Introduction
Diagnosis of paratuberculosis infection can be made either by detection of the causative agent, Mycobacterium avium subsp. paratuberculosis (MAP), or through detection of a biological response made by an infected animal, for example using a serum antibody enzyme-linked immunosorbent assay (ELISA) to detect a humoral immune response.
Culture methods for MAP are the gold standard diagnostic test, however the time it takes to grow the bacteria is a major issue for producers and in the control of disease spread. Nucleic acid amplification based tests (NAATs) such as the polymerase chain reaction (PCR) are an alternative, far more rapid method to detect MAP DNA in a sample matrix; most commonly this is faeces due to the pathobiol- ogy of paratuberculosis, but other sample types including milk, tissue, semen, blood, lymphatic fluid and culture media can also be examined.PCR has, and will continue, to make a significant contribution to the detection of pathogens associated with infectious disease. Direct PCR- based diagnostics can have higher sensitivity than culture for the detection of pathogens and have the advantage of being rapid and unaffected by antibiotics (Obara et al., 2011; Ljungstrom et al., 2015). Conventional PCR (PCR followed by gel-based detection) was first integrated into the diagnostic regime for paratuberculosis in the early 1990s and has continued, using a range of MAP target sequences. Most notably, IS 900 has been used for the detection of MAP (Green et al., 1989; Moss et al., 1991) and IS1311 for strain typing applications (Collins et al., 199 7; Marsh et al., 1999). Following conventional PCR was the introduction of real-time or quantitative PCR (qPCR) in the late 1990s∕early 2000s.
For MAP PCR-based diagnostic tests, though certainly much faster than culture, lack of diagnostic sensitivity has been a major issue, particularly when comparing the sensitivity of faecal PCR and faecal culture.
The previous edition of this book concluded with a statement that progress was being made on this front and that ‘PCR holds more promise now for the diagnosis of paratuberculosis’ (Bolske and Herthnek, 2012). In the past 10 years, there have indeed been marked advancements, moving from a consensus in the literature that culture is the more sensitive detection method (Eamens et al., 2007; Douarre et al., 2010), to the current status where the majority of countries that have implemented a control programme for paratuberculosis employ a faecal PCR diagnostic test in that programme (Yamamoto et al., 2018b; Whittington et al., 2019). There is support for a move towards increased PCR-based diagnostics in human tuberculosis medicine to ‘rule in' cases and enable more rapid treatment, though sputum culture remains the gold standard in most low-burden settings (Babafemi et al., 2017; Vinuesa et al., 2018). Developments in MAP PCR-based diagnostics have been made possible by advancements within the paratuberculosis field, as well as those in research on related mycobacterial diseases and more distant areas of applied science such as forensics.The ability to detect MAP DNA in a sample by PCR is governed by a number of factors: the disease pathogenesis, which impacts the likelihood that MAP will be present in a sample from an infected animal at the time that the diagnostic test is applied, the type of sample tested and the resilience of the MAP organism. Paratuberculosis is a chronic infection, characterized by intermittent faecal shedding and dissemination events by the MAP bacterium, particularly during the subclinical stages of the disease. This means that, while many antemortem diagnostic tests including those based on PCR perform well at the herd level, individual animal diagnosis is complicated by the stage of disease. Diagnosis becomes progressively easier as an animal approaches clinical stages of disease due to more consistent and high levels of faecal shedding of MAP and increased incidence of dissemination and presence in the milk and blood (Alonso-Hearn et al., 2009; Bower etal., 2011; Stabel etal., 2014). The sample type is an important consideration as this can impact the ability to extract useable DNA (Stevenson and Sharp, 199 7), one of the main impediments to the application of PCR directly to sample material from infected animals. The nature of MAP is also important to consider, in relation to the extraction approach required to lyse the tough outer cell wall of the bacterium in order to release the DNA (Odumeru et al., 2001).
This chapter will examine recent developments in PCR-based diagnostic tests for paratuberculosis and the application and interpretation of these tests at the individual and herd level.
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