Using ELISA for detection of antibodies against MAP

ELISA is one of the most common serological tests used for diagnosis of MAP and it is the most sensitive and specific test for serum antibodies to MAP in cattle (SOCKETT at al.

1992). It was designed by YOKOMIZO et al. (1985), modified by MILNER et al. (1988), and was developed into a commercial kit by COX et al. (1991). Serological tests are not suitable for newly infected animals because anti-MAP antibodies are not usually produced in the early stage of infection. These tests for MAP detection are more revealing when used to carry out a preliminary investigation of the disease prevalence in a herd and for confirmation of diagnosis in clinically sick animals (GUMBER et al. 2006). However, ELISA is considered to be the most suitable serological test for use as a screening test in subclinical paratuberculosis infected animals (COLLINS and SOCKETT 1993). ELISA-based testing is readily automated and low cost; however, it is less sensitive and specific than fecal culture-based tests for paratuberculosis (SWEENEY et al. 1995) and is less sensitive and specific than fecal culture.

ELISA detected about 30-40% of cattle identified as infected by culture of feces on solid media (WHITLOCK et al. 2000). Serology and fecal culture are the most commonly used tests in the field for MAP identification (WHITTINGTON and SERGEANT 2001).

Milk ELISA testing appears to be less sensitive and less specific than serum ELISA (SWEENEY et al. 1994), however, ELISA is more sensitive than the Agar gel

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immunodiffusion (AGID) and the complement fixation test (CFT) (COCITO et al. 1994).

HENDRICK et al. (2005) and SALGADO et al. (2005) applied the ELISA on milk and serum samples and found that the ELISA on bovine and caprine milk have specificity similar to that of serum ELISA, but is less sensitive. SAMARINEANU et al. (2007) developed two ELISA kits for serological diagnosis of bovine paratuberculosis. Milk ELISA performed equivalent to serum ELISA using fecal culture as a reference for MAP infection and has the advantage of decreased labor costs on farms that use dairy herd improvement association testing. At the cow level, the milk ELISA relative sensitivity was significantly different from that of the serum ELISA (21.2% and 23.5%, respectively). Estimates for herd-level sensitivity for the milk and serum ELISA relative to fecal culture results ranged from 56-83% (LOMBARD et al. 2006). FRY et al. (2008) compared the accuracy of four commercial ELISA for diagnosis of bovine paratuberculosis using sera from 53 MAP fecal culture-positive dairy cows (cases) and sera from 345 dairy cattle resident in 11 fecal-negative herds. The specificity of all four ELISA kits was 99% and their diagnostic sensitivity ranged from 30.2% to 41.5%. Several commercially available ELISA have similar sensitivities and specificities (COLLINS et al.

2005). ELISA sensitivity was as low as 15% in subclinical animals that were shedding low numbers of MAP (1-10 colonies per fecal culture tube) and as high as 88% in fecal culture positive cows with clinical signs of paratuberculosis. Specificity estimates ranged from 82.6%

in cows with clinical signs of paratuberculosis to 100% in cows from a herd without history of paratuberculosis, bulls from a commercial bull stud and cows from a closed, uninfected herd.

The overall sensitivity and specificity of the revised ELISA kit were 50% and 96.8%, respectively (DARGATZ et al. 2001).

Antibodies have an active role in MAP infection in vitro. MAP immune sera or purified specific antibodies enhance bacterial interaction with macrophages improve the activation of the nuclear factor NF-KB in infected cells and affect MAP intracellular viability (JOLLY et al. 2011). MUNDO et al. (2008) and FERNÁNDEZ et al. (2011) detected high levels of specific Immunoglobin G1 (IgG1) against several antigens in sera from MAP-infected cattle at a clinical stage of the disease. However, STABEL et al. (2003) found that antibody response for paratuberculosis was not detected in calves during the 6 month infection period of the experiment.

LITERATURE REVIEW

17 The sensitivity of the ELISA as a culture method depends on the level of MAP shedding in feces and the age of animals. A large study in Australia showed that actual sensitivity of the ELISA in 2, 3, and 4 years old cows was 1.2%, 8.9%, and 11.6%, respectively, but remained 20% and 30% in older age-groups. The overall actual sensitivity for all age-groups was calculated to be about 15% (JUBB et al. 2004). ELISA sensitivity is around 15% when animals are not shedding MAP in feces, while it achieves 87% in animals presenting clinical symptoms of paratuberculosis (COUSSENS 2004). Both the rate of detecting animals shedding MAP in feces (sensitivity relative to culture) and the magnitude of ELISA results (S/P or optical density readings) are directly related to the probability that the tested animal is infected and shedding the organism in feces. Furthermore, the ELISA is sufficiently precise for herd screenings (COELHO et al. 2007). GILARDONI et al. (2012) found that although conventional ELISA (detecting IgG) has low sensitivity during the subclinical stage of the infection, it is the test most used for paratuberculosis control due to its low cost, high throughput, standardized protocols and correlation with MAP fecal shedding levels.

MARASSI et al. (2005) reported a sensitivity of 76.7% and a specificity of 70% in an improvement of a previously described ELISA recommended as screening test for herds by FERREIRA et al. (2002). ELISA, PCR and culture are used to increase the sensitivity of MAP detection, in order to confirm whether herds with history of paratuberculosis or MAP diagnosis are truly infected (PINEDO et al. 2008).

The principal diagnostic test for herd classification and screening in some countries, including Australia, is the absorbed ELISA for paratuberculosis. (KENNEDY and ALLWORTH 1998).

JAKOBSEN et al. (2000) found that higher assay sensitivity than that available with current ELISAs for detection of infected individual animals would be desirable, but herd-level sensitivity is the more critical factor, and this can be improved by increasing the number of animals tested per herd and by focusing testing on the older animals since they have had sufficient time for a M. paratuberculosis infection to progress and induce antibody production. Herd screening for possible M. paratuberculosis infection by ELISA demands use of high specificity assays to limit false-positive herd classifications, and the time and effort spent trying to confirm the diagnosis (JORDAN 1996).

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The advantages of ELISA testing are automation, objective result interpretation, repeatability, multiple sample evaluation and availability of sensitivity or specificity cut-offs. It is relatively inexpensive, has good sensitivity and specificity and is useful for determining the herd prevalence of paratuberculosis. However, the disadvantages are that antigenic variability and disparate animal ages can lead to sensitivity and specificity errors in the tests (ALINOVI et al.

2009; GILARDONI et al. 2012).