‘Nano-immuno test’ for the detection of live Mycobacterium avium subspecies paratuberculosis bacilli in the milk samples using magnetic nano-particles and chromogen

Abstract

Early rapid detection of Mycobacterium avium subspecies paratuberculosis (MAP) bacilli in milk samples is the major challenge since traditional culture method is time consuming and laboratory dependent. We report a simple, sensitive and specific nano-technology based 'Nano-immuno test' capable of detecting viable MAP bacilli in the milk samples within 10 h. Viable MAP bacilli were captured by MAP specific antibody-conjugated magnetic nano-particles using resazurin dye as chromogen. Test was optimized using true culture positive (10-bovine and 12-goats) and true culture negative (16-bovine and 25-goats) raw milk samples. Domestic livestock species in India are endemically infected with MAP. After successful optimization, sensitivity and specificity of the 'nano-immuno test' in goats with respect to milk culture was 91.7% and 96.0%, respectively. Whereas, it was 90.0% (sensitivity) and 92.6% (specificity) with respect to IS900 PCR. In bovine milk samples, sensitivity and specificity of 'nano-immuno test' with respect to milk culture was 90.0% and 93.7%, respectively. However, with respect to IS900 PCR, the sensitivity and specificity was 88.9% and 94.1%, respectively. Test was validated with field raw milk samples (goats-258 and bovine-138) collected from domestic livestock species to detect live/viable MAP bacilli. Of 138 bovine raw milk samples screened by six diagnostic tests, 81 (58.7%) milk samples were positive for MAP infection in one or more than one diagnostic tests. Of 81 (58.7%) positive bovine raw milk samples, only 24 (17.4%) samples were detected positive for the presence of viable MAP bacilli. Of 258 goats raw milk samples screened by six diagnostic tests, 141 (54.6%) were positive for MAP infection in one or more than one test. Of 141 (54.6%) positive raw milk samples from goats, only 48 (34.0%) were detected positive for live MAP bacilli. Simplicity and efficiency of this novel 'nano-immuno test' makes it suitable for wide-scale screening of milk samples in the field. Standardization, validation and re-usability of functionalized nano-particles and the test was successfully achieved in field samples. Test was highly specific, simple to perform and easy to read by naked eyes and does not require laboratory support in the performance of test. Test has potential to be used as screening test to estimate bio-load of MAP in milk samples at National level.

Preparation of immunomagnetic beads coupled with a rhodamine hydrazine immunosensor for the detection of Mycobacterium avium subspecies paratuberculosis in bovine feces, milk, and colostrum

“…Immunomagnetic beads prepared using a mixture of mouse monoclonal and polyclonal antibodies against MAP increased the test sensitivity of IS900 PCR detection of MAP in bovine milk samples, presumably by reducing the concentration of PCR inhibitory substances (Gilardoni et al, 2016). Immunomagnetic beads prepared using goat polyclonal antibodies against MAP and resazurin dye as a chromagen provided a useful field test for detecting MAP in bovine milk samples (Singh et al, 2018). In contrast, the combination of a polyclonal-antibodybased IMB with culture in Herrold's egg yolk medium did not provide acceptable specificity for isolating MAP from milk-based samples (Stephan et al, 2007).…”

“…Magnetic separation methods have been used in the microbiology laboratory for the isolation and detection of pathogenic organisms, particularly when combined with methods such as culture, ELISA, or PCR (Grant et al, 1998;Foddai et al, 2010;Husakova et al, 2017). Specific antibodies coupled with magnetic beads, known as immunomagnetic beads (IMB), can enhance analytical sensitivity by selectively isolating and concentrating a microbiological target into a more desirable volume and matrix (Gilardoni et al, 2016;Singh et al, 2018).…”

Toward paratuberculosis control in animals: current updates and future perspectives

“…Although some agglutination tests are commercially available as latex agglutination tests (Singh et al, 2018), ELISA has become the method of choice for primary screening in MAP control programs due to the suitability of sample collection, ability to screen a large number simultaneously, and rapid laboratory turnaround time (Salem et al, 2013a).ELISA shows a good agreement between the results of serum and individual milk samples taken simultaneously (Geisbauer et al, 2007). The sensitivity of ELISA ranges from 0.15 in infected, asymptomatic, non-shedders, and 0.47 in infected, asymptomatic, shedders up to 0.71 in infected, symptomatic shedders.…”

“…Currently, several commercial ELISA systems are available as indirect fluorescent antibody test (i_FAT), Indigenous ELISA test (i_ELISA), and Dot-ELISA 6293 (d_ELISA) (Singh et al, 2018). The improvement of test specificity and sensitivity can be achieved by (1) the involvement of MAP species-specific surface antigens in the manufacturing process (EVELISA system), or the utilization of antigens secreted by young MAP-culture (JTC-ELISA).…”

Broad-Spectrum Theranostics and Biomedical Application of Functionalized Nanomaterials

“…A nano-immuno test detects the Mycobacterium avium subspecies paratuberculosis (MAP) in the milk samples by using MAP-specific antibody-conjugated magnetic nanoparticles with resazurin dye as a chromogen. A ‘nano-immuno test’ in goat milk was 90.0% (sensitivity) and 92.6% (specificity) when compared to an IS900 PCR test [ 214 ]. An in-situ immuno-AuNP network-based ELISA biosensor was developed to detect pathogenic microorganisms with high sensitivity based on immuno-magnetic separation.…”