Tests Used in Prevalence Studies

An essential step in understanding and quantifying the magnitude of the problem with paratuberculosis requires identification of infected animals. Diagnostic tests mainly approach identification of MAP infection with two distinct methods.

The first method is detection of the organism itself (bacterial culture or PCR), whereas the other is detection of immunological response to the organism using ELISA, agar gel immunodiffusion (AGID) or complement fixation tests (CFTs). The latter two tests are less commonly used compared with ELISA (Tiwari et al., 2006; Whittington et al., 2019). Further details on these specific tests are described in Chapters 18, 19 and 20 of this book. These tests can either be performed as individual tests on single animals, or as tests on pooled samples (including bulk milk). The high cost and long diagnostic waiting time are disadvantages of faecal culture; to offset costs, a pooled sampling approach with five cows/sample is used (Collins et al., 2006). There is also the approach to using ELISA tests in pooled milk samples such as the bulk tank milk (BTM) ELISA. The major limitation in the use of these diagnostic tools for identifying MAP infection is low sensitivity. That diagnostic test evaluations are often done in comparison to faecal culture, which has variable sensitivity (1970%) (Whitlock et al., 2000; Tiwari et al., 2006;

Nielsen and Toft, 2008), represents a challenge. Lack of a standardized test with 100% sensitivity makes it difficult to assess true prevalence of disease.

The use of the ELISA on its own results in only 10-25% of infected animals being detected; therefore, most estimates of animal and herd level prevalence based on these numbers are likely underestimates (Whitlock et al., 2000; Barkema et al., 2010). Collins et al. (2006) reported higher sensitivities for milk and serum ELISA between 25 and 35%.

The sensitivity of faecal culture is slightly higher at 55-65% and faecal PCR tests have reported sensitivities in the same range as ELISA (25-35%) (Collins et al., 2006). To maximize sensitivity, the recommended time to conduct milk ELISA is either within the first 2 weeks or after the 45 th week of lactation (Lombard et al., 2006). This is an attempt to capture increased immunoglobulin production in colostrum or to avoid the dilution effects of peak milk production. Sensitivity of ELISA does increase as disease progresses, with a positive correlation between ELISA OD and MAP faecal shedding (Lombard et al., 2006). The probability of detecting infected animals also increased with age (Nielsen and Toft, 2008; Zare et al., 2013; Sun et al., 2015). Due to the overall low sensitivity of ELISA, it is often recommended that positive ELISA animals are confirmed with faecal culture. Apart from imperfect sensitivity, a limitation of faecal culture for identifying paratuberculosis is intermittent shedding (Donat et al., 2015). It is important to note that the performance of these tests is dependent on within-herd prevalence; therefore, as prevalence decreases during a control programme, diagnostic test performance also decreases.

The use of faecal culture and PCR on environmental samples has been proposed to help identify a herd's paratuberculosis status, but there are false-negatives when within-herd prevalence is low (Donat et al., 2015). To maximize sensitivity, site selection of samples is important; therefore, it is recommended to collect from manure storage and high-traffic areas (Raizman et al., 2004; Smith et al., 2011; Lavers et al., 2013; Wolf et al., 2015). Klawonn et al. (2016) reported that environmental cultures had a sensitivity of ~64% for beef cow-calf herds. Whereas specificity is often cited to be 98-100%, there is some concern over cross-reactivity with other mycobacteria and ‘passive shedding' of MAP (Nielsen and Toft, 2008; Nielsen, 2014).

BTM ELISA tests have also been evaluated for paratuberculosis monitoring/screening. However, results will be influenced by the number of shedders relative to the overall herd size, the proportion of BTM that infected cows contribute and the stage of lactation of the infected animals. The reported range in sensitivity of bulk tank ELISA is similar to that of individual ELISA (25%) (Stabel et al., 2002). To address this low sensitivity, there have been modified techniques of current bulk tank ELISA tests proposed (Nielsen et al., 2000; Slana et al., 2008). It is recommended that these BTM tests be used for monitoring regional/national prevalence, but their interpretation on an individual-farm basis should be used with caution.

Interferon-γ assays are a newer diagnostic approach for detection of cell-mediated responses to paratuberculosis. Estimates for the sensitivity ranged from 13-85% (Nielsen and Toft, 2008; Nielsen, 2014).

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Source: Behr Marcel A., Stevenson K., Kapur V. (eds.). Paratuberculosis: Organism, Disease, Control. 2nd edition. — CAB International,2020. — 439 p.. 2020

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