Presence, characterization, and genotype profiles of Mycobacterium avium subspecies paratuberculosis from unpasteurized individual and pooled milk, commercial pasteurized milk, and milk products in India by culture, PCR, and PCR-REA methods

Open ArchivePublished:July 03, 2009DOI:https://doi.org/10.1016/j.ijid.2009.03.031

      Summary

      Background

      Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants, a chronic enteritis evocative of human inflammatory bowel disease. In industrialized countries MAP has been cultured from pasteurized milk, compounding the increasing concern that MAP may be zoonotic. The purpose of this study was to evaluate commercially available unpasteurized and pasteurized milk and its products for the presence of viable MAP or MAP DNA from an area of northern India with a population of 150 million people.

      Methods

      We studied 43 samples (16 unpasteurized, 27 pasteurized) purchased in Mathura, Agra, or New Delhi, for the presence of MAP by culture or by PCR for IS900 MAP DNA. Positives results were confirmed as MAP by restriction endonuclease analysis and/or DNA sequencing.

      Results

      Colonies appeared in 1.5–20 months post-inoculation. Of the unpasteurized samples, 44% (7/16) were MAP culture-positive and 6% (1/16) were positive for IS900 MAP DNA. Of the pasteurized samples, 67% (18/27) were MAP culture-positive and 33% (9/27) were IS900-positive. Subsequently, 100% (25/25) of the cultured colonies were IS900 and IS1311 MAP DNA-positive.

      Conclusions

      This is the first report from a developing country of MAP cultured from both pasteurized and unpasteurized milk and milk products. Thus we corroborate the presence of viable MAP in the food chain reported from industrialized countries. With the increasing concern that MAP may be zoonotic, these findings have major implications for healthcare in India. The decreased sensitivity in detecting MAP DNA by PCR directly from milk should be ascribed to our employing only one set of PCR primers.

      Keywords

      Introduction

      In ruminants, Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of Johne's disease (JD),
      • Johne H.A.
      • Frothingham L.
      Ein eigenthumlicher fall von tuberculose beim rind (A particular case of tuberculosis in a cow).
      a chronic enteric diarrheal disease that is evocative of human inflammatory bowel disease (IBD).

      Dalziel TK. Chronic intestinal enteritis. Br Med J 1913; ii:1068-70.

      JD is increasing in incidence in both the developed
      • Collins M.T.
      Mycobacterium paratuberculosis: a potential food-borne pathogen?.
      and the developing world, and in India.
      • Kumar P.
      • Singh S.V.
      • Bhatiya A.K.
      • Sevilla I.
      • Singh A.V.
      • Whittington R.J.
      • et al.
      Juvenile Capri-paratuberculosis (JCP) in India: incidence and characterization by six diagnostic tests.
      Apparently unrelated, the incidence of IBD is increasing in the industrialized world
      • Bernstein C.N.
      • Blanchard J.F.
      • Rawsthorne P.
      • Collins M.T.
      Population-based case control study of seroprevalence of Mycobacterium paratuberculosis in patients with Crohn's disease and ulcerative colitis.
      as well as in India,
      • Venugopalen S.
      • Radhakrishnan S.
      • Rajachandrasekharan R.
      Crohn's disease (a study of 21 cases).
      • Venkatkrishnan L.
      • Thakaran A.
      • Francis J.
      • Ramesh G.N.
      • Philip M.
      • Augustine P.
      Crohn's disease—analysis of 26 cases.
      • Amarapurkar D.
      • Patel N.D.
      • Amarapurkar A.D.
      • Agal S.
      • Baigal R.
      • Gupta P.
      Tissue polymerase chain reaction in diagnosis of intestinal tuberculosis and Crohn's disease.
      • Desai H.G.
      • Gupte P.A.
      Increasing incidence of Crohn's disease in India: is it related to improved sanitation?.
      • Makharia G.K.
      Rising incidence and prevalence of Crohn's disease in Asia: is it apparent or real?.
      where gastroenteritis and colitis are common health problems.
      • Tandon B.N.
      • Upadhyaya A.K.
      • Tandon H.D.
      • Gadekar N.G.
      Ulcerative colitis in North Indian subjects: a preliminary report.
      • Khosla S.N.
      • Girdhar N.K.
      • Lal S.
      • Mishra D.S.
      Epidemiology of ulcerative colitis in hospital and select general population of northern India.
      • Sood A.
      • Midha V.
      • Sood N.
      • Bhatia A.S.
      • Avasthi G.
      Incidence and prevalence of ulcerative colitis in Punjab.
      MAP has been cultured from chlorinated potable municipal water in the USA
      • Mishina D.
      • Katsel P.
      • Brown S.T.
      • Gilberts E.C.
      • Greenstein R.J.
      On the etiology of Crohn disease.
      and Europe,
      • Hermon-Taylor J.
      • El-Zaatari F.A.
      The Mycobacterium avium subspecies paratuberculosis problem and its relation to the causation of Crohn disease.
      pasteurized milk in the USA
      • Ellingson J.L.E.
      • Anderson J.L.
      • Koziczkowski J.J.
      • Radcliff R.P.
      • Sloan S.J.
      • Allen S.E.
      • et al.
      Detection of viable Mycobacterium avium subsp. paratuberculosis in retail pasteurized whole milk by two culture methods and PCR.
      and Europe,
      • Grant I.R.
      • Hitchings E.I.
      • McCartney A.
      • Ferguson F.
      • Rowe M.T.
      Effect of commercial-scale high-temperature, short-time pasteurization on the viability of Mycobacterium paratuberculosis in naturally infected cows’ milk.
      • Ayele W.Y.
      • Svastova P.
      • Roubal P.
      • Bartos M.
      • Pavlik I.
      Mycobacterium avium subspecies paratuberculosis cultured from locally and commercially pasteurized cow's milk in the Czech Republic.
      the intestine
      • Chiodini R.J.
      • Van Kruiningin H.J.
      • Thayer Jr., W.J.
      • Coutu J.
      Spheroplastic phase of mycobacteria isolated from patients with Crohn's disease.
      and stool
      • Bull T.J.
      • McMinn E.J.
      • Sidi-Boumedine K.
      • Skull A.
      • Durkin D.
      • Neild P.
      • et al.
      Detection and verification of Mycobacterium avium subsp. paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn's disease.
      of patients with Crohn's disease (CD), the breast milk of mothers with Crohn's disease,
      • Naser S.A.
      • Schwartz D.
      • Shafran I.
      Isolation of Mycobacterium avium subsp paratuberculosis from breast milk of Crohn's disease patients.
      and from the blood of patients with IBD.
      • Naser S.A.
      • Ghobrial G.
      • Romero C.
      • Valentine J.F.
      Culture of Mycobacterium avium subspecies paratuberculosis from the blood of patients with Crohn's disease.
      MAP is not reproducibly killed by standard pasteurization procedures.
      • Singh A.V.
      • Singh S.V.
      • Makharia G.K.
      • Singh P.K.
      • Sohal J.S.
      Presence and characterization of Mycobacterium avium subspecies paratuberculosis from clinical and suspected cases of Crohn's disease and in the healthy human population in India.
      • Grant I.R.
      • Ball H.J.
      • Rowe M.T.
      Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis.
      Although prevailing medical dogma considers MAP to be an innocent commensal,
      • Selby W.
      • Pavli P.
      • Crotty B.
      • Florin T.
      • Radford-Smith G.
      • Gibson P.
      • et al.
      Two-year combination antibiotic therapy with clarithromycin, rifabutin, and clofazimine for Crohn's disease.
      there are increasing concerns that MAP may be zoonotic,
      • Greenstein R.J.
      • Collins M.T.
      Emerging pathogens: is Mycobacterium avium subspecies paratuberculosis zoonotic?.

      Nacy C, Buckley M. Mycobacterium avium paratuberculosis: infrequent human pathogen or public health threat? Washington DC, USA: American Academy of Microbiology; 2008, p. 1-37. Available at: http://www.asm.org/ASM/files/ccLibraryFiles/Filename/000000004169/MAP.pdf (accessed May 2009).

      and at a minimum responsible for IBD
      • Greenstein R.J.
      • Su L.
      • Haroutunian V.
      • Shahidi A.
      • Brown S.T.
      On the action of methotrexate and 6-mercaptopurine on M. avium subspecies paratuberculosis.
      • Greenstein R.J.
      • Su L.
      • Shahidi A.
      • Brown S.T.
      On the action of 5-aminosalicylic acid and sulfapyridine on M. avium including subspecies paratuberculosis.
      .
      The Republic of India has more than 400 million milk producing animals

      FAOSTAT. Rome, Italy: Food and Agriculture Organization, 2005. www.FAOSTAT.org.

      providing approximately 86.9 million tonnes of milk annually. Buffalo milk is the most preferred and costliest, and accounts for 54.5% of the total production; cow milk accounts for 41% and goat milk the remaining 4.5%.

      Hemme T, Garcia O, Saha A. A review of milk production in India with particular emphasis on small-scale producers. PPLPI working paper No. 2. International Farm Comparison Network, Pro-Poor Livestock Policy Initiative; 2003. Available at: http://www.fao.org/ag/againfo/programmes/en/pplpi/docarc/wp2.pdf (accessed May 2009).

      Approximately 35% of Indian milk is pasteurized, predominantly by state cooperatives, multinational companies, or government dairy plants.

      India in business. Investment and technology promotion and energy security. Delhi: Ministry of External Affairs, Government of India; 2008.

      Usually pasteurized products are packaged milk, milk powders, cheese, and ice cream. Of the unpasteurized remainder, 33% remains in the farm or village of production and 51% is distributed by unregulated small vendors, sweet makers, and small dairy units.

      Khanna AK. Dairy industries in India. Presented at the XXXIIIth Industry Conference, New Delhi, India, 2004.

      In India, MAP has been cultured from the unpasteurized milk of goats
      • Kumar P.
      • Singh S.V.
      • Bhatiya A.K.
      • Sevilla I.
      • Singh A.V.
      • Whittington R.J.
      • et al.
      Juvenile Capri-paratuberculosis (JCP) in India: incidence and characterization by six diagnostic tests.
      • Singh S.V.
      • Vihan V.S.
      Detection of Mycobacterium avium subspecies paratuberculosis in goat milk.
      and cattle.
      • Sharma G.
      • Singh S.V.
      • Sevilla I.
      • Singh A.V.
      • Whittington R.J.
      • Juste R.A.
      • et al.
      Evaluation of indigenous milk ELISA with m-culture and m-PCR for the diagnosis of bovine Johne's disease (BJD) in lactating Indian dairy cattle.
      • Singh S.V.
      • Singh A.V.
      • Singh R.
      • Sandhu K.S.
      • Singh P.K.
      • Sohal J.S.
      • et al.
      Evaluation of highly sensitive indigenous milk ELISA kit with fecal culture, milk culture and fecal-PCR for the diagnosis of bovine Johne's disease (BJD) in India.
      We hypothesized that viable MAP may be entering the human food chain in India from both pasteurized as well as unpasteurized milk and its products. We additionally hypothesized that we could identify loco-regional sources of our isolates by performing genotypic analyses on MAP that we isolated. Accordingly, we evaluated milk and milk products for the presence of viable MAP (by culture) and MAP DNA in a region of northern India with a population of 150 million people.

      Materials and methods

       Origin of milk samples

      Forty-three randomly selected commercially available milk samples were purchased in three major cities (Mathura, Agra, and New Delhi) in northern India. Of these, 16 were of unpasteurized milk (500 ml), 18 of pasteurized milk (500 ml), and nine of pasteurized milk products (50 to 500 g). Of the 16 unpasteurized milk samples, six were from dairy farms in the cities of Farah and Mathura that sold milk directly to consumers and 10 were from lactating cows on a dairy farm in the district of Mathura. Milk was aseptically collected from all four quarters of the udder by cleaning and drying each teat and discarding the first expressed samples of milk.

       Preparation of samples for MAP culture and PCR

      Each liquid sample was centrifuged (3000 rpm, 20 min) providing three layers: supernatant, pellet, and remainder. The resulting final 215 samples were then tested for the presence of MAP using two methods. The first was direct PCR of the milk. The second was by culture of the milk or milk product for MAP. When a culture manifested growth, the DNA from the colony was subsequently extracted and subjected to MAP-specific (IS900) PCR.

       Culture of fat and sediment layer

      The supernatant and pellet were decontaminated in 0.9% hexadecylpyridinium chloride (HPC) for 18–24 hours at room temperature. The resulting liquid layer was decanted and discarded, and 0.02 ml of the residual mucilaginous sludge was inoculated onto modified Herrold's egg yolk medium (HEYM)
      • Merkal R.S.
      • Richards W.D.
      Inhibition of fungal growth in the cultural isolation of mycobacteria.
      • Whipple D.L.
      • Callihan D.R.
      • Jarnagin J.L.
      Cultivation of Mycobacterium paratuberculosis from bovine fecal specimens and a suggested standardized procedure.
      with and without mycobactin J (Allied Monitor Inc., MO, USA). Culture vials were observed for 1.5–20 months. Colonies were compared for morphological characteristics with MAP previously obtained from the screening of feces,
      • Kumar P.
      • Singh S.V.
      • Bhatiya A.K.
      • Sevilla I.
      • Singh A.V.
      • Whittington R.J.
      • et al.
      Juvenile Capri-paratuberculosis (JCP) in India: incidence and characterization by six diagnostic tests.
      tissues,
      • Singh N.
      • Singh S.V.
      • Gupta V.K.
      • Sharma V.D.
      • Sharma R.K.
      • Katoch V.M.
      Isolation and identification of Mycobacterium paratuberculosis from naturally infected goatherds in India.
      and milk.
      • Singh A.V.
      • Singh S.V.
      • Makharia G.K.
      • Singh P.K.
      • Sohal J.S.
      Presence and characterization of Mycobacterium avium subspecies paratuberculosis from clinical and suspected cases of Crohn's disease and in the healthy human population in India.
      • Singh S.V.
      • Vihan V.S.
      Detection of Mycobacterium avium subspecies paratuberculosis in goat milk.

       Identification of MAP by IS900 on milk products

      The remainder of the post-decontamination mucilaginous sludge that was not inoculated was then processed for PCR analysis for MAP DNA
      • van Soolingen D.
      • de Haas P.E.
      • Hermans P.W.
      • Groenen P.M.
      • van Embden J.D.
      Comparison of various repetitive DNA elements as genetic markers for strain differentiation and epidemiology of Mycobacterium tuberculosis.
      with minor modifications.
      • Kumar P.
      • Singh S.V.
      • Bhatiya A.K.
      • Sevilla I.
      • Singh A.V.
      • Whittington R.J.
      • et al.
      Juvenile Capri-paratuberculosis (JCP) in India: incidence and characterization by six diagnostic tests.
      DNA from colonies was isolated
      • Singh S.V.
      • Singh P.K.
      • Singh A.V.
      • Sohal J.S.
      • Subodh Swati
      • Narayanasamy K.
      Non-chemical method of DNA recovery and characterization of Mycobacterium avium subspecies paratuberculosis using IS900 PCR.
      and amplified using specific IS900 primers,
      • Vary P.H.
      • Andersen P.R.
      • Green E.
      • Hermon-Taylor J.
      • McFadden J.J.
      Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne's disease.
      which results in a 229-bp amplicon, and subjected to 1.8% agarose ethidium bromide gel electrophoresis. Our positive control was a well-characterized MAP ‘bison type’ strain.
      • Whittington R.J.
      • Marsh I.B.
      • Whitlock R.H.
      Typing of IS 1311 polymorphisms confirms that bison (Bison bison) with paratuberculosis in Montana are infected with a strain of Mycobacterium avium subsp. paratuberculosis distinct from that occurring in cattle and other domesticated livestock.
      As the negative control we used sterilized Liquipure® water (Sigma) that had been subjected to the identical PCR procedures as the samples and positive control.

       IS1311 PCR and restriction endonuclease analysis (REA)

      IS1311 PCR was carried out using M56 and M119 primers as previously described.
      • Whittington R.J.
      • Marsh I.B.
      • Whitlock R.H.
      Typing of IS 1311 polymorphisms confirms that bison (Bison bison) with paratuberculosis in Montana are infected with a strain of Mycobacterium avium subsp. paratuberculosis distinct from that occurring in cattle and other domesticated livestock.
      • Sevilla I.
      • Singh S.V.
      • Garrido J.M.
      • Aduriz G.
      • Rodriguez S.
      • Geijo M.V.
      • et al.
      Molecular typing of Mycobacterium avium subspecies paratuberculosis strains from different hosts and regions.
      In brief, in a 25 μl volume: 0.5–1.0 ng template DNA, 2.5 μl of 10X PCR buffer (Promega), 1.5 mM MgCl2 (Promega), 0.2 mM dNTPs, and 1 unit Taq (Promega). Thermal cycling conditions were: initial denaturation at 94 °C for 3 min followed by 37 cycles of denaturation at 94 °C for 30 s, annealing at 62 °C for 30 s, and extension at 72 °C for 1 min. The final extension was 72 °C for 10 min. PCR products were subjected to electrophoresis (2% agarose gel stained with ethidium bromide). Amplicons that co-migrated at 608 bp were considered positive results for IS1311 PCR.

       Differentiating MAP into ‘bison type and ‘cattle type’ using IS1311 PCR-REA

      IS1311 PCR-REA-purified MAP DNA was subjected to HinfI and MseI (Fermentas) restriction endonuclease digestion at 37 °C for 1.5 hours as previously described.
      • Sevilla I.
      • Singh S.V.
      • Garrido J.M.
      • Aduriz G.
      • Rodriguez S.
      • Geijo M.V.
      • et al.
      Molecular typing of Mycobacterium avium subspecies paratuberculosis strains from different hosts and regions.
      Following incubation, fragments were separated on 4% agarose gel. Genotypes were identified by fragment migration patterns as described.
      • Whittington R.J.
      • Marsh I.B.
      • Whitlock R.H.
      Typing of IS 1311 polymorphisms confirms that bison (Bison bison) with paratuberculosis in Montana are infected with a strain of Mycobacterium avium subsp. paratuberculosis distinct from that occurring in cattle and other domesticated livestock.
      To confirm that we had isolated MAP, DNA sequencing was performed on 13 isolates and compared to K10.
      • Li L.
      • Bannantine J.P.
      • Zhang Q.
      • Amonsin A.
      • May B.J.
      • Alt D.
      • et al.
      The complete genome sequence of Mycobacterium avium subspecies paratuberculosis.

      Results

      Positive colonies were detected at between 1.5 and 20 months post-inoculation.

       Unpasteurized milk (individual cows and pooled milk)

      In our unpasteurized milk samples, 44% (7/16) were MAP culture-positive and 6% (1/16) were MAP IS900 PCR-positive (Table 1). Of the 7/16 that were MAP culture-positive, all (7/7) were MAP-positive by IS900 PCR (Figure 1). The proportional agreement
      • Landis J.R.
      • Koch G.G.
      The measurement of observer agreement for categorical data.
      between culture and direct PCR showed significant concordance (kappa PA value = 62.0).
      Table 1Culture of MAP, confirmed by IS900 PCR from unpasteurized milk, pasteurized milk, and milk products
      SpecimenNumber of samplesCulture-positiveIS900 PCR
      SupernatantPelletCombined supernatant and pelletOriginal pelletsCulture-positive
      n (%)n (%)n (%)n (%)n (%)
      Unpasteurized milk samples165 (31)3 (19)7 (44)1 (6)7 (100)
      Pasteurized milk samples1811 (61)9 (50)13 (72)7 (39)13 (100)
      Commercial milk products
      Milk products include ice cream and flavored milk drinks.
      93 (33)3 (33)5 (56)2 (22)5 (100)
      Total4319 (44)15 (35)25 (58)10 (23)25 (100)
      MAP, Mycobacterium avium subspecies paratuberculosis.
      a Milk products include ice cream and flavored milk drinks.
      Figure thumbnail gr1
      Figure 1Agarose gel with the IS900 specific primers, which generate a fragment that migrates at a molecular weight of 229 bp. Lane 1 = markers; lanes 2–10 are all samples that had been grown in culture, had had DNA extracted, and were now tested for the presence of IS900-specific MAP DNA sequences. All samples shown here tested positive for the presence of IS900.

       Commercially available pasteurized liquid milk

      Of the 18 brands of commercially available pasteurized liquid milk samples, 72% (13/18) were MAP culture-positive and 39% (7/18) were directly positive for the presence of MAP by IS900 PCR (Table 1). Of the samples from which we cultured MAP, all (13/13) were MAP-positive by IS900 PCR (Figure 1, Figure 2, Table 1).
      Figure thumbnail gr2
      Figure 2Agarose gel with the IS900 specific primers, which generate a fragment that migrates at a molecular weight of 229 bp. Lane 1 = markers; lane 2 is the positive MAP DNA control; lane 3 is the negative control (water processed identically with the other samples); lanes 4–10 are all samples that had been isolated from the pellet of samples in this study. All samples show a positive signal.

       Commercially available pasteurized milk products

      Of the nine samples of milk products tested, 56% (5/9) were MAP culture-positive and 22% (2/9) were directly positive for the presence of MAP by IS900 PCR. Of the samples that were positive in culture, all (5/5) were MAP-positive by IS900 PCR (Figure 1, Table 1).

       MAP in supernatant and pellet by culture

      MAP culture was positive in 44% (19/43) of supernatant (fat) samples and 35% (15/43) of pellet samples (Table 1, Table 2).
      Table 2Comparison of concordance of presence or absence of MAP by culture in supernatant and pellet from unpasteurized milk, pasteurized milk, and milk products
      Supernatant MAP culture
      PositiveNegative
      n%n%
      Pellet MAP culture
      Positive9/43216/4314
      Negative10/432318/4342
      MAP, Mycobacterium avium subspecies paratuberculosis.
      Proportional agreement = 0.62 (substantial).

       Overall detection of MAP by culture and IS900 PCR

      For all samples, detection of MAP by culture was 58% (25/43) and by IS900 PCR was 23% (10/43). Of all 43 samples, 40% (17/43) were only positive by culture and 5% (2/43) were only positive by PCR. Overall, when culture and PCR positivity were combined, we detected MAP in 63% (27/43) of our samples (Table 3).
      Table 3Comparison of MAP PCR and MAP culture
      Culture
      PositiveNegative
      n%n%
      PCR
      Positive8/43192/435
      Negative17/434016/4337
      MAP, Mycobacterium avium subspecies paratuberculosis.
      Twenty-seven positive (63%) in both culture and PCR; 25 live MAP (58%); proportional agreement = 0.55 (moderate).

       Genotype profiles of MAP

      IS1311 REA fragment analysis performed on positive MAP cultures showed that 70% (14/20) were ‘bison type’ and 30% (6/20) were ‘cattle type’ (Figure 3). When stratified by region of origin, ‘bison type’ was most prevalent in bovine milk procured from Mathura (100%; 8/8), followed by Agra (71%; 5/7), and New Delhi (20%; 1/5). In contrast, ‘cattle type’ was more prevalent in the regions of New Delhi (80%; 4/5), followed by Agra (29%; 2/7). Interestingly, in the New Delhi region the ‘bison type’ MAP was found in one commercial brand that has a chilling plant in the city of Mathura, whereas all the ‘cattle type’ isolates were from a different commercial brand that has its production facilities in the city of Anand in Gujarat State.
      Figure thumbnail gr3
      Figure 3Differentiating MAP into ‘bison type’ and ‘cattle type’ using HinfI and MseI restriction endonuclease analysis (REA) and IS1311 PCR. Shown is a 4% agarose gel of digested DNA subjected to MAP-specific IS1311 PCR. Lane 1 = molecular weight markers; ‘cattle type’ MAP results in three fragments (lanes 2, 3, and 5);
      • Whittington R.J.
      • Marsh I.B.
      • Whitlock R.H.
      Typing of IS 1311 polymorphisms confirms that bison (Bison bison) with paratuberculosis in Montana are infected with a strain of Mycobacterium avium subsp. paratuberculosis distinct from that occurring in cattle and other domesticated livestock.
      in contrast, ‘bison type’ MAP generates only the two larger bands (lanes 4, 6, 7, and 8).
      • Whittington R.J.
      • Marsh I.B.
      • Whitlock R.H.
      Typing of IS 1311 polymorphisms confirms that bison (Bison bison) with paratuberculosis in Montana are infected with a strain of Mycobacterium avium subsp. paratuberculosis distinct from that occurring in cattle and other domesticated livestock.
      .
      DNA sequencing performed on 13 of our isolates showed 100% homology of a 267-bp fragment of the AV1/AV2 region of IS900,
      • Li L.
      • Bannantine J.P.
      • Zhang Q.
      • Amonsin A.
      • May B.J.
      • Alt D.
      • et al.
      The complete genome sequence of Mycobacterium avium subspecies paratuberculosis.
      confirming that our isolates were MAP (data not presented).

      Discussion

      India produces more milk products than any other country in the world,

      Hemme T, Garcia O, Saha A. A review of milk production in India with particular emphasis on small-scale producers. PPLPI working paper No. 2. International Farm Comparison Network, Pro-Poor Livestock Policy Initiative; 2003. Available at: http://www.fao.org/ag/againfo/programmes/en/pplpi/docarc/wp2.pdf (accessed May 2009).

      the majority of which are not subjected to pasteurization.

      India in business. Investment and technology promotion and energy security. Delhi: Ministry of External Affairs, Government of India; 2008.

      We cultured MAP from 63% of milk and milk products obtained from an area of northern India that has a population of 150 million. MAP was cultured from both pasteurized as well as unpasteurized samples. Thus, we have shown that present Indian pasteurization standards do not reliably render MAP non-viable. Our data show that the population in India is repetitively exposed to viable MAP, probably more frequently and at higher inoculation counts than are the populations of such countries as the USA
      • Ellingson J.L.E.
      • Anderson J.L.
      • Koziczkowski J.J.
      • Radcliff R.P.
      • Sloan S.J.
      • Allen S.E.
      • et al.
      Detection of viable Mycobacterium avium subsp. paratuberculosis in retail pasteurized whole milk by two culture methods and PCR.
      and Ireland.
      • Grant I.R.
      • Hitchings E.I.
      • McCartney A.
      • Ferguson F.
      • Rowe M.T.
      Effect of commercial-scale high-temperature, short-time pasteurization on the viability of Mycobacterium paratuberculosis in naturally infected cows’ milk.
      Paradoxically, we identified MAP more frequently by culture (58%) than by IS900 PCR (23%). The most plausible explanation for the lower detection of MAP DNA that we find is a methodological one. We employed a single set of primers for our IS900 PCR. Others have used nested primer PCR because of the difficulty of detecting the low copy number of MAP DNA templates.
      • Naser S.A.
      • Ghobrial G.
      • Romero C.
      • Valentine J.F.
      Culture of Mycobacterium avium subspecies paratuberculosis from the blood of patients with Crohn's disease.
      Often in employing the nested primer strategy, the first set of primers fails to detect any signal (RJG unpublished observations). Unfortunately, the probability of contamination-induced false-positives increases exponentially when the nested primer strategy is used. Nevertheless, we suggest that in future studies such as ours, with appropriate precautions against contamination, the use of nested PCR primers may be preferred over the single set of primers that we employed.
      We additionally need to consider the possibility that some of our MAP culture results may be ascribable to contamination in culture. However, our data indicate that contamination does not account for our findings. First, the DNA sequencing analysis on the isolates that we cultured shows that they are not from a single MAP strain. Second, the IS1311 REA fragment analysis on DNA isolated from the culture-positive colonies shows that there are distinct geographical differences in the incidence of ‘bison type’ and ‘cattle type’ strains. For example, when the ‘cattle type’ genotype was identified in the Delhi area, those isolates were from a processing plant located in Anand in the State of Gujarat. We conclude that our culture data cannot be ascribed to contamination.
      The possibility that MAP may be zoonotic
      • Greenstein R.J.
      • Collins M.T.
      Emerging pathogens: is Mycobacterium avium subspecies paratuberculosis zoonotic?.
      remains to be clarified.

      Nacy C, Buckley M. Mycobacterium avium paratuberculosis: infrequent human pathogen or public health threat? Washington DC, USA: American Academy of Microbiology; 2008, p. 1-37. Available at: http://www.asm.org/ASM/files/ccLibraryFiles/Filename/000000004169/MAP.pdf (accessed May 2009).

      However, until the MAP zoonosis conundrum is definitively resolved we suggest that the precautionary principle, instituted by the British Government for MAP in the UK

      Advisory Committee on the Microbiological Safety of Food. Mycobacterium avium subspecies paratuberculosis: review of advice. ACM 487. Food Standards Agency; 2001. Available at: http://archive.food.gov.uk/pdf_files/papers/acm487.pdf (accessed May 2009).

      should be applied in India and that MAP should be removed from the food chain. We conclude that alternative strategies to diminish the viable MAP burden to which the Indian population is exposed need to be considered and introduced. We suggest that a concerted, Government mandated and directed anti-MAP vaccination program

      Singh SV, Singh PK, Singh AV, Sohal JS, Gupta VK, Vihan VS. Comparative efficacy of an indigenous ‘inactivated vaccine’ using highly pathogenic field strain of Mycobacterium avium subspecies paratuberculosis ‘bison type’ with commercial vaccine for the control of Capri-paratuberculosis in India. Vaccine 2007; 25:7102-10.

      should be amongst the contingencies considered.

      Conflict of interest

      RJG has submitted provisional patents on hypotheses that were previously tested, and published, concerning MAP. He has no direct conflict of interest on any data published in this manuscript. All other authors declare that they have no conflict of interest.

      References

        • Johne H.A.
        • Frothingham L.
        Ein eigenthumlicher fall von tuberculose beim rind (A particular case of tuberculosis in a cow).
        Dtsch Zeitschr Tiermed Vergl Pathol. 1895; 21: 438-454
      1. Dalziel TK. Chronic intestinal enteritis. Br Med J 1913; ii:1068-70.

        • Collins M.T.
        Mycobacterium paratuberculosis: a potential food-borne pathogen?.
        J Dairy Sci. 1997; 80: 3445-3448
        • Kumar P.
        • Singh S.V.
        • Bhatiya A.K.
        • Sevilla I.
        • Singh A.V.
        • Whittington R.J.
        • et al.
        Juvenile Capri-paratuberculosis (JCP) in India: incidence and characterization by six diagnostic tests.
        Small Rumin Res. 2007; 73: 45-53
        • Bernstein C.N.
        • Blanchard J.F.
        • Rawsthorne P.
        • Collins M.T.
        Population-based case control study of seroprevalence of Mycobacterium paratuberculosis in patients with Crohn's disease and ulcerative colitis.
        J Clin Microbiol. 2004; 42: 1129-1135
        • Venugopalen S.
        • Radhakrishnan S.
        • Rajachandrasekharan R.
        Crohn's disease (a study of 21 cases).
        Indian J Surg. 1980; 42: 388-396
        • Venkatkrishnan L.
        • Thakaran A.
        • Francis J.
        • Ramesh G.N.
        • Philip M.
        • Augustine P.
        Crohn's disease—analysis of 26 cases.
        Indian J Gastroenterol. 1995; 14: A58
        • Amarapurkar D.
        • Patel N.D.
        • Amarapurkar A.D.
        • Agal S.
        • Baigal R.
        • Gupta P.
        Tissue polymerase chain reaction in diagnosis of intestinal tuberculosis and Crohn's disease.
        J Assoc Physicians India. 2004; 52: 863-867
        • Desai H.G.
        • Gupte P.A.
        Increasing incidence of Crohn's disease in India: is it related to improved sanitation?.
        Indian J Gastroenterol. 2005; 24: 23-24
        • Makharia G.K.
        Rising incidence and prevalence of Crohn's disease in Asia: is it apparent or real?.
        J Gastroenterol Hepatol. 2006; 21: 929-931
        • Tandon B.N.
        • Upadhyaya A.K.
        • Tandon H.D.
        • Gadekar N.G.
        Ulcerative colitis in North Indian subjects: a preliminary report.
        J Assoc Physicians India. 1964; 12: 43-52
        • Khosla S.N.
        • Girdhar N.K.
        • Lal S.
        • Mishra D.S.
        Epidemiology of ulcerative colitis in hospital and select general population of northern India.
        J Assoc Physicians India. 1986; 34: 405-407
        • Sood A.
        • Midha V.
        • Sood N.
        • Bhatia A.S.
        • Avasthi G.
        Incidence and prevalence of ulcerative colitis in Punjab.
        North India. Gut. 2003; 52: 1587-1590
        • Mishina D.
        • Katsel P.
        • Brown S.T.
        • Gilberts E.C.
        • Greenstein R.J.
        On the etiology of Crohn disease.
        Proc Natl Acad Sci U S A. 1996; 93: 9816-9820
        • Hermon-Taylor J.
        • El-Zaatari F.A.
        The Mycobacterium avium subspecies paratuberculosis problem and its relation to the causation of Crohn disease.
        in: Pedley S. Bartram J. Rees G. Dufour A. Cotruvo J. Pathogenic mycobacteria in water: a guide to public health consequences, monitoring and management.1st ed. IWA Publishing, London, UK2004: 74-94
        • Ellingson J.L.E.
        • Anderson J.L.
        • Koziczkowski J.J.
        • Radcliff R.P.
        • Sloan S.J.
        • Allen S.E.
        • et al.
        Detection of viable Mycobacterium avium subsp. paratuberculosis in retail pasteurized whole milk by two culture methods and PCR.
        J Food Prot. 2005; 68: 966-972
        • Grant I.R.
        • Hitchings E.I.
        • McCartney A.
        • Ferguson F.
        • Rowe M.T.
        Effect of commercial-scale high-temperature, short-time pasteurization on the viability of Mycobacterium paratuberculosis in naturally infected cows’ milk.
        Appl Environ Microbiol. 2002; 68: 602-607
        • Ayele W.Y.
        • Svastova P.
        • Roubal P.
        • Bartos M.
        • Pavlik I.
        Mycobacterium avium subspecies paratuberculosis cultured from locally and commercially pasteurized cow's milk in the Czech Republic.
        Appl Environ Microbiol. 2005; 71: 1210-1214
        • Chiodini R.J.
        • Van Kruiningin H.J.
        • Thayer Jr., W.J.
        • Coutu J.
        Spheroplastic phase of mycobacteria isolated from patients with Crohn's disease.
        J Clin Microbiol. 1986; 24: 357-363
        • Bull T.J.
        • McMinn E.J.
        • Sidi-Boumedine K.
        • Skull A.
        • Durkin D.
        • Neild P.
        • et al.
        Detection and verification of Mycobacterium avium subsp. paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn's disease.
        J Clin Microbiol. 2003; 41: 2915-2923
        • Naser S.A.
        • Schwartz D.
        • Shafran I.
        Isolation of Mycobacterium avium subsp paratuberculosis from breast milk of Crohn's disease patients.
        Am J Gastroenterol. 2000; 95: 1094-1095
        • Naser S.A.
        • Ghobrial G.
        • Romero C.
        • Valentine J.F.
        Culture of Mycobacterium avium subspecies paratuberculosis from the blood of patients with Crohn's disease.
        Lancet. 2004; 364: 1039-1044
        • Singh A.V.
        • Singh S.V.
        • Makharia G.K.
        • Singh P.K.
        • Sohal J.S.
        Presence and characterization of Mycobacterium avium subspecies paratuberculosis from clinical and suspected cases of Crohn's disease and in the healthy human population in India.
        Int J Infect Dis. 2008; 12: 190-197
        • Grant I.R.
        • Ball H.J.
        • Rowe M.T.
        Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis.
        Lett Appl Microbiol. 1998; 26: 166-170
        • Selby W.
        • Pavli P.
        • Crotty B.
        • Florin T.
        • Radford-Smith G.
        • Gibson P.
        • et al.
        Two-year combination antibiotic therapy with clarithromycin, rifabutin, and clofazimine for Crohn's disease.
        Gastroenterology. 2007; 132: 2313-2319
        • Greenstein R.J.
        • Collins M.T.
        Emerging pathogens: is Mycobacterium avium subspecies paratuberculosis zoonotic?.
        Lancet. 2004; 364: 396-397
      2. Nacy C, Buckley M. Mycobacterium avium paratuberculosis: infrequent human pathogen or public health threat? Washington DC, USA: American Academy of Microbiology; 2008, p. 1-37. Available at: http://www.asm.org/ASM/files/ccLibraryFiles/Filename/000000004169/MAP.pdf (accessed May 2009).

        • Greenstein R.J.
        • Su L.
        • Haroutunian V.
        • Shahidi A.
        • Brown S.T.
        On the action of methotrexate and 6-mercaptopurine on M. avium subspecies paratuberculosis.
        PLoS ONE. 2007; 2: e161
        • Greenstein R.J.
        • Su L.
        • Shahidi A.
        • Brown S.T.
        On the action of 5-aminosalicylic acid and sulfapyridine on M. avium including subspecies paratuberculosis.
        PLoS ONE. 2007; 2: e516
      3. FAOSTAT. Rome, Italy: Food and Agriculture Organization, 2005. www.FAOSTAT.org.

      4. Hemme T, Garcia O, Saha A. A review of milk production in India with particular emphasis on small-scale producers. PPLPI working paper No. 2. International Farm Comparison Network, Pro-Poor Livestock Policy Initiative; 2003. Available at: http://www.fao.org/ag/againfo/programmes/en/pplpi/docarc/wp2.pdf (accessed May 2009).

      5. India in business. Investment and technology promotion and energy security. Delhi: Ministry of External Affairs, Government of India; 2008.

      6. Khanna AK. Dairy industries in India. Presented at the XXXIIIth Industry Conference, New Delhi, India, 2004.

        • Singh S.V.
        • Vihan V.S.
        Detection of Mycobacterium avium subspecies paratuberculosis in goat milk.
        Small Rumin Res. 2004; 34: 231-235
        • Sharma G.
        • Singh S.V.
        • Sevilla I.
        • Singh A.V.
        • Whittington R.J.
        • Juste R.A.
        • et al.
        Evaluation of indigenous milk ELISA with m-culture and m-PCR for the diagnosis of bovine Johne's disease (BJD) in lactating Indian dairy cattle.
        Res Vet Sci. 2007; 84: 30-37
        • Singh S.V.
        • Singh A.V.
        • Singh R.
        • Sandhu K.S.
        • Singh P.K.
        • Sohal J.S.
        • et al.
        Evaluation of highly sensitive indigenous milk ELISA kit with fecal culture, milk culture and fecal-PCR for the diagnosis of bovine Johne's disease (BJD) in India.
        Comp Immunol Microbiol Infect Dis. 2007; 30: 176-186
        • Merkal R.S.
        • Richards W.D.
        Inhibition of fungal growth in the cultural isolation of mycobacteria.
        Appl Microbiol. 1972; 24: 205-207
        • Whipple D.L.
        • Callihan D.R.
        • Jarnagin J.L.
        Cultivation of Mycobacterium paratuberculosis from bovine fecal specimens and a suggested standardized procedure.
        J Vet Diagn Invest. 1991; 3: 368-373
        • Singh N.
        • Singh S.V.
        • Gupta V.K.
        • Sharma V.D.
        • Sharma R.K.
        • Katoch V.M.
        Isolation and identification of Mycobacterium paratuberculosis from naturally infected goatherds in India.
        Indian J Vet Pathol. 1996; 20: 104-108
        • van Soolingen D.
        • de Haas P.E.
        • Hermans P.W.
        • Groenen P.M.
        • van Embden J.D.
        Comparison of various repetitive DNA elements as genetic markers for strain differentiation and epidemiology of Mycobacterium tuberculosis.
        J Clin Microbiol. 1993; 31: 1987-1995
        • Singh S.V.
        • Singh P.K.
        • Singh A.V.
        • Sohal J.S.
        • Subodh Swati
        • Narayanasamy K.
        Non-chemical method of DNA recovery and characterization of Mycobacterium avium subspecies paratuberculosis using IS900 PCR.
        Indian J Exp Biol. 2007; 45: 812-816
        • Vary P.H.
        • Andersen P.R.
        • Green E.
        • Hermon-Taylor J.
        • McFadden J.J.
        Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne's disease.
        J Clin Microbiol. 1990; 28: 933-937
        • Whittington R.J.
        • Marsh I.B.
        • Whitlock R.H.
        Typing of IS 1311 polymorphisms confirms that bison (Bison bison) with paratuberculosis in Montana are infected with a strain of Mycobacterium avium subsp. paratuberculosis distinct from that occurring in cattle and other domesticated livestock.
        Mol Cell Probes. 2001; 15: 139-145
        • Sevilla I.
        • Singh S.V.
        • Garrido J.M.
        • Aduriz G.
        • Rodriguez S.
        • Geijo M.V.
        • et al.
        Molecular typing of Mycobacterium avium subspecies paratuberculosis strains from different hosts and regions.
        Rev Sci Tech. 2005; 24: 1061-1066
        • Li L.
        • Bannantine J.P.
        • Zhang Q.
        • Amonsin A.
        • May B.J.
        • Alt D.
        • et al.
        The complete genome sequence of Mycobacterium avium subspecies paratuberculosis.
        Proc Natl Acad Sci U S A. 2005; 102: 344-349
        • Landis J.R.
        • Koch G.G.
        The measurement of observer agreement for categorical data.
        Biometrics. 1977; 33: 159-174
      7. Advisory Committee on the Microbiological Safety of Food. Mycobacterium avium subspecies paratuberculosis: review of advice. ACM 487. Food Standards Agency; 2001. Available at: http://archive.food.gov.uk/pdf_files/papers/acm487.pdf (accessed May 2009).

      8. Singh SV, Singh PK, Singh AV, Sohal JS, Gupta VK, Vihan VS. Comparative efficacy of an indigenous ‘inactivated vaccine’ using highly pathogenic field strain of Mycobacterium avium subspecies paratuberculosis ‘bison type’ with commercial vaccine for the control of Capri-paratuberculosis in India. Vaccine 2007; 25:7102-10.