Proceedings of the 10th International Colloquium on Paratuberculosis

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#52 Is ‘Indian Bison Type’ Mycobacterium avium subspecies paratuberculosis represents‘ a new MAP Biotype’? Jagdeep Singh Sohal, Shoor Vir Singh, Pravin Kumar Singh, Ajay Vir Singh, M C Sharma Indian Council <strong>of</strong> Agricultural Research, India; Central Institute for Research on Goats, India Objective: In India, first case <strong>of</strong> Johne’s disease was identified in 1913, but active research was started only after 1984. Indian MAP has been found to be highly pathogenic and endemic in domestic animals. First time culture <strong>of</strong> Indian MAP isolates in 1988 by our group, helped in initial characterization using radio-active probes, protein and lipid pr<strong>of</strong>iles. Genotypic analysis with help <strong>of</strong> Spanish and Australian labs, first time indicated that ‘Indian MAP strains’ were similar to ‘Bison type’ MAP, but possibly represents a new biotype. Present study aimed to identify <strong>the</strong> genetic variations <strong>of</strong> ‘Indian MAP strains’ and <strong>the</strong>ir evolution compared with o<strong>the</strong>r MAP strains Materials and Methods: Different molecular markers (IS elements, LSPs and SSRs) <strong>of</strong> native MAP were analyzed in order to study above objectives. Results and Conclusion: Sequencing <strong>of</strong> different markers identified SNPs/indels unique to ‘Indian Bison Type’ MAP with potential to be used as ‘Molecular Signatures’ for native strains <strong>of</strong> MAP. Positional polymorphism was also identified in few IS elements. Studies on ‘Phylogenetic Analysis’ indicated that <strong>the</strong> ‘Indian Bison Type’ MAP may be most recent to evolve in <strong>the</strong> evolutionary ladder, when compared with MAP strains reported outside India. Molecular phylogeny studies also indicated that <strong>the</strong> o<strong>the</strong>r strains <strong>of</strong> MAP were perhaps evolutionary intermediates during its’ journey from M. avium to ‘Indian Bison Type’ MAP through ‘Sheep type’, ‘Cattle type’ and ‘US Bison type’ strains. Study also identified genetic rearrangements in <strong>the</strong> genome <strong>of</strong> ‘Indian Bison Type’ MAP that resulted in duplications <strong>of</strong> LSP regions in it’s genome. These ‘duplicated regions’ encode for important proteins including virulence factors, antigenic proteins and enzymes. The diagnostic kit (ELISA based) and ‘indigenous vaccine’ developed using ‘Indian Bison Type’ strain have been reported to be highly efficacious. Increased gene dosages due to duplicated regions may be responsible for over-production <strong>of</strong> virulence factors and antigens that may have bearing on over all virulence and efficacy <strong>of</strong> vaccine prepared using ‘Indian Bison Type’ MAP strains Fur<strong>the</strong>r studies at proteome and transcriptome level and complete sequencing <strong>of</strong> ‘Indian Bison Type’ MAP, will impart novel insights into <strong>the</strong> biology <strong>of</strong> native MAP. Documentation <strong>of</strong> new ‘Indian Bison Type’ MAP strain, (reported to be highly pathogenic) will help initiating efforts towards <strong>the</strong> control <strong>of</strong> paratuberculosis in <strong>the</strong> country. 249
#53 Novel IS900 sequence polymorphisms in Indian isolates <strong>of</strong> Mycobacterium avium subspecies paratuberculosis <strong>of</strong> human and bovine origin Ajay Vir Singh, Shoor Vir Singh, Pravin Kumar Singh, Jagdeep Singh Sohal, M C Sharma Indian Council <strong>of</strong> Agricultural Research, India; Central Institute for Research on Goats, India Objective: Study aimed to characterize clinical isolates <strong>of</strong> Mycobacterium avium subspecies paratuberculosis (MAP) <strong>of</strong> humans and bovine origin by sequencing two regions <strong>of</strong> IS900 elements. Materials and Methods: DNA was extracted from representative 11 native isolates <strong>of</strong> MAP <strong>of</strong> human and bovine origin from North India. Stool samples from human beings (N=2) exhibiting clinical symptoms indistinguishable to Crohn’s disease (CD) and were employed in goa<strong>the</strong>rds endemic for Johne’s disease (JD). Two DNA belonged to Crohn’s disease patients. Seven DNA were recovered from commercially pasteurized bovine milk samples. All <strong>the</strong> DNA samples were characterized by IS900 PCR at two regions (A and B) and PCR products were subjected for sequencing using big dye terminator chemistry (courtesy Tim Bull, UK). Using online and <strong>of</strong>fline alignment tools, sequences were analyzed for <strong>the</strong> global and local sequence comparisons. Results and Conclusions: Sequencing confirmed that region B <strong>of</strong> <strong>the</strong>se native MAP isolates <strong>of</strong> human and bovine origin were identical to MAP K10 and o<strong>the</strong>r sequences deposited in GenBank database. However, region A <strong>of</strong> <strong>the</strong>se isolates carried a stretch <strong>of</strong> nucleotides (from region 62 to 121) having number <strong>of</strong> sequence variations compared to MAP K10 and o<strong>the</strong>r sequences deposited in GenBank. Within native isolates <strong>the</strong>re were no variations (homologous) and had identical sequences. However, earlier studies reported highly conserved nature <strong>of</strong> IS900 in MAP isolates recovered from different species and geographical locations outside India. Previous studies by this group also reported presence <strong>of</strong> new ‘Indian Bison type’ genotype from different species <strong>of</strong> animals (domestic and wild) and human beings. This study also exhibited that ‘Indian MAP’ isolates may probably represent a new group <strong>of</strong> genotypes. Variations in <strong>the</strong> A region <strong>of</strong> IS900 sequences <strong>of</strong> ‘Indian MAP’ isolates may be used for designing one step assays to discriminate Indian and non-Indian MAP isolates. However, more extensive validation <strong>of</strong> <strong>the</strong> above observations is needed. 250
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Book of Abstracts
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Proceedings <stron
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Grant Support and Sponsorship <stro
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Scott Wells - Planning Committee Ch
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Monday, August 10, 2009 - All sessi
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Kari Røste Lybeck, Anne Kristine S
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1120-1140 #88: Influence of
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Concurrent Session B - Rm 175 Wille
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Thursday, August 13 Thursday, Augus
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Diagnostics and Genotyping Convenor
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#229 Detecting Johne’s Disease he
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Kalis CHJ, Hesselink JW, Barkema HW
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MATERIALS AND METHODS Sampling For
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To better determine the</st
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#107 Multilocus Short Sequence Repe
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Fig. 1. Dendogram (showing genetic
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#105 MIRU-VNTR genotyping o
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Diagnostics and Genotyping Poster A
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Missouri). Serum from a cow with do
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#7 Significance of
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RESULTS Of the 327
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Direct fecal nested Map PCR testing
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#16 Histopathological and immunohis
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#19 Development of
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#36 Genetic diversity of</s
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#60 Evaluation of
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#66 Comparison of
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(b) IS900 Nested PCR, using p90-p91
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(d) f57 Real Time PCR. The figure o
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• MIRU (3 loci); • VNTR-MIRU (7
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indexes, compared to each single an
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#108 Evaluation of
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#110 Detection and molecular confir
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endoscopic criteria and histopathol
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Bull TJ, McMinn EJ, Sidi-Boumedine
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#131 Seroprevalence of</str
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could be partly attributed to a sma
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#146 Analysis of v
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#162 Comparison of
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#187 Comparison of
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#191 Potentiating day-old blood sam
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samples from the s
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(MAA) (MAP87 and MAP3776) and 1 fro
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#196 Inter- and intra-subtype genot
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RESULTS In 601 (29%) of</st
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#211 Culture of my
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#242 Application and field validati
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#251 Diagnosis of
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Table 2. Paratuberculosis PCR resul
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#254 Programmes on Paratuberculosis
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Fig. 1. European countries specifyi
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A bulk milk quality assurance progr
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Republic of Lithua
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#276 Elimination of</strong
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other projects and
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Woodbine KA, Schukken YH, Green LE,
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Host Response and Immunology Keynot
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#33 MERKAL AWARD LECTURE No interfe
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#75 Interleukin 17 and interleukin
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#173 Local and systemic roles for b
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#23 Role of Mycoba
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Host Response and Immunology Poster
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#28 Intestinal MAP Infection via Pe
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#73 Age susceptibility of</
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#95 Role of nitric
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#134 Analysis of <
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#157 Study of <str
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#163 Immunohistochemical expression
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#193 Development of</strong
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#209 Murine macrophages carrying an
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#224 Application of</strong
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Table 1 UK and Cyprus MAP survey Re
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#228 Mycobacterium avium ssp. parat
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#145 MERKAL AWARD LECTURE Multinomi
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#50 Assessment of
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Table 1. Posterior median (CrIs) <s
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#55 Birth clusters of</stro
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from their dam, se
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#88 Influence of b
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#104 Relation between faecal shedde
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Epidemiology Poster Abstracts 109 1
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#22 The Prevalence of</stro
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CONCLUSION IS1311-based nested Ma/M
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#42 Seroprevalence survey o
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#77 Characterisation of</st
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Environmental samples were collecte
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REFERENCES 1) Chiodini RJ, Van Krui
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#113 Effect of soi
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#136 Error-adjusted, variance parti
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Latium Region Viterbo districts RES
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#144 The suitability of</st
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#178 Age structure of</stro
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A statistically significant lower f
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#183 Sero-prevalence of</st
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#218 Johne’s disease in t
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#244 A survey to estimate t
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Control Programs Keynote Lecture Re
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#202 Milk quality assurance for par
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Page 208 and 209: #40 Managing Bovine Johnes Disease
Page 210 and 211: #74 Economic analysis of</s
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Page 214 and 215: separately for each existing herd <
Page 216 and 217: #98 An inter-laboratory ring-trial
Page 218 and 219: #120 Paratuberculosis vaccine inter
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Page 222 and 223: COMPLEMENTARY ASPECTS On-Farm Disea
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Page 238 and 239: Pathogenomics and MAP Biology Conve
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Page 246 and 247: Pathogenomics and MAP Biology Persp
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Page 252 and 253: #69 In vitro susceptibility <strong
Page 254 and 255: #118 Cloning, expression and purifi
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Page 276 and 277: A total of 206 qua
Page 278 and 279: Feller, M., K. Huwiler, R. Stephan,
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Page 282 and 283: #115 Crohn’s disease and ruminant
Page 284 and 285: International John
Page 286 and 287: #76 Towards improved reporting stan
Page 288 and 289: #97 The EU ParaTBTools Project - Th
Page 290 and 291: #241 US Vaccine Initiative through
Page 292 and 293: Paratuberculosis ELISA Reliable, si
Page 294 and 295: PRIONICS AG WAGISTRASSE 27A PHONE +
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