A four year longitudinal sero-epidemiological study of bovine herpesvirus type-1 (BHV-1) in adult cattle in 107 unvaccinated herds in south west England

Background Bovine herpesvirus type-1 (BHV-1) is an important pathogen of cattle that presents with a variety of clinical signs, including the upper respiratory tract infection infectious bovine rhinotracheitis (IBR). A seroepidemiological study of BHV-1 antibodies was conducted in England from 2002 – 2004: 29,782 blood samples were taken from 15,736 cattle from 114 herds which were visited on up to three occasions. Antibody concentration was measured using a commercial ELISA. Farm management information was collected using an interview questionnaire, and herd size and cattle movements were obtained from the cattle tuberculosis testing database and the British Cattle Movement Service. Hierarchical statistical models were used to investigate associations between cattle and herd variables and the continuous outcome percentage positive (PP) values from the ELISA test in unvaccinated herds. Results There were 7 vaccinated herds, all with at least one seropositive bovine. In unvaccinated herds 83.2% had at least one BHV-1 seropositive bovine, and the mean cattle and herd BHV-1 seroprevalence were 42.5% and 43.1% respectively. There were positive associations between PP value, age, herd size, presence of dairy cattle. Adult cattle in herds with grower cattle had lower PP values than those in herds without grower cattle. Purchased cattle had significantly lower PP values than homebred cattle, whereas cattle in herds that were totally restocked after the foot-and-mouth epidemic in 2001 had significantly higher PP values than those in continuously stocked herds. Samples taken in spring and summer had significantly lower PP values than those taken in winter, whereas those taken in autumn had significantly higher PP values than those taken in winter. The risks estimated from a logistic regression model with a binary outcome (seropositive yes/no) were similar. Conclusion The prevalence of BHV-1 seropositivity in cattle and herds has increased since the 1970s. Although the study population prevalence of BHV-1 was temporally stable during study period, the associations between serological status and cattle age, herd size, herd type, presence of young stock and restocked versus continuously stocked herds indicate that there is heterogeneity between herds and so potential for further spread of BHV-1 within and between herds.

BHV-1 generally infects cattle greater than 6 months of age once maternal immunity has waned [3]. Clinical signs associated with infection include nasal discharge, conjunctivitis, fever, inappetance, milk drop, abortion and, sometimes, death, although sub-clinical infection is possible [4,5]. The virus is shed in secretions from the eyes, nose and reproductive organs. After initial infection and disease, cattle become carriers of the virus which becomes latent in the trigeminal or sacral ganglia. Reactivation of the virus may occur when cattle are stressed [4] and virus can then be transmitted to susceptible cattle. Infection can occur indirectly through contaminated material and wind borne particles [2].
BHV-1 has been in Great Britain (GB) since the 1960s [6]. IBR was first confirmed in GB in 1961 [7,8], but was not considered clinically important in GB until an outbreak of disease in Scotland in the late 1970s caused by a virulent strain of BHV-1 (subtype 1) [9][10][11]. In England and Wales, the overall prevalence of BHV-1 seropositive cattle was 2.1% in 1963 [6], rising to 15.1% of dairy and 16.8% of suckler cattle over 2 yrs of age in Norfolk in 1991 [12], and by 1992 BHV-1 antibodies were detected in 34% of cattle herds in the UK [13].
The reported risk factors for the presence of BHV-1 antibodies in cattle in the Netherlands included a large herd size, dairy herds with beef/veal cattle, a high density of herds in the municipality [14], purchasing cattle, cattle participating in shows, professional visitors not using farm protective clothing and herds situated close to other BHV-1 positive herds [15]. In Belgium, seropositivity was associated with increasing cattle age and herd size and purchased cattle had a higher probability of being infected than homebred cattle [16,17]. This paper presents the largest longitudinal study of BHV-1 seropositivity in cattle in the UK, and its association with cattle age, birthplace, herd type and size.

Source of data
The data used in this paper came from 114 dairy and/or suckler herds in south west England. Farms were visited between 2002 and 2006 and were located within the Randomised Badger Culling Trial (RBCT) [18]. In addition, the study included some farms that had been depopulated in 2001 as a result of the foot-and-mouth disease (FMD) epidemic [19] and subsequently repopulated. All the farmers agreed to participate and allow their breeding cattle to be sampled.
Samples of blood (up to 10 ml) were collected under Home Office licence from all accessible cattle over 2 years of age at three routine visits, at approximately one year intervals. A subset of herds (n = 15) had a whole herd test, i.e. blood samples were collected from cattle of all ages, prompted by the presence of an animal persistently infected (PI) with bovine viral diarrhoea virus (BVDV). Four herds had a whole herd test instead of a routine third visit; otherwise whole herd tests were additional to routine tests.
All blood samples were centrifuged at the University of Warwick at 3220 g for 15 minutes, and serum was removed and frozen at -20°C.
Farmers completed an interview questionnaire between 17 th June 2003 and 18 th February 2004 to obtain information on clinical signs of infectious bovine rhinotracheitis (IBR) disease and use of a BHV-1 vaccine. Four out of 114 farmers did not complete the questionnaire.
At each visit, the unique identity of cattle was recorded from their ear tag and linked to the relevant blood sample. When appropriate, freeze brand numbers were also recorded. The ear tag was matched to the individual cattle data on the British Cattle Movement Service (BCMS) to obtain/confirm the following: the date of birth, whether the animal was homebred or purchased, breed and sex. More than 99% of cattle were matched within the BCMS dataset. The main reason that cattle were not matched was because they were born before 2001, when it was not compulsory to record cattle birth dates. Herd size was estimated from the cattle tuberculosis testing data (VetNet database), and validated during farm visits.
All data were entered into a relational database (Post-greSQL, PostgreSQL Global Development Group) using Microsoft Access (Microsoft Corp. US) as a front end. All data were screened for errors. When data mismatches were detected, data were re-checked to determine the source of the mismatch and where possible this was corrected.

Serological test and interpretation
The presence of BHV-1 specific antibodies (IgG 1 ) was detected using the SVANOVIR ® IBR-Ab (Svanova Biotech AB, Uppsala Sweden) indirect enzyme-linked immunosorbent assay (ELISA). All testing was completed accord-ing to the kit instructions. Internal controls were included on each plate to control for batch to batch variation. All samples were run in duplicate, and all testing was completed at the University of Warwick. Positive samples were re-tested when the duplicates were more than 0.25 optical densities (OD) units apart or when the OD of the positive controls was more than 0.2 OD units apart. The sensitivity and specificity of the serological kit to the serum neutralisation test have been reported as 97.4% and 92.4% respectively [20]. BHV-1 antibody concentration was calculated as the percentage of the positive control serum (PP) using the following calculation: The PP values ranged from -1.01 to 2.81. A sample was defined as positive when the OD > cut-off and negative when the OD < cut-off. The cut offs were:

Vaccination
It was not possible to differentiate antibodies stimulated through vaccination from those from natural infection and so the 7 vaccinated herds were excluded from the analysis.

Datasets
The full serological dataset consisted of 29

Outcome variables
A herd was defined as BHV-1 seropositive when at least one cow in the herd was above the cut off for BHV-1 on at least one occasion.
Cattle were defined as BHV-1 seropositive when above the cut off for BHV-1 at least once.
The herd BHV-1 seroprevalence was calculated at each visit as the number of seropositive cattle in the herd divided by the total number of cattle tested.
Seroconversion was defined in a herd when at least one cow changed from being seronegative to seropositive between two visits.
Two outcomes were modelled to investigate associations between BHV-1 seropositivity and herd and cow variables. These were the continuous outcome PP, and a binary (positive/negative) outcome (seropositive yes/no).

The explanatory variables
The variables listed in Table 1 were tested as fixed effects. The variables triplet code (i.e. the form of badger control in the RBCT), herd restocking status after the 2001 FMD epidemic and farm geographical location were forced into the models because they formed part of the study design.

Statistical analysis
Associations between the outcomes and herd and animal factors were initially screened using Mann-Whitney and chi-squared tests. Variables with a significance value of <0.20 in the univariate analysis were tested in two multilevel models: one with a continuous outcome of PP value and one with a binary outcome of seropositive yes/no. There were three hierarchical levels in the models: repeated measure at each visit (level 1), clustered by cow (level 2), and herd (level 3) to control for the dependency between repeated measures within cattle and cattle within herds. The multivariable models were built using both manual forward selection and backwards elimination.
The models took the form: where Y ijk is the PP value or seropositivity value at visit i from cow j in herd k. βX 0 is the intercept, βX is a series of vectors of fixed effect varying at the herd (k), cattle (jk) and visit (ijk), ν k + u jk are the variances at the herd and cattle levels, and e ijk is the residual variance.
The model fit was assessed by plotting the raw, standardised and deletion residuals, and the leverage and influence for each random effect and all fixed effects were also assessed [21]. The detection of outliers in the models was completed using a top-down approach [22]: outliers at level 3 (herds) were examined first, and then the lower level clusters (cattle and visits). A model which included only the seropositive herds in Dataset B was also built using the procedure above.
Correlations between the explanatory variables were investigated using chi-squared tests. Variables that were significantly (P < 0.01) associated with each other were noted to help with interpretation. All analyses were done using MLwiN version 2.02 (Centre for Multilevel Modelling, Bristol, UK) [23].

Descriptive statistics Study visits
All 107 unvaccinated herds were visited at least once, 96 were visited twice and 95 were visited on three occasions, generating 9136, 8099 and 7850 serum samples, respectively. A further 1833 blood samples were collected from additional visits or because of a whole herd test and/or cattle were under 2 years of age.

Vaccination and clinical signs
The median herd size of vaccinated herds was 274; in contrast, the median herd size of unvaccinated herds was 185.
The median herd size of unvaccinated seronegative and seropositive herds was 58 and 193, respectively. Nine percent (10/114) of farmers reported that they had seen clinical signs of IBR in their herd in the past, and two of these farmers also reported that they now vaccinated their cattle. All vaccinated herds had at least one seropositive animal compared with 83.2% of unvaccinated herds with at least one seropositive animal. The mean within-herd seroprevalence in vaccinated herds was 74.9% compared with 43.2% in unvaccinated herds.

Herd and cattle BHV-1 seroprevalence
There were 89 herds (83.2%, 95% CI, 77.6% -88.8%) with at least one BHV-1 seropositive bovine on at least one occasion (  Table 2). When only the herds where seroconversion had occurred were analysed, the mean and median within herd seroprevalence were 52.9% and 57.1% (range, 2.1% -99.2%) ( Table 2). There was a large statistically significant difference between the median seroprevalence in herds where cattle had seroconverted (57.1%) and those where they had not (10.6%): U = 1908; P <0.05, (Mann-Whitney Utest). Given the reported sensitivity and specificity of the ELISA, approximately 700 and 2,046 cattle tests during the study are expected to be false negatives and false positives, respectively. There were 124 cattle that tested positive and then negative at subsequent visits. In addition, 85 cattle changed serological status twice: 25 cattle tested positive, negative then positive and 60 cattle tested negative, positive then negative. Note that we assume that antibody positivity remains for life after infection; if antibody titre wanes with time since infection, then a conversion from positive to negative might reflect a true status rather than a test failure.

Univariate associations with BHV-1 antibody seroprevalence
The seroprevalence of antibodies against BHV-1 increased with age ( Figure 1). The seroprevalence was consistently higher in adult cattle in herds where there was seroconver-sion but the same trend with age was present in seropositive herds where no cattle seroconverted (Figure 1). The decline in antibody from 6 months of age and seroconversion at approximately 13 months of age is indicative of loss of maternal immunity followed by active seroconversion ( Figure 1). The seroprevalence of antibodies was higher in large herds where cattle seroconverted compared with large herds where no cattle seroconverted. There was also a trend that seroprevalence was higher as herd size increased in herds where cattle seroconverted; this was not apparent where there was no seroconversion (Figure 2). There was a statistically significant difference between the medians of the two groups in Figure 2 (U = 0; P < 0.05, Mann-Whitney U-test).
Of the 3,452 cattle with no data on their place of birth, 2,101 (60.9%) were seropositive. Of the 10,791 cattle Unadjusted age-specific profile of the mean BHV-1 antibody seroprevalence and 95% CI by quarter year (up to 2 years of old) and year (after 2 years old) for seropositive herds Figure 1 Unadjusted age-specific profile of the mean BHV-1 antibody seroprevalence and 95% CI by quarter year (up to 2 years old) and year (after 2 years old) for seropositive herds. Herds are dichotomised by presence of seroconversion in the period of study. All cattle greater than 10 years of age were categorised in one age group. Data came from all visits to BHV-1 unvaccinated herds (Dataset A, see methods section).
with data, purchased cattle were significantly more likely to be seropositive (54.1%) than homebred cattle (43.6%). The mean PP value was higher for cattle purchased into dairy herds, and increased at a greater rate over time from purchase in cattle ≥ 2 years of age at purchase (Figure 3). There was a highly significant departure from homogeneity between the four categories in Figure 3, χ 2 = 12137, P < 0.01. There was a significant association between season of visit and PP values. Samples taken in the spring and summer had significantly lower PP values than samples taken in winter; whereas those taken in autumn had significantly higher PP values than those taken in winter.
There was no significant association between restocked/ continuously stocked herds and herd seroprevalence in the univariate analysis: 16.7% of restocked herds and 16.9% continuously stocked herds had no seropositive cattle. The herd type with the highest seroprevalence was dairy cattle only (Table 3). There were no herds with dairy cattle and young stock (e.g. dairy with grower herds) that were seronegative.  Mean and 95% CI BHV-1 PP value by the number of years after purchase for age group at purchase and herd purpose Figure 3 Mean and 95% CI BHV-1 PP value by the number of years after purchase for age group at purchase and herd purpose. seropositive herds only (data not shown). When a variable indicating that cattle on a farm had/had not seroconverted was included (as in Figures 1 and 2) in the final model, all other variables became non-significant.

Hierarchical model results
The sum of the level 1, level 2, and level 3 variances was 0.28. The greatest unexplained variance in PP values in the final multi-level model was between cattle, making up 48% of the total variance. However, the variances were significant at all three levels ( Table 4).
The normal probability plots of the standardised residuals for all three levels were linear (e.g. Figure 4), indicating that the normality assumption was met. The deleted residuals did not differ greatly from the respective standardised residual values, indicating that there were no large outliers in the data at any level. When the herds, cows or samples with the largest influence were removed, the fit of the model worsened but the results of the model remained unchanged.
The estimated risks were similar when a multi-level binary logistic regression model (outcome seropositive yes/no) was fitted ( Table 4). The variables that did not remain significant were whether cattle were homebred or purchased and whether herds had grower cattle.

Discussion
This is the largest seroepidemiological study of BHV-1 carried out in cattle herds in the UK, and the first for more than 15 years. The seroprevalence of BHV-1 has increased dramatically since the 1960s in both cattle and herds. In the 1960s the seroprevalence in cattle was 2.1% [6], whilst in the current study, 42.5% of cattle always tested positive. Similarly, the herd prevalence was 20% in 1974 [24], and 83.2% in the current study. The differences in herd seroprevalence between the current and previous studies could be explained in part if the herds in this study were biased towards a particular location, age or cattle purpose since these were factors that influenced herd seroprevalence (Table 4). However, the consistency of seropositivity across the farms in this study over the 3 year time period suggests that infection has reached a temporal equilibrium, or possibly that seropositivity is increasing slowly.
A particularly interesting result is the higher seroprevalence in herds that were restocked after FMD. Carrique- [25] reported that bovine tuberculosis could be introduced into a farm at restocking when cattle were bought from herds with high incidence of infection. It is highly likely that purchase will have brought BHV-1 into restocked herds. The reason for the apparent increase in seroprevalence of BHV-1 is unknown but it might be that movement and mixing in new herds is stressful for cattle, resulting in recrudescence of virus in infected cattle that then infect susceptible cattle in the newly formed herd. In non-restocked herds, purchased cattle had a lower seroprevalence, which is contradictory to this explanation. It should also be remembered that the vaccination status of individual cattle is unknown so that the difference in seroprevalence might be explained by differences in purchasing vaccinated cattle, especially in dairy herds. There may also be complex interactions between cattle age, purchasing and restocking, since restocking of herds Normal probability plot of the standardised residuals for the herd level in Model 1 where all unvaccinated herd were included Table 3  Figure 4 Normal probability plot of the standardised residuals for the herd level in Model 1 where all unvaccinated herd were included Table 3.

Mas et al. (2008)
is very different to "routine" purchase. Additionally, the existence of multiple antigenic types (which cannot be distinguished serologically) means that interpretation of seroepidemiology is not straightforward. Whatever the cause, these results suggest that these purchased cattle were associated with a high herd seroprevalence and highlight the point that farmers should consider the antibody BHV-1 status of cattle before introduction, to prevent concomitant introduction or re-introduction of BHV-1 of the same or a different antigenic type, that might influence disease presentation.
The seroprevalence of BHV-1 increased with herd size in the univariate analysis and in the hierarchical model; this is consistent with other studies [16,[26][27][28]. The association with herd size was greater in herds where seroconversion occurred during the study (Figure 2), suggesting that active transmission is related positively to herd size, perhaps because larger herds have more potential transmission contacts both within the herd and with other herds (e.g. from veterinarians, other farmers and purchased cattle). However, the association between BHV-1 PP values and herd size might be confounded with other factors associated with herd size, e.g. recrudescence of infection through stress [29], or exposure to more viral types.
The age serological profile is consistent with life-long seropositivity. Antibodies to BHV-1 in calves of ≤ 6 months of age probably correspond to colostrum derived maternal immunity. This wanes by 1 year of age, and starts to rise by 2 years of age because of active infection. This suggests that the optimum age for vaccination of cattle is 10 -14 months of age, although with some active seroconversion observed in adult cattle and a seroprevalence only exceeding 50% in older animals, vaccination is likely to have a positive impact when administered at any age. All the vaccinated herds had seropositive cattle, while approximately 80% of the unvaccinated herds had seropositive cattle. On average, less than 50% of the cattle in the unvaccinated herds tested positive at each visit, whilst >75% of the cattle from vaccinated herds were positive. These data suggest that either some cattle in vaccinated herds were not vaccinated, that vaccination does not always induce antibody or that antibody titre induced by vaccination wanes over time. It is possible that antibody titre generated by natural infection also wanes over time, but there is no previous work on this hypothesis.
In the current study, when the herd was a dairy herd rather than a suckler herd, the rate of seroconversion in adults was greater. The average age of cattle did not differ between dairy and suckler herds, and so this is a true effect, possibly due to winter housing or other stressors related to dairy cow management.
Housing and winter stressors are also an explanation for the small but significant effect of season on PP values. However, 31% and 47% of visits were 11-13 months apart for routine visits 1 and 2 and routine visits 2 and 3, respectively, so that there was a tendency for the same herds to be sampled in the same season and so herd effect is interpreted as an apparent seasonal effect. Other explanations are that seasonal culling and replacement of cattle might influence herd prevalence of BHV-1 antibodies.
All the variables above became non-significant when the binary variable seroconversion (indicating whether seroconversion was observed in the herd) was introduced into the final model. This indicates that the model risk factors are those associated with virus circulation between cattle on the farm.
The use of different modelling approaches has been discussed elsewhere [28,30] but not modelling different distributions of the same outcome. We compared these two outcomes because we wanted to investigate change in antibody level that was not necessarily occurring around the cut off. The associated risks were similar to those in Table 4 whether the data were modelled using a continuous or binary outcome, but the presence of grower cattle and purchased or homebred were not statistically significant in the binomial model, although the trends were in the same direction of risk. This suggests that these variables alter the antibody titre either always above or below the cut off and that this change was detectable in the continuous outcome model but not in the discrete model.
In the discrete model, we assumed that the serological measurement of seropositive/seronegative had perfect sensitivity and specificity. It is possible that bias could be introduced into the analysis if the sensitivity or specificity of the test varied with other variables, for example, with cattle age, or time since infection, however this has not been reported for BHV-1 antibody tests. The continuous outcome (PP) calculated from the ELISA will have had some random error (Figure 4), but avoided the need to categorise cattle as positive or negative to BHV-1 antibody, and avoided the possibility of misclassification due imperfect sensitivity and specificity, although would have retained a bias if serological measurement varied with other variables. However, the consistency between the discrete and continuous outcome models suggests that sensitivity and specificity do not alter with any of the explanatory variables in the model.
The results of this study do not necessarily represent all cattle herds in England, because of the locality of the farms sampled and because the analysis was based primarily on the serological values for BHV-1 from adult cattle. Using only unvaccinated herds in the study may have reduced the extrapolation of the results, because these herds may have had different management practices from vaccinated herds, but there were only 7 herds that reportedly used vaccination and so analysis of these herds' managements was not possible. In addition, there might have been an overestimation of the infection derived seroprevalence because purchased cattle might have been sourced from a vaccinated herd and so had antibodies derived from vaccination: we do not know the vaccination status of source herds. The serological test used in the current study did not distinguish between vaccine derived and pathogen derived antibody so this was unavoidable. It is interesting that only 7 herds were vaccinated and that farmers reported little clinical disease from BHV-1, suggesting that this infection is not an obvious production constraint or clinical disorder in most herds.
Finally, in the current analysis we could not distinguish between BHV-1 sub-types, the epidemiology of individual types might be different and this might account for some of the unexplained heterogeneity. BHV-1 subtype 3 is now classified as a new species (bovine herpesvirus-5 or BHV-5) [31]. It is probable that we were detecting antibodies to BHV-5 as well as BHV-1 because these viruses are antigenically related.

Conclusion
In conclusion, the level of BHV-1 infection has increased dramatically since previous estimates from the UK, with approximately 83% of the unvaccinated herds in the south west of England seropositive. This study adds to previous work with the finding that infection might have reached a temporal but not a spatial equilibrium and that between herd variability in PP might arise through variability in cattle age, herd size, herd purpose and the formation of new herds.

Authors' contributions
KAW helped collect samples, performed the data analysis and drafted the manuscript. GFM and LEG are the funded co-applicants who designed the study, supervised data collection and analysis and finalised the manuscript. SJM coordinated and completed ELISA testing. ARV assisted in design of the questionnaire and coordinated data collection and supervised collection of blood samples. SM setup and maintained the study database and downloads. All authors read and approved the final manuscript.