Identification of risk factors associated with disclosure of false positive bovine tuberculosis reactors using the gamma-interferon (IFNγ) assay

The gamma-interferon assay (IFNγ) is often used as an ancillary diagnostic test alongside the tuberculin skin test in order to detect Mycobacterium bovis infected cattle. The performance of the IFNγ test has been evaluated in many countries worldwide and wider usage as a disease surveillance tool is constrained due to the relatively low and inconsistent specificity at a herd and area level. This results in disclosure of a higher proportion of false positive reactors when compared with the skin test. In this study, we used cohorts of animals from low prevalence tuberculosis herds (n = 136) to assess a range of risk factors that might influence the specificity of the test. Univariate and multivariate logistic generalised estimating-equation (GEE) models were used to evaluate potential risk factors associated with a false positive IFNγ test result. In these herds, the univariate model revealed that the region of herd origin, the time of year when the testing was carried out, and the age of the animal were all significant risk factors. In the final multivariate models only animal age and region of herd origin were found to be significant risk factors. A high proportion of herds with multiple IFNγ false positive animals were located in one county, with evidence of within-herd clustering, suggesting a localised source of non-specific sensitization. Knowledge of the underlying factors influencing the IFNγ test specificity could be used to optimize the test performance in different disease level scenarios in order to reduce the disclosure rate of false positive reactors.Deposited by bulk impor

Characteristics of Mycobacterium bovis infected herds tested with the interferon-gamma assay

The IFN-γ (interferon gamma) assay is used in Ireland as an ancillary diagnostic test to the single intradermal comparative tuberculin test (SICTT) to maximise the detection of Mycobacterium bovis infected animals (bTB) in cattle herds. Understanding the relationships between herd and animal risk factors and IFN-γ test results is critical to enable the development and evaluation of policy measures on how best to use the test. In this study, we set out to characterise Irish herds with IFN-γ test positive animals in terms of herd size, number of SICTT reactors and number of IFN-γ positive tests, and to evaluate the IFN-γ test in terms of the test cut-off values. The results showed that larger herds with more SICTT reactors were likely to have more IFN-γ positives in the herd, and herds with an IFN-γ test positive animal that was also positive for bTB lesions at post-mortem had higher numbers of IFN-γ positive animals in the herd. Raising the cut-off values for the IFN-γ test only marginally decreased the combined sensitivity of the IFN-γ and the SICTT for diagnosis of bTB lesioned animals. The analysis has provided valuable information on the performance of the IFN-γ test as it is used under current bTB infection levels in Ireland.Department of Agriculture, Food and the Marin

Tuberculosis in cattle and its control: limitations to the use of the interferon-gamma assay in attested herds

Department of Agriculture, Food and the MarineTeagascDeposited by bulk impor

The performance of the interferon gamma assay when used as a diagnostic or quality assurance test in Mycobacterium bovis infected herds

There are two different contexts in the Irish bTB eradication programme in which the interferon-gamma assay (IFN-γ) is applied. Firstly, the IFN-γ assay is applied routinely to high risk cohorts in herds with four or more reactors to the SICTT. The IFN-γ test is then carried out on blood samples submitted to the laboratory within 8 h of collection (diagnostic testing). Secondly, the use of the IFN-γ assay has recently been extended to test SICTT reactors as part of a general quality assurance (QA) scheme to monitor the performance of the SICTT. Blood samples from reactors are tested one day after blood collection (QA testing). In this study, we analysed the relative performance of the SICTT and IFN-γ when used in parallel as an 8 h diagnostic test and as a 24 h QA test on SICTT reactors. A total of 17,725 IFN-γ tests were included in the analysis (11,658 diagnostic tests and 6067 QA tests). Of the samples submitted for diagnostic testing, the proportion positive to IFN-γ decreased with the severity of interpretation of the SICTT result. Of the standard reactors that were tested with IFN-γ in the QA programme, 92.2% were positive to the IFN-γ test. Among animals that were SICTT −ve/IFN-γ +ve, 18.9% were positive at post-mortem compared to 11.8% of those that were SICTT +ve (standard reactor)/IFN-γ −ve. These results highlight the risk associated with retaining SICTT −ve/IFN-γ +ve animals, and suggest that prompt removal of these animals is necessary to reduce the potential for future transmission

Antigen-specific IP-10 release is a sensitive biomarker of mycobacterium bovis infection in cattle

CITATION: Parsons, S. D. C., et al. 2016. Antigen-specific IP-10 release is a sensitive biomarker of mycobacterium bovis infection in cattle. PLoS ONE, 11(5):e0155440, doi:10.1371/journal.pone.0155440.The original publication is available at http://journals.plos.org/plosoneThe most widely used ante-mortem diagnostic tests for tuberculosis in cattle are the tuberculin skin test and the interferon-gamma (IFN-γ) release assay, both of which measure cell-mediated immune responses to Mycobacterium bovis infection. However, limitations in the performance of these tests results in a failure to identify all infected animals. In attempting to increase the range of diagnostic tests for tuberculosis, measurement of the cytokine IP-10 in antigen-stimulated blood has previously been shown to improve the detection of M. tuberculosis and M. bovis infection, in humans and African buffaloes (Syncerus caffer), respectively. In the present study, 60 cattle were identified by the single intradermal comparative tuberculin test as tuberculosis reactors (n = 24) or non-reactors (n = 36) and the release of IFN-γ and IP-10 in antigen-stimulated whole blood from these animals was measured using bovine specific ELISAs. There was a strong correlation between IP-10 and IFN-γ production in these samples. Moreover, measurement of the differential release of IP-10 in response to stimulation with M. bovis purified protein derivative (PPD) and M. avium PPD distinguished between reactor and non-reactor cattle with a sensitivity of 100% (95% CI, 86%–100%) and a specificity of 97% (95% CI, 85%–100%). These results suggest that IP-10 might prove valuable as a diagnostic biomarker of M. bovis infection in cattle.http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0155440Publisher's versio

Correlation between IFN-γ and IP-10 release.

<p>Whole blood from non-reactor and reactor cattle was stimulated with either <i>M</i>. <i>bovis</i> PPD or <i>M</i>. <i>avium</i> PPD for 24 h at 37°C. The release of IFN-γ and IP-10 was highly correlated in these samples (r = 0.65; p < 0.0001).</p

Antigen-Specific IP-10 Release Is a Sensitive Biomarker of <i>Mycobacterium bovis</i> Infection in Cattle

<div><p>The most widely used ante-mortem diagnostic tests for tuberculosis in cattle are the tuberculin skin test and the interferon-gamma (IFN-γ) release assay, both of which measure cell-mediated immune responses to <i>Mycobacterium bovis</i> infection. However, limitations in the performance of these tests results in a failure to identify all infected animals. In attempting to increase the range of diagnostic tests for tuberculosis, measurement of the cytokine IP-10 in antigen-stimulated blood has previously been shown to improve the detection of <i>M</i>. <i>tuberculosis</i> and <i>M</i>. <i>bovis</i> infection, in humans and African buffaloes (<i>Syncerus caffer</i>), respectively. In the present study, 60 cattle were identified by the single intradermal comparative tuberculin test as tuberculosis reactors (n = 24) or non-reactors (n = 36) and the release of IFN-γ and IP-10 in antigen-stimulated whole blood from these animals was measured using bovine specific ELISAs. There was a strong correlation between IP-10 and IFN-γ production in these samples. Moreover, measurement of the differential release of IP-10 in response to stimulation with <i>M</i>. <i>bovis</i> purified protein derivative (PPD) and <i>M</i>. <i>avium</i> PPD distinguished between reactor and non-reactor cattle with a sensitivity of 100% (95% CI, 86%–100%) and a specificity of 97% (95% CI, 85%–100%). These results suggest that IP-10 might prove valuable as a diagnostic biomarker of <i>M</i>. <i>bovis</i> infection in cattle.</p></div

IP-10 release in response to antigenic stimulation after (A) 24 h and (B) 48 h.

<p>Whole blood from bTB-positive cattle (n = 12) was incubated at 37°C with saline (PBS), <i>M</i>. <i>avium</i> PPD (PPDa), <i>M</i>. <i>bovis</i> PPD (PPDb) and ESAT-6/CFP-10 peptides (EC). IP-10 release in whole blood in response to PPDb and EC was significantly greater than that in blood co-incubated with PBS and significantly greater in response to PPDb than in response to PPDa. Median values and the differential IP-10 responses to PPDa and PPDb (ΔPPD) and PBS and EC (ΔEC) are shown (*, p < 0.01; **, p < 0.05).</p

Spontaneous IP-10 release in whole blood from reactor cattle.

<p>Whole blood from cattle (n = 12) was incubated with phosphate buffered saline for 24 and 48 h. IP-10 release showed a significant increase over time (* < 0.005).</p

IP-10 release in response to antigenic stimulation in reactor and non-reactor cattle.

<p>Whole blood from non-reactor (n = 16) and reactor (n = 12) cattle was incubated with <i>M</i>. <i>avium</i> PPD (PPDa) and <i>M</i>. <i>bovis</i> PPD (PPDb) for 24 h at 37°C. For reactor cattle, IP-10 release in response to PPDb was significantly greater than that in response to PPDa. Median values are shown (*, p < 0.0005).</p