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Currently available tests are mainly based on detection of antibodies on serum (e.g. ELISA or AGID) or milk (milk-ELISA), faecal culture (FC) or tissue culture, in solid or liquid media (Bactec), and PCR testing on faeces, milk or tissue samples for the detection of the unique DNA sequence IS900 (Nielsen and Toft, 2008). Those tests, in individual animals, present generally a poor performance, especially lacking sensitivity (Se), primarily due to the chronic nature of the infection and potential latency within herds and animals (Whittington and Sergeant, 2001). Truly positive animals include latently infected, shedding and/or clinically affected animals (Nielsen and Toft, 2008). The Se of diagnostic tests strongly depends on the disease stage, where infected, infectious and/or affected animals need to be differentiated. The Se for latently infected animals (no shedding/true-latent) is generally low (Nielsen and Toft, 2008). Infected herds can be misclassified as non-infected if prevalence is low (<5%), only a fraction of the herd is tested, and/or diagnostic tests with low to moderate Se for detecting infected animals are used. Non-infected herds can be misclassified as infected when tests with imperfect specificity (Sp) are used, with the exception of culture (Berghaus et al., 2006).

Three different tests are currently available for measuring antibodies against M. paratuberculosis in the serum of infected animals. These are the complement fixation (CF) test, the agar gel immunodiffusion (AGID) test, and ELISA (Harris and Barletta, 2001). The Se of these tests is high for animals with clinical symptoms, or for those that shed large numbers of bacteria (Nielsen and Toft, 2008). Therefore, the main limitation

of an infection. In naturally infected cows, seroconversion has been shown to occur in 95–98% of animals shedding MAP (Nielsen and Ersboll, 2006). Seroconversion has been observed in 2.2 to 11.7 years old cows under field conditions and studies using fixed dosages and known age at infection have also found a great variation in the time to seroconversion (Nielsen and Toft, 2008). In cattle, Se of ELISA was 87% in clinical cases compared to 75% in subclinical heavy faecal shedders and 15% in subclinical light faecal shedders (Sweeney et al., 1995). The usual mix of animals in a subclinically infected herd renders Se of ELISA about 45% (Collins and Sockett, 1993) although further analysis by Whitlock et al. (1999) suggests that Se in cattle may be about 35%. Antibodies against MAP could also be detected in milk of lactating cows or bulk tank milk, using ELISA. This ‘milk-ELISA’ represents a cost effective tool for MAP surveillance in dairy cattle herds (Sergeant et al., 2008). Validation studies of milk- ELISA estimated a Se between 29% to 61% and a Sp in the range of 83% to 100% (Nielsen and Toft, 2008). In large, endemically infected sheep flocks the Se has been estimated to be about 25% (Whittington and Sergeant, 2001), although Hope et al. (2000) have reported Se to be between 35-54% for ELISA. The reason for the imperfect Se of serological tests in sheep, even in late stages of the disease, is the variability in the immune response of individuals. A significant proportion of clinically affected sheep, with well-developed histological lesions could have negative results in serological tests (Clarke and Little, 1996). In deer, previous studies by Griffin et al. (2003) and Rodgers et al. (2005) have shown that subclinically infected deer generally produce higher levels of antibody than previously reported in cattle (Collins et al., 2005) or sheep (Sergeant et al., 2003). Under field conditions, where deer have been infected naturally and vary in age, the estimated Se of an IgG1 ELISA (ParalisaTM) was about 67% for infected deer showing minimal pathology, if any (Griffin et al., 2005). A recent test validation using

Bayesian latent class analysis estimated a Se of 19% (95% CI 10-30%) on subclinically infected deer using the ParalisaTM(Stringer, 2010). In general, ELISA is a fast and low- cost serology test; however, it is less sensitive and specific than faecal culture (Whitlock et al., 1999).

Bacterial culture of tissues or faeces is a highly specific diagnostic test (Nielsen and Toft, 2008). However, it is costly and MAP culture requires long incubation periods, taking from 4-6 weeks, using automated liquid-culture systems (Bactec), until 12-16 weeks based on conventional growth on solid media (Berghaus et al., 2006). Additionally, in cattle, sero-conversion can be detected prior to MAP shedding when antibodies are tested in milk rather than serum (Nielsen, 2008). Culture of intestinal tissue is more sensitive than FC. It was observed that animals with repeated negative FC samples while being alive had positive culture results from intestinal tissues at abattoir (Whittington and Sergeant, 2001). The Sp of FC is considered to be almost 100%, if the isolates obtained at culture are confirmed to be MAP by molecular methods such as IS900-PCR. Although the potential pass-through phenomenon (Sweeney et al., 1992a) could cause non-infected animals testing FC-positive on contaminated premises, leading to false-positive reactions (Nielsen and Toft, 2008). However, Pradhan et al. (2011) in a longitudinal study on three dairy cattle farms in USA, reported that 80% of tested animals with at least a single positive faecal culture were also tissue culture positive, indicating that they were truly infected. Interestingly, it has been observed that MAP can be unevenly distributed within the faeces, contributing to possible false negative results due to intermittent shedding (Whittington and Sergeant, 2001). One way to overcome the FC costs of individual animals is to pool faecal samples. Currently, this is the most cost-effective option with acceptable Se for herd testing (Benedictus et al.,

1999; Kalis et al., 2000; van Schaik et al., 2003; Weber et al., 2004). Pooled culture has been used for identification of infected-herds, but could also be used for certification of low-risk herds and flocks (van Schaik et al., 2003). A study conducted by Van Schaik et al. (2007) determined that pools of 10 cows were the preferred option to determine the herd infection status, maximizing Se and minimizing costs, with no significant difference in Se between pools of five or ten cows. Faecal pooling has also been recommended to assess the MAP herd-level prevalence (Wells et al., 2002; Raizman et al., 2004). In sheep, pool FC with sizes of 10, 30 and 50 samples have been used to estimate animal level prevalence, reporting Se of 91%, 85% and 77%, respectively (Dhand et al., 2007). In deer, pool size of 10 samples has been used for vaccine assessment in naturally infected animals (Stringer et al., 2011) and for the herd level evaluation of risk factors for clinical disease (Glossop et al., 2007).

Molecular analysis of nucleic acids using polymerase chain reaction (PCR) assays have been applied for MAP detection in faecal, milk and tissue samples (Fang et al., 2002; O'Mahony and Hill, 2004; Bogli-Stuber et al., 2005; Stabel and Bannantine, 2005), commonly targeting the IS900 insertion element sequence (Vansnick et al., 2004; Ravva and Stanker, 2005; Rowe and Grant, 2006). These assays have demonstrated to be sensitive and specific, and have reduced the detection time (Bogli-Stuber et al., 2005; Stabel and Bannantine, 2005). However, PCR has not achieved the same Se as culture when applied directly to tissues or faeces (Collins et al., 1993). This lack of Se of the PCR test was explained by the presence of PCR inhibitors which are difficult to remove from faecal samples (Harris and Barletta, 2001). In recent years, new diagnostic protocols have removed PCR inhibitors from faecal samples, allowing the direct application of quantitative, real time PCR to faeces samples (Kawaji et al., 2007).

Current evidence indicates a greater diagnostic Se of this tool in comparison with tissues culture (Kawaji et al., 2011).

Surveillance programs have been designed to account for the relationship between test Se and disease stage. For example, only animals >3 years of age were sampled in a US programme, and >2 years for the initial test and >4 years in subsequent tests in an Australian programme (Sergeant et al., 2008). Additionally, abattoir surveillance has been implemented by the Australian sheep industry and by the New Zealand deer industry. It involves visual examination of viscera and their regional lymph nodes to identify gross pathological lesions attributable to MAP infection. Suspect lines were followed by a histological assessment (Abbott and Whittington, 2003) and possible follow-up by Bactec culture and/or PCR (Glossop et al., 2005). Abattoir surveillance has the advantage of a wide cross-section sample, where several regions can be inspected at relatively low cost, compared to expensive farm-based surveillance methods (Abbott and Whittington, 2003). However, gross lesions of lymph nodes are not specific for paratuberculosis in sheep, and such lesions are not always present in all infected animals (Fodstad and Gunnarsson, 1979; Hope et al., 2000; Abbott and Whittington, 2003). Moreover, an unknown proportion of animals affected by paratuberculosis on-farm are not submitted for slaughter. Uncertainty therefore exists about the ability of this surveillance method to detect the infection in flocks, particularly those with a low prevalence. Abbott and Whittington (2003), using a mathematical simulation model, estimated an abattoir HSe of 75% for sheep surveillance in Australia, assuming an intra-flock TP of 2%. Moreover, Bradley and Cannon (2005), using 1,200 sheep sourced from known highly infected farms in Australia, estimated an inspector level Se between 53 to 87% and a Sp between 97 to 100%. Conversely, In New Zealand

Hunnam (2011) estimated a meat inspector Se for deer lines, of only 13.3% although a high Sp of 99.9% was reported. Despite of both studied used histology to compare meat inspectors assessment, Hunnan (2011) cover a greater number of inspectors and abattoirs, and used normal animal submission as test subjects, in contrast to the known farms status from animals sourced in the Australian study, which could have biased their results. However, differences in estimates could also be attributed to patho- biological differences of disease manifestation between those two species.