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the domestic livestock population of the country. In order to prevent the human infection through consumption of commercially marketed milk and milk products (flavoured milk and milk powder), it is essential to control bio-load of MAP in the domestic livestock population at the National level.    

1 Introduction   Mycobacterium avium subspecies paratuberculosis (MAP), the cause of chronic enteritis called Johne’s disease (JD) in domestic livestock, has also been associated with number of incurable, auto-immune diseases (Inflammatory Bowel disease or Crohn’s disease, etc.) in human beings (McFadden et al., 1992; Bull et al., 2003; Abubakar et al., 2007; Scanu et al., 2007; Cirone et al., 2007; Naser et al., 2014; Banche et al., 2015; McNees et al., 2015). Live bacilli (MAP) have been recovered from milk and milk products made from laboratory scale and commercial size pasteurization facilities for liquid milk in Czech Republic, UK, USA and India (Grant et al., 1996; Shankar et al., 2010). Country has huge population (>500 million) of domestic livestock, endemically infected with MAP (Singh et al., 2014; Rawat et al., 2014). We have reported high to very high bio-incidence of MAP in the raw milk (individual animals and pooled milk) of domestic livestock (Singh et al., 2007; Shankar et al., 2010; Singh et al., 2014). At 155.5 million tonnes (MT) of milk produced in 2016-17, India is leading milk producer in the world (Annual Report, DADF, 2016-2017) and approximately 40.0% of milk is pasteurized, predominantly by state cooperatives, multi-national companies, or government dairy plants (India in business, Investment and technology promotion and energy security, Delhi: Ministry of External Affairs, Government of India, 2008) for the preparation of various milk products. Traditionally raw milk also used as base for many Ayurvedic medicines (Tursun et al., 2015). Un-like, M.bovis, the cause of bovine TB, MAP is able to resist pasteurization (Lund et al., 2002) and live bacilli have been cultured from commercial pasteurized milk supplies (Grant et al., 2002), retailed pasteurized milk (Singh et al., 2009; Paolicchi et al., 2012;Eftekhari & Mosavari, 2016) and milk products like milk powder (Hruska et al., 2011; Botsaris et al., 2016; Acharya et al., 2017) including retail cheese (Ikonomopoulos et al., 2005; Raghuvanshi et al., 2014; Eftekhari & Mosavari, 2016). Retailed cheese, powdered milk and ice-creams are important milk products that are being consumed by large percentage of human population both in India and globally. Consumption of milk and milk products {liquid pasteurized milk (for drinking purpose), milk powder (as infant supplement), cheese (as top-up and main ingredient of highly popular pizza) and ice-creams (as sweet dish during personal, family, official and group celebrations and marriage ceremonies etc.) made from pasteurized milk} without boiling has increased the risk of infection manifold. Popularity of milk and milk products (flavoured milk and milk powder) is not restricted to urban areas but also reached in rural areas. Since, MAP is not in-activated during pasteurization, therefore routine daily consumption of these milk products has led to increased compromise on 'public health concerns' and milk as important vehicle of some of the dangerous 'food borne pathogens' like MAP, in the country (Singh et al., 2016d) and globally (Cirone et al., 2007; Eltholth et al., 2009; Patel & Shah, 2011). In-activated MAP bacilli can also be a risk for number of health problems due to structural components (Grant, 2006). Despite direct effect on human health, consumption of milk and milk / dairy products {flavoured milk and powdered infant formula (PIF)} laden with MAP bacilli are not legally banned by any country in the world. Many food safety authorities in UK advised government to adopt precautionary steps to minimize the entry of MAP into the human food chain (Grant, 2006). Today milk and dairy products received most attention as vehicles for transmission of MAP, an important food borne pathogen, since MAP is secreted in the milk of infected animals. Raw milk may also be contaminated with fecal material during milking. Though number of studies in developed countries reported presence of MAP bacilli or DNA in the raw milk samples (Stabel et al., 2002; Stephan et al., 2002), but studies are extremely limited in other parts of the world. MAP infection, the cause of incurable chronic enteritis is endemic in nearly 500 million population of domestic livestock in the country (Singh et al., 2010; Singh et al., 2013b; Singh et al., 2014; Chaubey et al., 2016), however, information on the bio-incidence of MAP in commercially retailed pasteurized milk and milk products in the country is almost non-existent (Shankar et al., 2010; Raghuvanshi et al., 2014).  Bio-safety of food items and bio-incidence of MAP in milk and milk products is matter of intense research in developed countries and a battery of tests have been used to demonstrate the presence of MAP. Though, culture is ‘Gold standard’ test but takes long time (6-8 weeks). PCR based assays rapidly confirm this fastidious slow-growing bacilli in clinical samples (Millar et al., 1996; Singh et al., 2013a; Singh et al., 2014; Nielsen & Toft, 2014; Garg et al., 2015). In our laboratory besides traditionally used microscopy, we have developed and standardized number of diagnostic assays like Indigenous ELISA kit (i_ELISA), indirect fluorescent antibody test (i_FAT), indigenous dot-ELISA (d_ELISA), IS900 PCR and latex agglutination test (LAT) for the screening of milk and milk products and to estimate bio-incidence of MAP (Sharma et al., 2008; Singh et al., 2016a; Singh et al., 2016b; Singh et al., 2016c). Present study first time used both traditional and newly developed and standardized serological and molecular tests (microscopy, IS900 PCR, i_ELISA, d_ELISA, LAT and i_FAT) to estimate bio-incidence of MAP in liquid pasteurized milk and milk products (flavoured milk and milk powder) made from pasteurized milk and sold by 10 leading commercial market brands in Mathura and Agra districts of South UP in North India. Figure 1 MAP bacilli as seen in acid fast staining in                         commercial milk samples
2 Materials and Methods 2.1. Collection and processing of milk samples: Commercial food items of 'mass consumption' {liquid pasteurized milk and milk products (flavoured milk and milk powder) made from 'pasteurized milk'} were screened to estimate the bio-incidence of MAP using six tests. For this purpose 133 commercial milk samples (100 liquid milk from eight market brands, 19 flavoured milk from five market brands and 14 milk powder from two market brands) belonged to 10 leading commercial market brands (Amul, Ananda, Nestle India, Gyan, Mother dairy, MTR, Namaste India, Nova, Paras and Sanchi) were purchased from local markets in Mathura (Farah and Kosi) and Agra districts of South Uttar Pradesh. First time milk samples were screened using whole milk as 'test sample' without any processing. In our previous studies, milk samples were first processed by centrifugation, wherein each milk samples was separated in to whey, fat and sediment layers and three fractions of each milk samples were processed independently. Samples were screened both by antigen {(microscopy, i_FAT and IS900
PCR) and antibody (i_ELISA, d_ELISA and LAT)} detection tests to estimate bio-incidence of MAP. Each of the 133 milk samples were screened six times by each of the six diagnostic tests (3 antigen and 3 antibody detection tests). IS1311 PCR_RE and milk culture were performed on limited number of IS900 PCR positive samples, to know the 'bio-type profile' (molecular epidemiology) and demonstrate live MAP bacilli in commercial milk samples. In the present study each 'milk sample' was treated as representing 'single animal' and a sample positive in any of the six tests was considered positive for 'bio-incidence' of MAP. Approximately, 10-12 ml of milk (liquid milk and flavoured milk) and 2.0 grams of milk powder / dairy creamer (homogenized in 10–12 mL of autoclaved distilled water) were collected from sachet / packet / container and stored. Two milli-liters of these samples were used as 'test sample' and was sufficient to perform all the six tests. 'Test samples' (liquid milk, flavoured milk and homogenized milk powder) were subjected to microscopy, i_FAT and IS900 to detect MAP bacilli and LAT, i_ELISA and d_ELISA tests to detect anti-MAP lacto-globulins (antibodies). 2.2. Microscopy Smears were prepared from 20 µl of commercial milk samples, heat fixed, stained by Ziehl Neelsen (ZN) staining (Singh et al., 2008) and examined under oil immersion (×100) for acid-fast bacilli (AFB) indistinguishable to MAP (Figure 1). 22.3. Indirect Fluorescent Antibody Test (i_FAT) Test was performed as per Singh et al. (2016a). Briefly, smears were prepared on clean slides from (20 µl commercial milk) samples, 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 minutes at 37?C, followed by second dipping in phosphate-citrate buffer (2.1% citric acid and 3.56% disodium hydrogen phosphate in 100 ml triple distilled water, pH- 5) and were heated to boiling in microwave for 30 seconds (15 cycles) with rest of 20 seconds after each heating cycle (total time 10 minutes). Slides were then air dried at room temperature. Then primary antibody (whey as control in ratio of 1:4 and serum in ratio of 1:50) in serum dilution buffer (1% BSA in PBST) was added on the slides. Slides were incubated for 1 hour at 37?C in BOD 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 hour 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. One smear prepared from the heat killed MAP culture was used as control for the comparison of results. Slides positive for MAP infection exhibited green fluorescence under fluorescent microscope (Figure 2). Figure 3 Agarose gel electrophoresis of PCR products obtained by IS900 PCR (A) performed on commercial milk samples [Lane M: 100bp DNA ladder; Lane C: MAP DNA (positive control, 229bp); Lane 1: Negative control and Lane 2–12: Commercial milk test samples]  
Figure 2 Green fluorescence indicating the presence of MAP bacilli by Indirect Fluorescent antibody test (i_FAT).
a: Positive control; b:  Commercial milk sample; c: Negative control
2.4. DNA Isolation   DNA isolation from commercial milk was carried out as per Van Soolingen et al. (1991) with some modifications. Briefly, 500 μl commercial 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 RT for 15 min. After that 100 μl of 24% sodium dodecyl sulfate (SDS) was added and incubated at RT for 10 min, followed by heating at 80°C for 10 min. Then, 32.5 μl of proteinase K (10mg/ml) was added to above sample and incubated at 55°C for 2 hrs., 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 15800 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 hours. DNA was pelleted out by centrifugation at 15800 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.   2.5. IS900
PCR DNA isolated from commercial milk samples, was subjected to specific IS900 PCR using 150C and 921 primers of Vary et al. (1990). Presence and yield of specific PCR product (229bp) was considered as positive for MAP infection (Figure 3). DNA isolated from MAP culture was taken as control for comparison of results. Figure 4 Molecular bio-typing of IS900 positive MAP DNA by IS1311 PCR (B) and IS1311 PCR-REA analysis (C)  
  2.6. IS1311 PCR and restriction endonuclease analysis (IS1311 PCR _REA) IS1311 PCR of IS900 PCR positive DNA samples were performed using M56 and M119 primers as per Sevilla et al. (2005). Resultant PCR products (608bp) were subjected to electrophoresis (2% Agarose gel stained with Ethidium bromide). Amplicons that co-migrated at 608bp were taken as positive. The 608bp IS1311 PCR products were restricted by restriction endonuclease digestion with HinfI and MseI enzymes (Fermentas) for 1.5 hrs by incubating at 37°C (Sevilla et al., 2005). Fragments (amplicons) were separated on 3.0% agarose gel and genotypes were identified on the basis of fragment migration patterns (Whittington et al., 2001) (Figure 4). 2.7. Indigenous ELISA test Test was performed as per Singh et al. (2016b) and instead of whey, commercial milk sample (liquid milk and flavoured milk) and homogenized milk powder were used as test sample. Briefly, each well of flat bottom 96 well ELISA plate was coated with 0.1 µg of protoplasmic antigen in 100 µl of carbonate-bicarbonate buffer, (pH 9.6) per well and incubated at 4°C overnight. Plates were washed thrice with PBST (1X PBS with 0.05% Tween 20) followed by blocking in 100 µl of 3.0% skimmed milk in 1X PBS, incubated for one hour at 37°C. Plates were washed three times with PBST and then 100 µl of commercial milk diluted in PBST with 1.0% BSA in ratio of 1:2 was added as sample in duplicate wells and incubated for 2 hrs at 37°C. Plates were washed thrice 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). Whole milk from weak and culture positive and healthy and culture negative goat were used as positive and negative controls, respectively. Optical densities (OD) values were transformed and expressed as sample-to-positive (S/P) ratios (Collins, 2002). 2.7.1. Analysis of OD values Sample to positive ratios were derived to estimate corresponding status of JD in goats was determined as per Collins (2002). Samples in low positive (LP), positive (P) and strong positive (SP) categories in sample to positive ratios were considered positive for MAP infection (Collin, 2002). Figure 5 Dot-ELISA screening of commercial milk samples (1-10) showing presence of MAP antibodies as positive brown dot; +ve: Positive control (brown dot); -ve: Negative control (no brown dot)  
S/P ratio value = [(Sample OD – Negative OD) / (Positive OD - Negative OD)].   2.8. Dot- 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 (2µg 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 hours at 37°C. Combs were dipped in blocking solution (3.0% skimmed milk powder in 1X PBS) for 1 hr at 37°C. After washing in PBST combs were dipped in test samples (100 µl commercial milk in 1:2 dilution in 1% BSA in 1X PBST) for one hr followed by again washing. Further combs were incubated with 200 µl of rabbit anti-bovine (1:3000 in 1X PBS) / caprine (1:3000 in 1X PBS) HRP conjugate solution at 37°C for 30 min. Finally, combs were dipped in 200 µl of 3, 3'-Diaminobenzidine (6mg / 10 ml of 1X PBS), at room temperature till development of colour (1-2 min) (Figure 5). Once the spot was visible combs were dipped in water to stop the reaction. Milk positive and negative controls used in the study were confirmed by IS900
PCR and microscopy, were used on two legs of each comb to assist in reading of test samples. 2.9. Latex Agglutination test 2.9.1 Preparation of latex beads Commercial milk samples (n-133) were screened using LAT 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 gm glycine, 0.07 gm Sodium Hydroxide, 1.7 gm Sodium Chloride, 0.1 gm Sodium Azide in 100 mL of triple distilled water) (pH- 8.6), then 20 µl of antigen (4mg / mL) was added and incubated for 3 hours 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 1X PBS) and mixed in shaker incubator for 45 min at 37?C. Finally beads were washed twice in 1X PBS.   Table 1 Bio-incidence of MAP in commercial pasteurized milk samples of leading Indian brands sold in the local markets using ‘Indigenous milk ELISA kit’  
i_ELISA kit, % (n) Categories Strong Positive (SP) Positive (P) Low Positive (LP) Suspected (S) Negative (N) False (F) Sub-total Nil 15.7 (21/133) 11.2 (15/133) 11.2 (15/133) 60.9 (81/133) 0.7 (1/133) Total (n-133)  
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