the different regional PI prevalence scenarios were grouped into three broad groups of high, medium and low prevalence, within which were the simulated herd seroprevalence spot samples. The TDC for each region and herd grouping was compared to the mean of the lowest TDC (low regional PI prevalence and low herd seroprevalence).

The TDC in regions of high PI prevalence is as much as 7.3 times the value in regions with low PI prevalence. This result holds irrespective of the herd seroprevalence category. The variance of the TDC is a consequence of the variable epidemiological outcomes, and reflects the processes of infection, self-clearance and re-infection, or a failure to self-clear BVDV. Re-infection into susceptible herds dramatically increases the cost of BVDV in the low prevalence scenarios, increasing the costs as much as 7 times the mean for less than a quarter of holdings whilst the median cost is zero (most farms have self-cleared BVDV and subsequently not re-introduced PIs). A higher proportion (>25%) of farm iterations did not achieve a zero cost in the high regional prevalence A c c e p t e d M a n u s c r i p t 16 scenarios. The range of TDC is highest in the high PI prevalence scenarios, with an interquartile range of as much as 2 to 10 times the mean TDC of the low prevalence scenarios.

Discussion

BVDV infection has been shown to be costly to individual farmers, though the financial argument at the farm level in mainland Scotland is apparently inadequate to persuade farmers [START_REF] Gunn | Assessing economic and social pressure for the control of bovine viral diarrhoea virus[END_REF] to embark upon systematic control as has been adopted in other regions [START_REF] Lindberg | The control of bovine viral diarrhoea virus in Europe: today and in the future[END_REF].

The results of simulating BVDV dynamics using the initial conditions was highly variable and produced results that contrasted markedly with results of the endemic state of BVDV in subsequent years of the simulation. This observation challenges the reliance on previous models [START_REF] Cherry | Evaluation of bovine viral diarrhea virus control using a mathematical model of infection dynamics[END_REF][START_REF] Gunn | Modelling and costing BVD outbreaks in beef herds[END_REF][START_REF] Innocent | A computer simulation of the transmission dynamics and the effects of duration of immunity and survival of persistently infected animals on the spread of bovine viral diarrhoea virus in dairy cattle[END_REF][START_REF] Viet | A model of the spread of the bovine viral-diarrhoea virus within a dairy herd[END_REF]) that have reported findings for which interpretation should be restricted to 'epidemic' BVDV, that is to say, the spread of BVDV in a wholly susceptible population.

In Scotland, BVDV is considered an endemic disease and modeling farms that interact with one another and thereby retain a variable degree of herd immunity, is more realistic than a susceptible and closed herd. The nature of stochastic models may also yield variable results at the outset of a simulation that do not adequately represent patterns that form later in the model as the simulation begins to equilibrate. This is a danger if a sufficient burn-in period is not used and also if it is not discarded from subsequent analysis.

A c c e p t e d M a n u s c r i p t 17 Modelling with the possibility of BVDV re-introduction via replacements is something novel in BVDV literature, which has tended to focus on closed herds. The dynamics presented here do not capture the trading patterns of any given farm, and are in fact rudimentary. They do, however, allow for the potential re-introduction of BVDV through interaction with an external source of infection. In the case that the trading, here described as purchasing replacements, is deemed inadequate, the principal of interacting with a wider environment may be interpreted as any introduction of PI animals. Future work will undoubtedly focus on the interaction of farms, given that for individuals there is already a known financial imperative to act. Examination of the role of neighbours, and therefore the necessity to cooperate is now required.

The regional prevalence of PIs dominated the financial consequence of BVDV in this simulation. This was a surprising result given the high cost to a single farm [START_REF] Gunn | Modelling and costing BVD outbreaks in beef herds[END_REF]) and the potential for BVDV to linger in herds for protracted periods of time [START_REF] Brownlie | Pathogenesis and Epidemiology of Bovine Virus Diarrhea Virus-Infection of Cattle[END_REF]. However, previous studies both observational [START_REF] Stahl | Self-clearance from BVDV infections -A frequent finding in dairy herds in an endemically infected region in Peru[END_REF] and simulated [START_REF] Humphry | Modelling BVD at herd level compared with individual animal level[END_REF] have identified the natural extinction of BVDV. This process, dubbed 'self-clearance' (Lindberg and Houe 2005) is likely to reduce the prevalence of BVDV in a closed system, however the re-introduction of infection will either maintain a low level of virus circulating or produce repeated epidemic outbreaks.

For any given farm the classification derived from the spot test would change over time, thus the prevalence for the population of farms would be consistent and for the individual farms would fluctuate. At low regional prevalence of PIs the chance of re-introducing infection is low (or zero) such that the cost of BVDV to the region is negligible as farms A c c e p t e d M a n u s c r i p t 18 successively self-clear. At higher regional prevalence the regional cost is much higher because farms are more prone to re-introducing BVDV. Given the regional cost of BVDV there is a strong financial incentive for regions, over and above individual holdings, to cooperate to eradicate BVDV. This is an interesting finding since for individuals there may be high disease cost, but then the vast majority (almost 70% in Scottish Beef suckler herds) are BVDV-free and then have no cost. The increase in the variability of the TDC for the high regional PI prevalence suggests that the cost of BVDV across the region became more widely distributed across herds, as more herds showed a sustained cost of endemic BVDV over the 15 years of the simulation and fewer herds sustained a disease-free status. Wholly susceptible farms are, however, at risk of epidemic BVDV, which has been shown to incur high costs [START_REF] Gunn | Modelling and costing BVD outbreaks in beef herds[END_REF]). This dramatic, but brief cost was reflected by the very high TDC for a small minority of farms (<5%) even in the low regional prevalence scenarios compared to highly skewed distribution of zero cost (~95%), which skewed the mean cost for the whole region to a non-zero amount.

If BVDV is therefore treated as a regional cost, irrespective of any given individual herd's disease status, then encouraging participation and compliance with an eradication scheme may require pump-priming. With two thirds of farms not currently suffering costs of disease ensuring their active involvement would likely necessitate regional investment (where a region is deliberately left undefined). When BVDV is reduced to low levels modelled here (~0.5% of the population as PIs) the issue is more to do with compliance, A c c e p t e d M a n u s c r i p t 19 as the proportion of farms with active infection drops to less than 5%. The precise balance of cost-sharing may depend on the scale of the eradication and the mixture of cattle enterprises. Cost-sharing would also require scaling as an eradication scheme progresses since unlike the models presented here, the prevalence of PIs in the regional pool would be a function of the eradication scheme rather than a static risk of BVDV reintroduction.

Conclusions

We have presented a model of endemic BVDV that has explored the increased persistence of this disease on a farm that interacts with a region. Within any given region, for many farms BVDV may not incur a cost given that there is no active transmission of the virus, however the consequence of re-introduction of BVDV to a fully susceptible herd is epidemic BVDV infection with associated increased costs.

The disease status of any single herd may fluctuate over time, but when iterations were aggregated based on the prevalence scenario, the risk of (re-)introduction of BVDV appears relatively stable assessed across many farms with a similar regional prevalence and has a linear function with the regional prevalence of PIs.

BVDV can be considered a public issue beyond the financial incentives for individuals to manage the disease. At a regional level, BVD costs all individual farmers (the mean cost to farmers in the region) in higher proportions than the seroprevalence on any given holding. This is likely to result from the dynamic nature of the disease and the balance of There is a pressing need for greater data to parameterise and structure models for the beef suckler cow industry as most current information is heavily biased towards dairy systems.

The relative longevity of animals and the increased mixing both lend themselves to the persistence of BVDV. This is balanced by the dynamics of trading patterns and seasonal calving rather than year-round calving, which may support eradication schemes as potential PIs may be kept apart from pregnant cows at risk of foetal infection.

  farm and have explored the consequences of introducing BVDV into a wholly susceptible farm.

  during pregnancy resulted in foetal consequences dependent on the month of gestation. The probability of different outcomes (generation of a PI calf, abortion, the birth of a weak calf with congenital defects or no deleterious effect) were based on calculations made by[START_REF] Gunn | Estimating the losses associated with bovine viral diarrhoea (BVD) within the Scottish cow-calf herd[END_REF] using data fromHoue (1996) (see the supplementary information).

  each of the model scenarios was significantly different (Kolmogorov-Smirnov test at 5%

  herd immunity, suffering disease losses and self-clearance of the disease (on top of active BVDV control).

  PIs), medium (2-4% PIs) and low (0-2% PIs), and herd seroprevalence was based on a single spot sample of youngstock, with seroprevalence cohorts defined by the BVDV survey (low, 0 seropositive youngstock; medium, 10-80% seropositive; high, 90-100% seropositive).

Acknowledgements

This work was supported by the Scottish Executive Environment and Rural Affairs Department.


Patrick Presi

D Heim

BVD eradication in Switzerland -A new approach

Keywords: Bovine viral diarrhea, eradication, Switzerland, PI animals

A c c e p t e d M a n u s c r i p t Abstract Diverse concepts for BVD eradication or control have been applied in several countries with varying success. Results of previous studies conducted in Switzerland have shown that the prevalence of antibody-positive animals is high and that BVDV is wide spread in the country causing serious economic losses. A new approach to eradicate BVD in the cattle population in Switzerland was chosen. It consists in testing the whole Swiss cattle population for virus detection in a short period of time, without initial antibody screening.

Identified persistently infected (PI) animals have to be slaughtered, and new herd infections should be avoided by movement restrictions. Ear-notches are collected using special tags for labeling the animals, and are analyzed using ELISA or rtRT-PCR Methods. Confirmatory tests if needed are performed on blood samples using rtRT-PCR.

The eradication program is divided in four phases: 1) Pre-pasturing phase: all young bovines going to transhumance in summer have to be negative tested before. 2) Initial phase: all non-tested bovines have to be tested. Movement restrictions are effective at the same time. 3) Calves phase: all new-borne calves have to be tested 4) Surveillance phase: several strategies will be compared using a modeling approach.

After the pre-pasture phase already 595'230 animals (37% of the livestock) have been tested within four months. A prevalence of 1.1% of PI's was observed. The average age of infected animals is 403 days compared to 794 days for non-infected animals, with the oldest PI animal being over 11 years old. On average PI animals are slaughtered within 18 days after the last positive result.

The pre-pasture phase has shown that sampling and testing a high number of animals in a short time is challenging but possible. The next phase will deal with a double amount of animals in a similar time frame. The coordination between all partners as well as the collaboration of farmers is the key factor for ensuring the success of the program. et al., 2003). In Switzerland, based on a cross-sectional study, 57.6% of animals had antibodies to BVD virus (BVDV) and a within-herd prevalence of 55%; none of the farms investigated was free of antibody-positive animals [START_REF] Rüfenacht | Prevalence of cattle infected with bovine viral diarrhoea virus in Switzerland[END_REF]. With different production structures and diverse initial situations, many European countries have initiated national and regional control-and-eradication campaigns for BVDV [START_REF] Greiser-Wilke | Bovine viral diarrhoea eradication and control programmes in Europe[END_REF][START_REF] Moennig | BVD control in Europe: current status and perspectives[END_REF].

The first systematic eradication schemes were launched in 1993-1994 in the Shetland

Islands, Denmark, Finland, Norway and Sweden [START_REF] Bitsch | Experiences from the Danish programme for eradication of bovine virus diarrhoea (BVD) 1994-1998 with special reference to legislation and causes of infection[END_REF][START_REF] Bitsch | Control of bovine viral diarrhea virus infection without vaccines[END_REF][START_REF] Lindberg | The control of bovine viral diarrhoea virus in Europe: today and in the future[END_REF][START_REF] Synge | The control of bovine virus diarrhoea virus in Shetland[END_REF]. Regional approaches to control the BVD have been undertaken in various countries such as Germany [START_REF] Gaede | Elimination of persistently BVDV infect animals: efficient herd screening using RT-PCR and antigen ELISA in milk and serum samples[END_REF][START_REF] Greiser-Wilke | Bovine viral diarrhoea eradication and control programmes in Europe[END_REF], Italy [START_REF] Ferrari | Bovine virus diarrhoea (BVD) control programme in an area in the Rome province (Italy)[END_REF] and France (Joly et al., 2005;[START_REF] Viet | Assessment of strategies to control BVDV spread in a dairy herd using computer simulation[END_REF]. In Austria, a programme was first implemented at a regional level and in 2004 extended to the whole country [START_REF] Obritzhauser | BVDV infection risk in the course of the voluntary BVDV eradication program in Styria/Austria[END_REF]Rossmanith et al., 2005).

The Scandinavian BVD-eradication models build mainly upon the initial classification of the population in non-infected and infected herds, the surveillance and certification of non-infected herds and the virus elimination from infected herds [START_REF] Lindberg | Principles for eradication of bovine viral diarrhoea virus (BVDV) infections in cattle populations[END_REF]. Herd-level tests for the determination of probable BVDV status are performed either on bulk milk or on serum or milk from a small number of animals of a certain age group. Enzyme-linked immunosorbent assays (ELISAs) is used for the detection of antibodies.

A c c e p t e d M a n u s c r i p t Regardless of the prerequisites like legal support and varying initial prevalences of herds with PI animals, it has taken the Scandinavian countries approximately ten years to reach their final phases [START_REF] Hult | control scheme in Brittany (western France)[END_REF][START_REF] Nyberg | Eradication of BVDV-infection in norwegian cattle 1992-2003 -a success story[END_REF][START_REF] Rikula | grassland-the key for successful BVDV-eradication in Lower Austria[END_REF][START_REF] Voss | The BVD situation in Denmark, development and surveillance program[END_REF]).

An alternative, when the prevalence of antibodies is high, is to focus the program on the direct identification and removal of PI animals. This option was adopted in Lower Saxony in Germany [START_REF] Greiser-Wilke | Bovine viral diarrhoea eradication and control programmes in Europe[END_REF].

Due to the high density of farms and high level of contact between farms, as well as the prevalence situation of antibody positives, Switzerland opted for an intensive compulsory eradication program without initial antibody screening. The improvement of diagnostic tests for the detection of PI-animals in recent years in terms of early detection of young infected animals and the possibility to use ear-notch samples made the Swiss approach feasible [START_REF] Hilbe | Comparison of five diagnostic methods for detecting bovine viral diarrhea virus infection in calves[END_REF][START_REF] Houe | Test strategies in bovine viral diarrhea virus control and eradication campaigns in Europe[END_REF]. In 2008, all cattle will be tested.

Movement restrictions will take place in order to speed up the eradication program and minimize the apparition of new or re-infection of herds during the process.

The present paper describes the eradication program implemented in Switzerland and provides preliminary results as well as discusses some critical points imperative for its success.

Material and Methods

Structure of the livestock in Switzerland

The bovine livestock in Switzerland consists of 1.572 million animals distributed in 43'722 farms with 55% of farms present in the plain and 45% in the mountains. The average herd size is of 36 cattle. Every type of stock-farming is present in Switzerland from purely fattening farms to dairy farms with the majority of farms being a mix of both.

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

The animal movement is very intense and is characterized mostly by the pasture that takes place every summer in various mountainous regions of the country. Around 25% of cattle are going to transhumance in the mountain with a mix between animals from many places of origin. The animals stay during summer in the mountains and return in autumn to their farm of origin. In addition, due to the specificity of the market, it is frequent that animals travel all over the country to be sold. Animal movements are therefore dense and consist of a complex network.

Eradication program

The key points of the eradication program in Switzerland are the followings:

-Compulsory for every farmer -Individual identification and elimination of PI animals in the whole Swiss cattle population -Action conducted in a short period (a year for the testing of the population)

-Movement restrictions -Continuous information and education of farmers

The program is divided in four phases (Table 1): 1.Phase before pasturing (January to July 2008)

In Switzerland, pasturing has an important role in maintaining and transmitting the virus, inducing new persistently infected calves. Therefore, all cattle younger than 2 years had to be tested antigen negative before going to summer pasturing. In some regions this was extended to older cattle as well. The number of animals to be tested was estimated at 350'000 based on data of previous years.

2.Initial phase (October to December 2008)

All not tested cattle will be tested. An exception is made for cattle from pure fattening farms, where all cattle on the farm go directly to slaughter. The animal movements in the After the initial phase all cattle alive should have been tested and all positive animals culled. However, there might still be newborn calves that are persistently infected. Therefore, all newborn calves have to be sampled and tested. The farmer, while marking the calves, will take ear tissues and send them for analysis. In case of positive testing, the calf is slaughtered and the movement of every pregnant female on the farm is blocked till calves were born.

4.Monitoring phase (from October 2009 to 2011)

In October 2009 the major part of the Swiss cattle population will be free from BVD infected animals. In this period it is essential to monitor and to verify the entire population of Swiss cattle.

It is planned to keep on testing the new born calves for a period of at least a year and then target the monitoring on antibodies in milk from first lactating cows (starting in 2011). Modeling will be used to compare the different monitoring strategies and to better allocate resources to ensure a cost-efficient surveillance program. without confirmatory test or a blood sample is taken and tested using rtRT-PCR. If this result is negative, the sample is considered as negative; if positive, the sample is considered as positive; if not interpretable, the sample is sent to the reference laboratory for retesting. The reference laboratory partly confirms positive results as well as clarifies problems. The specificity and sensitivity of the tests are between 98% and 99.8% respectively 97.1% and 100% based on literature [START_REF] Wolf | BVDV: Diagnostische Erfahrungen aus der bayerischen Modellstudie[END_REF][START_REF] Wolf | BVDV: Diagnostische Erfahrungen aus der bayerischen Modellstudie[END_REF][START_REF] Fux | Ohrgewebediagnostik von BVDV-PI-Kälbern: Ist eine Sensitivität von 100% möglich? 27[END_REF][START_REF] Hilbe | Comparison of five diagnostic methods for detecting bovine viral diarrhea virus infection in calves[END_REF].

[Insert Table I about here]

Type of analyses

Data flow

The success of a national program of such a magnitude highly depends on the coordination and communication between every party involved. The Federal Veterinary Office is coordinating this program and has developed a database (ISVET) to connect the different partners (Figure 1). At the regional level, cantonal veterinarians are organizing the sampling process and are controlling the implementation of the measures. The BVD-Web is a website showing information from ISVET that lists all individual animals per farm, including their eartag-number, birthdate and test-result if existing. BVD-Web is accessible for the sample takers; lists of animals can be printed through the central laboratory database to the ISVET. During the phase before pasturing all negative results for individual animals were announced on the movement animal database, which is accessible for all farmers. In the following phase the movement restrictions for the individual farms and animals will be displayed on this database.

[Insert Figure 1 about here] The analyses were performed using the statistical computing environment R (R Development Core Team,2008).

Results

Before the initial phase, a total of 595'230 animals (37% of the livestock) from 33'617 farms (77% of the farms) have been tested for BVDV (as of 25 th September 2008). 81% of these animals were under three years old. The proportion of infected animals distributed in age categories compared to the proportion of animals sampled and the age distribution in livestock is shown in Figure 2.

[Insert Figure 2 about here] Of the 6988 positive tested in the initial test, 3883 underwent a confirmation test of which 3427 were confirmed positive, corresponding to a confirmatory rate of 88%. The total of 6532 PI animals gave a prevalence of 1.1% [95% confidence interval: 1.07, 1.14]. Under the assumption that the 3092 animals that were tested once would undergo a confirmation test and also 12% would be negative, results in a corrected PI-share of 1.03% [95% confidence interval: 1.00; 1:07]. Regional fluctuation in the proportion of PIanimals ranged between zero and 1.54%.

The average age of infected animals is 403 days compared to 794 days for non-infected animals (Figure 3). The oldest PI animal was over 11 years old.

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

On average PI animals had been slaughtered within 18 days after the last laboratory result. The maximal number of days elapsed before slaughtering was 242 days (Figure 4).

[Insert Figure 3 and 4 about here]

Discussion

The high number of animals tested for BVD in a few months was demanding for all partners. The challenge for the sample takers was to handle a new electronic tool (BVD-Web) and to take ear-notch-samples with a new technique in a limited time. After a short adaption phase all sample takers appreciated the use of the BVD-Web, which let them have an overview of the program as well as simplifying the labeling process. The sampling resulted in a high number of empty samples arriving to the laboratories, which decreased from 4% at the beginning of the phase up to 1.2%. Laboratories also gained expertise and efficacy in analyzing large amounts of samples in a short period.

The pre-pasture phase allowed the detection and elimination of 6532 PI animals.

Although the impact on the reduction of inducing new PI-animals cannot be assessed before having analyzed the results of the newborn calves in the following nine months, past studies clearly indicated the impact of BVD-negative transhumant on the reduction of new infections [START_REF] Bodmer | BVD-free transhumance in the summer of 2006[END_REF][START_REF] Siegwart | Increased risk of BVDV infection of calves from pregnant dams on communal Alpine pastures in Switzerland[END_REF]. Pilot studies conducted in Switzerland [START_REF] Rüfenacht | Prevalence of cattle infected with bovine viral diarrhoea virus in Switzerland[END_REF] combined with coming results of the calve phase will allow quantifying the impact of the reduction of new PI-animals.

Targeting the first phase of the eradication program on young animals and animal populations at risk in terms of intensity of contact of animals from different herds, the probability of detecting PI animals was higher than in the average cattle population. In consequence, the overall prevalence of PI animals should decrease once all animals in the cattle population have been tested. Although on average PI's are younger than non-PI animals, about 20% of PI's are over two years old, with a maximum of 11 years old. This is consistent with results obtained from a monitoring program conducted in Germany [START_REF] Gaede | Drei Jahre staatliche BVD-Tilgungspflicht in Sachsen-Anhalt: Rahmenbedingungen, Zwischenbilanz und Optionen für die Fortführung[END_REF]. Targeting a control program only on young animals can result in missing PI's. Those animals will then have a strong effect on maintaining the infection in the population. This effect will grow, as the prevalence of antibodies against BVDV decreases.

The time elapsed between PI's detection and slaughter can slow down the progress of the eradication program [START_REF] Lindberg | Characteristics in the epidemiology of bovine viral diarrhea virus (BVDV) of relevance to control[END_REF]. By restricting animal movements we minimize this effect. Animals without negative test results, are not allowed to go to summer pasture. In addition, in the initial phase, all herds are blocked from the beginning of sample-taking until all results are negative and the PI-animals are slaughtered respectively. Moreover, movements are not allowed from a not-tested herd to a tested one. In herds with PI-animals, all pregnant animals are blocked until the calf born is tested negative.

Considering logistical problems occurring around the sampling process and results of analysis combined with the sensitivity of the tests and the detection of acutely infected animals, the confirmation rate of PI animals is still 88%. This is indicating a high specificity of the tests. The 12% false positives caused the slaughter of about 400 animals although they were negative. However, animals slaughtered after the first test had often some clinical signs, were weak or had not a high value for the farmer.

Therefore, the number of misclassified animals is probably reduced. The level of false negatives cannot be assessed, because only a few cases based on clinical signs or other suspicions (negative offspring of a PI-cow) were retested.

The database provides a better coordination, overview, control and automation at all levels. Veterinarians for instance can control whether all animals on a farm are sampled The automatic availability of the BVD-status of all farms and cattle on the animal movement database supports the farmers to make correct decisions, which is a central point in biosecurity [START_REF] Lindberg | The control of bovine viral diarrhoea virus in Europe: today and in the future[END_REF].

Conclusion

The BVD-eradication program was initiated by the farmers' associations. These stakeholders were also involved in the development of the eradication-program. Before and during the program a lot of emphasis was put on the information and education of farmers by sending leaflets, videos, creating a website (www.stopbvd.ch) and presentations for farmers. Although time consuming and cost-intensive, the awareness of the farmers is one of the key factors for the success of this program.

The results and observations of the pre-pasture phase have shown that sampling and testing a high number of animals in a short time is challenging but feasible. The next phase will deal with the double amount of tests in a similar time frame. In that context, the collaboration of farmers is very important, not only for ensuring the success of the program but also for avoiding costs increase. Another crucial point is the blocking of farms and animals, which will reduce the risk of BVD-free herds from being reinfected.

Currently it is planned to begin in 2011 with antibody screening in milk of first calving cattle. Alternatives in consideration are: 1) to conduct antigen screening in blood of calves over 6 months old, 2) to combine the two surveillance approaches, 3) to define risk population on which to target the sampling. An epidemiological model will help to assess the best surveillance strategy to be undertaken at the end of the eradication programme.

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

The Swiss BVD-eradication program will cost approximately 40 millions €; a third is paid by the farmers. Although the costs are initially high, with the short and intensive approach economically drastic re-infections can be probably reduced, having for the next years still a cattle population with a high sero-prevalence. Therefore, we assume that the long term costs will not differ substantially from other BVD-eradications programs. The costs will be quantified in the years following the active eradication.

  that in European countries the prevalence of bovine viral diarrhea (BVD) antibodies at the herd level before starting any eradication or control program ranged from 95% in England and Wales down to less than 1% in Finland (Greiser-Wilke

  A c c e p t e d M a n u s c r i p t herds are restricted from the time of sample taking until the results are available (15 days on average). Farms with all animals tested negative have no further restrictions. In farms with positive tested animals, all pregnant females have movement restrictions until calving. Calves are tested immediately after they were born and are blocked until the negative result is released.3.Secondary or calve phase(October 2008 to October 2009)

For

  this eradication program, official sample takers use special ear-tags, to collect tissue sample while simultaneously identifying the animals. The sample is sent to one of the nine certified laboratories involved in the program the same day. In the laboratories A c c e p t e d M a n u s c r i p t either real time reverse transcriptase PCR (rtRT-PCR) or Enzyme-Linked-Immunosorbent-Assay (ELISA) is used to detect virus-positive animals. The initial analyses are performed using one of the four tests: 1) HerdCheck Ag/Serum Plus from IDEXX Laboratories based on the identification of the E rns -antigen; 2) PrioCheck BVDV Ag PI focus ELISA from Prionics AG, based on the identification of the p80-or other expressions of the NS2/3-antigen (only for animals older than 60 days) ; 3) cador BVDV RT-PCR Kit from Qiagen GmbH; 4) BoVir-SL® BVDV TaqMan RT-PCR from AnDiaTec GmbH & Co. KG. All initially positive as well as not interpretable results can be verified through a confirmation test. It can be decided by the farmer to cull the animal

  overview of the situation. The laboratories communicate the test-results

  s c r i p t or not. The laboratories transmit the test-results to the central laboratory database and thus avoid sending papers with test-results to farmers and veterinarians. Herds and individual animals have automatically movement restrictions based on the test results.

  Figure 1Structure of the data flow in the frame of the BVD-eradication program.

Acknowledgements

We thank all the parties involved in this complex program, the farmers, the sample takers, the veterinarians, the laboratories and the cantonal veterinary offices.

Conflict of Interest

Monitoring phase (October 2009) Approach analogous to the calve phase during 2 years, then the status might be monitored via antibody detection in milk from first lactating cows (from 2011). Different strategies are to be tested through modeling.

A c c e p t e d M a n u s c r i p t Time elapsed between last result and slaughter of PI animals.


W Rossmanith
email: wigbert.rossmanith@noel.gv.at

M Deinhofer

R Janacek

R Trampler

E Wilhelm

Voluntary and compulsory eradication of bovine viral diarrhoea virus in Lower Austria

Keywords:

A voluntary BVDV eradication program without vaccination was introduced in Austria in 1997, according to the Swedish model employing herd-level antibody tests. Since this time, identified persistently infected (PI) animals have to be slaughtered. In order to protect noninfected herds, the major routes of introduction of BVDV infection into a herd, such as communal grazing and livestock trade, have to be controlled. In 1998 PI animals were identified in 7.5% of affiliated herds of herd book breeders. During the voluntary program, the majority of the 2455 herd book breeders who actively participated in the BVDV program had cleared up their herds. Hence, in 2005 only nine infected herds A c c e p t e d M a n u s c r i p t 2 (0.36%) remained under clearance. These data show that BVDV control can be achieved at the farm level without a nation-wide BVDV eradication program.

A federal law was passed in 2004, obligating all herd owners to follow the BVDV eradication program.

Between 2005 and 2007, the number of herds with a certified BVDV-free status increased from 7931 to 9952 (2006) and 11,166 (2007), respectively. Currently (2008), 11,017 of 12,031 existing herds have been certified as BVDV-free herds (91.57%). Nearly all certified BVDV-free dairy herds can now be monitored by testing milk samples because the cows have no antibodies to BVDV. This signifies a marked improvement since 1998, when 46% of the 5024 tested dairy herds had a high level of BVDV antibodies in bulk tank milk.

Total eradication of BVDV-infected herds will need a further one or two years.

Introduction

Infection with the bovine viral diarrhoea virus (BDVD) was first reported in 1946 (Olafson et al., 1946). In Austria, BVDV was first described to have occurred in a mountainous region in the year 1972 [START_REF] Bürki | Studies on European malignant catarrh and bovine virus diarrhea in the mountain regions of Austria. I. Search for the virus causing malignant catarrh[END_REF].

Economic losses result from prenatal infections [START_REF] Houe | Attempts at preventing further spread of bovine diarrhoea virus (BVDV) infection in 5 Danish dairy herds in which BVDV had been isolated[END_REF], which cause abortions, infertility, malformations in calves, and the birth of persistently infected immunotolerant calves (PI) that often die of mucosal disease (MD) (Brownlie et al., 1984, Roeder andDrew, 1984). Diarrhoea and symptoms of respiratory disease are observed more frequently in infected herds [START_REF] Baker | The clinical manifestations of Bovine Viral Diarrhea infection[END_REF].

Vaccines against BVDV infection have rarely been used for prevention purposes in Austria.

Their sporadic use is limited to herds in which significant damage has already occurred. A A c c e p t e d M a n u s c r i p t 3 successful model of a vaccination strategy that entirely eliminates the pathogen has not been reported thus far. In other words, the vaccination must be continued for an indefinite period of time. Comprehensive country-wide implementation of this measure is not conceivable for economic reasons. Moreover, there have been reports of live vaccines contaminated with BVDV [START_REF] Kreeft | Attempts to characterise bovine viral diarrhea virus isolated from cattle after immunisation with a contaminated vaccine[END_REF] as well as severe outbreaks of type 2 BVDV infections [START_REF] Lindberg | Principles for eradication of bovine viral diarrhoea virus (BVDV) infections in cattle population. In: Proceedings of the European Symposium on Control of Bovine Viral Diarrhoea Virus in Cattle[END_REF].

Knowledge about how BVDV behaves in the host, the pregnant host and its foetus has been utilised to establish a systematic eradication strategy [START_REF] Alenius | A national approach to the control of bovine viral diarrhoea virus[END_REF].

This approach serves as an example to combat other viral infections and confirms the importance and utility of knowledge of epidemiological associations. Eradication strategies for BVDV without vaccination were started in Sweden andNorway in 1993, andin Finland andDenmark in 1994. These strategies have already led to eradication of the pathogens (Houe et al., 2006). The central aspect of a BVDV eradication strategy is to identify born and unborn persistently infected animals and to eliminate these from the herds (Radosits and Littlejohn, 1988).

Persistently infected animals and dams pregnant with persistently infected calves must be excluded from contact between herds. Protection and control of non-infected herds is as important for eradication programs as is disease clearance in infected herds [START_REF] Alenius | A national approach to the control of bovine viral diarrhoea virus[END_REF].

In order to classify herds as being non-infected, they must be re-examined at least once within a period of twelve months. This minimum period of one year is derived from the successful strategy of the pathogen: it withdraws from the infected dams mature and intact immune system, infects the foetus, and remains concealed here for six to eight months.

Simultaneously, an immature immune system is damaged and creates immunotolerance, which causes the pathogen to spread after birth (Peterhans et al., 2004). Without persistently infected animals in the herd, no further infections or seroconversions can occur because A c c e p t e d M a n u s c r i p t 4 animals transiently infected with BVDV are likely not able to infect neighbouring animals under routine conditions (Niskanen et al., 2002). This article will highlight the advantages of the Scandinavian BVDV eradication model and the advancements made during the initially voluntary, and later compulsory, phase of the eradication program in Lower Austria.

Material and Methods

Herds involved in the study

At the start of the voluntary BVDV eradication program (1997) 5,024 herds participated in the project. Nearly all of them were from herd book breeders who performed at least one or several herd-level tests for determination of the animals' BVDV status. From the year 1999 onward, the organizers of breeding cattle auctions decided to prohibit pregnant dams with BVDV antibodies from entering the livestock trade market. From this time on, the 2,455 farms that regularly participated in the market made efforts to keep their herds free of BVDV infection or initiated virus clearance.

From the year 2005 onward, nearly all herds in Lower Austria (13,382) with animals for breeding have been included in the compulsory BVDV eradication program while feeder herds are excluded from the program.

Herd-level test for determination of BVDV status and follow-up monitoring

All aspects of the Swedish eradication strategy [START_REF] Alenius | A national approach to the control of bovine viral diarrhoea virus[END_REF] implemented during the voluntary eradication program in Lower Austria (1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004) have been incorporated into the Austrian BVD regulation. The initial herd investigation is complete and the herds are A c c e p t e d M a n u s c r i p t 5 considered to be free of BVDV when tests for specific antibodies to BVDV in bulk milk or milk samples from young cows or spot tests of the replacement stock at three investigations performed over a period of at least twelve months yield appropriate results. After this time, yearly follow-up investigations are required.

Initial herd investigation and follow-up for virus clearance

If the spot test of the replacement stock reveals a current BVDV infection or if a PI animal is found in the herd, an initial herd investigation must be performed. The initial herd investigation must include all animals in the herd. The follow-up investigation should be performed after the initial examination and include testing of all animals in the herd that were born within one year after the initial herd investigation or after eradication of the last PI animal. The follow-up investigation must be performed at the earliest one year after the initial herd investigation or one year after elimination of the last PI animal and must be concluded by investigating a group of young animals aged 6 to 12 months. All blood samples of these animals must be free of specific antibodies to BVDV.

Rules and regulations for owners of herds prior to transaction of cattle

Prior to transaction of cattle, the owners are obliged to conduct appropriate control investigations of their herds (bulk milk or a group of young cows or replacement stock).

Animals from suspicious herds must be subjected to individual testing. From the beginning of the voluntary program until August 2004, BVDV-specific antigen testing was done on leukocyte preparations (HerdCheck BVDV antigen/leucocytes, IDEXX Scandinavia, Österbybruck, Sweden). Modified leukocyte preparations were used from 1999 onward [START_REF] Rossmanith | Improved antigen and nucleic acid detection in a bovine virus diarrhoea eradication program[END_REF] while the BVDV antigen/serum ELISA is in use since 2004 (HerdCheck BVDV antigen/serum, IDEXX). The results (positive and negative) of the Ag-ELISA are checked by RT-PCR (Vilcek et al., 1994). Samples testing positive on the Ag/serum ELISA are re-tested with the Ag/leukocyte ELISA to confirm the result of the Ag/serum ELISA.

Laboratory methods

Administration of relevant data for the diagnosis, eradication and monitoring of BVD is done by the use of a database that permits web-based as well as laboratory application.

The animals and their samples are registered and administered, and the obtained test readings are assigned to the samples, by the use of the laboratory application.

The web-based application shows the animals of a herd, their data, and their examination results. Authorised users can perform herd classifications.

Results

Detection of specific antibodies to BVDV

Since the beginning of the voluntary eradication program and during the compulsory BVDV eradication program, more than 400,000 blood and milk samples have been examined by the indirect ELISA and no false negative results have been obtained. Based on the last 20 herds in which PI animals were found in the year 2008, Table 1 shows that seroconversions were diagnosed in 15 of 20 herds before the occurrence and identification of PI animals. Thus, it was possible to classify a herd as being possibly infected before the birth of a PI animal.

Table 1:

Detection of BVDV antigen

The commercially available antigen test used during the first two years of the voluntary eradication program in Lower Austria were, in part, not sensitive enough to detect PI animals.

By modifying the leukocyte preparation and testing the results of the Ag-ELISA by RT-PCR from the year 1999 onward, the sensitivity of the antigen test could be significantly improved.

The number of samples that showed a negative or ambiguous result on Ag-ELISA and a positive result on RT-PCR could be reduced from 4.71% to 0.82% [START_REF] Rossmanith | Improved antigen and nucleic acid detection in a bovine virus diarrhoea eradication program[END_REF].

After the manufacturer had introduced a new configuration of the Ag-ELISA in the year 2000, nearly identical results were achieved with the new Ag-ELISA and RT-PCR.

From 2004 onward, the HerdCheck BVDV Ag/serum ELISA is used. This ELISA is simple in terms of handling and permits a high throughput of samples. In the year 2005, for instance, 59,626 samples were tested for the BVDV antigen and 511 PI animals were identified. In order to avoid potentially false positive results of the Ag/serum ELISA or positive results from samples of transiently infected animals, samples whose positive outcome on the Ag/serum ELISA could not be confirmed in the other two test systems were re-tested at the earliest after three weeks. For 52 samples the Ag/serum ELISA was positive, but the Ag/leukocyte ELISA and RT-PCR were negative. Of these, six samples were repeated and seroconversion was detected for all of them. The initial investigation yielded a positive result Worthy of note is the high percentage (54%) of proven seroconversions in samples that were positive on the Ag/serum ELISA and negative on the Ag/leukocyte ELISA at the initial investigation.

Results of the voluntary eradication program

At the start of the voluntary eradication program in 1997, bulk milk samples were tested for specific antibodies to BVDV in 5,024 dairy farms of Lower Austria, nearly all of which were herd book breeders.

Either no BVDV antibodies or only small quantities of BVDV antibodies were found in 54% of the investigated bulk milk samples. In sections of the province north of the Danube, with no common grassland and very little private grassland, clearly unsuspicious results were obtained in 59%. In areas with common grassland and private grassland, a markedly lesser proportion of bulk milk samples, namely 49%, were unsuspicious.

Blood testing of replacement stock was performed in 759 herds with suspicious bulk milk samples. The replacement stock had no BVDV antibodies in 76.8% of the herds whereas they did have antibodies to BVDV in 23.2% of the herds. Only some (58%) of the 4,220 herd book breeders were actively involved in the voluntary program. Of the 4,220 herd book breeders, about 1.9% still had PI animals in the year 2005.

Results of investigations and eradication of BVDV infection on common grassland

In 1999, 4,630 animals from 732 herds with an unknown BVD status were investigated before transhumance. At this investigation, 24.4% of the animals had specific antibodies to BVDV and 33 PI animals were identified. The investigation program for animals prior to transhumance to common grassland was continued during the next few years. At the end of the respective grazing season, 10% of the animals on the respective common grassland were tested for specific antibodies to BVDV. The last increase in antibodies was observed at the end of the grazing season in the year 1999; it originated from a PI animal that had been moved to the common grassland without having undergone the examination. In the year 2002 the seroprevalence in animals examined prior to transhumance was only 12.96%.

Table 3:

In administrative districts of grassland areas, in which about 960 farms have some of their animals grazing on common grassland and about 50% of all farms (8,084) have their animals Currently, of 11,017 BVD-free herds 6,709 can be monitored by examinations of bulk milk because bulk milk samples contain no or only small quantities of BVDV antibodies. A further 1,453 herds are monitored by milk samples of young cows whose samples are free of BVDV antibodies. The remaining 2,855 herds must be monitored by blood samples because a large number of these comprise no milkable cows or the herds contain older dairy cows with antibodies to BVDV, which renders monitoring by bulk milk impossible. The indirect ELISA to detect specific antibodies to BVDV is a reliable procedure whose results provide a large quantity of essential information. False negative results were only registered at the initial investigations of sour milk samples in 1996. The samples had been inadequately cooled and brought for investigation in a non-preserved state. The fact that the milk had turned sour was not realised before and during the investigation. Even a small shift in pH due to milk turning sour (and not identifiable by the naked eye) disturbs the antigenantibody reaction of the indirect ELISA and leads to false negative results.

Shortly afterwards, the use of ProClin TM for preservation of milk samples became a mandatory measure prescribed by law.

Re-testing of bulk milk or groups of young animal permits classification of herds into noninfected and infected ones [START_REF] Niskanen | Relationship between the levels of antibodies to bovine viral diarrhoea virus in bulk tank milk and the prevalence of cows exposed to the virus[END_REF][START_REF] Alenius | A national approach to the control of bovine viral diarrhoea virus[END_REF]. The prevalence of specific antibodies to BVDV in bulk milk samples and particularly in the samples of young animals is highly over-estimated and employed as a justification for not using this economical method to monitor herds. From Bavaria it has been reported that, in 42.6% of 777 non-vaccinated herds, no or only small quantities of BVDV-specific antibodies were found [START_REF] Brendel | Prevalence and epidemiology of bovine virus diarrhea virus (BVDV) in Bavarian cattle herds[END_REF]. In the Bavarian study, less than one half of herds with antibody-positive bulk milk samples had BVDV antibodies in samples of young animals as well. Therefore, 70-80% of the herds had to be monitored by the serological method. This coincides with our experience from Lower Austria. In a study from Switzerland as well, only 22.9% of samples from animals aged 7 to 12 months were found to be antibody-positive [START_REF] Rüfenacht | Prevalence of cattle infected with bovine viral diarrhoea virus in Switzerland[END_REF]. Examination of bulk milk is the most sensitive method to detect new infection of herds in dairy farms [START_REF] Lindberg | Principles for eradication of bovine viral diarrhoea virus (BVDV) infections in cattle population. In: Proceedings of the European Symposium on Control of Bovine Viral Diarrhoea Virus in Cattle[END_REF]. This method registers all lactating cows; the latter constitute 50% of herds with young animals in Lower Austria. Once a lactating cow has been infected with BVDV, the A c c e p t e d M a n u s c r i p t 12 infection can be identified after 14 days. BVDV antigen testing of a PI calf born afterwards can be successfully performed at the earliest five months after infection of the dam.

Even in the last 20 herds in Lower Austria, which had PI animals in the year 2008, seroconversion was diagnosed in 15 of 20 herds before the birth and identification of the first PI animal.

Small quantities of BVDV antibodies found on indirect ELISA in bulk milk tests, although the herd contained only one PI animal, was reported just once (Obritzhauser et al., 2002).

Inclusion of the strongly viraemic milk of the PI cow was considered to have facilitated the formation of antibody-antigen complexes, which negatively influenced the analysis of antibodies. This situation was never encountered during the eradication program in Lower Austria.

Detection of specific antibodies to BVDV also provides essential information for virus clearance. Animals that developed antibodies after an infection are considered immunocompentent and are themselves not persistently infected under routine conditions. BVDV antigen testing need not be performed in these animals. If antibody-negative, sexually mature heifers are found at initial herd investigations, one may expect virus clearance to take longer. If the entire herd is infected and antibody-positive, the continuance of BVDV in the herd is limited [START_REF] Lindberg | Principles for eradication of bovine viral diarrhoea virus (BVDV) infections in cattle population. In: Proceedings of the European Symposium on Control of Bovine Viral Diarrhoea Virus in Cattle[END_REF].

As actively acquired antibodies remain detectable for an entire lifetime and no seroconversion occurs without the presence of a PI animal in the herd [START_REF] Houe | Serological analysis of a small herd sample to predict presence or absence of animals persistently infected with bovine viral diarrhoe virus (BVDV) in dairy herds[END_REF], the age of the youngest animal that is still antibody-positive is an indicator of how long ago the PI animal was present in the herd.

The examination of a group of young animals with an antibody-negative outcome is therefore a prerequisite for completing virus clearance in the herd or, at worst, indicates that virus clearance must be continued for a longer period of time. In 1997 and 1998, before the Ag-ELISA was rendered more sensitive by modification of the leukocyte preparation and before Experience has also shown that not every calf that dies early or is stillborn is submitted for investigation. Infections may spread even from these animals -which remain in the herd for a very short period of time -provided there are enough susceptible animals in the herd.

However, the results of the indirect ELISA also permit valuable interpretations with regard to the individual animal; these interpretations could be significant for an eradication program.

Animals that have developed active antibodies are immunocompetent; they are not PI animals. Degradation of maternal antibodies in PI calves is completed at the age of 10 weeks [START_REF] Houe | Attempts at preventing further spread of bovine diarrhoea virus (BVDV) infection in 5 Danish dairy herds in which BVDV had been isolated[END_REF]. Only in exceptional cases do PI animals have low titres of maternal antibodies until the fourth month of their lives. In cases of non-persistently infected calves, maternal antibodies can be detected by indirect ELISA until the sixth to eight month of their lives.

Antibody-negative pregnant dams tested after the 150 th day of pregnancy do not produce PI calves if they are not persistently infected themselves. Animals that have actively formed antibodies through infection with BVDV prior to sexual maturity are highly protected from new infection of their foetus.

Due to the poor sensitivity of the commercially available BVDV antigen ELISA which was used in 1997 and 1998, at herd investigations one frequently found seroconversions caused by undetected PI animals. This has been reported by other authors as well [START_REF] Shannon | 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].

These undetected PI animals might be the reason for the more prolonged circulation of BVDV reported in herds without the presence of PI animals [START_REF] Barber | Disease in a dairy herd associated with the introduction and spread of bovine virus diarrhoea virus[END_REF][START_REF] Moermann | A long term epidemiological study of bovine viral diarrhoea infections in a large herd of dairy cattle[END_REF][START_REF] Moen | Indication of transmission of BVDV in the absence of persistently infected (PI) animals[END_REF].

Alternatively, calves that were aborted or died early, or strongly viraemic lochia, might have infected susceptible animals. Modification of the leukocyte preparation of the Ag-ELISA and re-testing its results with RT-PCR were able to improve the sensitivity of antigen detection A c c e p t e d M a n u s c r i p t 14 [START_REF] Rossmanith | Improved antigen and nucleic acid detection in a bovine virus diarrhoea eradication program[END_REF]. The new configuration of the Ag/leukocyte ELISA introduced in the market at the beginning of 2000 led to nearly identical results being obtained with Ag-ELISA and RT-PCR.

Based on Ag/serum ELISA testing it may be concluded that the sensitivity of this antigen detection procedure renders it very useful for an eradication program.

The seroconversions demonstrated by repeated investigation make it clear that the Ag/serum ELISA also detects transient BVDV infections. When this ELISA is used without confirmation by Ag/leukocyte ELISA or without a repeat investigation, it is not possible to distinguish between persistently and transiently viraemic animals.

Therefore, it is essential to test for specific antibodies to BVDV at the initial investigation, followed by the repeat investigation. A proven seroconversion excludes the fact that the sample originates from a PI animal.

Progress of the eradication program in Lower Austria

Nearly all participants of the voluntary eradication program from 1997 to 2004 were herd book breeders, who keep records of their operational results and fertility parameters. One study from Lower Austria reports on herds with PI animals that had significantly poorer inseminations per pregnancy, non-return rates, and service periods, compared to control groups [START_REF] Wetchy | Influence of bovine viral diarrhoea virus infections upon reproductive performance of cattle herds[END_REF].

The voluntary nature of the program was changed from 1998 onward because the compulsory examination for common grassland was introduced at this time. In 1999 the organisers of breeding cattle auctions imposed a compulsory examination for livestock being traded at the auctions. Pregnant dams from infected herds were prohibited from the market. Antibodypositive pregnant dams had to be from unsuspicious herds.

A c c e p t e d M a n u s c r i p t 15 At the beginning of the inspection regulation for animals on common grassland, the seroprevalence on common grassland was 24.4% (1999). In the year 2002 only 12.96% seropositive animals were found on transhumance.

Reliable detection of PI animals before transhumance can prevent the spread of BVDV on grazing land even if some of the herded animals originate from infected farms and the presence of a transiently infected animal on transhumance cannot be excluded [START_REF] Deinhofer | Successful control of BVD-infection on common grassland[END_REF].

Circulation of BVDV without the presence of a persistently infected animal was never observed on common grassland in Lower Austria. Ten per cent of the animals that had been antibody-negative at transhumance in the spring were re-tested for antibodies when they returned from transhumance in the autumn.

In Switzerland the animals were tested for BVDV-specific nucleic acids by real-time PCR one week before transhumance, and for BVDV antibodies at the time of transhumance.

Although no PI animal was found, four seroconversions were detected in the autumn (Bodmer et al., 2008).

Abortions were offered as one explanation for the seroconversions. Transient infections that were possibly not detected by RT-PCR, and seroconversion that had possibly occurred later than one week, may be an alternative explanation. Inquiring whether there had been a PI animal in the farm of origin, which had caused the transient infection, might help to clarify this situation.

An increased risk of infection on common grassland and private grassland no longer exists in Lower Austria.

The percentage of herds without BVDV in 2008 was more or less equal in districts with common grassland and private grassland (91.52%) and in districts without these types of grassland (91.66%). The 2,455 farms that regularly brought their stock to breeding cattle auctions tried to achieve disease clearance in their herds or keep them free of BVDV infection. Among the 2,455 farms that regularly traded their cattle at the auctions, only nine farms had a PI animal in 2005 (0.36%). Nevertheless, the mean number of farms with PI animals among 4,220 herd book breeders was still 1.9% in 2005. Thus, the mean number of farms with PI animals of herd book breeders (1,765) who did not actively participate in the voluntary program was about 4%.

The small number of infected herds in these 2,455 farms is a strong indicator of the fact that the BVDV eradication program can be successfully implemented in some herds even if the remaining farms do not participate in the program. Our experience from Lower Austria showed that maintaining the status of a BVDV-free herd is not as labour-intensive or expensive as farmers in other countries believe it is [START_REF] Gunn | Assessing economic and social pressure for the control of bovine viral diarrhoea virus[END_REF].

The proportion of non-herd-book breeders (9,162) in which a PI animal was found in the year 2005 was 1.83%. The somewhat higher percentage of herds with PI animals among herd book breeders can be explained by the more intensive exchange of animals for breeding purposes.

During the next three years, the BVDV status of all herds was markedly improved. If the eradication program can be continued, a similar BVDV status as that in Sweden will be within easy reach [START_REF] Hult | Experiences from BVDV control in Sweden[END_REF]. No PI animal has been found in Finland since the year 2003 (Rikula et al., 2005).

A large percentage of herds with milkable cows can be monitored by serology testing of bulk milk samples or milk samples from groups of young cows. The cost of the BVDV program will thus be reduced because blood tests will not be needed. Additionally, the status of the herds can be monitored with just one sample. A cost-benefit analysis performed in Norway applying bulk milk sampling approach, yielded very good results [START_REF] Valle | Ten years of bovine virus diarrhoea virus (BVDV) control in Norway: a cost-benefit analysis[END_REF].

In conclusion, the voluntary program demonstrated that BVDV can be controlled at the farm

  of the program, specific antibodies to BVDV in blood and milk samples are detected by the use of an indirect ELISA (Svanovir TM , Svanova, Biotech, Uppsala, Sweden). Potassium-EDTA tubes are used for blood samples and polypropylene tubes for milk samples; the tubes contain ProClin TM (Supelco, Bellaforte, USA) as preservative.

  35 samples only on the Ag/Serum ELISA and RT-PCR. At re-testing, seroconversion was proved for 19 samples. A further seven repeated samples were positive on the Ag/serum ELISA and simultaneously on RT-PCR, but remained negative on the Ag/leukocyte ELISA. For the remaining 9 of 35 repeated samples, the Ag/serum ELISA, the Ag/leukocyte ELISA and RT-PCR were positive. At the initial investigation, three samples tested positive on RT-PCR but remained negative on the two ELISA test systems. The repeat investigation showed seroconversion for all three samples.In the year 2005, only one sample was positive on the Ag/leukocyte ELISA and RT-PCR but negative on the Ag/serum ELISA. Regrettably, this sample was not sent for re-testing.

  the initial herd investigations in 1998 and 1999, PI animals were found in about 7.5% of the 2,455 actively involved herds. By 2002 this percentage had reduced to 3.4%. In 2005 only nine (0.36%) of these 2,455 breeders who regularly traded their livestock at the market had PI animals.

  grassland, the percentage of certified herds (7,399) is currently(2008) 91.52%, which is about the same as that in the remaining administrative districts with no transhumance to common grassland and very little private grassland. Of 3,947 farms, 3,618 (91.66%) have achieved a certified BVDV-free status. Table 4: 3.5. Results of the compulsory BVDV eradication program Progress in BVDV eradication resulted in a decline of PI animals between 2005 and 2007. 511 PI animals in 248 herds were found in 2005; 269 PI animals in 124 herds were found in 2006 and, finally, 115 PI animals in 46 herds were found in 2007. This trend continued in the year 2008. On 31 st August 2008, only 30 PI animals were found in 20 herds. During the same time period, the number of herds with a certified BVDV-free status increased from 7,931 (2005) to 9,952 (2006) and finally to 11,166 (2007).

  re-tested by RT-PCR, detection of unexpected seroconversions at the final examination of the group of young animals was a saving indicator of the fact that the Ag-ELISA had yielded a false negative result and one PI animal had remained undetected.

  limitations on transaction of cattle from infected herds, designated for breeding cattle auctions, have exerted a positive effect on the progress of the voluntary eradication program.

  In the year 2005, 511 PI animals were still found in 248 herds. In 2006, only 269 PI animals were identified in 124 herds. In 2007, 115 PI animals were detected in 46 farms and in 2008until 31 st August 2008 -only 30 PI animals have been found in 20 herds. Currently (2008), 11,017 of 12,031 existing herds subject to the BVDV regulation have been certified as BVDV-free herds (91.57%). The decline in the absolute number of certified BVDV-free herds can be explained by the declining number of cattle herds. Nevertheless, the percentage of certified herds in 2008 has increased.

  level without a nation-wide BVDV eradication program. The promising results of the voluntary eradication program were also seen during the compulsory program. Using low-cost serological screening methods on milk samples keeps costs low for the farmers participating in the program. It would be realistic to estimate that complete extermination of BVDV in Lower Austria will be achieved by 2009 or 2010.

Table 5 : 4. Discussion

 5

Table 1 :

 1 Comparison of the respective time point of established seroconversion and the birth of the first PI animal in 20 herds in which PI animals were found in 2008. * No seroconversion was identified before the birth of first PI animal.

	22

Table 2 :

 2 BVDV antibodies in bulk milk samples (OD ≥ 0.24): herds with antibody-positive spot tests of replacement stock (8 to 10 blood samples per herd) in 1997.

	Bulk milk samples	% positive	Herds in which spot	% positive
		(OD≥0.24)	tests of replacement
			stock were
			performed
	5,024	45.7	759	23.2

Table 3 :

 3 Transhumance examinations in 1999 and 2002.

	Year	Number of herds	Number of animals	% antibody positive
	1999	732	4630	24.40
	2002	745	3163	12.96

Table 4 :

 4 Comparison of percentages of certified herds in grassland areas and the remaining

	areas
	2008	Total number of	Certified herds	Percentage of
		herds		certified herds
	Grassland areas	8,084	7,399	91.52
	Remainder	3,947	3,618	91.66

Acknowledgements

The authors would like to thank Professor Stefan Alenius, DVM, PhD, at Department of Ruminant Medicine and Veterinary Epidemiology, Faculty of Veterinary Medicine, Uppsala, for his valuable support in introducing the BVDV eradication program in Lower Austria.

A c c e p t e d M a n u s c r i p t 20 Niskanen, R., Lindberg, A., Traven, M., 2002. Failure to spread bovine virus diarrhoea virus infection from primarily infected calves despite concurrent infection with bovine coronavirus. Vet. J. 163, 251-259. Obritzhauser, W., Obritzhauser, G., Deutz, A., Köfer, J., Möstl, K., and Scheibner, H., 2002. Influence of cows persistently infected with Bovine Virus Diarrhoea Virus (BVDV) on BVD bulk milk diagnosis.