Optimising and Evaluating the Characteristics of a Multiple Antigen ELISA for Detection of Mycobacterium bovis Infection in a Badger Vaccine Field Trial
Aznar, I. ; Frankena, K. ; More, S.J. ; Whelan, C. ; Martin, W. ; Gormley, E. ; Corner, L.A.L. ; Murphy, D. ; Jong, M.C.M. de - \ 2014
PLoS ONE 9 (2014)7. - ISSN 1932-6203
gamma-interferon assay - meles-meles - endobronchial inoculation - experimental tuberculosis - protective immunity - cattle herds - bcg - sensitivity - challenge - pathology
A long-term research programme has been underway in Ireland to evaluate the usefulness of badger vaccination as part of the national bTB (bovine tuberculosis) control strategy. This culminated in a field trial which commenced in county Kilkenny in 2009 to determine the effects of badger vaccination on Mycobacterium bovis transmission in badgers under field conditions. In the present study, we sought to optimise the characteristics of a multiplex chemiluminescent assay for detection of M. bovis infection in live badgers. Our goal was to maximise specificity, and therefore statistical power, during evaluation of the badger vaccine trial data. In addition, we also aimed to explore the effects of vaccination on test characteristics. For the test optimisation, we ran a stepwise logistic regression with analytical weights on the converted Relative Light Units (RLU) obtained from testing blood samples from 215 badgers captured as part of culling operations by the national Department of Agriculture, Food and the Marine (DAFM). The optimised test was applied to two other datasets obtained from two captive badger studies (Study 1 and Study 2), and the sensitivity and specificity of the test was attained separately for vaccinated and non-vaccinated badgers. During optimisation, test sensitivity was maximised (30.77%), while retaining specificity at 99.99%. When the optimised test was then applied to the captive badger studies data, we observed that test characteristics did not vary greatly between vaccinated and non-vaccinated badgers. However, a different time lag between infection and a positive test result was observed in vaccinated and non-vaccinated badgers. We propose that the optimized multiplex immunoassay be used to analyse the vaccine trial data. In relation to the difference in the time lag observed for vaccinated and non-vaccinated badgers, we also present a strategy to enable the test to be used during trial evaluation.
Molecular typing of Dutch isolates of Xanthomonas arboricola pathovar pruni isolated from ornamental cherry laurel
Bergsma-Vlami, M. ; Martin, W. ; Koenraadt, H. ; Teunissen, H. ; Pothier, J.F. ; Duffy, B. ; Doorn, J. van - \ 2012
Journal of Plant Pathology: rivista di patologia vegetale 94 (2012)Supp.1. - ISSN 1125-4653 - p. S29 - S35.
pectate lyase secretion - acutatum-sensu-lato - colletotrichum-acutatum - c-gloeosporioides - sp-nov - fruit - diversity - infection - susceptibility - pathogenicity
Xanthomonas arboricola pv. pruni (Xap) has been found in several cherry laurel (Prunus laurocerasus) nurseries in the Netherlands, causing leaf spot. As no information is available yet about the epidemiology of this quarantine bacterium in cherry laurel, molecular typing of Xap isolates can considerably improve our understanding of pathogen spread between various cherry laurel production systems in different regions of the Netherlands and pathogen relatedness among different disease outbreaks. In this study, the genotypic diversity within a population of 25 Xap isolates isolated from different cherry laurel cultivars grown in different locations in the Netherlands between 2008-2010, was assessed using Multiple-locus variable-number analysis (MLVA). The identity of these Xap isolates was initially determined based on the EPPO standard PM 7/64. Confirmation of the identity of these Xap isolates was additionally achieved with diverse methodologies, including gyrase subunit B (gyrB) sequence typing, BOXand ERIC-PCR, AFLP, and Xap-specific PCR’s: one based on the previously described Pagani primers (2004) (conventional-PCR and its TaqMan-PCR variant) and one based on the recently described Pothier primers (2011c). Based on the results of the MLVA analysis, the Dutch population of Xap isolates could be divided into two groups; however no correlation with the geographical origin or any other character of these isolates could be established. Additionally, based on colony morphology, a panel of 5 look-a-likes were isolated from symptomatic leaves of P. laurocerasus which reacted in the Xap-specific PCR described by Pagani (2004) but that did not react in the Xap-specific PCR described by Pothier et al. (2011c). Further characterisation of these look-a-like isolates with AFLP, BOXand ERIC-PCR, and gyrB sequencing showed that the Xap-specific PCR described by Pagani does not discriminate between Xap and the look-a-like isolates. Similarly to Pagani PCR, the performance of a pathogenicity test with a pure culture of the isolate was not always discriminative between Xap and the look-a-like isolates, unraveling a complexity in Xanthomonas pathogenicity. Therefore, in routine screening based on the EPPO standard PM 7/64, complementary techniques such as BOX- ERIC-PCR, gyrB sequencing, Xap-specific PCR described by Pothier (2011c), MLVA and AFLP should be used to obtain a reliable diagnosis of Xap and avoid false positive results.
Relative effectiveness of Irish factories in the surveillance of slaughtered cattle for visible lesions of tuberculosis, 2005-2007
Olea-Popelka, F. ; Freeman, Z. ; White, P. ; Costello, E. ; O'Keeffe, J. ; Frankena, K. ; Martin, W. ; More, S. - \ 2012
Irish Veterinary Journal 65 (2012). - ISSN 2046-0481
Background In Ireland, every animal is examined at slaughter for its fitness for human consumption. The aim of this study was to determine the relative effectiveness of factories in submitting and subsequently in having suspect lesions confirmed as bovine tuberculosis (TB) lesions during the years 2005-2007. This work provides an update from previously published data for years 2003-2004. During 2005-2007 data were available on 4,401,813 cattle from attested herds (i.e. herds classified free of bovine TB), from which data for potential confounding factors were available for 3,344,057 slaughtered animals at one of the 37 export-licensed factories. Findings From these animals, 8,178 suspect lesions were submitted for laboratory confirmation. Lesions from 5,456 (66.7%) animals tested as positive, and 269 (3.2%) were inconclusive for bovine TB. Logistic regression was used to determine adjusted submission and confirmation risks for each factory while controlling for confounding factors. Factory rankings based on adjusted and crude risks were similar. The average crude submission risk for all the factories was 25 lesions per 10,000 animals slaughtered, ranging from 0 to 52. The crude confirmation risk varied between 30.3% and 91.3%. Conclusions Substantial variation in the effectiveness of lesion submission and subsequent confirmation as bovine TB was found among the 37 factories. Compared to previous years (2003-2004), there was an increased bovine TB lesion submission and confirmation risk. Continued monitoring of the effectiveness of slaughter surveillance in Ireland is recommended; emphasis should be placed on efforts to improve bovine TB surveillance in factories with lower rankings.
Trial design to estimate the effect of vaccination on tuberculosis incidence in badgers
Aznar, I. ; McGrath, G. ; Murphy, D. ; Corner, L.A.L. ; Gormley, E. ; Frankena, K. ; More, S.J. ; Martin, W. ; O'Keeffe, J. ; Jong, M.C.M. de - \ 2011
Veterinary Microbiology 151 (2011)1-2. - ISSN 0378-1135 - p. 104 - 111.
formulation induces resistance - mycobacterium-bovis bcg - bacille calmette-guerin - brushtail possums - oral vaccination - meles-meles - pulmonary tuberculosis - trichosurus-vulpecula - protective immunity - southwest england
The principal wildlife reservoir of Mycobacterium bovis in Ireland is the European badger. Studies in the Republic of Ireland (RoI) have shown that badgers culled in association with cattle herd tuberculosis breakdowns (focal culling) have a higher prevalence of infection than the badger population at large. This observation is one rationale for the medium term national strategy of focal badger culling. A vaccination strategy for the control of bovine tuberculosis (bTB) in badgers is a preferred long-term option. The Bacillus Calmette-Guérin (BCG) vaccine has been shown to decrease disease severity in captive badgers under controlled conditions. As the vaccine has been tested in a controlled environment with precise information on infection pressure, it cannot be assumed a priori that the effects of vaccination are similar in the wild, where other environmental and/or ecological factors prevail. For this reason we have designed a vaccine field trial to assess the impact of vaccination on the incidence of TB infection in a wild badger population. The selected study area for the vaccine trial (approximately 755 square kilometers) is divided into three zones each of which has similar characteristics in terms of size, number of main badger setts, cattle herds, cattle and land classification type. Three vaccination levels (100%, 50% and 0%) will be allocated to the three zones in a way that a gradient of vaccination coverage North to South is achieved. The middle zone (zone B) will be vaccinated at a 50% coverage but zone A and C will be randomly allocated with 100% or 0% vaccination coverage. Vaccination within zone B will be done randomly at individual badger level. The objective of this paper is to describe the design of a field tuberculosis vaccination trial for badgers, the epidemiological methods that were used to design the trial and the subsequent data analysis. The analysis will enable us to quantify the magnitude of the observed vaccination effect on M. bovis transmission in badgers under field conditions and to improve our knowledge of the biological effects of vaccination on susceptibility and infectiousness.