Introduction

Increasing evidence for the presence of pathogenic Mycobacterium avium ssp. paratuberculosis (MAP) in pasteurized / commercial milk samples has led research efforts to focus on diagnostic methods that can intervene at early stages of production of dairy products and prevent further spread of MAP infection to human beings (Singh et al. 2016d). MAP is weakly gram-positive, Ziehl Neelsen (ZN) positive acid fast bacilli. This highly pathogenic mycobacterium is the causative agent of chronic incurable enteritis known as Johne’s disease, which is responsible for mal-absorption of nutrients in the gut and swelling and enlargement of mesenteric lymph nodes. This highly infectious disease affects productivity of both small and large ruminants (Chaubey et al. 2016). Clinically infected animals shed large quantities of MAP bacilli. However, sub-clinically infected animals shed much lesser quantitites of bacilli. Recently large volume of evidences point towards MAP bacilli as the causative agent of a similar inflammatory disease involving intestines of the human population and is known as Crohn’s disease (Chamberlin et al. 2007; Singh et al. 2016e). Bacilli have been reported to infect mucosal layer of the gut and peripheral blood mono-nuclear cells (PBMCs) in the blood of Crohn’s patients (Naser et al. 2014). Besides inflammatory bowel disease (Crohn’s disease), MAP has also been suggested to be involved in diseases like irritable bowel syndrome (Scanu et al. 2007) and ulcerative colitis (Pierce 2010), among others. Presence of live MAP bacilli in the milk of infected lactating animals leads to contamination of milk and milk products (Acharya et al. 2017; Grant et al. 2017; Stephen et al. 2016). Ability of MAP bacilli to survive the pasteurization temperatures is a matter of great concern, since MAP is now a major ‘food borne pathogen’, which lends support to the ‘zoonotic potential’ of bacilli (Cirone et al. 2007; Patel and Shah 2011; Stabel and Lambertz 2004; Wynne et al. 2011).

Nano-technology based approaches for the detection of live MAP bacilli is a relatively unexplored field, with untapped ability as potential ‘Gold standard’ test (Stephen et al. 2015). Existing methods like ELISA, PCR and microscopy lack in specificity and sensitivity or have low reproducibility (Singh et al. 2016d). Hence, new diagnostic approaches are currently being investigated with the aim to find a single ‘point of care’ method that can accurately detect the presence of live bacilli, that can replace current dependence on series of diagnostic tests that are currently being used.

The core aim of this study was ‘selective separation’ of MAP bacilli from the milk samples. This was achieved by the conjugation of the live MAP bacilli with specific antibodies immobilized on the magnetic nano-particle (MNPs) by the application of externally applied magnetic field. This process significantly increased the specificity of this ‘nano-immuno test’. The test can accurately detect live MAP bacilli based on visual change in colour, indicating the presence of the viable MAP bacilli in the milk samples.

Materials and methods

Iron (III) chloride hexahydrate, Iron (II) chloride tetrahydrate, (3-amino) propyltriethoxysilane (APTES), glutaraldehyde and resazurin sodium salt were purchased from Sigma-Aldrich (St. Louis, MO) and used as received unless otherwise stated. All reagents were of analytical grade.

Preparation of magnetic nano-particles

Fe3O4 ‘nano-particles’ were synthesized by chemical co-precipitation as per Joo et al. (2012) with some minor modifications. Briefly, 2.92 g of FeCl3.6H2O and 1.15 g FeCl2.4H2O were added to 150 mL of distilled water. The solution was incubated at 60 °C for 20 min with vigorous shaking after which 40 mL of (1.5 M) NaOH was added to the solution drop wise and allowed to react for 1 h. A black precipitate developed immediately (magnetic particles) which was then incubated for 30 min at room temperature to cool. The solution was finally sonicated (55% amplitude, 0.5 s) for 30 min. Afterwards with the help of the magnet, MNPs were separated and washed with acetone and left to dry.

Production of polyclonal antibodies against MAP antigens

Hyper-immune sera was raised against MAP antigens in goat kids in the experimental shed of Animal Health Division at Central Institute for Research on Goats (CIRG) by injecting 1.0 mL of ‘Indigenous Vaccine’ against Johne’s disease at 0, 2, 4 and 6 weeks. This vaccine was developed using native strain (‘S 5’) of a novel bio-type (‘Indian Bison Type’) of MAP (Singh et al. 2007), which is also source of antigens for development of different diagnostic tests. After the completion of 4 doses schedule, serum samples were collected at weekly intervals up to 60 days and were stored at −200 C, till further screening using ‘indigenous ELISA kit’.

Immobilization of polyclonal antibodies and attachment of MAP bacilli on MNPs

MNPs were functionalized with glutaraldehyde as per Joo et al. (2012). Briefly 0.4 mL of (3-amino) propyl tri-ethoxy silane (APTES) was added to 1 mg of MNPs in 40 mL ethanol and incubated at room temperature for 1 h in a shaker. Nano-particles were separated using a magnet, removing the solvent and re-dispersing in 10 mL ethanol 3 times. The 100 μL of 5.0% glutaraldehyde was added in water at room temperature for 30 min with shaking after which the nano-particles were separated using a magnet, removing the solvent and re-dispersing in water. Finally 10 μL of 1 mg/mL antibodies were added at RT for 1 h while shaking. Afterwards the MNPs were separated using magnet and were dispersed in 1X PBS.

Visual colorimetric determination of viable MAP bacilli (chromogen detection)

Functionalized MNPs were added to 3.0 mL of spiked milk and incubated at room temperature in a shaker for 1 h. By magnetic decantation the MNPs were separated out and washed in 1X PBS. To the MAP-MNPs,7.0 mL of 1X PBS was added followed by the addition of 1 mL resazurin dye. The suspension was incubated at room temperature in the dark until the appearance of change in colour from blue to pink.

Optimization and validation of ‘nano-immuno test’

‘Nano-immuno test’ was optimized with true culture positive (10-bovine and 12-goats) and true culture negative (16-bovine and 25-goats) laboratory tested raw milk samples collected from domestic livestock species. However, MAP infection is endemic in the domestic livestock population of the coutnry (Singh et al. 2014) from domestic livestock animals.

For the validation of ‘nano immuno-test, 396 raw milk samples (goats-258 and bovine-138) belonging to farmer’s herds in the districts of Mathura and Agra and farm goats (Central Institute for Research on Goats - CIRG) in North India. Milk samples were screened without initial processing (separating whey, fat and sediment layers by centrifugation) to estimate the ‘bio-load of MAP’, using 3 traditional (microscopy, IS900 PCR, indigenous ELISA kit), 3 newly standardized tests (Dot-ELISA, Latex Agglutination and indirect Fluorescent Antibody tests) and Nano-immuno test. Each of the milk samples was screened by each of the 7 diagnostic tests including ‘nano-immuno test’.

Acid fast staining (microscopy)

Smears were made from 20 μl of milk, heat fixed, stained by Ziehl Neelsen (ZN) staining and examined under oil immersion (×100) for acid-fast bacilli (AFB) indistinguishable to MAP (Singh et al. 2008).

Indirect fluorescent antibody test (i_FAT)

The test was performed as per Singh et al. (2016a). Briefly, smears were prepared on clean slides from 2 μl milk), air dried at room temperature and heat fixed. Slides were dipped in solution of 30.0% H2O2 in 90.0% methanol (3:7 ratio) and incubated for 10 min at 37 °C, followed by second dipping in phosphate-citrate buffer (2.1 g citric acid and 3.56 g disodium hydrogen phosphate in 100 mL of triple distilled water, pH- 5). The mixture was heated to boiling in microwave for 30 s (15 cycles) with the rest of 20 s after each heating cycle (total time 10 min). Slides were then air dried at room temperature. Afterwards primary antibodies (whey in ratio of 1:4 and serum in ratio of 1:50) in serum dilution buffer (1% BSA in PBST) were added on the slides. Slides were then incubated for 1 h at 37 °C in BOD (biological oxygen demand) incubator, followed by washing of slides in 1X PBS (3 times). Anti-species secondary antibody (FITC conjugate) was added in the ratio 1:750 in 1X PBS (pH -7.6). Slides were incubated in dark for 1 h at 37 °C followed by washing of slides 5 times in 1X PBS in dark. Slides were air dried in dark at room temperature. Finally slides were mounted with glycerine and covered with cover slip and then observed immediately under fluorescent microscope. Slides positive for MAP infection exhibited green fluorescence.

DNA isolation

DNA isolation from whole milk was carried out as per Van Soolingen et al. (1991) with some modifications. Briefly, to 500 μl milk sample, 100 μl of lysis buffer (50 mM NaCl, 125 mM EDTA, 50 mM Tris-HCl; pH 7.6) was added and incubated at room temperature for 15 min. After that 100 μl of 24% sodium dodecyl sulfate (SDS) was added and incubated at room temperature for 10 min., followed by heating at 80 °C for 10 min. Then 325 μg of proteinase K was added to above sample and incubated at 55 °C for 2 h., followed by addition of 115 μl of 5 M NaCl and 93 μl CTAB-NaCl with proper mixing and incubated at 65 °C for 30 min. Equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added to sample and centrifuged at 15,800 g for 5 min. After centrifugation, resulting aqueous phase from sample was transferred to sterilized eppendrof tube and DNA was precipitated by adding 0.8 volume of chilled iso-propanol and kept at −20 °C for 2 h. DNA was pelleted out by centrifuging tube at 15,800 g for 10 min at 4 °C and then supernatant was discarded. Finally, pellet was washed with 500 μl of 70% ethanol and re-suspended in 30 μl TE buffer/ nuclease free water and stored at −20 °C.

IS900 PCR

DNA isolated from whole milk was subjected to specific IS900 PCR using 150C and 921 primers of Vary et al. (1990) Presence and yield of specific PCR product (229 bp) was considered as positive for MAP infection.

Indigenous ELISA test (i_ELISA)

Test was performed as per Singh et al. (2016b) using whole milk as test sample. Briefly, each well of flat bottom 96 well ELISA plate (Greiner Bione and Nunc) was coated with 0.1 μg of protoplasmic antigen (procured from Microbiology laboratory of Animal Health Division at Central Institute of Research on Goats (CIRG), Makhdoom in 100 μl of carbonate-bicarbonate buffer, (pH 9.6) per well and incubated at 4 °C overnight. Plates were washed 3 times with 1X PBST (1X PBS with 0.05% Tween 20) followed by blocking in 100 μl of 3.0% skimmed milk in 1X PBS, incubated for 1 hour at 37 °C. Plates were washed three times with PBST and then 100 μl of whole milk diluted in PBST with 1.0% BSA in ratio of 1:1 was added as sample in duplicate wells and incubated for 2 h at 37 °C. Plates were washed 3 times followed by addition of 100 μl of optimally diluted rabbit anti-bovine (1:6000 in 1X PBS) / caprine (1:5000 in 1X PBS) conjugate and again incubated for one hour at 37 °C. Finally after five times washing, 100 μl of freshly prepared OPD substrate was added and incubated till colour developed (3–5 min) at 37 °C. Absorbance was read at 450 nm in ELISA reader (i-Mark micro-plate reader, Biorad). Milk whey from weak and culture positive and healthy and culture negative goats were used as positive and negative controls, respectively. Optical densities (OD) values were transformed and expressed as sample-to-positive (S/P) ratios as per Collins (2002). All the chemicals and conjugates used in this protocol were of molecular grade procured from the standard commercial supplier such Sigma-Aldirch, USA.

Analysis of OD values

S/P ratio value = [(Sample OD – Negative OD)/(Positive OD - Negative OD)]. Sample to positive ratios were derived to estimate corresponding status of Johne’s disease (JD) in goats was determined as per Collins (2002). Samples in low positive (LP), positive (P) and strong positive (SP) categories were considered positive for MAP infection.

Dot- ELISA (d_ELISA)

Test was performed as per Singh et al. (2016b). Briefly, tips of 12 legged immune-diffusion combs (Advanced Microdevices pvt. ltd., Ambala, Haryana) fixed with nitrocellulose membrane were coated with 1 μl (2 μg of sPPA in 1 μl of carbonate-bi-carbonate buffer, pH 9.6) of sPPA spot in middle of nitrocellulose paper and incubated for 2 h at 37 °C. Combs were dipped in blocking solution (3.0% skimmed milk powder in PBS) for 1 h at 37 °C. After washing in PBST combs were dipped in test samples (100 μl whole milk in 1:2 dilution in 1% BSA in 1XPBST) for 1 hour followed by again washing. Combs were incubated with 200 μl of rabbit anti-goat HRP conjugate solution at 37 °C for 30 min. Finally, combs were dipped in 200 μl of 3, 3′-Diaminobenzidine (6 mg / 10 mL of 1X PBS), at room temperature till development of colour (1–2 min). Once the spot was visible combs were dipped in water to stop the reaction. Positive and negative controls used in the study were confirmed by IS900 PCR and microscopy and were used on two legs of each comb to assist in reading of ‘test samples’.

Latex agglutination test (LAT)

LAT was performed as per Singh et al. (2016c). Briefly, MAP antigen coated latex beads were prepared using 10 μl of polystyrene latex beads (3.0 μm mean size, Sigma Aldrich). Beads were washed four times in distilled water and re-suspended in 20 μl of 0.5 M glycine saline buffer (1.4 g glycine, 0.07 g Sodium Hydroxide, 1.7 g Sodium Chloride, 0.1 g Sodium Azide in 100 mL of triple distilled water) (pH- 8.6), then 20 μl of antigen (4 mg / mL) was added and incubated for 3 h at 37 °C in shaker incubator. Mixture was centrifuged at 5000 rpm for 10 min and after aspirating the supernatant, mixture was re-suspended in blocking buffer (1% BSA in 1 X PBS) and mixed in shaker incubator for 45 min at 37 °C. Finally beads were washed twice in 1 X PBS. LAT was performed by mixing 4 μl of milk sample with 2 μl of antigen coated latex beads with tip of a pipette on a glass slide. Slide was shaken gently for 2 min, and milk sample was considered positive, if agglutination was observed within 2 min and negative if no agglutination was observed after 2 min.

Nano-immuno test

Test was performed as per protocol standardized in our laboratory. Briefly, 72.0 mg of MAP antibody functionalized magnetic nano-particles (MAbs-MNPs complex) were suspended in 1 mL of raw milk. To this suspension, 140 μl of prepared resazurin dye (Dissolve 100 mg of resazurin salt in 50 mL of hot triple distilled water in a dark bottle) was added. The solution was then incubated at room temperature in a shaker incubator in dark and observed for the change in color after 10 h.

Statistical analysis

To measure statistical significance between two tests, Mc Nemar’s test and kappa agreement statistical analysis methods were applied by Graph Pad software, USA. Sensitivity and specificity of the tests was measured by Med-Calc software, Belgium.

Results

Magnetic nano-particles were prepared and separated successfully with the help of external magnetic field (Fig. 1a, b). Properties of magnetic nano-particles were tested through FTIR and confirmed with TEM and SEM imaging (Fig. 1c, d).

Fig. 1
figure 1

a Magnetic separation of MNPs was achieved within 1 minute using permanent magnet. b Drying of MNPs, c TEM image of MNPs, d A SEM image of MNPs

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FTIR spectra were obtained to study the various methods of functionalization of the MNPs which confirmed the binding of MNPs to APTES. In Fig. 2 Fe-O bonding at 588 cm−1, CH2 stretching at 2854 cm−1, Si-O-Si vibration at 1081 cm−1 which are characteristics of silane layers and C-O-O bonding at 1461 cm−1 confirms the binding of MNP on APTES while peak at 1637 cm−1 confirms the loading of glutaraldehyde onto APTES. From the graph the presence on C=N bond confirms the binding of antibodies onto glutaraldehyde. The binding of antibody conjugated MNP to MAP was investigated through TEM imaging. Figure 3 shows the binding of MNPs to MAP.

Fig. 2
figure 2

FTIR graph depicting the bonding visualized due to the attachment of cross linkers to MNPs and binding of antibodies onto the MNP

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Fig. 3
figure 3

TEM images of MAP bacilli after binding to (a) bare MNPs, (b) antibody-immobilized MNPs

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After successfully achieving the preparation of MAb-MNPs complexes, laboratory tested culture positive goat milk (37) and bovine milk (26) samples were screened by ‘nano-immuno test’ for its optimization and validation. Change in the color from blue to purple of the milk samples indicated presence of live MAP bacilli after 6–7 h of incubation, followed by pink after 10 h, thereby confirming the presence of live/viable MAP bacilli (Fig. 4). Test gives qualitative estimation on presence or absence of live MAP bacilli.

Fig. 4
figure 4

Visual (colorimetric) detection of MAP antigen in milk by nano-immuno test, Color changes after incubation of: a 0 h, b 4 h, c 7 h and (d) 10 h

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Of the 37 goats milk samples, 12 (32.4%) were culture positive. Of these 12 (32.4%) milk samples, 9 (24.3%) and 11 (29.7%) were also positive in IS900 milk PCR and Nano-immuno test, respectively (Tables 1 and 2). Of the 26 bovine milk samples, 10 (38.5%) were culture positive. Of these 10 (38.5%) milk samples, 8 (30.8%) and 9 (34.6%), were positive in milk-PCR and Nano-immuno test, respectively (Tables 1 and 2). Sensitivity and specificity of the ‘nano-immuno test’ with respect to milk culture was 91.7 and 96.0%, respectively. With respect to IS900 PCR, it was 90.0 (sensitivity) and 92.6% (specificity). In bovine milk samples, sensitivity and specificity of ‘nano-immuno test’ with respect to milk culture was 90.0% and 93.7%, respectively. However, sensitivity and specificity was 88.9 and 94.1% with respect to IS900 PCR (Table 3).

Table 1 Optimization of ‘Nano-immuno test’ with culture and IS900 PCR positive milk samples of goats and bovines to detect live / viable MAP bacilli in raw milk samples
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Table 2 Comparative study of different tests for Nano-immuno test optimization to detect viable MAP bacilli in 63 MAP positive raw milk samples
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Table 3 Statistical analysis using McNemar’s test and kappa values between Nano-immuno test & Culture and Nano-immuno test & IS900 PCR tests
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Color intensity of Nano-Immuno test (Fig. 4) is purely qualitative since the tests was developed to cater the need of diagnosis of presence and absence of live MAP bacilli in the milk samples in the field area. In this ‘pilot scale study’ focus was on to establish proof of concept. Since test is read by naked eyes (visually), therefore it can only detect presence or absence of live MAP bacilli in the milk samples. However color intensity with respect to positive and negative control can provide an indication of the concentration of bacilli in test samples, if the test is standardized using positive control milk samples spiked with measured quantities of MAP bacilli. Test can be easily adapted as semi-quantitative, on the basis of intensity of color change (time and intensity vis a vis positive and negative control), the milk samples can be categorized on the scale of +1 to +5. To develop it as fully quantitative we have to use qPCR additionally on the MAP bacilli attached to MNPs coated with polyclonal MAP antibodies.

Of 138 bovine milk samples screened, 81 (58.7%) were positive for MAP infection and average bio-load of MAP ranged from 13.0 to 52.2%, since number of positive bovine milk samples in each test was variable. Using six diagnostic tests, 17.4, 36.9, 39.1, 13.0, 43.5, 52.2, 41.3% milk samples were positive in i_FAT, microscopy, IS900 PCR, i_ELISA, d_ELISA and LAT, respectively (Table 4). Of the 258 goat milk samples screened, 141 (54.6%) were positive for MAP infection and average bio-load of MAP ranged from 17.4 to 46.5% depending on the positivity of goat milk samples in each test. Using six diagnostic tests, 18.6, 32.5, 32.5, 17.4, 44.2, 46.5 and 45.3% milk samples were positive in i_FAT, microscopy, IS900 PCR, i_ELISA, d_ELISA and LAT, respectively (Table 4).

Table 4 Screening of raw milk samples of bovines by Nano Immuno rapid test vis a vis six other diagnostic tests for the detection of MAP infection
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Of bovine (138) and goat (258) raw milk samples screened statistically, kappa values and two-tailed p values were calculated (Table 6). Nano-immuno test had moderate (with respect to i_FAT, microscopy, i_ELISA and LAT), almost perfect (with respect to IS900 PCR), fair (with respect to i_ELISA) level of agreement using 138 bovine milk samples. Nano-immuno test had 100.0% specificity when compared to all five diagnostic tests except IS900 PCR (95.0%) and sensitivity of nano-immuno test ranged from 33.3%–47.1% in all five diagnostic tests except IS900 PCR (100.0%) using 138 bovine milk samples (Table 6). Using 258 goats milk samples, nano-immuno test had substantial (with respect to i_FAT, microscopy, i_ELISA and LAT), almost perfect (with respect to IS900 PCR), fair (with respect to i_ELISA, d_ELISA and LAT) level of agreement. When compared to all six diagnostic tests, nano-immuno test had varying specificities of 100.0% (with respect to i_FAT and microscopy), 98.6% (with respect to IS900 PCR), 97.9% (with respect to i_ELISA and LAT) and 95.6% (with respect to d_ELISA) using 258 goats milk samples (Table 6). Of 258 goats milk samples screened, sensitivity of nano-immuno test ranged from from 35.0%–57.1% in all five diagnostic tests except IS900 PCR (100.0%) (Table 6).

Discussion

Global studies report high bio-load of MAP in commercial milk and milk products (Acharya et al. 2017; Stephen et al. 2016; Alajmi et al. 2016; Smith et al. 2016). Nano-based approach in diagnosis has so far never been actively explored, though it has potential to address many of the critical issues currently being faced in the diagnosis of chronic Mycobacterial infections in general and MAP in particular. First time, a Nano based test was hypothesized and validated for the detection of viable MAP bacilli in milk samples in the field. In our study, Fig. 1c shows the TEM image of the synthesized MNPs. These spherical MNPs had an average diameter of 30 nm and exhibited good paramagnetic properties in the influence of externally applied magnetic force (Fig. 1c, d). Joo and co workers designed similar approach for the detection of pathogenic bacteria using magnetic nano-particles and optical nano-crystals probes (Joo et al. 2012). Attachment of MAP through the linkage of antibody - MNPs was facilitated by the APTES, glutaraldehyde cross linking as observed by Joo et al. (2012). FTIR was carried out to confirm the binding of MNPs to APTES (Fig. 2). Fe-O bonding at 588 cm−1, CH2 stretching at 2854 cm−1, Si-O-Si vibration at 1081 cm−1 which are characteristics of silane layers and C-O-O bonding at 1461 cm−1 confirmed binding of MNP on APTES while peak at 1637 cm−1 confirmed the loading of glutaraldehyde on to APTES. The presence of C=N bond on the graph confirmed the binding of antibodies onto glutaraldehyde.

Binding of antibody-conjugated MNPs to MAP bacilli were investigated through TEM imaging. Figure 3a, b showed bare MNPs and antibody-conjugated MNPs exposed to MAP bacilli. As observed from the image (Fig. 3) ‘antigen-antibody’ binding facilitated attachment of MNPs with the bacilli. Resazurin (7-hydroxy-3H-74 phenoxazin-3-one-10-oxide), a dye that got reduced in the presences of metabolically active cells was used in the study. Resazurin viability assay have previously been reported for MAP detection (Carroll et al. 2009; Van den Driessche et al. 2014). In this viability assay, major drawback was the fastidious and extremely slow growing nature of MAP bacilli takes time in facilitating the reduction of dye (Fig. 4).

Screening of 138 bovine raw milk samples by seven diagnostic tests, 81 (58.7%) were positive for presence of MAP either in one or more than one diagnostic tests (Table 5). Of the total 81 positive samples, only 6 (4.3%) were detected by one test (dot_ELISA), rest 75 (54.3%) were detected by two or more than two tests, thus confirming the high (58.7%) bio-load of MAP in the raw milk samples driven from bovine population (Table 5). Six (4.3%) samples that were detected by six tests were missed only by IS900 PCR, which may be due to the lower concentration of MAP bacilli in milk samples and lower detection limit of PCR (approx 100 bacilli per 1 mL sample).

Table 5 Comparison of seven diagnostic tests for the detection of Mycobacterium avium subspecies paratuberculosis infection in the raw milk samples {bovine (138) and goats (258)}
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Of the 138 bovine milk screened, nano-immuno test detected, 24 (17.4%) positive for presence of live MAP bacilli which was 29.6% of total 81 (58.7%) milk samples positive for MAP using seven tests. Of the 24 (29.6%) samples positive in nano-immuno test, 18 (75.0%) were also positive by the other six tests. However, there were 6 bovine samples tested (25.0%) which were positive with all the tests except the IS900 PCR (Table 5). Screening of raw milk samples of 258 goats by seven diagnostic tests, 141 (54.6%) were positive for the presence of MAP bacilli in one or more than one test. Of the total 258 milk samples only 6 (2.3%) were detected by single test (dot-ELISA) and rest 135 (52.3%) were detected by two or more than two tests, thus confirmed high bio-load (54.6%) of MAP in raw milk samples of goats (Table 5). Of 258 raw milk samples of goats, only 48 (18.6%) were positive by Nano test which was 34.0% of the total 141 (54.6%) raw milk samples positive for MAP. ‘Nano-immuno test’ confirmed the presence of live MAP bacilli in 48 (18.6%) raw milk of goats. Since ‘Nano-immuno test’ is a qualitative test and will depend on the number of MAP bacilli in milk samples. Quantity of MAP bacilli is usually low in milk samples as compared to other samples (feces, tissues etc.). However, presently in Indian conditions in the absence of control measures, the bio-load of MAP in milk samples is usually very high (Singh et al. 2016d) and the test may prove to be highly successful in the Indian situation of Johne’s disease in domestic livestock (Singh et al. 2014). Therefore results of the test may vary person to person (human factor) and may compromise the sensitivity of test to certain extent.

Unlike our study, previous studies utilized magnetic nano-particles in MAP DNA isolation and immune-magnetic PCRs to detect the presence of MAP in milk samples (Djonne et al. 2003; Gilardoni et al. 2016; Grant et al. 2000; Metzger-Boddien et al. 2006). However, no study has been reported the use of magnetic nano-particles as the spot and field based test to detect viable MAP bacilli in the milk samples of domestic livestock. Presence and transmission of MAP bacilli through milk constitutes a serious ‘public health issue’ and has confirmed MAP as an important ‘food borne’ pathogen of high significance in human population, if not a typical zoonotic agent. Since MAP escapes pasteurization temperature, therefore is continually entering into human food chain by the consumption of infant milk powder and ice-creams etc., (Acharya et al. 2017).

To prove the utility, re-usability and novelty of nano-immuno test, the results were compared statistically with six other diagnostic tests (3 traditional and 3 newly standardized tests) for the detection of MAP bacilli in milk samples. The test was validated and statistically significant results obtained having <0.0001 two tailed P values. Using 138 bovine raw milk samples, nano-immuno test had kappa values of 0.528, 0.528, 0.832, 0.430, 0.324 and 0.461, when compared with i_FAT, microscopy, IS900 PCR, i_ELISA, d_ELISA and LAT, respectively. Using 258 goats raw milk samples, nano-immuno test had kappa values of 0.643, 0.643, 0.961, 0.384, 0.319 and 0.383 when compared with i_FAT, microscopy, IS900 PCR, i_ELISA, d_ELISA and LAT, respectively (Table 6).

Table 6 Statistical analysis using McNemar’s test and kappa values between Nano-immuno and other six diagnostic tests for the screening of milk samples
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In our study, change in colour from blue to purple was observed after 6–7 h of incubation, followed by appearance of pink colour after 10 h, confirming the presence of viable MAP bacilli in milk samples. A study in 2015 observed that at higher concentration (3.43 × 106 MAP cells/mL), a greater difference between live and heat-killed cells/bacilli were observed from day 7, onwards (Pooley et al. 2016). Standardization, validation and re-usability of test were successfully achieved in the field samples. Positive samples in Nano-immuno test were also detected by other diagnostic tests. Test was highly specific, simple to perform and easy to be read by eye and does not require laboratory support. It can be used as mass screening test involving large number of samples. In Table 7, different attributes of the ‘Nano-immuno test’ were compared with number of other tests employed by other workers ((Slana et al. 2008; Husakova et al. 2017; Foddai et al. 2010; Grant 2016; Stratmann et al. 2006; O'Brien et al. 2017) etc.) for the concentration of live MAP bacilli in clinical samples (milk) using bead based immuno-magnetic, peptide based, etc., tests.

Table 7 Comparison of nano-immuno test with other magnetic beads separation assays
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Conclusions

In view of the lack of attention and priority, efforts to control Johne’s disease in domestic livestock are non-existent in India despite high bio-load in domestic livestock. MAP besides gaining in pathogenicity due to repeated passage from infected parents to new generation, it continues to spread. Infected lactating female progressively shed MAP in their milk, thus reaching human population, through consumption of milk and milk products. In existing diagnostic tests, only culture can detect live bacilli, but it takes long time (6–8 weeks) to grow MAP. This led scientists to search for alternative tests to detect live pathogen in clinical samples. This novel nano-technology based chromogenic method for the detection of viable MAP bacilli in milk samples is a step in that direction. Test offer high specificity and simple approach that does not require sophisticated instrumentation or laboratory support and can be used for the screening of large number of milk samples in the field area. This is the first report offering a novel alternative approach to conventional methods using ‘Nano-immuno’ based diagnostic test for the detection of live MAP bacilli in milk samples in the field.