Tracy A. Clegg

Using latent class analysis to estimate the test characteristics of the γ-interferon test, the single intradermal comparative tuberculin test and a multiplex immunoassay under Irish conditions

Considerable effort has been devoted to improving the existing diagnostic tests for bovine tuberculosis (single intradermal comparative tuberculin test [SICTT] and γ-interferon assay [γ-IFN]) and to develop new tests. Previously, the diagnostic characteristics (sensitivity, specificity) have been estimated in populations with defined infection status. However, these approaches can be problematic as there may be few herds in Ireland where freedom from infection is guaranteed. We used latent class models to estimate the diagnostic characteristics of existing (SICTT and γ-IFN) and new (multiplex immunoassay [Enferplex-TB]) diagnostic tests under Irish field conditions where true disease status was unknown. The study population consisted of herds recruited in areas with no known TB problems (2197 animals) and herds experiencing a confirmed TB breakdown (2740 animals). A Bayesian model was developed, allowing for dependence between SICTT and γ-IFN, while assuming independence from the Enferplex-TB test. Different test interpretations were used for the analysis: SICTT (standard and severe interpretation), γ-IFN (a single interpretation), and a range of interpretations for the Enferplex-TB (level-1 [high sensitivity interpretation] to level-5 [high specificity interpretation]). The sensitivity and specificity (95% posterior credibility intervals; 95% PCI) of SICTT[standard] relative to Enferplex-TB[level-1] and γ-IFN were 52.9-60.8% and 99.2-99.8%, respectively. Equivalent estimates for γ-IFN relative to Enferplex-TB[level-1] and SICTT were 63.1-70.1% and 86.8-89.4%, respectively. Sensitivity of Enferplex-TB[level-1] (95% PCI: 64.8-71.9%) was superior to the SICTT[standard], and specificity of the Enferplex-TB[level-5] was superior to γ-IFN (95% PCI: 99.6-100.0%). These results provide robust measures of sensitivity and specificity under field conditions in Ireland and suggest that the Enferplex-TB test has the potential to improve on current diagnostics for TB infection in cattle. The extent of that potential will be assessed in further studies.

Potential infection-control benefit for Ireland from pre-movement testing of cattle for tuberculosis

Pre-movement testing for bovine tuberculosis (BTB) was compulsory in Ireland until 1996. We determined the proportion of herd restrictions (losing BTB-free status) attributable to the recent introduction of an infected bovid; described events between restoration of BTB-free status (de-restriction) and the next herd-level test for BTB; estimated the proportion of undetected infected cattle present at de-restriction; identified high-risk movements between herds (movements most likely to involve infected cattle); and determined the potential yield of infected cattle discovered (or herds that would not lose their BTB-free status) by pre-movement testing, relative to the numbers of cattle and herds tested. We used national data for all 6252 herds with a new BTB restriction in the 12 months from 1 April 2003 and 3947 herds declared BTB-free in the 12 months from 1 October 2001. We identified higher-risk animals from our logistic generalized estimating-equation models. We attributed 6-7% of current herd restrictions to the recent introduction of an infected animal. There were considerable changes to herd structure between de-restriction and the next full-herd test, and infection was detected in 10% of herds at the first assessment (full-herd test or abattoir surveillance) following de-restriction. Following movement from a de-restricted herd, the odds of an animal being positive at the next test increased with increasing time in the source herd prior to movement, increasing time between de-restriction and the next full-herd test and increasing severity of the source herd restriction. The odds decreased with increasing size of the source herd. We estimated that 15.9 destination-herd restrictions per year could be prevented for every 10,000 cattle tested pre-movement and that 3.3 destination-herd restrictions per year could be prevented for every 100 source herds tested pre-movement. The yield per pre-movement test can be increased by focusing on high-risk movements; however, this would result in a substantial decrease in the total number of potential restrictions identified.

Prevalence of Coxiella burnetii (Q fever) antibodies in bovine serum and bulk-milk samples

Q fever (Coxiella burnetii) is a zoonotic disease of increasing public health importance. The objective of this study was to estimate the prevalence of, and risk factors associated with, exposure to C. burnetii in cattle in the Republic of Ireland. Bulk-tank milk samples from 290 dairy herds and 1659 sera from 332 dairy and beef herds, randomly sampled, were tested by indirect ELISA to detect antibodies to C. burnetii. In total, 37·9% of bulk-milk sample herds and 1·8% of sera (from 6·9% of herds) were antibody positive. Of risk factors tested using logistic regression analysis, only large herd size (bulk-milk analysis) and dairy breed (serum analysis) significantly increased the odds of being positive for antibodies to C. burnetii. Herds with positive milk or serum samples were randomly distributed throughout the Republic of Ireland and no clustering was observed. The use of an ELISA to test bulk-milk samples collected by randomized stratified sampling is a cost-effective method by which national herd prevalence can be estimated by active surveillance.

Prevalence and distribution of paratuberculosis (Johne's disease) in cattle herds in Ireland.

A simple random survey was conducted in Ireland during 2005 to estimate the ELISA-prevalence of paratuberculosis, commonly called Johnes disease (JD), in the cattle population. Serum samples were collected from all 20,322 females/breeding bulls over 12 months-of-age in 639 herds. All samples were tested using a commercially available absorbed ELISA. The overall prevalence of infected herds, based on the presence of at least one ELISA-positive animal, was 21.4% (95% CI 18.4%-24.9%). Herd prevalence levels amongst dairy herds (mean 31.5%; 95% CI: 24.6%, 39.3%) was higher than among beef herds (mean 17.9%; 95% CI: 14.6%-21.8%). However, the animal level prevalence was similar. The true prevalence among all animals tested, was calculated to be 2.86% (95%CI: 2.76, 2.97) and for animals >= 2 yrs, it was 3.30% (95%CI: 3.17, 3.43). For animals in beef herds, true prevalence was 3.09% (95%CI: 2.93, 3.24), and for those in dairy herds, 2.74% (95%CI: 2.59, 2.90). The majority of herds had only one ELISA-positive infected animal. Only 6.4% (95% CI 4.7%-8.7%) of all herds had more than one ELISA-positive infected animal; 13.3% (CI 8.7%-19.7%) of dairy herds ranging from two to eight ELISA-positive infected animals; and, 3.9% beef herds (CI 2.4%-6.2%) ranging from two to five ELISA-positive infected animals. The true prevalence of herds infected and shedding Mycobacterium avium subspecies paratuberculosis is estimated to be 9.5% for all herd types; 20.6% for dairy herds; and 7.6% for beef herds. If ELISA positive animals <2-years-of-age are excluded, the true herd prevalene reduces to: 9.3% for all herd types; 19.6% for dairy herds; and 6.3% for beef herds based on a test specificity (Sp) of 99.8% and test sensitivity (Se) (i.e., ability to detect culture-positive, infected animals shedding at any level) of 27.8-28.9%.

Risk factors associated with increased mortality of farmed Pacific oysters in Ireland during 2011

The Pacific oyster, Crassostrea gigas, plays a significant role in the aquaculture industry in Ireland. Episodes of increased mortality in C. gigas have been described in many countries, and in Ireland since 2008. The cause of mortality events in C. gigas spat and larvae is suspected to be multifactorial, with ostreid herpesvirus 1 (OsHV-1, in particular OsHV-1μvar) considered a necessary, but not sufficient, cause. The objectives of the current study were to describe mortality events that occurred in C. gigas in Ireland during the summer of 2011 and to identify any associated environmental, husbandry and oyster endogenous factors. A prospective cohort study was conducted during 2010-2012, involving 80 study batches, located at 24 sites within 17 bays. All 17 bays had previously tested positive for OsHV-1μvar. All study farmers were initially surveyed to gather relevant data on each study batch, which was then tracked from placement in the bay to first grading. The outcome of interest was cumulative batch-level mortality (%). Environmental data at high and low mortality sites were compared, and a risk factor analysis, using a multiple linear regression mixed effects model, was conducted. Cumulative batch mortality ranged from 2% to 100% (median=16%, interquartile range: 10-34%). The final multivariable risk factor model indicated that batches imported from French hatcheries had significantly lower mortalities than non-French hatcheries; sites which tested negative for OsHV-1μvar during the study had significantly lower mortalities than sites which tested positive and mortalities increased with temperature until a peak was reached. There were several differences between the seed stocks from French and non-French hatcheries, including prior OsHV-1μvar exposure and ploidy. A range of risk factors relating to farm management were also considered, but were not found significant. The relative importance of prior OsHV-1μvar infection and ploidy will become clearer with ongoing selection towards OsHV-1μvar resistant oysters. Work is currently underway in Ireland to investigate these factors further, by tracking seed from various hatchery sources which were put to sea in 2012 under similar husbandry and environmental conditions.

Aspects of bovine herpesvirus-1 infection in dairy and beef herds in the Republic of Ireland

BackgroundInfection with bovine herpesvirus-1 (BHV-1) causes a wide range of disease manifestations, including respiratory disease and abortion, with world-wide distribution. The primary objective of the present study was to describe aspects of BHV-1 infection and control on Irish farms, including herd-level seroprevalence (based on pooled sera) and vaccine usage.MethodsThe characteristics of a diagnostic indirect BHV-1 antibody ELISA test when used on serum pools were evaluated using laboratory replicates for use in the seroprevalence study. The output from this indirect ELISA was expressed as a percentage positivity (PP) value. A proposed cut off (PCO) PP was applied in a cross-sectional study of a stratified random sample of 1,175 Irish dairy and beef cattle herds in 2009, using serum pools, to estimate herd seroprevalence. The study was observational, based primarily on the analysis of existing samples, and only aggregated results were reported. For these reasons, ethical approval was not required. Bulk milk samples from a subset of 111 dairy herds were analysed using the same ELISA. Information regarding vaccine usage was determined in a telephone survey.ResultsA PCO PP of 7.88% was determined to give 97.1% sensitivity and 100% specificity relative to the use of the ELISA on individual sera giving maximization of the prevalence independent Youdens index, on receiver operating characteristics analysis of replicate results. The herd-level BHV-1 seroprevalence was 74.9% (95% CI - 69.9%-79.8%), with no significant difference between dairy and beef herds. 95.5% agreement in herd classification was found between bulk milk and serum pools. Only 1.8 percent of farmers used BHV-1 marker vaccine, 80% of which was live while 75% of vaccinated herds were dairy.A significant association was found between herd size (quartiles) and seroprevalence (quartiles).ConclusionsThe results from this study indicate BHV-1 infection is endemic, although BHV-1 vaccines are rarely used, in the cattle population in Ireland.

Herd-level factors associated with the presence of bovine viral diarrhoea virus in herds participating in the voluntary phase of the Irish national eradication programme

A risk factor study was conducted to identify variables associated with initial positive or inconclusive results for bovine viral diarrhoea virus (BVDV) in ear punch samples collected from calves between 1st January and 15th July 2012 (the study period) as part of the voluntary phase of the Irish national BVD eradication programme based on testing of ear tag tissue samples from calves born in participating herds. Univariable analysis indicated significant associations with the following factors: herd type; the number of cows in the herd; the number of calves born in the study period; the number of calves tested in the study period; the number of cattle purchased in 2011, between 2009 and 2011 and between 2007 and 2011; the number of tested calves whose dams had been purchased within 9 months of their calving date; and the percentage of calf mortality within 28 days of birth. The percentage of the cows in each herd that was homebred, location (province) the number of separate land parcels used by each herd, the presence of an associated sheep enterprise and the purchase of cattle through marts were not found to be significant. An initial logistic regression model was developed to model the probability of a herd having one or more BVD virus-positive or inconclusive calves. When vaccination status was initially excluded, province, log of the numbers of cows in the herd, the number of cows purchased between 2009 and 2011, the number of tested calves whose dams had been purchased within 9 months of their calving date and calf mortality were significant. When vaccination status was included, using a subset of the data based on farmers responding to a survey on vaccination status, it was retained as a significant variable along with the same variables already listed, showing a significant 2-way interaction with the log of the number of cows. There was not a significant association between an initial positive or inconclusive result and the length of time for which herds had been vaccinating. The study provides a series of key communication messages relating to both the delivery of, and benefits from, the national eradication programme.

Comparison of the Immulite and RIA assay methods for measuring peripheral blood progesterone levels in Greyhound bitches

Determination of optimal breeding time in bitches earmarked for single insemination only is based on measurement of peripheral blood serum or plasma progesterone concentration. In this paper a comparison is made between radioimmune assay (RIA) and chemoluminescent assay (Immulite) for determination of P4 concentrations in the bitch. The Immulite assay is shown to be an accurate and reliable method for serum or plasma P4 measurement. It compares favourably with other methods in terms of turn-around time, cost and accessibility for veterinarians in practice.

The comparative performance of the single intradermal comparative tuberculin test in Irish cattle, using tuberculin PPD combinations from different manufacturers

Ireland currently obtains its avian and bovine tuberculin purified protein derivatives (PPDs) from a single source. Because problems of supply or quality cannot be discounted, it is prudent that Ireland identify alternative supplier(s) as part of a broad risk management strategy. Therefore, the aim of this study was to compare the performance of a number of different tuberculin combinations (that is, pairings of bovine and avian PPD; with different manufacturers) in the single intradermal comparative tuberculin test (SICTT), as currently performed in Ireland. The study was randomised, controlled and double-blinded. A total of 2172 cattle were used in the study. Each animal was tested using two SICTTs, the first based on the tuberculin combination in current use, and the second using one of six trial tuberculin combinations. Analyses were conducted to compare both reactor-status and skin increase. For each control/trial tuberculin combination, there was good agreement between the control and trial reactor-status. Differences in skin increases were mainly confined to animals categorised as either negative or severe inconclusive. However, the measured differences were minor, and unlikely to have a significant impact on the actual test outcome, either for individual animals or for herds. In conclusion, while further studies determining sensitivity and specificity in Ireland would have to be done in the event of a change in tuberculin PPD there should be minimal disruption of the national programme if alternative tuberculin PPDs meeting WHO, OIE and EU regulations were used. In this study, the precision of the guinea pig bio-assay to assess tuberculin potency was low and therefore Ireland should maintain its practice of periodically assessing potency in naturally infected cattle, even though this is not currently required under WHO, OIE or EU Regulations.

Bovine viral diarrhoea virus seroprevalence and vaccination usage in dairy and beef herds in the Republic of Ireland

BackgroundBovine viral diarrhoea (BVD) is an infectious disease of cattle with a worldwide distribution. Herd-level prevalence varies among European Union (EU) member states, and prevalence information facilitates decision-making and monitoring of progress in control and eradication programmes. The primary objective of the present study was to address significant knowledge gaps regarding herd BVD seroprevalence (based on pooled sera) and control on Irish farms, including vaccine usage.MethodsPreliminary validation of an indirect BVD antibody ELISA test (Svanova, Biotech AB, Uppsala, Sweden) using pooled sera was a novel and important aspect of the present study. Serum pools were constructed from serum samples of known seropositivity and pools were analysed using the same test in laboratory replicates. The output from this indirect ELISA was expressed as a percentage positivity (PP) value. Results were used to guide selection of a proposed cut-off (PCO) PP. This indirect ELISA was applied to randomly constructed within-herd serum pools, in a cross-sectional study of a stratified random sample of 1,171 Irish dairy and beef cow herds in 2009, for which vaccination status was determined by telephone survey. The herd-level prevalence of BVD in Ireland (percentage positive herds) was estimated in non-vaccinating herds, where herds were classified positive when herd pool result exceeded PCO PP. Vaccinated herds were excluded because of the potential impact of vaccination on herd classification status. Comparison of herd-level classification was conducted in a subset of 111 non-vaccinating dairy herds using the same ELISA on bulk milk tank (BMT) samples. Associations between possible risk factors (herd size (quartiles)) and herd-level prevalence were determined using chi-squared analysis.ResultsReceiver Operating Characteristics Analysis of replicate results in the preliminary validation study yielded an optimal cut-off PP (Proposed Cut-off percentage positivity - PCO PP) of 7.58%. This PCO PP gave a relative sensitivity (Se) and specificity (Sp) of 98.57% and 100% respectively, relative to the use of the ELISA on individual sera, and was chosen as the optimal cut-off since it resulted in maximization of the prevalence independent Youden’s Index.The herd-level BVD prevalence in non-vaccinating herds was 98.7% (95% CI - 98.3-99.5%) in the cross-sectional study with no significant difference between dairy and beef herds (98.3% vs 98.8%, respectively, p = 0.595).An agreement of 95.4% was found on Kappa analysis of herd serological classification when bulk milk and serum pool results were compared in non-vaccinating herds. 19.2 percent of farmers used BVDV vaccine; 81% of vaccinated herds were dairy. A significant association was found between seroprevalence (quartiles) and herd size (quartiles) (p < 0.01), though no association was found between herd size (quartiles) and herd-level classification based on PCO (p = 0.548).ConclusionsThe results from this study indicate that the true herd-level seroprevalence to Bovine Virus Diarrhoea (BVD) virus in Ireland is approaching 100%. The results of the present study will assist with national policy development, particularly with respect to the national BVD eradication programme which commenced recently.