Genotype profiles of Mycobacterium avium subspecies paratuberculosis isolates recovered from animals, commercial milk, and human beings in North India

  • S.V. Singh
    Correspondence
    Corresponding author. Tel.: +91 9719072856; fax: +91 565 2763246.
    Affiliations
    Microbiology Laboratory, Animal Health Division, Central Institute for Research on Goats, Makhdoom, PO – FARAH, District - Mathura, Uttar Pradesh, 281 122, India
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  • J.S. Sohal
    Affiliations
    Microbiology Laboratory, Animal Health Division, Central Institute for Research on Goats, Makhdoom, PO – FARAH, District - Mathura, Uttar Pradesh, 281 122, India
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  • P.K. Singh
    Affiliations
    Microbiology Laboratory, Animal Health Division, Central Institute for Research on Goats, Makhdoom, PO – FARAH, District - Mathura, Uttar Pradesh, 281 122, India
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  • A.V. Singh
    Affiliations
    Microbiology Laboratory, Animal Health Division, Central Institute for Research on Goats, Makhdoom, PO – FARAH, District - Mathura, Uttar Pradesh, 281 122, India
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Open ArchivePublished:February 24, 2009DOI:https://doi.org/10.1016/j.ijid.2008.11.022

      Summary

      Objective

      To understand the genetic diversity of Mycobacterium avium subspecies paratuberculosis (MAP) isolates recovered from domestic and wild ruminants, commercial milk, and human beings in North India.

      Methods

      Genotyping of MAP isolates (N = 117) recovered from animals, commercial milk, and human beings in different regions of North India between 1998 and 2007 was carried out using IS1311 PCR-restriction enzyme analysis (REA) and short sequence repeat (SSR) typing (G and GGT repeat loci).

      Results

      Of the 117 MAP isolates recovered from North India, bison-type was the predominant (83.8%) genotype followed by cattle-type (16.2%). Bison-type was the exclusive genotype recovered from goats, sheep, buffaloes, and blue bulls. However, both bison-type and cattle-type genotypes were recovered from cattle, humans, and commercial bovine milk samples. The relative distribution of the two genotypes was different in the different regions. Bison-type was the major genotype at the Central Institute for Research on Goats (CIRG), Akos, Ajmer, and Mathura, whereas, cattle-type was the major genotype from New Delhi and Agra. SSR typing of these isolates revealed that all MAP bison-type isolates had an identical profile (7g4ggt) with respect to G and GGT repeat SSR loci. In this study the sheep-type genotype was not found in North India.

      Conclusions

      This study is the first from India to report the presence of two kinds of MAP genotypes (cattle-type and bison-type). However, non-reporting of the sheep-type genotype may not mean that it is absent in North India; the use of multiple culture media to recover MAP from clinical samples for future investigations is advised.

      Keywords

      Introduction

      Mycobacterium avium subspecies paratuberculosis (MAP) is the cause of paratuberculosis or Johne's disease (JD) in a wide range of species.
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      The disease is a serious animal health problem worldwide, adversely affecting productivity, and also has public health significance. It is the most costly infectious disease of dairy cattle, resulting in annual losses to the tune of $250 million in the USA alone.
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      The National Paratuberculosis Program in Norway.
      Information on the prevalence of MAP genotypes in India is not currently available.
      There are three main MAP genotypes: ‘sheep-type’ (S) or type I, ‘cattle-type’ (C) or type II, and ‘intermediate’ or type III based on IS900 restriction fragment length polymorphism (RFLP), pulsed-field gel electrophoresis (PFGE), and culture characteristics.
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      Cattle-type has primarily been isolated from cattle and other domestic and wild ruminants, non-ruminants, and human beings.
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      • Pavlas M.
      • Rozsypalova Z.
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      Characterization by restriction endonuclease analysis and DNA hybridization using IS900 of bovine, ovine, caprine and human dependent strains of Mycobacterium paratuberculosis isolated in various localities.
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      Sheep-type strains are extremely slow growers and have been isolated primarily from sheep.
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      Molecular characterization of Mycobacterium paratuberculosis isolates from sheep, goats, and cattle by hybridization with a DNA probe to insertion element IS900.
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      • Hope A.
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      DNA fingerprinting of Australian isolates of Mycobacterium avium subsp. paratuberculosis using IS900 RFLP.
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      • Marsh I.
      Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: IS900 restriction fragment length polymorphism and IS1311 polymorphism analyses of isolates from animals and a human in Australia.
      Some of the ovine
      • Collins D.M.
      • Gabric D.M.
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      Identification of two groups of Mycobacterium paratuberculosis strains by restriction endonuclease analysis and DNA hybridization.
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      • Collins D.M.
      • Huchzermeyer H.F.
      Characterization of ovine strains of Mycobacterium paratuberculosis by restriction endonuclease analysis and DNA hybridization.
      • de Lisle G.W.
      • Yates G.
      • Collins D.M.
      Paratuberculosis in farmed deer: case reports and DNA characterization of Mycobacterium paratuberculosis.
      and caprine isolates
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      • Mateos A.
      • Domínguez L.
      • Sharp J.M.
      • Stevenson K.
      Genetic diversity of Mycobacterium avium subspecies paratuberculosis isolates from goats detected by pulsed-field gel electrophoresis.
      have been clubbed together as the intermediate group. Types I and III are difficult to culture and subculture compared to type II strains. The major difficulty with IS900 RFLP and PFGE analysis is the requirement for large quantities of good quality DNA, which is a difficult proposition in the case of extremely slow growing type I/III isolates. IS1311 PCR-REA (restriction enzyme analysis),
      • Marsh I.
      • Whittington R.
      • Cousins D.
      PCR-restriction endonuclease analysis for identification and strain typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium based on polymorphisms in IS1311.
      RDA-PCR (representational difference analysis),
      • Dohmann K.
      • Strommenger B.
      • Stevenson K.
      • de Juan L.
      • Stratmann J.
      • Kapur V.
      • et al.
      Characterization of genetic differences between Mycobacterium avium subsp. paratuberculosis type I and type II isolates.
      and DMC-PCR
      • Collins D.M.
      • De Zoete M.
      • Cavaignac S.M.
      Mycobacterium avium subsp. paratuberculosis strains from cattle and sheep can be distinguished by a PCR test based on a novel DNA sequence difference.
      can offset these problems. PCR-based tests subdivide MAP strains into cattle or type II and sheep or type I/III. IS1311 PCR-REA
      • Whittington R.J.
      • Marsh I.B.
      • Whitlock R.H.
      Typing of IS1311 polymorphisms confirms that bison (Bison bison) with paratuberculosis in Montana are infected with strain of Mycobacterium avium subspecies paratuberculosis distinct from that occurring in cattle and other domestic livestock.
      is able to further divide type II isolates into cattle-type (C) and bison-type (B) based on C/T polymorphisms at position 223 of IS1311. However, IS1311 PCR-REA is not able to distinguish type I and III isolates and detects them either as S-type or type I/III.
      The recent use of short sequence repeat (SSR) typing has made it possible to determine that certain MAP genotypes are involved in overt paratuberculosis and others result in subclinical infection.
      • Amonsin A.
      • Li L.L.
      • Zhang Q.
      • Bannantine J.P.
      • Motiwala A.S.
      • Sreevatsan S.
      • et al.
      Multilocus short sequence repeat sequencing approach for differentiating among Mycobacterium avium subsp. paratuberculosis strains.
      • Ghadiali A.H.
      • Strother M.
      • Naser S.A.
      • Manning E.J.
      • Sreevatsan S.
      Mycobacterium avium subsp. paratuberculosis strains isolated from Crohn's disease patients and animal species exhibit similar polymorphic locus patterns.
      SSR typing has revealed that certain MAP genotypes are host-restricted and others are shared.
      • Ghadiali A.H.
      • Strother M.
      • Naser S.A.
      • Manning E.J.
      • Sreevatsan S.
      Mycobacterium avium subsp. paratuberculosis strains isolated from Crohn's disease patients and animal species exhibit similar polymorphic locus patterns.
      • Motiwala A.S.
      • Strother M.
      • Amonsin A.
      • Byrum B.
      • Naser S.A.
      • Stabel J.R.
      • et al.
      Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: evidence for limited strain diversity, strain sharing, and identification of unique targets for diagnosis.
      • Motiwala A.S.
      • Amonsin A.
      • Strother M.
      • Manning E.J.
      • Kapur V.
      • Sreevatsan S.
      Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild animal species.
      • Motiwala A.S.
      • Strother M.
      • Theus N.E.
      • Stich R.W.
      • Byrum B.
      • Shulaw W.P.
      • et al.
      Rapid detection and typing of strains of Mycobacterium avium subsp. paratuberculosis from broth cultures.
      The results of SSR analysis by Amonsin et al.
      • Amonsin A.
      • Li L.L.
      • Zhang Q.
      • Bannantine J.P.
      • Motiwala A.S.
      • Sreevatsan S.
      • et al.
      Multilocus short sequence repeat sequencing approach for differentiating among Mycobacterium avium subsp. paratuberculosis strains.
      indicated that the mononucleotide G repeat (AAK46234) and the trinucleotide GGT repeat (CAB06859) were the most discriminatory of the 11 SSRs analyzed. Therefore, in the present investigation these two loci were selected for fingerprinting of representative MAP bison-type isolates.
      In India JD is endemic and highly prevalent in domestic ruminant species. MAP has also been isolated from wild animals and human beings with typical or suspected cases of Crohn's disease (CD).
      • Kumar P.
      • Singh S.V.
      • Bhatiya A.K.
      • Sevilla I.
      • Singh A.V.
      • Whittington R.J.
      • et al.
      Juvenile Capri-Paratuberculosis in India: incidence and characterization by six diagnostic tests.
      • Kumar S.
      • Singh S.V.
      • Sevilla I.
      • Singh A.V.
      • Whittington R.J.
      • Juste R.A.
      • et al.
      Lacto-incidence and evaluation of 3 tests for the diagnosis of Johne's disease using milk of naturally infected goatherds and genotyping of Mycobacterium avium subspecies paratuberculosis.
      • 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 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.
      • Singh N.
      • Vihan V.S.
      • Singh S.V.
      • Gupta V.K.
      Prevalence of Johne's disease in organized goat herds.
      • Singh S.V.
      • Singh A.V.
      • Singh P.K.
      • Gupta V.K.
      • Kumar S.
      • Vohra J.
      Sero-prevalence of paratuberculosis in young kids using ‘bison type’, Mycobacteriumavium subsp. paratuberculosis antigen in plate ELISA.
      • 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.
      • Singh S.V.
      • Singh A.V.
      • Singh R.
      • Sharma S.
      • Shukla N.
      • Misra S.
      • et al.
      Sero-prevalence of Johne's disease in buffaloes and cattle population of India by indigenous ELISA kit using PPA from Mycobacterium avium subspecies paratuberculosis ‘bison type’ genotype of goat origin.
      • Yadav D.
      • Singh S.V.
      • Singh A.V.
      • Sevilla I.
      • Juste R.A.
      • Singh P.K.
      • et al.
      Pathogenic ‘bison type’ Mycobacterium avium subspecies paratuberculosis genotype characterized from riverine buffalo (Bubalus bubalis) in North India.
      Despite the high endemicity of MAP in domestic ruminants, the resulting economic losses have never been estimated. At present the country lacks JD control programs to reduce the increasing burden of this disease. Information on the prevalence of MAP genotypes in India is non-existent. This pilot study aimed to type MAP strains using IS1311 PCR-REA to determine the genotype profiles of MAP prevalent in North India and to study the genetic homogeneity of bison-type genotype using SSR typing.

      Materials and methods

       MAP isolates

      Genotype profiles of a panel of 117 MAP isolates recovered from animals (domestic and wild), bovine milk (pasteurized, unpasteurized pooled, and individual cow samples), and human beings were screened using IS1311 PCR-REA. Animal isolates were recovered from clinical cases of JD. Human MAP isolates were from patients either with CD or suspected CD. MAP isolates from milk were recovered from randomly collected milk samples from commercial and private milk supplies. All of the MAP isolates were isolated and subcultured on Herrold's egg yolk medium (HEYM) supplemented with mycobactin J.
      • 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.
      Fecal,
      • 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.
      milk,
      • Kumar S.
      • Singh S.V.
      • Sevilla I.
      • Singh A.V.
      • Whittington R.J.
      • Juste R.A.
      • et al.
      Lacto-incidence and evaluation of 3 tests for the diagnosis of Johne's disease using milk of naturally infected goatherds and genotyping of Mycobacterium avium subspecies paratuberculosis.
      • 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.
      and tissue
      • Yadav D.
      • Singh S.V.
      • Singh A.V.
      • Sevilla I.
      • Juste R.A.
      • Singh P.K.
      • et al.
      Pathogenic ‘bison type’ Mycobacterium avium subspecies paratuberculosis genotype characterized from riverine buffalo (Bubalus bubalis) in North India.
      samples were processed as per standardized Microbiology Laboratory methods (Central Institute for Research on Goats, Makhdoom). Primary MAP colonies were characterized on the basis of acid-fastness, slow growth, mycobactin dependency, and IS900 PCR.
      • Vary P.H.
      • Andersen P.R.
      • Green E.
      • Hermon-Talyor J.
      • McFadden J.J.
      Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne's disease.
      These MAP isolates were obtained from different samples (feces, tissues of intestine, mesenteric lymph nodes, supramammary lymph nodes, milk, vaginal secretions, human biopsies, and human stool samples) processed in the Microbiology Laboratory from 1998 to 2007.

       IS1311 PCR

      IS1311 PCR was carried out using M56 and M119 primers as per Sevilla et al.
      • 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.
      with some modifications. Briefly, PCR were set up in volume of 25 μl, using 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 U Taq (Promega). Thermal cycling was as follows: 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, extension at 72 °C for 1 min, and final extension at 72 °C for 10 min. Amplicon sizes of 608 bp were considered positive in IS1311 PCR, after separation on 2% agarose gel stained with ethidium bromide.

       IS1311 PCR-REA

      IS1311 PCR-REA was carried out as per Sevilla et al.
      • 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.
      Briefly, the reaction was carried out in a volume of 30 μl, containing 20 μl positive IS1311 PCR product, 3 μl 10X buffer (Fermentas), and 2 U of each endonuclease HinfI and MseI (Fermentas). The reaction mixture was incubated at 37 °C for 1.5 h. Band patterns were visualized after electrophoresis on 4% agarose gel and staining with ethidium bromide. Genotype profiles were interpreted as per Whittington et al.
      • Whittington R.J.
      • Marsh I.B.
      • Whitlock R.H.
      Typing of IS1311 polymorphisms confirms that bison (Bison bison) with paratuberculosis in Montana are infected with strain of Mycobacterium avium subspecies paratuberculosis distinct from that occurring in cattle and other domestic livestock.

       SSR typing

      G and GGT repeat loci of a few representative MAP bison-type isolates (from different regions and host species) were analyzed to study the homogeneity of these isolates. Amplification of SSR loci was carried out as per conditions described by Amonsin et al.
      • Amonsin A.
      • Li L.L.
      • Zhang Q.
      • Bannantine J.P.
      • Motiwala A.S.
      • Sreevatsan S.
      • et al.
      Multilocus short sequence repeat sequencing approach for differentiating among Mycobacterium avium subsp. paratuberculosis strains.
      PCR products were purified by plate purification, and sequencing was done using Big Dye Terminator (BDT) chemistry. Sequences were analyzed using an automated DNA sequencer (ABI Prism 3700 DNA analyzer). The quality of sequencing data was analyzed using ABS sequence scanner v. 1.0 software, and the number of repeats at each locus for every isolate was determined.

      Results

       Culture characteristics of MAP isolates

      MAP colonies were recovered between 1998 and 2007 and had similar incubation times and cultural and bacterial morphology. Colonies appeared between 45 and 120 days of incubation. Multibacillary colonies were seen in a few cultures and the rest were paucibacillary. Typical MAP colonies were straw-colored, consisting of a central raised nipple surrounded by a flat part. Colonies from commercial milk and human beings appeared slightly later compared to other colonies.

       Genotyping of MAP isolates

      The bison-type genotype was predominant based on IS1311 PCR-REA genotyping of the MAP isolates recovered from North India (Table 1, Table 2). Of 117 MAP isolates, 83.8% belonged to the bison-type genotype and 16.2% belonged to the cattle-type genotype. Herds located at the town of Farah (CIRG, Makhdoom) and Akos village of Mathura District (Uttar Pradesh State) and Ajmer District (Rajasthan State) were infected only with bison-type genotype regardless of the species (Table 2). Bison-type genotype was the major genotype observed in samples (feces, milk, and tissues) belonging to goats, sheep, blue bulls, and human beings located at the CIRG campus (Makhdoom) of Mathura District (97.7% bison-type and 2.3% cattle-type). MAP isolates from Mathura city were also predominantly bison-type (bison-type 90.9% and cattle-type 9.1%; Table 2). However, in samples from Agra city (52.6% cattle-type and 47.4% bison-type) and New Delhi (63.6% cattle-type and 36.4% bison-type), cattle-type was the major MAP genotype (Table 2). The sheep-type genotype was not found in the present study.
      Table 1Genotype profile of Mycobacterium avium subspecies paratuberculosis isolates by host species and milk samples
      Host species/sampleRegionNo. of isolatesYear(s) of isolationNo. of cattle-type isolatesNo. of bison-type isolatesNo. of sheep-type isolates
      GoatAkos, Agra32006–2007Nil3Nil
      Ajmer, Rajasthan112005–2007Nil11Nil
      CIRG, Makhdoom121998–2007Nil12Nil
       Total26-26 (100%)-
      SheepAjmer, Rajasthan142005–2007Nil14Nil
      CIRG, Makhdoom91998–2006Nil9Nil
       Total23-23 (100%)-
      CattleAgra72004–200561Nil
      Mathura82005–2006-8Nil
       Total156 (40%)9 (60%)-
      BuffaloAgra62004–2007Nil6Nil
       Total6-6 (100%)-
      Blue bullCIRG, Makhdoom42005–2007Nil4Nil
       Total4-4 (100%)-
      Bovine milkFarah, Mathura52006–2007Nil5Nil
      Agra52006–200732Nil
      Mathura12006–2007Nil1Nil
      Delhi62006–200742Nil
       Total177 (41.2%)10 (58.8%)-
      HumanMathura2200711Nil
      Agra120071NilNil
      Delhi52005–200732Nil
      CIRG, Makhdoom182004–2007117Nil
       Total266 (23.1%)20 (76.9%)-
      Total11719 (16.2%)98 (83.8%)-
      CIRG, Central Institute for Research on Goats.
      Table 2Genotype profile of Mycobacterium avium subspecies paratuberculosis isolates by region
      RegionSpecies/sample screenedNo. of isolatesNo. of cattle-type isolatesNo. of bison-type isolates
      CIRG, MakhdoomGoat12Nil12
      Sheep9Nil9
      Blue bull4Nil4
      Humans18117
       Total431 (2.3%)42 (97.7%)
      AkosGoat3Nil3
       Total3-3 (100%)
      Ajmer, RajasthanGoat11Nil11
      Sheep14Nil14
       Total25-25 (100%)
      MathuraCattle8Nil8
      Pooled bovine milk samples1Nil1
      Humans211
       Total111 (9.1%)10 (90.9%)
      AgraCattle761
      Buffalo6Nil6
      Pasteurized milk samples532
      Humans11Nil
       Total1910 (52.6%)9 (47.4%)
      DelhiPasteurized milk samples642
      Humans532
       Total117 (63.6%)4 (36.4%)
      FarahIndividual cow milk samples5Nil5
       Total5-5 (100%)
      Total11719 (16.2%)98 (83.8%)

       Genotyping of goat and sheep isolates

      All MAP isolates (26 from goat and 23 from sheep) recovered from clinically sick and advanced cases of JD in goats and sheep, regardless of the region of origin in North India, were of bison-type (B) genotype (Table 1).

       Genotyping of cattle isolates

      MAP isolates recovered from cattle with clinical JD and from dairy herds located in Agra and Mathura city of North India were genotyped. Of the total of 15 MAP isolates genotyped (seven from Agra and eight from Mathura), 40% were cattle-type and 60% were bison-type. All MAP isolates recovered from dairy cattle of Mathura city were bison-type. However, six (85.7%) out of seven isolates recovered from dairy cattle of the Agra region belonged to cattle-type (C) genotype and one (14.3%) was bison-type (Table 1, Table 2).

       Genotyping of buffalo isolates

      Buffaloes screened in the present study were all from Agra city. They belonged to individual farmers and were driven from Agra District before being sacrificed for meat production in unauthorized cottage slaughterhouses. In these slaughterhouses only one or two buffaloes were sacrificed per day. All the MAP isolates (n = 6) recovered from buffaloes were genotyped as bison-type (Table 1, Table 2).

       Genotyping of blue bull (Boselaphus tragocamelus) isolates

      A herd of nearly 100 blue bulls (wild ruminant antelope) use the CIRG campus as a safe haven during the day, having been chased away from farms where they destroy crops. In the night they share grazing resources used by goats and sheep in the day (CIRG campus) and frequently move out of campus. Blue bulls are known to travel long distances in search of food. Fecal samples of blue bulls residing around the CIRG, Makhdoom campus were screened and all MAP isolates (n = 4) were identified as bison-type (Table 1, Table 2).

       Genotyping of bovine milk isolates

      The 17 MAP isolates from bovine milk samples screened (Table 1) originated from: Farah (five samples from unpasteurized milk of individual dairy herd cows), Mathura (one milk sample from pooled unpasteurized milk), and milk samples from Agra (five milk samples) and New Delhi (six milk samples) from pasteurized milk packs of 500 ml of different commercial brands sold in the market. Of these, 41.2% MAP isolates belonged to cattle-type and 58.8% were of bison-type genotype (Table 1). Farah and Mathura bovine milk isolates were typed as bison-type. From Agra city pasteurized milk samples, three of the five MAP isolates (60%) were cattle-type and two (40%) were bison-type. From New Delhi city commercial pasteurized milk samples, four of the six MAP isolates (66.7%) belonged to cattle-type genotype and two (33.3%) were bison-type.

       Genotyping of human isolates

      Twenty-six MAP isolates were recovered from confirmed and suspected cases of CD. Two MAP isolates from Mathura city were from persons suspected for CD. CIRG human MAP isolates (n = 18) were recovered from stool samples of persons working the goat and sheep herds (n = 17) and a laboratory worker (n = 1), all with suspected CD. One isolate from Agra was isolated from stool samples of a confirmed CD patient. Five isolates from New Delhi were recovered from biopsies of confirmed CD patients. Of the two isolates from Mathura (suspected cases of CD), one was cattle-type and one was bison-type. The isolate from the CD patient in Agra belonged to cattle-type. Of the 18 MAP isolates from CIRG, Makhdoom (suspected cases of CD), one (5.6%) was cattle-type (from the laboratory worker) and 17 (94.4%) were bison-type (from the animal attendees). Of the five isolates from CD patients from New Delhi, three were cattle-type and two were bison-type.

       SSR typing of MAP bison-type isolates

      All MAP bison-type isolates yielded detectable PCR products for G and GGT repeat loci. Sequence analysis of these two loci revealed that all MAP isolates recovered from different animal species and regions had the same profile of SSR repeats, 7G and 4GGT (7g4ggt). The accession numbers are given in the footnote to Table 3.
      Table 3SSR typing of Mycobacterium avium subspecies paratuberculosis bison-type isolates
      Serial No.MAP isolateHost species
      1MAP S5Goat
      2MAP S4Goat
      3MAP S12Sheep
      4MAP A35Goat
      5MAP C43Bovine
      6MAP C42Bovine
      7MAP B42Blue bull
      8MAP 120Goat
      9MAP 121Goat
      MAP, Mycobacterium avium subspecies paratuberculosis.
      Accession numbers: EF514848 , EF514849 , EF514851 , EF514852 , EF514853 , EF514856 , EF514857 , EF514858 , EF514859 , EF514860 , EF514861 .

      Discussion

      With the exception of a few reports,
      • 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.
      information on the distribution of MAP genotypes in India does not exist. This study is the first to report that bison-type genotype (which has only been reported so far from bison in theUSA
      • Whittington R.J.
      • Marsh I.B.
      • Whitlock R.H.
      Typing of IS1311 polymorphisms confirms that bison (Bison bison) with paratuberculosis in Montana are infected with strain of Mycobacterium avium subspecies paratuberculosis distinct from that occurring in cattle and other domestic livestock.
      ), is the dominant genotype in animals, human beings, and bovine milk in North India (Table 1, Table 2). Previous studies from India have also reported a high prevalence of bison-type MAP.
      • Whittington R.J.
      • Marsh I.B.
      • Whitlock R.H.
      Typing of IS1311 polymorphisms confirms that bison (Bison bison) with paratuberculosis in Montana are infected with strain of Mycobacterium avium subspecies paratuberculosis distinct from that occurring in cattle and other domestic livestock.
      • Kumar P.
      • Singh S.V.
      • Bhatiya A.K.
      • Sevilla I.
      • Singh A.V.
      • Whittington R.J.
      • et al.
      Juvenile Capri-Paratuberculosis in India: incidence and characterization by six diagnostic tests.
      • 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.
      • Yadav D.
      • Singh S.V.
      • Singh A.V.
      • Sevilla I.
      • Juste R.A.
      • Singh P.K.
      • et al.
      Pathogenic ‘bison type’ Mycobacterium avium subspecies paratuberculosis genotype characterized from riverine buffalo (Bubalus bubalis) in North India.
      • 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.
      Contrary to the present findings, cattle-type is the major genotype infecting livestock and wild animal populations worldwide.
      • Pavlik I.
      • Bejckova L.
      • Pavlas M.
      • Rozsypalova Z.
      • Koskova S.
      Characterization by restriction endonuclease analysis and DNA hybridization using IS900 of bovine, ovine, caprine and human dependent strains of Mycobacterium paratuberculosis isolated in various localities.
      • Pavlik I.
      • Horvathova A.
      • Dvorska L.
      • Bartl J.
      • Svastova P.
      • Du Maine R.
      • et al.
      Standardization of restriction fragment length polymorphism analysis for Mycobacterium avium subspecies paratuberculosis.
      • Whittington R.J.
      • Hope A.F.
      • Marshall D.J.
      • Taragel C.A.
      • Marsh I.
      Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: IS900 restriction fragment length polymorphism and IS1311 polymorphism analyses of isolates from animals and a human in Australia.
      • Marsh I.
      • Whittington R.
      • Cousins D.
      PCR-restriction endonuclease analysis for identification and strain typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium based on polymorphisms in IS1311.
      • Motiwala A.S.
      • Amonsin A.
      • Strother M.
      • Manning E.J.
      • Kapur V.
      • Sreevatsan S.
      Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild animal species.
      • Djonne B.
      • Pavlik I.
      • Svastova P.
      • Bartos M.
      • Holstad G.
      IS900 restriction fragment length polymorphism (RFLP) analysis of Mycobacterium avium subsp. paratuberculosis isolates from goats and cattle in Norway.
      Bison-type MAP has not been reported from livestock outside India.
      Bison-type genotype was predominant in cattle located in Mathura and Agra (Table 1, Table 2). Cattle in Mathura regions were infected with bison-type MAP exclusively (100%). Of the cattle screened from Agra, 85.7% were infected with cattle-type and the rest with bison-type genotype (Table 1, Table 2); cattle-type is also the principal genotype infecting cattle outside India.
      • Pavlik I.
      • Bejckova L.
      • Pavlas M.
      • Rozsypalova Z.
      • Koskova S.
      Characterization by restriction endonuclease analysis and DNA hybridization using IS900 of bovine, ovine, caprine and human dependent strains of Mycobacterium paratuberculosis isolated in various localities.
      • Pavlik I.
      • Horvathova A.
      • Dvorska L.
      • Bartl J.
      • Svastova P.
      • Du Maine R.
      • et al.
      Standardization of restriction fragment length polymorphism analysis for Mycobacterium avium subspecies paratuberculosis.
      • Whittington R.J.
      • Hope A.F.
      • Marshall D.J.
      • Taragel C.A.
      • Marsh I.
      Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: IS900 restriction fragment length polymorphism and IS1311 polymorphism analyses of isolates from animals and a human in Australia.
      • Marsh I.
      • Whittington R.
      • Cousins D.
      PCR-restriction endonuclease analysis for identification and strain typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium based on polymorphisms in IS1311.
      • Motiwala A.S.
      • Amonsin A.
      • Strother M.
      • Manning E.J.
      • Kapur V.
      • Sreevatsan S.
      Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild animal species.
      • Djonne B.
      • Pavlik I.
      • Svastova P.
      • Bartos M.
      • Holstad G.
      IS900 restriction fragment length polymorphism (RFLP) analysis of Mycobacterium avium subsp. paratuberculosis isolates from goats and cattle in Norway.
      Information on MAP genotypes infecting buffaloes in and outside India is limited. In this pilot study, buffaloes were found to be infected only with bison-type MAP (Table 1, Table 2). Two previous studies have also reported bison-type MAP from buffaloes in India.
      • Yadav D.
      • Singh S.V.
      • Singh A.V.
      • Sevilla I.
      • Juste R.A.
      • Singh P.K.
      • et al.
      Pathogenic ‘bison type’ Mycobacterium avium subspecies paratuberculosis genotype characterized from riverine buffalo (Bubalus bubalis) in North India.
      • Singh S.V.
      • Sharma G.
      • Yadav D.
      • Singh A.V.
      • Sevilla I.
      • Juste R.A.
      • et al.
      Prevalence of Mycobacterium avium subsp. paratuberculosis (bison type) in cattle and buffalo populations of Agra region in India.
      The blue bull herd sharing a grazing area with goats and sheep at the CIRG campus (Mathura) were also infected with bison-type MAP.
      • Kumar P.
      • Singh S.V.
      • Bhatiya A.K.
      • Sevilla I.
      • Singh A.V.
      • Whittington R.J.
      • et al.
      Juvenile Capri-Paratuberculosis in India: incidence and characterization by six diagnostic tests.
      • Kumar S.
      • Singh S.V.
      • Sevilla I.
      • Singh A.V.
      • Whittington R.J.
      • Juste R.A.
      • et al.
      Lacto-incidence and evaluation of 3 tests for the diagnosis of Johne's disease using milk of naturally infected goatherds and genotyping of Mycobacterium avium subspecies paratuberculosis.
      • 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.
      • Thoresen O.F.
      • Olsaker I.
      Distribution and hybridization patterns of the insertion element IS900 in clinical isolates of Mycobacterium paratuberculosis.
      MAP isolated from commercial bovine milk supplies (Table 1, Table 2), belonged to bison-type (58.8%) and cattle-type (41.2%). However, in bovine milk supplies from Farah and Mathura, bison-type was reported exclusively (Table 1, Table 2), and cattle-type genotype was predominant in commercial bovine milk supplies from Agra and New Delhi (Table 1, Table 2). In the confirmed and suspected cases of CD, bison-type (76.9%) was the major genotype followed by cattle-type (23.1%) (Table 1, Table 2). Goat and sheep attendants working with CIRG goatherds (endemic for infection with bison-type MAP
      • Kumar P.
      • Singh S.V.
      • Bhatiya A.K.
      • Sevilla I.
      • Singh A.V.
      • Whittington R.J.
      • et al.
      Juvenile Capri-Paratuberculosis in India: incidence and characterization by six diagnostic tests.
      • Kumar S.
      • Singh S.V.
      • Sevilla I.
      • Singh A.V.
      • Whittington R.J.
      • Juste R.A.
      • et al.
      Lacto-incidence and evaluation of 3 tests for the diagnosis of Johne's disease using milk of naturally infected goatherds and genotyping of Mycobacterium avium subspecies paratuberculosis.
      • 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.
      • Thoresen O.F.
      • Olsaker I.
      Distribution and hybridization patterns of the insertion element IS900 in clinical isolates of Mycobacterium paratuberculosis.
      ), were exclusively infected with MAP bison-type. The isolate from the laboratory worker at CIRG belonged to cattle-type genotype. This person had a history of consuming raw cow's milk. However, in CD patients from New Delhi, the major genotype was cattle-type. This study shows that the genotype profile in humans was the same as that seen in animals for that particular region (Table 1, Table 2). It proves that humans acquired MAP from the animals located in that area.
      Bison-type genotype was more pathogenic for cattle as compared to cattle-type.
      • Singh S.V.
      • Sevilla I.
      • Juste R.A.
      • Whittington R.J.
      • Kumar V.
      • Gupta V.K.
      • et al.
      Mycobacterium avium subsp. paratuberculosis (bison type) genotype infecting goat population in India.
      The predominance of MAP bison-type in cattle, buffaloes, goats, sheep, blue bulls, human beings, and commercial milk shows the ability of this genotype to infect multiple species. HEYM supplemented with mycobactin J was the only medium used for the isolation of MAP. But HEYM does support the growth of the slow growing sheep-type genotype.
      • de Juan L.
      • Álvarez J.
      • Romero B.
      • Bezos J.
      • Castellanos E.
      • Aranaz A.
      • et al.
      Comparison of four different culture media for isolation and growth of type II and type I/III Mycobacterium avium subsp. paratuberculosis strains isolated from cattle and goats.
      de Juan et al. suggested the use of four culture media to inoculate any single specimen.
      • de Juan L.
      • Álvarez J.
      • Romero B.
      • Bezos J.
      • Castellanos E.
      • Aranaz A.
      • et al.
      Comparison of four different culture media for isolation and growth of type II and type I/III Mycobacterium avium subsp. paratuberculosis strains isolated from cattle and goats.
      Sequence analysis of G and GGT repeat loci showed that MAP bison-type isolates had similar SSR repeat profiles, i.e., 7G and 4GGT (7g4ggt). No allelic variation in MAP bison-type isolates was indicative of interspecies transmission (cattle, goats, sheep, buffaloes, and blue bulls) and sharing of bison-type genotype. Motiwala et al.
      • Motiwala A.S.
      • Janagama H.K.
      • Paustian M.L.
      • Zhu X.
      • Bannantine J.P.
      • Kapur V.
      • et al.
      Comparative transcriptional analysis of human macrophages exposed to animal and human isolates of Mycobacterium avium subspecies paratuberculosis with diverse genotypes.
      reported an identical 7g4ggt profile for MAP bison-type recovered from bison in the USA. Though sheep are reported to be infected with MAP type I or sheep-type,
      • Motiwala A.S.
      • Amonsin A.
      • Strother M.
      • Manning E.J.
      • Kapur V.
      • Sreevatsan S.
      Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild animal species.
      in North India, bison-type MAP with identical SSR profiles has infected sheep and other animal species. Ghadiali et al.,
      • Ghadiali A.H.
      • Strother M.
      • Naser S.A.
      • Manning E.J.
      • Sreevatsan S.
      Mycobacterium avium subsp. paratuberculosis strains isolated from Crohn's disease patients and animal species exhibit similar polymorphic locus patterns.
      employing this highly polymorphic SSR (G and GGT repeat) typed MAP from domestic and wild animals and humans and concluded that the 7g4ggt genotype had the ability to infect multiple animal species and human beings. The study of Motiwala et al.
      • Motiwala A.S.
      • Strother M.
      • Theus N.E.
      • Stich R.W.
      • Byrum B.
      • Shulaw W.P.
      • et al.
      Rapid detection and typing of strains of Mycobacterium avium subsp. paratuberculosis from broth cultures.
      also supports the ability of 7g4ggt genotype to infect multiple host species. This study also shows the ability of 7g4ggt to cross the species barrier (goats, sheep, cattle, buffaloes, blue bulls, and human beings; Table 3).
      This study is the first from India to report the presence of two kinds of MAP genotypes (cattle-type and bison-type). However, non-reporting of the sheep-type genotype may not mean that it is absent in North India; the use of multiple culture media to recover MAP from clinical samples for future investigations is advised.

      Acknowledgements

      The authors are grateful to the Director, CIRG, Makhdoom for providing the necessary research facilities.
      Conflict of interest: No conflict of interest to declare.

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