Volume 6, Issue 1, February Issue - 2018, Pages:188-203 |
Authors: Manju Singh#, Shoor Vir Singh*, Saurabh Gupta#, Kundan Kumar Chaubey, Jagdip Singh Sohal, Kuldeep Dhama |
Abstract: In this study, 133 milk samples (100 commercial liquid milk, 19 flavored milk and 14 milk powder) made from pasteurized milk by 10 leading commercial brands were purchased from the markets in Mathura and Agra districts of South Uttar Pradesh in North India. These milk samples were screened to estimate ‘bio-incidence’ of Mycobacterium avium subspecies paratuberculosis (MAP) using multiple tests; 3 antibody {Indigenous ELISA (i_ELISA), dot ELISA (d_ELISA) and Latex agglutination test (LAT)} and 3 antigen {(microscopy, Indirect fluorescent antibody test (i_FAT) and IS900 PCR}. Of 133 samples screened, 42.8, 58.6, 9.0, 27.0, 49.6 and 42.8% were positive for MAP in microscopy, i_FAT, IS900 PCR, i_ELISA, d_ELISA, and LAT, respectively. i_FAT was most sensitive followed by d_ELISA, LAT, microscopy, i_ELISA and IS900 PCR. In general, i_FAT, d_ELISA, LAT and microscopy were significantly superior to i_ELISA and IS900 PCR for estimating bio-incidence of MAP in milk samples. High bio-incidence of MAP in food items of mass consumption (liquid milk, flavoured milk and milk powder) made from pasteurized milk and freely sold in local markets by leading commercial brands emphasized the need for the immediate implementation of programs for the control of MAP in domestic livestock. MAP, the cause of incurable Johne's disease is endemic in 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. |
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Full Text: 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. |
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