International Journal of Infectious Diseases
Volume 14, Issue 2 , Pages e121-e126, February 2010

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

  • H. Shankar

      Affiliations

    • Veterinary Microbiology Laboratory, Animal Health Division, Central Institute for Research on Goats, Makhdoom, PO Farah, Mathura, Uttar Pradesh, India
    • ,
  • S.V. Singh

      Affiliations

    • Veterinary Microbiology Laboratory, Animal Health Division, Central Institute for Research on Goats, Makhdoom, PO Farah, Mathura, Uttar Pradesh, India
    • Corresponding Author InformationCorresponding author. Tel.: +91 565 2763260x269; fax: +91 565 2763246.
    • ,
  • P.K. Singh

      Affiliations

    • Veterinary Microbiology Laboratory, Animal Health Division, Central Institute for Research on Goats, Makhdoom, PO Farah, Mathura, Uttar Pradesh, India
    • ,
  • A.V. Singh

      Affiliations

    • Veterinary Microbiology Laboratory, Animal Health Division, Central Institute for Research on Goats, Makhdoom, PO Farah, Mathura, Uttar Pradesh, India
    • ,
  • J.S. Sohal

      Affiliations

    • Veterinary Microbiology Laboratory, Animal Health Division, Central Institute for Research on Goats, Makhdoom, PO Farah, Mathura, Uttar Pradesh, India
    • ,
  • R.J. Greenstein

      Affiliations

    • Laboratory of Molecular Surgical Research, Veterans Affairs Medical Center, Bronx, New York, USA

Received 23 September 2008; accepted 31 March 2009. published online 03 July 2009.

Corresponding Editor: William Cameron, Ottawa, Canada

Article Outline

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: Johne's disease, Mycobacterium avium subspecies paratuberculosis, Milk, Dairy products, Pasteurized, Culture, IS1311 PCR-REA

 

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Introduction 

In ruminants, Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of Johne's disease (JD),1 a chronic enteric diarrheal disease that is evocative of human inflammatory bowel disease (IBD).2 JD is increasing in incidence in both the developed3 and the developing world, and in India.4 Apparently unrelated, the incidence of IBD is increasing in the industrialized world5 as well as in India,6, 7, 8, 9, 10 where gastroenteritis and colitis are common health problems.11, 12, 13

MAP has been cultured from chlorinated potable municipal water in the USA14 and Europe,15 pasteurized milk in the USA16 and Europe,17, 18 the intestine19 and stool20 of patients with Crohn's disease (CD), the breast milk of mothers with Crohn's disease,21 and from the blood of patients with IBD.22 MAP is not reproducibly killed by standard pasteurization procedures.23, 24 Although prevailing medical dogma considers MAP to be an innocent commensal,25 there are increasing concerns that MAP may be zoonotic,26, 27 and at a minimum responsible for IBD28, 29.

The Republic of India has more than 400 million milk producing animals30 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%.31 Approximately 35% of Indian milk is pasteurized, predominantly by state cooperatives, multinational companies, or government dairy plants.32 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.33 In India, MAP has been cultured from the unpasteurized milk of goats4, 34 and cattle.35, 36

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.

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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 (500ml), 18 of pasteurized milk (500ml), and nine of pasteurized milk products (50 to 500g). 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 (3000rpm, 20min) 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 18–24hours at room temperature. The resulting liquid layer was decanted and discarded, and 0.02ml of the residual mucilaginous sludge was inoculated onto modified Herrold's egg yolk medium (HEYM)37, 38 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,4 tissues,39 and milk.23, 34

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 DNA40 with minor modifications.4 DNA from colonies was isolated41 and amplified using specific IS900 primers,42 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.43 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.43, 44 In brief, in a 25μl volume: 0.5–1.0ng template DNA, 2.5μl of 10X PCR buffer (Promega), 1.5mM MgCl2 (Promega), 0.2mM dNTPs, and 1 unit Taq (Promega). Thermal cycling conditions were: initial denaturation at 94°C for 3min followed by 37 cycles of denaturation at 94°C for 30s, annealing at 62°C for 30s, and extension at 72°C for 1min. The final extension was 72°C for 10min. PCR products were subjected to electrophoresis (2% agarose gel stained with ethidium bromide). Amplicons that co-migrated at 608bp 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 1.5hours as previously described.44 Following incubation, fragments were separated on 4% agarose gel. Genotypes were identified by fragment migration patterns as described.43

To confirm that we had isolated MAP, DNA sequencing was performed on 13 isolates and compared to K10.45

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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 agreement46 between culture and direct PCR showed significant concordance (kappa PA value=62.0).

Table 1. Culture 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 productsa93 (33)3 (33)5 (56)2 (22)5 (100)
Total4319 (44)15 (35)25 (58)10 (23)25 (100)

MAP, Mycobacterium avium subspecies paratuberculosis.

aMilk products include ice cream and flavored milk drinks.

  • View full-size image.
  • Figure 1. 

    Agarose gel with the IS900 specific primers, which generate a fragment that migrates at a molecular weight of 229bp. 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).

  • View full-size image.
  • Figure 2. 

    Agarose gel with the IS900 specific primers, which generate a fragment that migrates at a molecular weight of 229bp. 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 2. Comparison 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 3. Comparison 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.

  • View full-size image.
  • Figure 3. 

    Differentiating 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);43 in contrast, ‘bison type’ MAP generates only the two larger bands (lanes 4, 6, 7, and 8).43.

DNA sequencing performed on 13 of our isolates showed 100% homology of a 267-bp fragment of the AV1/AV2 region of IS900,45 confirming that our isolates were MAP (data not presented).

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Discussion 

India produces more milk products than any other country in the world,31 the majority of which are not subjected to pasteurization.32 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 USA16 and Ireland.17

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.22 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 zoonotic26 remains to be clarified.27 However, until the MAP zoonosis conundrum is definitively resolved we suggest that the precautionary principle, instituted by the British Government for MAP in the UK47 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 program48 should be amongst the contingencies considered.

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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.

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PII: S1201-9712(09)00188-X

doi:10.1016/j.ijid.2009.03.031

International Journal of Infectious Diseases
Volume 14, Issue 2 , Pages e121-e126, February 2010

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