Introduction

Cultivation and identification of Mycobacterium avium subsp. paratuberculosis (MAP) remains the definitive diagnostic test for paratuberculosis. A positive culture provides information that no oth­er test can: viable pathogen present; potentially infectious.

Performing culture on clinical samples in­forms the case definitions that are vital in disease control (Whittington et al., 2017). Culture of MAP allows classification of animals using these definitions, which permeate research studies on topics ranging from genome-wide association (Purdie et al., 2011) through to diagnostic test validation (Alinovi et al., 2009; Gardner et al., 2011; Keller et al., 2014; Plain et al., 2014; Butot et al., 2019). Culture is required to propagate the organism for experimental infection models to study the disease and to enumerate viable cells in the inoculum in such models (Begg et al., 2010; Corbett et al., 2018). It is the basis for production of commercial vaccines and for studies of their efficacy (Reddacliff et al., 2006b; Mercier et al., 2014). In food safety and public health, culture of MAP is central to understanding exposure, dis­infection and thermal inactivation (Whittington et al., 2010; Van Brandt et al., 2011b; Faria et al., 2014; Botsaris et al., 2016; Galiero et al., 2016). In medicine, culture is used to assess the suscep­tibility of MAP to antimicrobials (Krishnan et al., 2009b). In epidemiology, culture informs our understanding of transmission and spread of MAP through data on its prevalence in livestock (Bauman et al., 2016; Noll et al., 2017; Selim et al., 2019), its infection of wild animals (Beard et al., 2001; Kumar et al., 2010; Matos et al., 2014; Salgado et al., 2016; Galiero et al., 2018), its persistence in a viable form in the environment (Eppleston et al., 2014), its fate and transport to waterways, in air and even to plants (Aboagye and Rowe, 2011; Eisenberg et al., 2011; Salgado et al., 2011; Kaevska et al., 2014).

The pathogenesis of MAP infection is such that subclinical paratuberculosis is always more common than clinical disease (Whittington and Sergeant, 2001; Vazquez et al., 2014).While paratuberculosis can be detected at post-mortem examination or in an abattoir during meat inspec­tion, MAP is present in the intestinal mucosa or mesenteric lymph nodes long before microscopic or gross lesions develop in these tissues, render­ing culture more sensitive than gross pathology (Elze et al., 2013; Stringer et al., 2013). For diag­nosis, the microscopic lesions induced by MAP in the intestinal tract of ruminants are quite characteristic, but even where acid-fast bacilli (AFB) are visualized in tissues associated with the granulomatous infiltrate, specificity is not assured because other mycobacterial species sometimes infect the gut and can cause similar changes. Furthermore, it is not possible to determine from mild lesions alone whether an animal is still in­fected because recovery is possible (Dennis et al., 2011; Begg et al., 2018). Immunohistochemistry and in situ hybridization reactions conducted on tissue sections containing AFB are not specific for MAP and neither are serological tests such as enzyme-linked immunosorbent assay (ELISA). MAP is defined as an obligate parasite and patho­gen; for this reason, its precise identification by cultivation is the benchmark for diagnosis of paratuberculosis. At herd/flock level, if MAP is de­tected there must be - or must recently have been - an infected animal somewhere nearby. Thus, it is standard practice to use culture to confirm a presumptive diagnosis in individual animals and for index cases. Furthermore, culture of samples pooled from more than one animal, or the envi­ronment, is now accepted for herd and flock sur­veillance. One area where the specificity of culture is yet to be widely applied is in the investigation of the proposed link between MAP and Crohn's disease in humans, where there is still an overreli­ance on molecular tests to demonstrate organism DNA.

Important aspects in the culture of MAP are the analytical sensitivity of the particular method, the contamination rate, the cost of the test and the time taken to report results. Unfortunately, this information is lacking for many culture pro­tocols. Culture of MAP is usually used as a gold standard to evaluate other types of diagnostic test, but unless the most sensitive culture protocols are employed, sensitivity estimates for tests such as direct polymerase chain reaction (PCR), serology and histopathology will be inflated. It is a fact that not all culture protocols are equally sensitive and therefore claims in many diagnostic test evalua­tions are flawed.

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