Diagnosis

The clinical diagnosis of paratuberculosis is challenging in goats; symptoms are vague and non-specific, as numerous other diseases pre­sent with weight loss. Goats with paratuber­culosis showed complete blood cell count and protein electrophoresis values within reference intervals, though some differences in protein electrophoresis were observed between healthy goats and goats with paratuberculosis (Bonelli et al., 2017).

2015). Generally, the choice of laboratory di­agnostic methods can vary according to the purpose of testing and the prevalence of MAP (Collins, 2011; Bauman et al., 2019).

Volatile organic compounds have been investigated as biomarkers of MAP infection, and could potentially be developed into new non-invasive diagnostic tests. Volatile organic compounds have been analysed in both exhaled breath and headspace of faeces, and significant differences have been detected between infected and non-infected goats (Purkhart et al., 2011; Bergmann et al., 2015).

13.7.1 Culture-based diagnosis

Cultivation of MAP is a highly specific method, but it is costly and generally requires from 8-16 weeks of incubation to produce visible colonies on solid media (de Juan et al., 2006). Using liq­uid culture media can significantly shorten the incubation period (Bauman et al., 2016).

The first detection of faecal shedding has varied considerably from 1.5 months and up to 1-2 years after infection (Storset et al., 2001; Stewart et al., 2007; Kohler et al., 2015). Intermittent faecal shedding is common, and the number of bacteria in faeces may be below detection level. Vaccination also reduces faecal shedding of MAP, interfering with diagnosis (Rosseels and Huygen, 2008; Hines et al., 2014; Mercier et al., 2014). Bacteriological culture is effectively 100% specific, as slow-growing, mycobactin-dependent, acid-fast bacteria that harbour the IS900 element are identified as MAP (Olsen et al., 2002).

As goats can be infected by different types of MAP strains, different media and an extended incubation period up to 8 months is suggested for solid media to detect MAP in new areas or flocks (de Juan et al., 2006). Extending of the in­cubation period in liquid media up to 240 days showed increase in the sensitivity of MAP detec­tion in goats (Bauman et al., 2016). Different selective and non-selective media containing mycobactin can be used to culture MAP from goats, including Lowenstein-Jensen, Herrold's egg yolk medium, Middlebrook 7H11 (M7H11) or Dubos medium (Saxegaard, 1985; de Juan et al., 2006). Middlebrook 7H11 supplement­ed with mycobactin j, OADC (Oleic Albumin Dextrose Catalase) and newborn calf serum is particularly suitable for the primary isolation of both MAP-C and MAP-S from small ruminants (Dimareli-Malli etal., 2013). A study showed the combination of M7H11 and Herrold's egg yolk medium supplemented with mycobactin J and sodium pyruvate allowed the detection of all MAP isolates (Dimareli-Malli et al., 2013).

Pooled faecal culture can be used for herd diagnosis if the animals are moderate or high shedders of MAP, but this would be unsuitable in herds with only a few low shedders (Eamens et al., 2007).

13.7.2 PCR-based methods

Different methods based on IS900 PCR have been used to detect MAP from different samples of naturally and experimentally infected goats, including faeces (Ikonomopoulos et al., 2007), milk (Djonne et al., 2003) and intestinal tissue (Whittington et al., 1999). The sensitivity and specificity of PCR vary between different meth­ods (see Chapter 19, this volume). MAP-S strains are very fastidious to culture and goats with a history of contact with MAP-positive sheep should be analysed using PCR (Collins, 2011).

13.7.3 Immunological methods

MAP antigens can be detected in paraffin- embedded tissue sections from goats by immu­nohistochemistry (IHC), and this method seems to be more sensitive than staining by the Ziehl- Neelsen technique (Thoresen et al., 1994). IHC however, seems to be less sensitive than bacte­rial culture in detecting MAP in tissues (Kruger et al., 2015b).

Cell-mediated immune response detected by the MAP-specific IFN-γ test can be useful to monitor the paratuberculosis status of non­vaccinated goat herds. The method seems to give fewer false-positive reactions in young goats compared with young cattle (Storset et al., 2005; Lybeck et al., 2011). IFN-γ responses can be found from about 2 months of age or 2 months after experimental infection (Storset et al., 2001; Stewart et al., 2007; Mercier et al.,

2016), but is often delayed for another 6-15 months or longer (Lybeck et al., 2011; Kohler et al., 2015; Mercier et al., 2016). Cellular im­mune responses are generally believed to occur prior to faecal shedding and antibody responses, but several exceptions have been seen (Storset et al., 2001; Stewart et al., 2007; Lybeck et al., 2011; Kohler et al., 2015; Mercier et al., 2016).

The detection of a delayed-type hypersen­sitivity reaction to johnin has been successfully applied in goats that are naturally infected by MAP (Tripathi et al., 2006), although the johnin skin test appears far less sensitive than faecal PCR (Rawther et al., 2012). Based on studies in cattle, the skin test results are considered to be unreliable (Kalis et al., 2003).

In contrast, antibody-detection-based tests for MAP infection are inexpensive and fast. Enzyme-linked immunosorbent assay (ELISA), agar gel immunodiffusion and complement fixation can be used in paratuberculosis control programmes for the goat industries without a major risk of generating large proportions of false-positive test results. Of these methods, the ELISA is most sensitive for detection of infected goats (Whittington et al., 2003; Gumber et al., 2006). All three methods show good specificity, although serological cross-reactions between MAP infections and other goat pathogens have been reported. False-positive reactions can oc­cur due to cross-reacting antibodies produced against Corynebacterium pseudotuberculosis (Manning et al., 2007) and Mycobacterium bovis (Alvarez et al., 2008).

Commercial bovine paratuberculosis ELISAs have also been used to test goat milk. ELISA testing of goat milk samples appears to offer a useful, low-cost alternative for detection of goats with paratuberculosis that have pro­gressed to the stage of shedding MAP in their faeces (Salgado et al., 2007). Conflicting results exist regarding whether testing of milk is less sensitive than testing of serum for presence of MAP antibodies (Salgado et al., 2007; Angelidou et al., 2014b). In one study, testing of bulk tank milk with ELISA demonstrated comparable sen­sitivity with detection of MAP antibodies in indi­vidual milk or serum, but was less sensitive than faecal culture and PCR (Bauman et al., 2019).

There is a continuous search for more spe­cific and sensitive antigens for use in both cell- mediated and antibody tests, such as peptides or recombinant proteins from MAP. The combined use of several such antigens could increase test sensitivity (Casey et al., 2011; Souriau et al.,

2017).

13.8