Full Text: 1 Introduction Mycobacterium avium subspecies paratuberculosis (MAP) is the major milk contaminant and is endemic in the domestic livestock population of the country (Singh et al., 2014a; Chaubey et al., 2017) and also globally (Geraghty et al., 2014). Recently, MAP the cause of Johne’s disease (JD) in animals has been associated with Crohn’s (CD) and other auto-immune disorders in human beings (Singh et al., 2008; Naser et al., 2014; Banche et al., 2015). Johne’s disease harms farm economics, low per animal productivity (Shephard et al., 2016; Cho et al., 2012), a serious threat to public health (Singh et al., 2016d), high costs of disease management (Johnson-Ifearulundu et al., 1999), reduced milk production, increased mortality and culling rates (Raizman et al., 2009), the in-utero transmission of MAP (Whittington & Windsor, 2009) and reduced average weaning weight of calves (Bhattarai et al., 2013) leading to huge losses in the dairy industry. Recently, ‘food safety’ has become a major concern. Therefore, milk and milk products received maximum attention as a potential source of MAP. In India, studies have limited on the bio-presence of MAP in the milk of cows (Sharma et al., 2008), goats (Singh & Vihan, 2004), in commercial milk (Singh et al., 2009; Raghuvanshi et al., 2010) and milk products (Singh et al., 2018; Shankar et al., 2010). Pasteurization and spray drying used in making of cheese (Faria et al., 2014), powdered infant formula/calf milk replacers (Botsaris et al., 2016; Acharya et al., 2017), yogurt (Cirone et al., 2013), sour cream, and curd (Klanicova et al., 2012; Messelhausser et al., 2012) are unable to kill MAP and viable bacilli have been recovered (Sharma et al., 2008). Pasteurization norms of heating milk to 72°C with a holding time of 15 sec are insufficient to inactivate MAP bacilli. MAP forms clump on heating due to high lipid contents (Mullan, 2015). High bio-presence in raw milk has also been reported by Grant (2006) and Hammer et al. (2006) in the United Kingdom (UK). Long incubation (4–6 months) and decontamination are major constraints in the culture of MAP (Singh et al., 2007). Molecular methods (IS900 PCR, Real-time PCR, and f57 quantitative PCR) have frequently been used for rapid detection of MAP with high sensitivity in powdered infant formula (Hruska et al., 2011; Botsaris et al., 2016; Acharya et al., 2017), cheese (Faria et al., 2014), dairy products like yogurt, sour cream, and curd (Klanicova et al., 2012; Messelhausser et al., 2012; Cirone et al., 2013). Both traditional (Indigenous Enzyme linked Immuno Sorbent Assay Kit (i_ELISA), microscopy and IS900 PCR) and recently standardized tests (dot-ELISA (d_ELISA), indirect Fluorescent antibody (i_FAT) and Latex Agglutination (LAT)) were used for the detection of MAP (Singh et al., 2016a; Singh et al., 2016b; Singh et al., 2016c) in milk products (Raghuvanshi et al., 2014; Stephen et al., 2016). Information on the bio-presence of MAP in commercially marketed ‘milk products’ is extremely limited in the country (Sharma et al., 2008; Raghuvanshi et al., 2014; Stephen et al., 2016). Therefore, the present study aimed to estimate the bio-presence of Mycobacterium avium subspecies paratuberculosis (MAP) infection as the major microbial contamination of milk products sold in the Indian markets by the major commercial market brands in India in Indian market using multiple tests {microscopy, serological (i_ELISA, d_ELISA, LAT), molecular (IS900 PCR), i_FAT and culture}. 2 MATERIALS AND METHODS 2.1. Profile of milk products Samples (276) of 12 types of milk products (Curd, Buttermilk, Ice cream, Lassi, Butter, Cheese, MatkaKulfi, Cream, Coffee milkshakes, Fruit Yoghurt, Makhana Kheer and Rabdi) made from pasteurized milk were purchased from local markets in Mathura (Farah and Kosi) and Agra districts of North India from 2015 to 2017. Milk products were driven from 22 leading commercial market brands (Table 1). Distribution of 276 milk products purchased for screening was: 36.2 (100), 26.0 (72), 15.2 (42), 7.2 (20), 5.8% (16), 3.6 (10) and 1.4% (4) of curd, buttermilk, ice-cream, lassi, butter, cheese, and Matka kulfi, respectively (Table 1). Milk products were of two types: (i) ‘Large sample sized’ {Curd (100), Buttermilk (72) and Ice cream (42)} and (ii) ‘Small sample sized’ {Lassi (20), Butter (16), Cheese (10), MatkaKulfi (4), Cream (4), Coffee milkshakes (2), Fruit Yoghurt, (2), Makhana Kheer (2), Rabdi (2)}. 2.2. Screening methods Cheese (2.0 gm) was finely grounded in 10–12 ml of autoclaved distilled water and a homogenized solution was used as a ‘test sample’. Curd, buttermilk, milkshakes, kheer, yogurt, lassi, rabari, and kulfi were used directly as ‘test sample’. Ice-creams were melted at room temperature and the melted liquid was used as ‘test sample’. Using tests like Microscopy, IS900 PCR, i_FAT we targeted bacilli, and using ELISA, d_ELISA, LAT, MAP lactoglobulin were targeted in the milk products. 2.2.1 Microscopy Microscopy was performed as per Singh et al. (2018). The presence of short acid-fast pink staining rods will be taken as positive (Figure 1). 2.2.2 Indirect Fluorescent antibody test Indirect Fluorescent antibody test (i_FAT) developed for tissues (D’Haese et al., 2005), was modified for milk samples (Singh et al., 2016b). Positive samples exhibiting green fluorescence were considered positive (Figure 2).   2.2.3 Deoxyribonucleic acid isolation Commercial milk product's DNA was isolated as per van Soolingen et al. (1991) with some modifications (Singh et al., 2018). 2.2.4 IS900 Polymerase Chain Reaction Isolated DNA was amplified by specific IS900 PCR (Singh et al., 2008) using Vary et al. (1990) primers. The yield of a specific PCR product (229 bp) will be taken as positive (Figure 3). 2.2.5 IS1311 assay and restriction endonuclease analysis IS900 PCR positive DNA samples were subjected to bio-typing using M56 and M119 primers (Sevilla et al., 2005) (Figure 3). 2.2.6 SYBR Green Real Time assay Targeting IS900 Gene MAP DNA from milk products (n=34) was screened by IS900 SYBR green Real time PCR (SYBR RT_PCR) following the Gupta et al. (2017) protocol (Figure 4). 2.2.7 Culture IS1311PCR positive samples were cultured as per Merkal & Richards (1972) and tubes screened at weekly intervals for the appearance of typical MAP colonies (Singh & Vihan, 2004). 2.2.8 Indigenous Enzyme Linked Immuno Sorbent Assay kit Milk-based indigenous ELISA’ kit (Singh et al., 2007) used for screening with modifications (Singh et al., 2018). OD values were expressed as sample-to-positive (S/P) ratios as per Collins (2002). Sample in low positive (LP), positive (P), and strong positive (SP) categories were considered positive for MAP infection. 2.2.9 Dot-Enzyme Linked Immuno Sorbent Assay Dot-ELISA was performed as per Singh et al. (2016a) (Figure 5). 2.2.10 Latex Agglutination test The Latex Agglutination test (LAT) was performed as per the method of Singh et al. (2016c) and positive results were noted within 2 min (Figure 6). 2.3 Statistical analysis Statistical significance was measured between two tests, McNemar’s test and Kappa agreement statistical analysis methods applied by Graph Pad software, California, United States of America (USA). Sensitivity and specificity were measured by Med-Calc software, Acacialaan 22 8400 Ostend, Belgium. 3 Results Samples from Cream (4), Coffee milkshakes (2), Fruit Yoghurt (2), MakhanaKheer (2), Rabdi (2) were negative in all diagnostic tests performed. Of 276 milk products screened, cumulative bio-presence was 52.8% (146) by six tests (Table 1). Product-wise, bio-presence of MAP was the highest in butter (75.0%), followed by curd (66.0%), buttermilk (52.9%), lassi (50.0%), cheese (40.0%) and ice-cream (28.5%). The sensitivity of the test for the detection of bio-presence of MAP was, 41.3 (114), 39.8 (110), 33.3 (92), 23.1 (64), 22.4 (62) and 7.9% (22) in microscopy, i_FAT, dot_ELISA, LAT, i_ELISA and IS900 PCR, respectively. None of the 47.1% (130) milk products (perfect negative) were detected by any of the six tests (Table 1). Samples (22) positive in IS900 PCR were bio-typed and bio-typing of seven types of milk products revealed the presence of ‘Indian Bison Type’ biotype in 5.7% (16) in five milk products (curd, buttermilk, ice-cream, butter, and cheese) (Table 2). 3.1 Large sample-sized milk products 3.1.1 Curd The Bio-presence of MAP was very high (66.0%) in curd samples (100) of 14 brands. The 50.0, 40.0, and 6.0% samples were found to be positive for the bio-presence of MAP using microscopy, i_FAT and IS900 PCR (antigen detection), and 34.0, 44.0 and 34.0% in i_ELISA, d_ELISA and LAT (antibody detection) tests, respectively. The 6.0, 18.0, 6.0, 6.0, 8.0, and 20.0%, samples were positive in six, five, four, three, two, and one test, respectively (Table 3). Microscopy was most sensitive followed by d_ELISA, i_FAT, i_ELISA, LAT, and IS900 PCR. Except, Namaste India and Nestle, the rest of 14 market brands, 85.7% (12) were positive and bio-presence was 76.9 and 46.1% in Mother dairy and Amul brands, respectively (Table 4). Bio-typing of 46.1% positive of Amul revealed 7.6% were ‘Indian Bison Type’. 3.1.2 Buttermilk Buttermilk from eight brands was next largest sample {37.5% (n-72)} and 50.0% were positive (Table 2). Bio-presence was 52.9, 28.5, and 50.0% in Amul, Mother Dairy, and Ananda brands, respectively (Table 4). All samples from Namaste India (4), Parag (2), and Sanchi dairy (2) were positive. Two samples each from Gyan and Payas diary were negative. The i_FAT was most sensitive (44.4%) followed by microscopy (38.8%), d_ELISA (33.3%), LAT (22.2%), i_ELISA (19.4%), and IS900 PCR (19.8%) for the detection of bio-presence of MAP in milk products. In all the six tests, 8.3 and 50.0% samples were positive and negative, respectively (Table 3). Except, 8.3% of samples detected by single test (microscopy, i_FAT, and d_ELISA), rest 41.6% were detected by multiple tests (two to six tests). Bio-typing of 36 (50.0%) positive samples showed, 16 (22.2%) were ‘Indian Bison Type’ where Amul (5.8%), Mother dairy (14.2% and Namaste India (100.0%) (Table 4). 3.1.3 Ice cream Of 42 ice-cream samples, 12 (28.5%) were positive (Table 2). The Bio-presence of MAP was 37.5, 25.0, 33.3, and 100.0% in Cream well, Sudha, Havmor and Say Natural brands, respectively. None of the samples from Amul, Vadilal, and Madhu brand was positive (Table 4). i_FAT was most sensitive (23.8%) followed by microscopy (19.0%), d_ELISA (9.5%), and in three tests (LAT, i_ELISA, and IS900 PCR-4.7% each). In i_FAT, 9.5% of samples were positive and 19.0% by multiple tests (two to six tests) (Table 3). Of two (4.7%) positive ice-cream (Say Natural brands) in IS900 PCR, both were ‘Indian Bison Type’ (Table 4). 3.2 Small sample size milk products 3.2.1 Lassi Out of 11.7% (20) lassi samples (six brands), 50.0% (10) were positive (Table 2). Bio-presence was 25.0, 100.0, 100.0, and 100.0% in samples from Mother dairy, Amul, Parag, and Sanchi dairies, respectively. None from Gopalji and Saras were positive (Table 4). i_FAT and microscopy were most sensitive (40.0%) followed by d_ELISA, LAT, and i_ELISA (20.0%). In IS900 PCR, none was positive. Except, 10.0% samples (positive in one test), rest 40.0% were detected by multiple tests (two to six tests) (Table 3). 3.2.2 Butter In the case of butter, cumulatively, 12.5% and none samples were positive and negative respectively, in all six tests. Out of 9.6% (16) butter samples (four brands), 75.0% (12) were positive (Table 2) and bio-presence was 100.0% in Amul, Mother dairy, Saras, and Sudha brands (Table 4). i_FAT and microscopy were most sensitive (87.5%) followed by d_ELISA (75.5%), i_ELISA (37.5%), LAT (25.0%) and IS900 PCR (12.5%). Except, 12.5% (positive in one test), rest 87.5% were positive by multiple tests (two to six tests) (Table 3). Screening of 16 positive samples in IS900 PCR, only two samples from Mother dairy were ‘Indian Bison Type’ (Table 4). 3.2.3 Cheese and paneer Of the total, 6.5% (10) cheese samples (four brands), four (40.0%) were positive in multiple tests (i_FAT, microscopy, d_ELISA, and LAT) (Tables 2 and 3) and bio-presence was 100.0% in Amul brand, and samples from Mother dairy, Pankajpaneer and Saras) were negative (Table 4). i_ELISA and IS900 PCR detected 20.0% samples. Bio-typing of four IS900 positive samples, 50.0% (2) from Amul were ‘Indian Bison Type’ (Table 4). 3.2.4 Matkakulfi and other milk products Of four Matkakulfi (Cream well and Vadilal), two (Cream well) were positive by i_FAT and microscopy. Fruit yogurt (Mother dairy), cream (Amul), coffee milkshake (Cavins), Rabdi, and Makhana Kheer(Sudha) were negative (Tables 3 and 4). 3.3 Screening by culture and IS900 Assay Of 276 milk products screened, 2.8% (lassi-1, ice-cream-1, butter-2, curd-2, and butter milk-2) were positive for live MAP bacilli. Culture positive milk products (2.8%) were also detected positive by six tests except for one sample of lassi which was negative in IS900PCR. Further, among the 7.9% (22/276) milk products positive in IS900PCR, 6.0 (6/100), 13.8 (10/72), 4.76 (2/42), 12.5 (2/16) and 20.0% (2/10) belonged to curd, butter-milk, ice cream, butter, and cheese, respectively (Table 2). 3.4 Sensitivity and specificity of six tests Based on kappa and two-tailed p values, the sensitivity and specificity of each test were calculated and has been provided in Table 5. d_ELISA had highest (100.0%) and IS900 PCR had lowest (19.3%) sensitivity while IS900 PCR found to be most specific (100.0%) and d_ELISA least specific (85.9%). Based on a statistical comparison of six tests, the strength of agreement was ‘good’ for d_ELISA concerning i_ELISA, with a kappa value of 0.734. A comparison of LAT with i_ELISA showed the strength of agreement was ‘very good’ with a kappa value of 0.815. Similarly, the strength of agreement was ‘good’ for i_FAT concerning microscopy with a kappa value of 0.700 (Table 5). 3.5 Screening of IS900 assay negative samples by SYBR green Real Time assay Screening of 34 samples, {negative in IS900PCR and positive in microscopy (n-18), in i_FAT (n-8) and d_ELISA (n-8)}, 20.6% (7/34) were positive using RT_PCR, which improved the detection rate of MAP infection in milk products (Table 6). 4 Discussion India has 17.0% of the world’s population of domestic livestock and JD is endemic in the country (Singh et al., 2014a; Gupta et al., 2019). Chaubey et al. (2017) in past 31 years (1985-2017) showed consistent increase in the bio-load from 11.4% (464/4057) in 1985–1990 to 44.2% (3832/8658) in 2011-2017, using multiple tests; {(average – 26.8% (6976/26009)}. Studies further showed that bio-presence increased from 44.2% (3832/8658) in 2011-2017 to 48.6% (5440/11180) in past nine months (April 2017-December 2017) (unpublished data). This is mainly due to low priority accorded to JD therefore there is absence of  control measures. Though at 132.43 million tonnes of milk production (FAOSTAT, 2018), India is the leading milk producer in the world, however, per animal productivity is very low mainly due to the high bio-presence of MAP in the domestic livestock. Studies from Shankar et al. (2010) reported 22.0 – 100.0% bio-presence of MAP in milk products and 56.0 – 78.0% in ice-creams and flavored milk in 2010, Raghuvanshi et al. (2014) observed 0.0 to 16.6% in paneer samples from goat milk and Stephen et al. (2016) reported, 32.7 – 74.5% in paneer samples from bovine milk revealed high bio-presence of MAP in milk and milk products sold in India for human consumption. The 276 milk products (12 types and 22 leading commercial brands) from Agra and Mathura were screened and seven (curd, buttermilk, ice cream, lassi, butter, cheese, and Matka kulfi) were positive for MAP. Milk products (cream, coffee milkshakes, fruit yogurt, makhana kheer, Rabdi) negative for MAP may be due to low sample size (12/276 or 4.3%) or required high temperature or long time heating (coffee milkshakes, makhana kheer, rabdi) during manufacturing. Makhana kheer a native porridge requires a long time boiling of milk. Rabdi is concentrated milk made by slow long time heating. Coffee milkshakes need boiling of milk. The cream is not boiled but in fruit yogurt, fruits are added to the cream. In large sample-sized milk products, the highest samples were positive in curd (66.0%), followed by buttermilk (50.5%) and ice-cream (28.5%), posing a serious threat to human health since products are of mass consumption. Human patients suffering from severe MAP infection had a weakness for ice-creams (Singh et al., 2016d). In small sample size products, the highest bio-presence was in butter (100.0%), followed by lassi (50.5%), Matka kulfi (50.0%), and cheese (40.0%). High bio-presence in milk products may be due to high bio-presence in domestic livestock (Singh et al., 2014a; Chaubey et al., 2017) and continue to increase (un-published data up to December 2017). A similar high bio-presence was reported in the human population of this region (Singh et al., 2014b), Study showed milk products sold by leading market brands were ‘unsafe’ for human consumption. Of 23,196 human beings reported for screening at pathology laboratories in Mathura, 28.8% against auto-immune disorders, 40.5% infectious diseases, and 40.9% non-infectious disorders in 2013 (Singh et al., 2014b). Screening of 23196 patients by i_ELISA, MAP was active in 34.0% population (Singh et al., 2014b). Screening of 3093 blood by IS900 blood PCR, 8.4% were positive for human paratuberculosis. These studies corroborated the high bio-presence of MAP in the human population as a consequence of the consumption of milk products contaminated with high bio-presence of MAP (Singh et al., 2014b). Highest positivity was in butter (100.0%), followed by curd (66.0%), buttermilk (50.5%), lassi (50.5%), Matka kulfi (50.0%), cheese (40.0%) and ice-cream (28.5%). Thick waxy wall of MAP bacilli has an affinity for fat molecules in butter, curd, lassi, buttermilk, and Matka kulfi (Mullan, 2015). Similarly, cheese (40.0%) has high-fat contents, wherein bacilli get partitioned with fat. Though fat contents are high in ice-creams due to heating during making MAP bio-presence may be low. Of the milk products screened, butter was most unsafe for human consumption. In the absence of control programs, MAP bio-presence was high in domestic livestock (Singh et al., 2014a). This study first time revealed that consumption of milk products not safe, though the human population including babies (infant milk formula), largely depends on milk products as food items of mass consumption and are continuously getting exposed to MAP. Depending on stress conditions several cases are culminating as patients of ‘human paratuberculosis’. Test-wise, the sensitivity of i_FAT, microscopy, and d_ELISA was highest and parallel. i_FAT was most sensitive followed by microscopy and d_ELISA. i_ELISA and LAT had similar sensitivity in curd, buttermilk, ice-cream, and lassi. i_ELISA was superior for the screening of butter as compared to cheese, where LAT was better. The study highlighted the selection of a test for screening milk products after considering the nature of milk products. Depending on resources, purpose, and sample we can choose the screening test(s). The use of RT_PCR and culture led to further confirmation of MAP in milk products. High bio-presence of MAP (28.5–100.0%) in the milk products in present findings were similar to our earlier reports of high bio-presence in domestic animals (Singh et al., 2014a) raw milk (personal communication) and commercial pasteurized liquid milk (Singh et al., 2018) and human population (Singh et al., 2014b) in North India. Unless MAP infection is controlled in animals, the human population will continue to get exposed to MAP. Chaubey et al. (2017) correlated that high bio-presence of MAP in the human population may have resulted in a sharp rise in auto-immune disorders (Crohn’s disease, inflammatory bowel disease, ulcerative colitis, diabetes type 1, thyroiditis, rheumatoid arthritis, Allergic rhinitis, milk allergies, etc.). MAP infection is incurable both in animals and human beings. Of the 12 types of milk products screened, consumption of butter, curd, buttermilk, lassi, and cheese was the most likely threat for human infection with MAP. Due to the presence of a thick waxy cell wall, MAP survives pasteurization temperatures by forming clumps (Donaghy et al., 2010). Viable MAP has been detected even after the application of pressure along with pasteurization (Donaghy et al., 2010). The available published reports provided evidence for the existence of live MAP in unpasteurized, pasteurized milk, colostrums, milk powder, and all types of fresh cheese by Bradner et al., (2013) and Eftekhari & Mosavari, (2016). Studies show high bio-presence of MAP in milk and milk products may be leading to the increased incidence of Crohn’s disease in the human population. Botsaris et al. (2010) in their report on the bio-presence of MAP DNA using quantitative real-time PCR (RT_PCR), showed that 25.0% cheese originating from sheep, goat, and mixed milk from farms and products in Cyprus were found positive. However, viable MAP bacilli were not detected either in the combined phage IS900 PCR or by conventional culture method. Faria et al. (2014) also reported MAP from 10.0 and 3.3% retail Coalho cheese samples in Brazil by PCR and culture, respectively, and sequenced the DNA from positive culture samples showing 99% identity with the insertion sequence IS900. Similarly, various researchers tested the efficacy of various tests in the detection of Mycobacterium avium paratuberculosis in milk products (Table 7). Using culture in the present study, live MAP colonies were recovered from 2.8% milk products (lassi, ice creams, butter, curd and butter, 0.3, 0.3, 0.7, 0.7 and 0.7%, respectively). Similar findings were reported by Van Brandt et al. (2011). The authors conducted a study to detect MAP in yogurt and commercial fermented milk products containing probiotic strains. They concluded the easy survivability of MAP in yogurt but MAP numbers decreased in fermented milk products containing probiotic cultures. Their results filled-up the knowledge gap on the behavior of MAP in yogurt and fermented milk products containing probiotic cultures. Though MAP could not be isolated from yogurt or fermented milk, their research was millstone and valuable in the context of the risk of MAP transmission to humans through yogurt and probiotic fermented milk products. Klanicova et al. (2012) and Cirone et al. (2013) confirmed the survival of MAP in fermented milk products (yogurt, acidophilus milk, and kefir) during fermentation conditions, low pH and refrigerated storage for at least 20 days. MAP in raw materials might reach consumers via consumption and emphasize on implementation of good manufacturing practices during the production and storage of fermented milk products. Singh & Vihan (2004) screened raw milk of five goats suffering from clinical Johne’s disease for the presence of MAP. Consistently high bio-presence of MAP in goat milk samples, authors concluded that MAP was endemic in goat herds located at CIRG, Makhdoom, India. This may be due to repeated transmission of MAP from infected mothers to the new generation through milk and colostrum. Despite regular screening and culling of suspected goats since the establishment of herds in 1979, the bio-presence of MAP could not be reduced and shedding MAP in feaces and milk continued. Screening of raw milk from a large number of goats, cows, and buffaloes the bio-presence was 9.4% in goats (Kumar et al., 2008), 6.0% in cattle (Sharma et al., 2008) and 48.0% in buffaloes (Yadav et al., 2008). Shankar et al. (2010) in 2008 first time screened pasteurized milk and milk products and samples were centrifuged to concentrate MAP bacilli, which got partitioned in fat or sediment layers depending on fat percent. Each layer was processed independently. However, in this study milk products were screened without centrifugation. The milk used in the preparation of these 12 milk products was mainly of bovine (buffaloes, cattle) origin and goat milk is sold as an adulterant. Therefore, the bio-presence of MAP estimated using milk products reflected the bio-presence of MAP in buffaloes and cattle and goats. After milk and ice-creams, soured milk products (curd, lassi, butter, and buttermilk) and fermented milk products (yogurt, cheese, and cream) were popular milk products. Curd, lassi, and buttermilk due to the presence of lacto-bacilliis prescribed for patients suffering from gastro-disorders and after antibiotic therapy. Mostly fresh curd is prepared in every home, however, in metropolitan cities, people depend on commercial curd, buttermilk, butter, and lassi. Mother dairy was the most popular brand sold in the capital city of New Delhi and Amul all over India. Bio-typing of milk products (curd, butter-milk, ice-cream, lassi, butter, and cheese) revealed the presence of ‘Indian Bison Type’ biotype of MAP. It is predominant bio-type reported from animals, milk and milk products (raw and pasteurized), and human population and also wild animals and environment indicating circulation of the bacilli in the ‘ecosystem’ infecting new population (animals and human beings) and at the same time increasing in the percent bio-presence continuously (Singh et al., 2014a; Singh et al., 2014b; Chaubey et al., 2016; Singh et al., 2018). Conclusion The study indicated that the high bio-presence of MAP in milk products was a result of similar high bio-presence of MAP in raw milk used for making these milk products, which enabled MAP to survive heat treatments during pasteurization. High bio-presence of MAP and presence of live MAP bacilli in milk products suggested MAP as high-risk pathogen for the human population and exposure to MAP infection and fragments/components of bacilli. Test combinations were superior to individual tests for the screening of milk products and test combinations can be decided depending on the type of sample, the number of bacilli, purpose study, resources available, etc. In-order to prevent transmission of MAP to the next generation of animals and human population control measures are urgently needed. To prevent the infection of farm animals will lead to a reduction in chances of contamination of milk and milk products, which may lead to infection of the human population. Milk products sold by leading market brands were un-safe for human use. Brands negative in this study may not be a true picture since the sample size was very low in the number of milk products, therefore the need for large scale study encompassing all the regions of the country. Acknowledgement Authors are thankful to Indian Council of Medical Research (ICMR) (grant No. 5/8/5/28/TF/2013/ECD-I) and Ministry of Food Processing Industries (MoFPI), New Delhi (Grant No. SERB/MOFPI/0029/2014) for providing funds and Director, Central Institute for Research on Goats, Makhdoom, Farah for providing laboratory facilities. Conflict Of Interest No potential conflict of interest to declare.