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Research in Veterinary Science ■■ (2015) ■■-■■
ELSEVIER
Prevalence of exposure to bovine viral diarrhoea virus (BVDV) and bovine herpesvirus-1 (BoHV-1) in Irish dairy herds
R.G. Sayersa*, N. Byrnea, E. O'Dohertya, S. Arkinsb
a Animal & Bioscience Research Department, Animal & Grassland Research and Innovation Centre, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland b Department of Life Sciences, University of Limerick, Limerick, Ireland
ABSTRACT
Bovine viral diarrhoea virus (BVDV) and bovine herpesvirus 1 (BoHV-1) are contagious bovine viral agents. The objectives of this study were to use quarterly bulk milk and 'spot' testing of unvaccinated youngstock to establish the national prevalence of exposure to BVDV and/or BoHV-1 in Irish dairy herds. Seasonality of bulk milk ELISA results was also examined. From a geographically representative population of 305 dairy herds, 88% and 80% of herds yielded mean annual positive bulk milk readings for BVDV and BoHV-1, respectively. Of these, 61% were vaccinated against BVDV and 12% against BoHV-1. A total of 2171 serum samples from weanlings having a mean age of 291 days yielded 543 (25%) seropositive for BVDV, and 117 (5.4%) seropositive for BoHV-1. A significant seasonal trend in bulk milk antibody ELISA readings and herd status was recorded for BVDV, with more herds categorised as positive in the latter half of the year.
© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Research in Veterinary Science
journal homepage: www.elsevier.com/locate/rvsc
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Article history: Received 29 May 2014 Accepted 22 February 2015
Keywords:
BoHV-1
Prevalence
Bulk milk ELISA
Spot test
Seasonality
1. Introduction
Bovine viral diarrhoea (BVD), caused by BVD virus (BVDV), and infectious bovine rhinotracheitis (IBR), caused by bovine herpesvirus 1 (BoHV-1), are highly contagious viral diseases of cattle (Moennig et al., 2005; Muylkens et al., 2007; Nandi et al., 2009). Both exhibit a worldwide distribution (Lindberg et al., 2006; Thiry et al., 2006) and are listed as notifiable diseases by the Office International des Epizooitic1 (OIE). Although OlE-listed diseases, compulsory national control programmes for BVDV and BoHV-1 do not exist in many countries (Ackermann and Engels, 2006; Heffernan et al., 2009).
Where regulation does exist, successful BVDV eradication has been achieved through the use of 'test and cull' protocols involving removal of persistently infected (PI) individuals (Heffernan et al., 2009; Lindberget al., 2006; Moennig et al., 2005; Presi et al., 2011; Ridpath, 2012; Stahl and Alenius, 2012; Valle et al., 2005). In the case of BoHV-1, vaccination with marker/DIVA (Differentiating Infected from VAccinated) vaccines (Mars et al., 2001; Nandi et al., 2009; Nardelli et al., 2008; van Oirschot, 1999) constitutes the primary method of control and eradication in high prevalence regions. In January 2013, a mandatory national eradication programme for BVD, coordinated by the Animal Health lreland (AHl),
* Corresponding author. Animal & Grassland Research and Innovation Centre, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland. Tel.: +353(0)2542215; fax: +353(0)2542385.
E-mail address: riona.sayers@teagasc.ie (R.G. Sayers).
1 www.oie.int.
was introduced in the Republic of Ireland (Graham et al., 2013). As yet, a co-ordinated approach to BoHV-1 control does not exist in Ireland.
In order to determine the necessity for, and measure ongoing success of an eradication programme, it is useful to conduct prevalence studies to obtain baseline data (Heffernan et al., 2009; Lindberg et al., 2006; Lindberg and Alenius, 1999; Paisley et al., 2001). National prevalence studies, however, are often prohibitively expensive (Thrushfield, 2005). The advent of bulk milk testing overcomes this issue and reliable antibody detection bulk milk test procedures have been developed for both BVDV and BoHV-1 (Beaudeau et al., 2001; Nylin et al., 2000). Bulk milk analysis for BVDV antibodies, however, does not readily distinguish between vaccinated and unvaccinated herds (Lindberg et al., 2006). This issue has been overcome in the case of BoHV-1 with the advent of BoHV-1 gE-deleted DIVA vaccines. Due to legislative requirements,2 all BoHV-1 vaccines administered in the Republic of Ireland since December 31, 2004 are DIVA vaccines (Simon, 2004).
Additionally, bulk milk BVD antibody readings may reflect historical rather than current herd viral status (Brulisauer et al., 2010; Lindberg and Alenius, 1999). To overcome this issue, it is useful to test unvaccinated homeborn youngstock (weanlings) for antibodies against BVDV, i.e. a 'spot test' (Houe, 1992,1994; Mars and Van Maanen, 2005). Positive antibody readings in this population, once maternal antibodies have dissipated, can be indicative of current
2 Diseases of Animals Act 1966; Control on Animal and Poultry Vaccines Order 2002; S.l. 528 of 2002 www.irishstatutebook.ie.
http://dx.doi.org/10.1016/j.rvsc.2015.02.011
0034-5288/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/).
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or recent viral circulation (Houe, 1992,1994; Lindberg and Alenius, 1999), and as such provide a useful adjunct to bulk milk testing.
Although preliminary surveillance studies have indicated high levels of both BVDV and BoHV-1 in the Irish national cattle population (Cowley et al., 2011, 2012; O'Grady et al., 2008; O'Neill et al., 2009), national prevalence data for BVD and BoHV-1 exposure among a geographically representative sample of Irish dairy farms are not available. In addition, evaluation of longitudinal BVD and BoHV-1 bulk milk data over a single lactation in a predominantly spring-calving dairy system has not been reported previously. The primary objective of this study, therefore, was to use bulk milk analysis and spot testing of Irish dairy herds to generate national prevalence data for both BVD and BoHV-1, while investigating the usefulness of this diagnostic strategy in an Irish context.
2. Materials and methods
2.1. Sample population and survey
The study was licenced by the Irish Department of Health and Children in 2009, meeting all legislative requirements for research involving animals in the Republic of Ireland at the time of the study.
A detailed description of the sample population used in this study is outlined in O'Doherty et al. (2013). Briefly, stratified proportional sampling based on herd size and geographical location was used to randomly select and invite 500 herds from the Irish Cattle Breeding Federations (ICBF) database to partake in the study on a non-incentivised basis. Over the 2009 lactation, four bulk milk samples (23 March, 8 June, 31 August and 2 November) were submitted by post in a standardised kit from each participating farm. Each study farm was visited between October 2009 and January 2010 to collect blood samples by coccygeal venepuncture from 20% of the replacement heifer group (weanlings for spot test) on each farm, with a minimum of five weanling heifers sampled on each farm. All heifers were homeborn and not vaccinated against BVDV. Where possible, only weanlings over 270 days of age were sampled, although not achievable in all cases. Accurate weanling age based on calf registration data was downloaded from the ICBF database.
2.2. Sample analysis
Commercially available enzyme linked immunosorbent assay (ELISA) kits were used to test bulk milk samples for the presence antibodies against: (i) BVD p80 (NS3) protein, (Institut Pourquier, France); (ii) Ultrapurified IBR lysate (Institut Pourquier, France) in BoHV-1 unvaccinated herds; and (iii) IBRgE, (IDEXX laboratories, USA) in BoHV-1 vaccinated herds. Weanling serum samples were also tested for antibodies against BVD p80, ultrapurified IBR lysate, and IBRgE with serum adapted positive cut-off values applied as outlined by kit manufacturers (Table 1). All analyses were completed by commercial accredited laboratories; BVD p80 and IBR lysate by
National Milk Laboratories Ltd. (UK), and IBR gE by Enfer Diagnostics Ltd. (Ireland).
2.3. Herd classification
Calving data from the ICBF were used to determine calving-season of each herd (spring-calving and non-spring-calving, i.e. spring-autumn [SA] and year-round [YR]) as described by O'Doherty et al. (2013). Vaccination status (vaccinated [V] and unvaccinated [UV]) was determined by questionnaire, with date of vaccination, product used, and class of animal vaccinated (cows, yearling-heifers, weanlings) recorded. In all cases, kit-manufacturer positive cut-off values were applied to ELISA outputs in order to classify herds as 'positive' or 'negative'. Herds were classified as positive or negative at each of the four sampling time points (longitudinal data). Additionally, a mean annual ELISA result for each herd (herd status data) was calculated to provide an overall bulk milk classification for each herd. Herds were also categorised on the basis of combined BVDV and BoHV-1 bulk milk antibody status, i.e. negative for both viral antibodies, positive for BoHV-1 and negative for BVD, negative for BoHV-1 and positive for BVD, and positive for both viral antibodies.
Finally, herds were classified with regard to the presence of seropositive unvaccinated weanlings. Two datasets were constructed with weanlings either categorised 'positive aged >180 days of age' or 'positive aged >270 days of age' to both assess and minimise potential interference from maternally derived antibodies (MDAs) (Fulton et al., 2004). Herds having at least one weanling serologi-cally positive for either BVDV or BoHV-1 were classified as having 'evidence of recent viral circulation' (RVC) (Houe, 1992; Handel et al., 2011). Herds not recording a positive weanling or recording a positive weanling under either 180 or 270 days of age, depending on the dataset, were classified as 'not having evidence of recent viral circulation' (NRVC).
2.4. Data analysis
Descriptive analysis and graphical representations were completed in Excel (MS Office 2010). Normality of the data was assessed visually using ladder of powers histograms, with normality of residuals assessed using normal probability plots and kernel density estimate plots constructed in Stata (Version 12). True prevalence was calculated using the Rogan-Gladen estimator in the survey toolbox version 1.04 (www.ausvet.com.au (Cameron, 1999)). Pearson's chi-squared, Fisher's exact, univariable and multivariable logistic regression, generalised estimating equations (GEE), multinomial logistic regression, Wilcoxon rank sum, and Hosmer-Lemeshow test of goodness of fit analyses were carried out using Stata (Version 12).
Seasonal trends in true prevalence for both diseases were tabulated. In addition, box plots of %inhibition, %S/P, and S/N ratio for BVDp80, IBR lysate, and IBR gE, respectively, at each sampling time
Table 1
ELISA kit performance data and positive cut-off values for BVD and BoHV-1assays used in this study.
Test BVD P80 IBR Lysate IBRgE BVD P80 IBR lysate IBR gE
Milk Milk Milk Serum Serum Serum
Sensitivity 95.0% 100% 72.0-88.4% 97.6% 98.7% 100%
Specificity 97.7% 99.6% 100% 97.3% 99.9% >99%
Positive cut-off >55 >25 <0.8 >60 >55 <0.60
(Kit) %Inhibitiona %S/Pb S/N ratioc %Inhibitiona % S/Pb S/N ratioc
Within-herd prevalence >30%d Not available 10.0-15.0%e n/a n/a n/a
a %Inhibition = [1 - (OD 450 of analysed sample / mean OD 450 of negative control)] x 100.
b %S/P = (OD 450 of sample - OD 450 of negative control) / (mean OD 450 of positive control - OD 450 of negative control) x 100. c S/N ratio = (sample mean - absorbance 650 nm)/negative control mean.
d Beaudeau et al., 2001.
e Wellenberg et al, 1998; Kramps et al., 1994.
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point were constructed. Two BVD datasets were examined by GEE and logistic regression, i.e. all study herds regardless of BVD vaccination status, and BVD unvaccinated herds only.
Longitudinal data were used for the purposes of GEE analysis. To examine seasonal effects on bulk milk analysis, a univariable analysis of bulk milk results (constructed as both categorical [positive vs. negative] and continuous [ELISA readings] variables) and sampling time point was completed (Woodbine et al., 2009).
Examination of additional influences on bulk milk longitudinal data (both categorical and continuous) by a number of independent variables was also completed. Independent variables examined included region (high density dairy vs. low density dairy), herd size (31-65 cows vs. 66-99 cows vs. > 99 cows), calving season (spring-calving vs. non-spring-calving), type of farming enterprise (dairy livestock only vs. mixed livestock), vaccination status (V vs. UV), and recent viral circulation (RVC >180 days or RVC >270 days vs. NRVC >180 days or NRVC >270 days). A total of four datasets were analysed by GEE to account for BVD vaccination status (V, UV) and differing weanling age groups (>180 and >270 days of age).
Logistic regression was used to examine associations between recent viral circulation status (RVC >180 or >270 days vs. NRVC >180 or >270 days) and vaccination status (V vs. UV), the dependent variables, and region, calving-season, enterprise-type, herd-size, and annual mean bulk milk herd status (independent variables). Four datasets were constructed to account for BVD vaccination status (V, UV) and positive-weanling age (>180 or >270 days).
Multinomial logistic regression analysis was completed on combined BVDV and BoHV-1 bulk milk status with region, herd-size, enterprise-type, calving-season and recent viral circulation status as independent variables.
For all GEE analyses, herd was included as a repeated measure and an exchangeable correlation used. A binomial distribution was assumed and a logit link function applied for categorical data; a Gaussian distribution and identity link function were used for continuous data. All regression models were constructed by first completing a univariable analysis. Those variables recording p values of <0.15 in univariable analyses were included in multivariable models. A manual backwards elimination with a forward step was used to build models with variables recording p values of <0.05 maintained. Second level interactions deemed biologically significant were also included. The overall fit of regression models was assessed using the Hosmer-Lemeshow good of fit test following ordinary logistic regression (categorical variables). Normality of residuals was assessed following logistic regression (categorical variables) and linear regression (continuous variables).
To examine differences in bulk milk readings between RVC herds and NRVC herds, a Wilcoxon rank sum analysis was completed for each sampling time point. Herds were examined based on vaccination status (V and UV) for both BVDV and BoHV-1, and a third analysis was completed for BVDV where all herds regardless of vaccination status were included. Both viral circulation infection
classifications were examined, i.e. herds with positive weanlings greater than 180 or 270 days of age.
3. Results
A total of 312 herds were recruited to the study (Fig. 1), yielding a sufficient sample size to achieve a 95% confidence level and precision of 5% for a national dairy herd population of approximately 18,000 herds with an expected national prevalence of 70%. A complete set of four bulk milk samples was not achieved for four farms and vaccination data were not returned by three farmers. Of the herds recruited to the study, 305 herds were therefore suitable for final analysis. Weanling ages were unavailable for eleven herds and these data were excluded from statistical analysis.
Study herds have previously been shown to geographically represent the Irish national dairy farm population (O'Doherty et al., 2013). The distribution of study herds across region, herd size, calving season, and type of enterprise is included in Table 2. Approximately 60% of study participants were vaccinated against BVDV using inactivated vaccines, with 12.5% vaccinating against BoHV-1 using DIVA vaccines. A total of 33 study farms administered vaccines for both BVDV and BoHV-1.
3.1. Prevalence of bulk milk positive herds
The apparent prevalence (Ap) of bulk milk antibody positive herds for BVDV and BoHV-1 was approximately 88% (80% in unvaccinated herds) and 80% (78% in unvaccinated herds), respectively. True prevalence (Tp) and 95% CI at each sampling time point and across vaccination status is outlined in Table 3. Concurrent exposure to BVDV and BoHV-1 was detected in 72% of herds, with only 10 herds recording bulk milk seronegative status for BVDV and BoHA-1. Seasonal trends in ELISA readings for each disease are included in Fig. 2.
3.2. Seasonal pattern ofbulk milk results
Univariable GEE analysis highlighted significant seasonal differences in BVDV and BoHV-1 herd status examined as both categorical and continuous variables (Supplementary Table S1).
Multivariable analysis of exposure to BVDV and BoHV-1 as continuous variables highlighted a general increase in ELISA readings as the year progressed for both BVD and BoHV-1 (Table 4). When examined as categorical variables, a significant seasonal effect was only observed for BVD bulk milk herd status and a significant interaction between enterprise type and sampling time point was highlighted (Table 4). Herds with a mixed livestock enterprise, in general, were more likely to record a BVD positive bulk milk result in the latter half of the year. This association was apparent regardless of BVD vaccination status. Visual examination of normal probability plots and kernel density plots of residuals did not highlight evidence of non-normality. Goodness of fit analyses for
Table 2
Distribution of study herds across region, herd size, calving-season and enterprise-type.
Region3 Counties represented Herd size (cows) Calving season Enterprisec
(density) 31-65 66-99 >99 Spring SA/YRb Dairy Mixed
Region 1 Carlow, Cavan, Clare, Donegal, Dublin, Galway, Kildare, Laois, Leitrim, n = 29 n = 26 n = 44 n = 75 n = 24 n = 52 n = 46
(Low) Longford, Louth, Mayo, Meath, Monaghan, Offaly, Roscommon, Sligo, 9.5% 8.5% 14.4% 24.6% 7.9% 17% 15.1%
32.5% Westmeath, Wexford, Wicklow
Region 2 Cork, Kerry, Kilkenny, Limerick, Tipperary, Waterford, Limerick n = 52 n = 72 n = 82 n = 190 n = 16 n = 88 n = 118
(High) 17.0% 23.6% 26.9% 62.3% 5.2% 28.9% 38.7%
a Regions were chosen to correspond with Irish dairy farm distribution (Sayers et al., 2013) and to represent a natural geographical spread. b SA/YR represents Spring-Autumn and Year-Round calving seasons. c Type of enterprise was not supplied by a single participant.
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0-20 _ 20 - 60 I 60-100 I 100-140 I 140-180 Study herds
Connaught
Munster
Leinster
Fig. 1. (a) Location of study herds and, (b) representation of the density of animals on dairy farms in Ireland.
categorical data using ordinary logistic regression yielded nonsignificant values ranging from p = 0.286 to p = 0.878.
Examination of individual herd results highlighted 15 herds which recorded elevated BVD readings in August and November samplings (Table 5), four of which (herds 67, 263, 273, 275) may be suggestive of introduction of BVD virus to the lactating herd. Herd 263 reported diarrhoea, fever, and milk drop across the lactating herd over the month of July prior to submission of the August bulk milk sample. An additional herd (herd 142) administered vaccine in September which may account for the elevated reading in November. Remaining herds, although having progressed to positive herd status in either August or November, did not record sufficiently elevated readings to be regarded as biologically significant given a positive cut-off of 55% inhibition.
3.3. Youngstock serological status
A total of 2171 serum samples from weanlings having a mean age of 291 days (range 109 to 549) were analysed, with 543 testing seropositive for BVDV and 117 testing seropositive for BoHV-1. The
Table 3
True prevalence (Tp) and 95% confidence interval (CI) of exposure to BVDV and BoHV-1 in Irish dairy herds of varying vaccination status at each sampling time point.
Sample date BVD IBR
Tp 95% CI (%) Tp 95% CI (%)
All herds n = 305
March 90.5 86.4,94.7
June 90.5 86.4,94.7
August 96.2 92.8,99.6
November 94.4 90.8,98.1
Unvaccinated herds only n = 113 n = 269
March 81.5 73.3,89.8 80.2 75.8,84.7
June 83.4 75.4,91.4 79.6 75.1,84.1
August 94.9 89.0,100.0 77.3 72.6,82.0
November 93.0 86.6,99.3 79.6 75.1,84.1
gE herds* n = 36
March 100 n/a
June 100 n/a
August 100 n/a
November 100 n/a
Describes herds vaccinated with a BoHV-1 DIVA vaccine and tested using a gE ELISA.
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100 S" 90
'■m J2
80 70 60 50 40 30 20 10 0
1.2 1 0.8 0.6
° 0.4 <
É E3 S S
Mar June Aug Nov Mar June Aug Nov (Pos) (Pos) (Pos) (Pos) (Neg) (Neg) (Neg) (Neg) Sample date (BVD herd status)
450 ) 400 350 <5 300
ut 250 p
i 200 S 150
ûu 100 50 0
E3 LJ LJ
+ + + +
March June August Nov March June Aug Nov (Pos) (Pos) (Pos) (Pos) (Neg) (Neg) (Neg) (Neg) Sample date (BoHV-1 unvaccinated herd status)
Mar June Aug Nov Mar June Aug Nov (Pos) (Pos) (Pos) (Pos) (Neg) (Neg) (Neg) (Neg) Sample date (BoHV-1 vaccinated herd status)
Fig. 2. Box plots outlining seasonal trends3 in bulk milk ELISA readings across positive (Pos) and negative (Neg) (i) BVD, (ii) BoHV-1 unvaccinated, and (iii) BoHV-1 vaccinated herds in 2009.
aMar: March; June: June; Aug: August; Nov: November.
3.4. Associations between herd demographics and bulk milk status
Regional differences in both BVDV and BoHV-1 herd classification were highlighted by multivariable GEE analysis (Table 4), though this was not consistent across all models. Study herds in the most dairy dense region of Ireland (Region-2) were almost twice as likely to be categorised as BVDV antibody positive over those in Region-1 when all herds, regardless of vaccination status, were included in the model. The reverse was highlighted for BoHV-1, where herds in Region-1 (the least dairy dense part of Ireland) were found to be almost twice as likely as those in Region-2 to be categorised as positive. Herd size was significantly associated with BoHV-1 herd status, with larger herds (>99 cows) approximately four times more likely than smaller herds to be categorised positive. Finally, vaccination was associated with positive herd status for both BVD (OR = 4.29) and BoHV-1 (OR = 31.88), with vaccinating herds more likely to be categorised positive.
3.5. Associations between recent viral circulation status, vaccination, and herd demographics
All models examined highlighted a significant association between BVDV bulk milk antibody status and BVDV RVC, herds having evidence of recent BVDV circulation at least three times more likely to be bulk milk positive than those herds recording no seropositive weanlings (Supplementary Table S2 and Table 6). No such association was highlighted in the case of BoHV-1 bulk milk antibody positive herds. A tendency for larger herds to have RVC for either BVDV or BoHV-1 was highlighted, with non-spring-calving herds also more likely to contain BVDV seropositive weanlings (Table 6).
Larger herds were more likely to vaccinate for both BVDV and BoHV-1 in this study population. In addition, herds vaccinating for BVDV were more likely to also vaccinate for BoHV-1 and vice versa (Table 7). There were tendencies for herds that were BoHV-1 bulk milk antibody positive to vaccinate for BVDV and for non-spring-calving herds to vaccinate for BoHV-1.
3.6. Multinomial logistic regression analysis
Multinomial logistic regression highlighted that compared to herds bulk milk antibody negative for both BVDV and BoHV-1, larger herds were more likely to be antibody positive for BoHV-1 and negative for BVDV (OR=3.70, 95% CI = 1.52, 9.04, P = 0.004) and antibody positive for both BVDV and BoHV-1 (OR = 2.67, 95% CI = 1.23, 5.81, P = 0.013). In addition, compared to antibody negative herds, those operating mixed livestock enterprises tended to be over three times more likely than dairy-only herds to present with exposure to one (OR = 4.04, P = 0.071, BoHV-1; OR = 3.35, P = 0.10, BVDV) or both viral pathogens (OR = 4.84, P = 0.024).
3.7. Wilcoxon rank sum analysis
age profile and BVDV/BoHV-1 serological test status of study weanlings is included in Fig. 3. At least one seropositive weanling over 180 days of age was identified in 119/294 study herds in the case of BVDV, and 24/294 in the case of BoHV-1. If an age limit of 270 days was applied, 96/294 herds recorded a single seropositive BVDV weanling and 18/294 a BoHV-1 seropositive weanling (Fig. 4). A total of 10 herds recorded weanlings >180 days old seropositive for both BVDV and BoHV-1, and 8 herds having concurrently seropositive weanlings if an age limit of >270 days was applied.
A significant difference was highlighted between RVC and NRVC herds in terms of BVDV bulk milk %inhibition readings when all herds were included in the analysis regardless of vaccination status, with z values ranging from -2.718 to -3.864 (Supplementary Table S3). A similar result was generated for BVDV unvaccinated herds alone, with z values ranging from -3.901 to -4.617. No significant difference in %inhibition was highlighted between BVDV vaccinated RVC vs. NRVC herds, however. An analysis of BoHV-1 yielded similar results, with a significant difference in ELISA outputs highlighted for unvaccinated herds (with the sole exception of the March sample), but no significant difference in ELISA readings between vaccinated RVC and NRVC herds (Supplementary Table S3).
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Table 4
Multivariable GEE analysis of BVDV and BoHV-1 herd classification.
Dependent variable Independent variable Odds ratio Confidence interval (95%) p value Model
Categorical (p value)
BVD status Region 2 vs. Region 1 2.02 1.09,3.84 0.027 Region
POSITIVE vs. NEGATIVE Vaccinated vs. Unvaccinated 4.29 1.09,8.06 <0.001 BVD vaccination
(All herds included regardless of Mixed August vs. Mixed March 3.19 1.54,6.58 0.002 Sampling time point
vaccination status) Mixed November vs. Mixed March 2.04 1.08,3.83 0.027 Enterprise
Mixed August vs. Mixed June 1.96 0.96,4.03 0.064* Sampling time point*enterprise (P < 0.001)
BVD classification Mixed August vs. Mixed March 3.46 1.45,8.25 0.005 Sampling time point
POSITIVE vs. NEGATIVE Mixed November vs. Mixed March 2.38 1.07,5.29 0.032 (P < 0.001)
(UV herds only)
IBR classification Region 1 vs. region 2 1.77 0.98,3.18 0.056* Region
POSITIVE vs. NEGATIVE Vaccinated vs. Unvaccinated 31.88 0.92,1102.57 0.057* IBR vaccination
(All herds included) >99cows vs. 31-65 cows 3.66 1.82,7.37 <0.001 Herd size
>99cows vs. 66-99cows 4.15 2.11,8.19 <0.001 (P < 0.001)
Continuous Coa CI p value Model
BVD ELISA readings Region 2 vs. Region 1 4.58 0.44,8.72 0.030 Region
Vaccinated vs. Unvaccinated 10.72 6.76,16.67 <0.001 BVD vaccination
August vs. March 4.53 2.66,6.39 <0.001 Enterprise
November vs. March 2.73 0.86,4.59 0.004 Sampling time point
Mixed June vs. Mixed March 4.26 1.71,6.81 0.001 Enterprise*samplingtime point
Mixed August vs. Mixed March 3.04 0.49,5.59 0.019 P < 0.001
Mixed November vs. Mixed March 4.30 1.75,6.84 0.001
BoHV-1 ELISA readings June vs. March 22.29 14.31,30.29 <0.001 Sampling time point
(excluding vaccinated herds i.e. those August vs. March 14.29 6.29,22.27 <0.001 Herd size
tested using gE) November vs. March 47.82 39.84,55.81 <0.001 P < 0.001
August vs. June -8.01 -16.00,-0.03 0.049
November vs. June 25.52 17.54,33.52 <0.001
November vs. August 33.54 25.55,41.53 <0.001
>99cows vs. 31-65 cows 68.33 40.82,95.83 <0.001
>99cows vs. 66-99cows 52.08 26.57,77.61 <0.001
a Co = Coefficient, i.e. the expected difference across the population. * Denotes an interaction between two independent variables.
Table 5
Herds recording a change (negative to positive) in BVD bulk milk antibody status in August and November.
Herd identification Calving season Enterprise BVD vaccination status Weanling status March June August Novemb
(Date of vaccination) 180 % inhibition
39 Spring Mixed UV Negative 27.93 43.45 58.37 60.42
54 Spring Dairy UV Positive 43.65 25.32 54.69 60.96
67 Spring Mixed UV Positive 19.59 49.72 72.74 62.59
92 Autumn Mixed UV Negative 51.5 38.11 71.62 19.45
139 Spring Dairy V (April) Negative 24.47 46.07 39.9 60.98
142 Spring Mixed V (September) Positive 51.26 48.52 61.65 84.84
152 Spring Mixed UV Negative 32.06 42.67 60.38 60.98
155 Autumn Mixed UV Negative 35.48 47.11 58.6 48.28
172 Spring Dairy V (March) Negative 39.58 43.92 46.71 57.75
184 Spring Mixed V January Positive 35.68 43.3 58.11 30.01
263 Spring Dairy UV Positive 51.35 54.17 81.58 83.54
273 Spring Mixed UV Positive 20.5 36.05 76.7 85.83
275 Spring Dairy V (March) Positive 54.24 35.95 71.09 74.98
278 Autumn Dairy V (January) Positive 52.76 31.74 55.1 53.43
291 Spring Dairy UV Negative 32.71 44.31 51.79 67.75
4. Discussion
The design of disease control strategies should be built upon local knowledge (Greiser-Wilke et al., 2003; Lindberg et al., 2006). This current study aimed to document levels of exposure to BVDV and BoHV-1 in the Irish dairy cattle population. Inclusion of both viruses allowed data on concurrent exposure to BVD and BoHV-1 to be investigated for the first time in Ireland. Although the current study has highlighted a high level of exposure to both BVDV and BoHV-1, levels are comparable to those reported for other regions (Garoussi
et al., 2008; Guarino et al., 2008; Paton et al., 1998; Stahl et al., 2002; Thobokwe et al., 2004; Van Wuijckhuise et al., 1998; Woodbine et al., 2009).
It has been reported that the level of antibodies to BVDV in milk is inversely related to the amount of milk produced (Niskanen et al., 1989). As the majority of herds included in the present study were spring-calving with peak lactation occurring approximately 9 weeks post-calving (Quinn et al., 2005), milk yield will reduce in the latter half of the year. An increase in bulk milk antibody readings might therefore be expected (Rikula et al., 2005), and indeed such a trend
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^ # & J? J? ¿P J? J? J? J? # s
f ^ ^ ^ # # # ¿Г & & -1
v v v v v v
Age category (days)
Fig. 3. Age and serological profile of study weanlings.
Positive weanlings <180 days Single positive weanling 180-270 days old Single positive weanling >270days old Multiple positive weanlings all 180-270 days old
Multiple positive weanlings all >270 days old
Multiple positive weanlings mixture of 180-270 and >270 days old
Positive weanlings - age unknown
hi BoHV-1 M BVD
10 20 30 40 Number of herds
Fig. 4. Number of herds containing varying ages and levels of seropositive youngstock.
was highlighted in the current study, particularly in the case of BVDV. The increase, however, was not large, and in the majority of cases could not be considered biologically significant. Woodbine et al. (2009) reported similar results for BoHV-1 in England where a small but significant seasonal association was detected. It has been reported, however, that the total amount of secreted daily IgG is higher during peak milk production than later in lactation which may counteract any dilution effect of increased milk production, thereby contributing to the lack of major seasonal trends (Guidry et al., 1980; Nylin et al., 2000).
It should be noted, however, that in the case of four farms, more dramatic increases in BVD bulk milk readings were recorded between the June and August sampling. Housing is more often associated with spread of viral disease than is outdoor grazing (Ampe et al., 2012). An increased risk of BVDV infection over the summer months was unexpected, therefore, as Ireland is predominantly a pasture-based livestock system (Shalloo et al., 2004), the vast majority of cattle outdoors during the summer period. Outdoor grazing may, however, present a greater opportunity for trans-boundary transmission of BVDV between neighbouring farms. In addition, livestock movements have been associated with increased risk of disease spread (Gates et al., 2013), and as many landholdings in Ireland are highly fragmented (O'Donnell et al., 2008), livestock movements over the summer months will increase.
With regard to application of a suitable disease monitoring strategies in regions of high bulk milk seroprevalence for BVDV and BoHV-1, testing of more than a single annual sample in bulk milk seropositive herds would appear excessive in the short to medium term. More frequent bulk milk surveillance would, however, be of benefit in BVDV antibody negative herds, and possibly those herds in the lowest positive quartile (Fig. 4; 55-78% inhibition), especially over the summer months, to allow rapid intervention in cases of viral introduction.
There is value in documenting concurrent exposure to both BVDV and BoHV-1, with studies such as that undertaken by Risalde et al. (2013) outlining an increased severity in BoHV-1-associated tissue lesions in the presence of sub-clinical BVDV infection. The present study has highlighted, however, that although generation of concurrent disease status data is useful, application of a single surveillance strategy for both is not appropriate. The results presented here highlighted an expected association between BVD bulk milk antibody status and youngstock seropositivity based on previous
Table 6
Logistic regression analysis of BVD and IBR recent infection status (180 days and 270 days).
Dependent variable Odds ratio Confidence interval (95%) p value Model (p value) Model goodness of fit
Independent variable (p value)
BVD recent infection 180"
66-99cows vs. 31-65 cows 1.80 0.93,3.47 0.079d Herd size p = 0.606
>99cows vs. 31-65cows 1.88 1.01,3.49 0.046 Calving season
SA/YR vs. Spring 2.44 1.19,5.04 0.015 Annual BVD herd status
BVD positive vs. BVD negativec 4.86 1.79,13.15 0.002 (p = 0.0002)
BVD recent infection 270b
Region 2 vs. region 1 0.56 0.32,0.98 0.041 Region p = 0.970
SA/YR vs. Spring 2.39 1.15,4.96 0.019 Calving season
BVD positive vs. BVD negativec 5.06 1.68,15.31 0.004 Annual BVD herd status (p = 0.0001)
BoHV-1 recent circulation 180"
66-99cows vs. 31-65 cows 7.11 0.86,58.57 0.068d Herd size p = 0.615
>99cows vs. 31-65cows 8.35 1.07,65.04 0.043 (p = 0.005)
BoHV-1 recent circulation 270b
Region 2 vs. region 1 0.40 0.15,1.06 0.066d Region p = 0.202
>99cows vs. 31-65 cows 7.52 0.91,62.03 0.061d Herd size
>99cows vs. 66-99cows 5.79 0.72,46.85 0.100d (p = 0.034)
a Recent infection herd classification based on the presence of at least one seropositive weanling over 180 days of age. b Recent infection herd classification based on the presence of at least one seropositive weanling over 270 days of age. c BVDV annual mean herd status. d Included for the purposes of highlighting a trend.
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Logistic regression analysis of BVD and IBR vaccination status.
Model goodness of fit (p value)
Dependent variable Odds ratio Confidence interval (95%) p value Model
Independent variable (p value)
BVDV vaccination 66-99cows vs. 31-65 cows >99cows vs. 31-65cows BVD positive vs. BVD negative2 BoHV-1 positive vs. IBR negativeb BoHV-1 vaccinated vs. unvaccinated BoHV-1 vaccination 66-99cows vs. 31-65 cows >99cows vs. 31-65cows SA/YR vs. Spring BVD vaccinated vs. unvaccinated
2.08 1.11,3.89
2.61 1.40,4.88
4.12 1.87,9.07
1.79 0.96,3.32
2.91 1.06,8.00
7.74 0.96,62.55
15.11 1.98,115.36
2.40 0.94,6.12
3.63 1.32,9.93
a BVD annual mean herd status.
b BoHV-1 annual mean herd status.
c Included for the purposes of highlighting a trend.
0.023 Herd size p = 0.299
0.003 Annual BVD herd status
<0.001 Annual IBR herds status
0.065c IBR vaccination
0.039 (p < 0.0001)
0.055c Herd size p = 0.494
0.009 Calving season
0.067c BVD vaccination
0.012 (p < 0.0001)
investigations of spot testing (Houe et al., 2006). The use of BVD spot test samples as a convenience sample for detecting the presence of BoHV-1 carriers in a herd, however, is not useful, with no association between BoHV-1 seropositive weanlings and BoHV-1 bulk milk antibody status having been highlighted. The prevalence of BoHV-1 seropositive youngstock in both dairy and beef herds tends to be low (Guarino et al., 2008; Romero-Salas et al., 2013; Waldner and Kennedy, 2008), thereby reducing the usefulness of this management group as herd sentinels. For the dairy herd therefore, where marker vaccines are used, bulk milk antibody status alone acts as a sufficient surveillance tool.
Given the similar methods of transmission of BVDV and BoHV-1, the occurrence of regional differences in a small country such as Ireland (agricultural land base is five million hectares approximately), having a large cattle population of approximately six million bovines (CSO, 2007) and poor farm-level biosecurity (Sayers et al., 2013), is noteworthy. Historically, BoHV-1 infections were very much associated with respiratory disease in beef units as opposed to dairy enterprises. Region-1, although the least cattle dense region of Ireland with regard to dairy livestock, has a much higher proportion of beef cattle, and farmers in this region are less likely to implement quarantine for purchased stock (Sayers et al., 2013). Both factors may contribute to the higher probability of being BoHV-1 bulk milk positive in this region. A similar conclusion has been suggested by Dias et al. (2013) where a predominantly dairying region of Brazil had the lowest apparent prevalence of BoHV-1 than other regions examined. This differs from BVDV, where it is accepted that the prevalence of BVDV is influenced by the regional cattle density (Almeida et al., 2013; Garoussi et al., 2008; Handel et al., 2011; Houe et al., 1995), and increased prevalence of BVDV is associated with higher cattle densities, supporting the findings of this current study.
Regional effects were also noted in RVC herds in models including youngstock of >270 days of age, with Region-2 less likely to have a seropositive weanling than Region-1. Although logical for BoHV-1 based on bulk milk findings, the increased likelihood of Region-1 herds having RVC for BVDV seems counterintuitive, and Region-2 having higher BVD bulk milk antibody readings. As foetuses of seropositive dams are rarely infected with BVDV, however (Brownlie et al., 1998), higher bulk readings should indicate greater foetal protection in these herds, leading to fewer PIs born and less youngstock exposed to BVDV. These findings are supported by a recent examination of the herd-level risk factors associated with the presence of BVDV on Irish farms, with herds in Munster (Region-2) being significantly less likely to produce one or more virus positive calves than Connaught (Region-1) (Graham et al., 2013). The model which included youngstock >180 days old did not yield a regional effect and may reflect interference from BVDV MDAs which have a relatively long half-life (Fulton et al., 2004).
In agreement with a number of other national and international studies (Boelaert et al., 2005; Cowley et al., 2011; Raaperi et al., 2010; Solis-Calderon et al., 2003; Van Wuijckhuise et al., 1998; Woodbine et al., 2009), a significant association between BoHV-1 infection/exposure and herd size was documented in this study, though not all countries report such a finding (Billinis et al., 2005). Larger herds also recorded higher ELISA readings in the current study. This may be due to increased within-herd prevalence in these herds as Raaperi et al. (2010) reported higher numbers of seropositive animals in larger herds. Additionally, larger herds have more susceptible animals available to maintain infection and herd size is a cluster variable for several biosecurity risks such as increased purchase of animals and increased visitors (veterinary practitioners, technicians, contract workers), all of which will increase the risk of disease introduction and maintenance (Boelaert et al., 2005; van Schaik et al., 1998). It is interesting to note that Sayers et al. (2013) reported that larger herds in Ireland are more likely to join a herd health scheme, which may reflect an awareness of the increased disease risk on these farms. Additionally, larger herds in the current study were also more likely to vaccinate for BVDV and BoHV-1, again highlighting an awareness of the increased disease risk in these herds.
The use of spot testing of youngstock is a well-established method of documenting recent BVDV circulation in a herd (Houe et al., 2006) to overcome the drawbacks of bulk milk analysis. To increase the usefulness of bulk milk analysis alone in highlighting recent BVDV circulation, Thobokwe et al. (2004) suggested a revised BVD ELISA positive cut-off of 80% inhibition using the Pourquier ELISA, a cutoff applied in a subsequent economic analysis (Heuer et al., 2007). In order to examine the applicability of such a strategy in another jurisdiction, a Wilcoxon rank sum analysis was applied in the current study to highlight differences in bulk milk analysis between herds recording positive and negative spot tests. While the findings in part aligned with those of Thobokwe et al. (2004), in that use of an elevated positive cut-off of 80% would be applicable to BVDV unvaccinated herds at certain times of the year, its use for vaccinated herds in Ireland is not appropriate. Interpretation of spot testing was stricter in this current study, however, which may account for the difference between both studies.
5. Conclusion
Prevalence studies have an important role to play in highlighting the necessity, or otherwise, for disease control and eradication schemes. This study highlighted high levels of exposure to BVDV and BoHV-1 in Irish dairy herds and also the lack of dramatic seasonal differences in bulk milk ELISA results. In the short to medium term, analysis of a single annual bulk milk sample would appear a
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suitable surveillance strategy for both BVDV and BoHV-1 in Ireland, with spot testing required to highlight recent viral circulation in BVDV vaccinated herds.
Acknowledgements
This research is an output from Irish Dairy Levy funding. The authors wish to acknowledge the assistance of Sean Coughlan, Martin Burke, and Kevin Downing, ICBF in sourcing study herds and supplying herd data; Dan Collins, CVERA for mapping the location of study herds; Yris Bloemhoff in co-ordinating sample collection. Finally, the authors wish to wholeheartedly thank the participating farmers
Appendix: Supplementary material
Supplementary data to this article can be found online doi:10.1016/j.rvsc.2015.02.011.
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