Sample-Specific Techniques and Challenges

samples)

MAP, Mycobacterium avium subsp. paratuberculosis; GE, genome equivalents; ASe, analytical sensitivity; ASp, analytical specificity; DSe, diagnostic sensitivity; DSP, diagnostic specificity; Com, commercial kit; FC, faecal culture; TC, tissue culture; LJ, Lowenstein-Jensen; HEYM, Herrold’s Egg Yolk Medium; RM, radiometric; IAC, Internal amplification control; PMS, peptide- mediated magnetic separation; ND, not determined.

312 K.M. Plainefa/.

culture as the reference method, ranged from 0.46-1.0; this varied depending on the DNA extraction method, sample selection approach, target gene(s) and culture method used for comparison. Diagnostic specificity ranged from 0.85-1.0. However, true diagnostic specificity can be difficult to assess when the population studied includes MAP-exposed animals, as qPCR can be more sensitive than other diagnostic test­ing methods, often detecting additional positive samples compared with faecal culture. Further, PCR methods detect non-viable bacteria and some low positives may be due to passive shed­ding (Kralik et al., 2014b). Hence, the analytical specificity of the qPCR assay may provide a more accurate assessment of the likelihood of obtain­ing false-positive results.

19.5.2 Milk and dairy products

Milk has been used as a test sample for both individual and herd-level diagnosis of para­tuberculosis. A range of technologies can be applied: culture of milk or milk fractions, mo­lecular methods (PCR, genotyping) and a rapid bacteriophage-based assay that specifically de­tects viable MAP, not just the presence of MAP DNA (Okura et al., 2012; Botsaris et al., 2016; Grant et al., 2017). The majority of recent pub­lications of PCR to detect MAP in dairy products have been related to the food safety aspects for human consumption, as a potential zoonotic infectious transmission route, or calf milk re­placers as a source of disease spread on-farm (Fig. 19.1, Table 19.4).

The occurrence of MAP in milk on-farm has been previously reviewed (Okura et al., 2012). MAP is generally present in low concen­trations in milk samples thus larger volumes are tested, with most methods incorporating a cen­trifugation step. Immunomagnetic or peptide- mediated separation prior to DNA extraction has also been used in order to ‘pull out' the MAP or­ganisms from the sample prior to the test being conducted (Grant et al., 2002; Metzger-Boddien et al., 2006; Ricchi et al., 2014; Gilardoni et al., 2016). The analytical limit of detection of MAP in milk by PCR has been reported to range be­tween 1 and 100 MAP/ml of milk, using qPCR methods with gene targets including IS 900, f57 ISMAP02 and ISMAV2 (Gao et al., 2007; Slana et al., 2009; Alajmi et al., 2016; Acharya et al., 2017b). The fraction of milk tested is significant; the majority of MAP is reported to be concen­trated in the casein pellet rather than the whey (Acharya et al., 2017b). However, the cream lay­er can contain significant numbers of MAP and may also be important to consider in testing re­gimes (Gao et al., 2007; Herthnek et al., 2008). Molecular techniques applied to milk samples in the diagnosis of bovine tuberculosis may also be of relevance to the diagnosis of paratuberculosis (Lermo et al., 2010).

PCR-based assays using milk as a sample may be best suited to the identification of highly

Fig. 19.1. Number of published studies by category using PCR methods to detect Mycobacterium avium subsp. paratuberculosis (MAP) in samples other than faeces (January 2008 - July 2019).

Table 19.4. Examples of recent published studies on PCR assays to detect Mycobacterium avium subsp. paratuberculosis (MAP) by sample type, for samples other than faeces.

infectious animals. Stabel et al. (2014) reported the detection of MAP by PCR in 13% of milk samples from subclinical cows compared with 49% of samples from clinically infected cows, with viable MAP mainly found in milk/colos- trum collected during early lactation (≤60 days in milk). If used for herd-level surveillance, indi­vidual animal milk testing would require large numbers of animals to be tested to generate con­fidence in detection of low prevalence farms and is unlikely to be cost-effective. However, if the information obtained from the extracted DNA is utilized in another way, for example for epi­demiological investigations (Kaur et al., 2010) or multiplex testing for a range of pathogens (Parker et al., 2017), then PCR-based methods may be suitable, otherwise they may be a more expensive test approach than a milk ELISA with similar diagnostic sensitivity.

19.5.2 Animal tissues and meat products

Confirmation of infection with MAP at the in­dividual animal level is commonly performed by culture of the MAP bacterium from the tis­sues of the intestine and mesenteric lymph nodes, which is considered the gold standard post-mortem diagnostic test for paratuberculo­sis. Molecular approaches can also be applied for detection of MAP in these same tissues, for rapid determination of the presence of MAP (Table 19.4).

Challenges with detection of a pathogen in host tissues include the extraction method used and the high quantity of host DNA (Park et al., 2014). This is a common issue for molecular ap­proaches attempting to identify a proportionally minute component of sequences specific for the pathogen in a high background of host DNA (Lee et al., 2017; Gu et al., 2019). High input quantities of DNA can cause inhibition of qPCR reactions and also interfere with the detection of the amplified product (Restrepo et al., 2006; Radomski et al., 2013). There are a number of ways to deal with this, such as target enrich­ment, simple dilution of the DNA extract and/ or optimizing the assay for input DNA quan­tity. Conventional PCR can provide an advan­tage over qPCR when applied to animal tissues as this method is less sensitive to the inhibition due to high levels of host DNA, thus researchers may choose conventional over qPCR for these applications (Zarei-Kordshouli et al., 2019). Validation is key to determine the limit of detec­tion of the assay used.

Tissue qPCR approaches may be of value in disease surveillance at slaughter or deter­mination of farm/regional prevalence levels, as many current abattoir surveillance pro­grammes involve examination of gross lesions and confirmation by histopathological and/or tissue culture, which is time-consuming and has relatively low sensitivity to detect infection (Munster et al., 2011; Acharya et al., 2017a). The majority of published studies on PCR-based detection of MAP in tissues relate to food safety or the detection of MAP DNA in human patient samples (Fig. 19.1). Methods applied in human research on Crohn's disease may utilize PCR due to anticipated low quantities of MAP in the in­testinal tissue sample (Banche et al., 2015) (see also Chapter 3, this volume). Such PCR assays face the same issues as detection of MAP in ani­mal tissues; greater cross-validation of PCR used in human and veterinary research would be of value to determine the analytical performance of these assays.

19.5.3 Other sample types

Methods for detecting MAP by PCR have been published for samples as diverse as semen, blood, lymphatic fluids, environmental samples and culture medium (Juste et al., 2008; Munster et al., 2013; Khol et al., 2014; Plain et al., 2015; Hahn et al., 2017). MAP has been detected in the blood of animals with late-stage paratuberculo­sis by PCR and bacteriophage assays (Munster et al., 2013; Swift et al., 2013), although MAP detection in the blood is not a common finding at a herd or flock level (Bower et al., 2011; Bower, 2011; Swift et al., 2016). Detection of MAP in culture medium in order to confirm specific growth once posed a significant challenge, par­ticularly for media containing egg (Whittington et al., 1998), though now commonly involves high-throughput approaches and/or commer­cial kits (Plain et al., 2015; Bauman et al., 2016; Rangel et al., 2017).

Methods to detect MAP by PCR in slurry/ liquid manure, boot swabs, dust and other types of environmental samples have been published (Aly et al., 2009; Eisenberg et al., 2012; Hahn et al., 2017; Donat et al., 2019). These are in­formative to the mechanisms of disease spread on-farm as well as potential avenues for herd­level diagnosis. The use of samples collected from the farm environment to identify infected herds is increasingly being investigated as an economical surveillance approach (Aly et al., 2009; Donat et al., 2016). There is also a sub­stantial component of the literature regarding PCR to detect MAP in environmental samples that pertains to the risks and prevalence of MAP contamination of the environment (Fig. 19.1).

A number of methods have been developed for water samples, for example to examine the ef­fect of farm runoff on the environmental load in local waterways (Richardson et al., 2019). In this study, 1 l of water was filtered (0.2 μM) and the pelleted material extracted using a method incorporating bead-beating and a magnetic bead extraction kit, followed by qPCR detection of both the IS900 and F57 genetic elements. Water from rivers and tributaries in catchment areas within regions used for grazing by live­stock (sheep and cattle) for both dairy farming and meat production exhibited up to 10⁸ cell equivalents/l of MAP.

19.6

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