Study farm selection
The study was carried out in 2009 and licensed by the Irish Department of Health and Children 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 has previously been outlined [15, 16]. Briefly, 500 herds from the Irish Cattle Breeding Federations (ICBF) database were invited to participate. Farms were randomly selected on the basis of a two-tier stratification protocol based on geographical location and herd size. All farms selected for inclusion were dairy farms and were registered on the ICBF’s database (a database of over 3,500 Irish dairy herds). Geographical regions were chosen based on the Irish Central Statistics Office (CSO) standard regions. Results of the national farm census conducted in 2000 were used to select farm size categoriesFootnote 1.
Sample collection and preparation
Study farms were visited to sample all bulls involved in the dairy breeding process. Bulls being raised for beef production were not included. Samples were taken by coccygeal venepuncture into plain glass vacutainers. Each sample was centrifuged at 5000 g for 5 min, serum aspirated and samples stored at −20 °C until analysed. Subsequently, a 100 μl aliquot of each serum sample was pooled in batches of ten for BVD virus testing. In addition, four bulk milk samples, and blood samples from 20 % of the replacement heifer group with a minimum of five heifers less than 9 months were sampled from each herd over the 2009 lactation These were used to determine herd BVD and BoHV-1 serostatus, both historical (bulk milk) and recent seroconversion (weanlings) [16].
Sample testing
Pooled serum samples were tested for the presence of BVD virus by commercial laboratory (Enfer Labs Ltd, Ireland) using a Real Time–Polymerase Chain Reaction (RT-PCR). The detection limit of this assay was 50 to 100 viral particles in 1 ml whole blood [17]. Where a pooled serum batch yielded a PCR positive result, individual samples were re-tested, again by PCR, to identify the positive sample within the batch. Positive samples were tested individually to confirm the virus-positive status of each animal. Sera were tested for BoHV-1 antibody using the appropriate ELISA method depending on individual vaccination status (for bulls status) and herd vaccination status (for herd status) i.e. IBRgB (Ultrapurified BoHV-1 lysate, Institut Pourquier, France) in BoHV-1 unvaccinated bulls/herds and IBRgE, (IDEXX laboratories, USA) in BoHV-1 vaccinated bulls/herds. Serum ELISA results were classified as positive or negative following kit-manufacturer positive cut-off values. BoHV-1 analyses were completed by commercial laboratories; BoHV-1 lysate by National Milk Laboratories Ltd. (NML UKAS) (UK), and BoHV-1 gE by Enfer Diagnostics Ltd. (Ireland, ISO 1509001/2000).
Descriptive data on the use of stock bulls on Irish dairy farms
Herds
Herds were classified according to a previous study [16] in different categories in relation to the calving season (spring-calving vs. non-spring-calving), number of bulls per farm (no bull vs. 1 bull vs. >1 bull), region (high density dairy vs. low density dairy), herd size (31–65 cows vs. 66–99 cows vs. >99 cows), type of farming enterprise (dairy livestock only vs. mixed livestock (farms with other cattle enterprises)), herd vaccination status (vaccinated vs. unvaccinated). Farms were also classified with regard to the origin of the bulls on each farm i.e. all bulls homebred vs. at least 1 bull purchased.
With regard to disease status, two herd classification methods were employed. Firstly herds were classified as seropositive or seronegative for each of the diseases BVD or BoHV-1 according bulk milk ELISA results [16]. For the purpose of this study, seasonal trends in bulk milk ELISA readings were not taken into account. Instead the average of all four ELISA readings was used for herd classification.
Secondly, herds were classified on the basis of having ‘evidence of recent viral circulation’ i.e. those herds having at least one weanling serologically positive for either BVDV or BoHV-1. Herds that did not record a positive weanling were classified as ‘not having evidence of recent viral circulation’ [16].
Finally, herds were classified according to ‘overall bull BoHV-1 status’, positive herds having at least one serologically positive bull in the farm.
Stock bulls
A questionnaire survey was used to determine the IBR vaccination status of each study farm, the vaccine product used, and the date the vaccine was administered. In the case of purchased bulls, the IBR vaccination history prior to purchase was not available. Any purchased bull testing positive for IBRgB, therefore, was re-tested using the IBRgE assay, as only marked vaccines (gE deleted) are available in republic of Ireland, in order to rule out prior vaccination as the source of seropositivity.
Bulls were categorised on the basis of breed and herd of origin (homebred vs. purchased). As a number of homebred bulls had moved off and back to the herd of origin, bulls were also classified according to their number of off-farm movements prior the time of sampling. Other categories included vasectomized status (vasectomized [Vas] vs. not vasectomized [NVas]), BoHV-1 individual vaccination status (vaccinated [Vacc] vs. unvaccinated [Unvacc]), and region in which the study herd was located (Region 2: high density dairy vs. Region 1: low density dairy). With regard to disease status, all individual animals were classified as positive or negative for BVD virus or BoHV-1 antibody.
Movement data of stock bulls
The national identification ear-tag number of each bull sampled was recorded during the farm visit. The availability of this unique and fully traceable animal identifier allowed specific demographic information on each bull to be sourced through the ICBF database. In addition, as all cattle movements in Ireland require a movement permit which is recorded to a central database, the number of between farm movements that each bull made prior to sampling were studied retrospectively.
Data analysis
Descriptive analyses were completed in Excel (MS Office 2010). Pearson’s chi-squared, Fisher’s exact, univariable and multivariable logistic regression, were carried out using Stata (Version 11).
Number of bulls per farm, number of vasectomized bulls, number of bulls per farm across regions, and number of non-homebred bulls was investigated. The age of each bull on the date of sampling was calculated, as well as the number of total movements recorded per bull and the time spent in the study herd prior to sampling.
Factors associated with BVD and BoHV1 status of stock bulls
Examination of relationships between BoHV-1 seropositivity of individual bulls and a number of independent variables was completed. Independent variables investigated comprised the age of the bulls at the sampling time, movements between farms pre-sampling, herd of origin (purchased vs. homebred) and reproductive use of the bulls (vasectomized vs. no vasectomized). Only BoHV-1 seropositivity of individual bulls was investigated using logistic regression due to the small number of BVD virus-positive bulls.
Factors associated with dairy herd status for BVD and BoHV1
Prior the construction of the final regression model an univariable analysis was completed. 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.
Logistic regression was used to examine the relationships of BVD herd status, BoHV-1 herd status, and evidence of recent viral circulation (categorical variables), as the dependent variables by a number of independent variables. Independent variables included having a bull, number of bulls, herd of origin of bulls and having vasectomized bulls in the farm. To investigate the relationships between the purchase of bulls and the herd status only herds with purchased bulls that had spent at least 3 months in the farm prior the sampling were considered.