Isolation of Mycobacterium avium subspecies paratuberculosis from Ugandan cattle and strain differentiation using optimised DNA typing techniques

BMC Veterinary Research

Lonzy Ojok³  Moses L Joloba²  David Patrick Kateete²  Ilias G Bouzalas¹  Panayiotis Loukopoulos¹  Chrysostomos I Dovas¹  Julius Boniface Okuni³ 

[1] Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, 54124, Greece;Department of Medical Microbiology, College of Health Sciences, Makerere University, P.O.Box 7062, Kampala, Uganda;College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, P.O.Box 7062, Kampala, Uganda

关键词: IS1311 PCR-REA;    Genotyping;    MIRUs;    SSR;    Uganda;    Cattle;    Mycobacterium avium subspecies paratuberculosis;   

Others  :  1119788
DOI  :  10.1186/1746-6148-8-99

 received in 2012-02-24, accepted in 2012-06-18,  发布年份 2012

【 摘 要 】

Background

The occurrence of paratuberculosis in Ugandan cattle has recently been reported but there is no information on the strains of Mycobacterium avium subspecies paratuberculosis (MAP) responsible for the disease. The aim of this study was to isolate and characterise MAP from seropositive cattle and paratuberculosis lesions in tissues obtained from slaughtered cattle in Uganda.

Results

Twenty one isolates of MAP were differentiated into 11 genotype profiles using seven genotyping loci consisting of Insertion Sequence 1311(IS1311), Mycobacterial interspersed repeat units (MIRU) (loci 2, 3), Variable number tandem repeats (VNTR) locus 32 and Short sequence repeats (SSR) (loci 1, 2 and 8). Three different IS1311 types and three MIRU 2 profiles (7, 9, 15 repeats) were observed. Two allelic variants were found based on MIRU 3 (1, 5 repeats), while VNTR 32 showed no polymorphism in any of the isolates from which it was successfully amplified. SSR Locus 1 revealed 6 and 7 G1 repeats among the isolates whereas SSR locus 2 revealed 10, 11 and 12 G2 repeats. SSR locus 8 was the most polymorphic locus. Phylogenetic analysis of SSR locus 8 sequences based on their single nucleotide polymorphisms separated the isolates into 8 genotypes. We found that the use of Ethylene glycol as a PCR additive improved the efficiency of the PCR reactions for MIRUs (2, 3), VNTR 32 and SSR (loci 1 and 2).

Conclusions

There is a high strain diversity of MAP in Uganda since 21 isolates could be classified into 11 genotypes. The combination of the seven loci used in this study results into a very precise discrimination of isolates. However analysis of SNPs on locus alone 8 is very close to this combination. Most of the genotypes in this study are novel since they differed in one or more loci from other isolates of cattle origin in different studies. The large number of MAP strains within a relatively small area of the country implies that the epidemiology of paratuberculosis in Uganda may be complicated and needs further investigation. Finally, the use of Ethylene glycol as a PCR additive increases the efficiency of PCR amplification of difficult templates.

【 授权许可】

   
2012 Okuni et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Clarke CJ: The Pathology and Pathogenesis of paratuberculosis in ruminants and other species. J Comp Pathol 1997, 116:217-261.
• [2]Collins MT: Update of Paratuberculosis: Epidemiology of Johne’s disease and biology of Mycobacterium paratuberculosis. Irish Vet J 2003, 56(11):565-574.
• [3]Naser SA, Ghobrial G, Romero C, Valentine JF: Culture of Mycobacterium avium subspecies paratuberculosis from the blood of patients with Crohn's disease. Lancet 2004, 364(9439):1039-1044.
• [4]Ghadiali AH, Strother M, Naser SA, Manning EJ, Sreevatsan S: Mycobacterium avium subsp. paratuberculosis strains isolated from Crohn's disease patients and animal species exhibit similar polymorphic locus patterns. J Clin Microbiol 2004, 42:5345-5348.
• [5]Okuni JB, Loukopoulos P, Reinacher M, Ojok L: Seroprevalence of Mycobacterium avium subspecies paratuberculosis antibodies in cattle from Wakiso, Mpigi and Luwero districts in Uganda. Int J Anim Vet Adv 2011, 3(3):156-160.
• [6]Bauerfeind R, Benazzi S, Weiss R, Schliesser T, Willems H, Baljer G: Molecular characterization of Mycobacterium paratuberculosis isolates from sheep, goats and cattle by hybridization with a DNA probe to insertion element IS 900. J Clin Microbiol 1996, 34:1617-1621.
• [7]Djonne B, Pavlik I, Svastova P, Bartos M, Holstad G: IS 900 restriction fragment length polymorphism (RFLP) analysis of Mycobacterium avium subsp. paratuberculosis isolates from goats and cattle in Norway. Acta Vet Scand 2005, 46:13-18. BioMed Central Full Text
• [8]Motiwala AS, Strother M, Amonsin A, Byrum B, Naser SA, Stabel JR, Shulaw WP, Bannantine JP, Kapur V, Sreevatsan S: Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: evidence for limited strain diversity, strain sharing, and identification of unique targets for diagnosis. J Clin Microbiol 2003, 41:2015-2026.
• [9]Motiwala AS, Amonsin A, Strother M, Manning EJB, Kapur V: Sreevatsan S: Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild animal species. J Clin Microbiol 2004, 42(4):1703-1712.
• [10]Whittington RJ, Marsh IB, Whitlock RH: 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(3):139-145.
• [11]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. Mol Cell Probes 1999, 13(2):115-126.
• [12]Bull TJ, Sidi-Boumedine K, McMinn EJ, Stevenson K, Pickup R, Hermon-Taylor J: Mycobacterial interspersed repeat units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of Mycobacterium avium complex. Mol Cell Probes 2003, 17(4):157-164.
• [13]Castellanos E, Romero B, Rodríguez S, de Juan L, Bezos J, Mateos A, Domínguez L, Aranaz A: Molecular characterization of Mycobacterium avium subspecies paratuberculosis Types II and III isolates by a combination of MIRU-VNTR loci. Vet Microbiol 2010, 144(1–2):118-126.
• [14]Thibault VC, Grayon M, Boschiroli ML, Hubbans C, Overduin P, Stevenson K, Gutierrez MC, Supply P, Biet F: New variable-number tandem-repeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing. J Clin Microbiol 2007, 45:2404-2410.
• [15]El-Sayed A, Hassan AA, Natour S, Abdulmawjood A, Bulte M, Wolter W, Zschock M: Evaluation of three molecular methods of repetitive element loci for differentiation of Mycobacterium avium subsp. paratuberculosis (MAP). J Microbiol 2009, 47(3):253-259.
• [16]Amonsin A, Li LL, Zhang Q, Bannantine JP, Motiwala AS, Sreevatsan S, Kapur V: Multilocus short sequence repeat sequencing approach for differentiating among Mycobacterium avium subspecies paratuberculosis strains. J Clin Microbiol 2004, 42:1694-1702.
• [17]Sohal JS, Singh SV, Singh PK, Singh AV: On the evolution of Indian Bison type' strains of Mycobacterium avium subspecies paratuberculosis. Microbiol Res 2009, 165(2):163-171.
• [18]Yadav D, Singh SV, Singh AV, Sevilla I, Juste RA, Singh PK, Sohal JS: Pathogenic Bison-type' Mycobacterium avium subspecies paratuberculosis genotype characterized from riverine buffalo (Bubalus bubalis) in North India. Comp Immunol Microbiol Infect Dis 2008, 31(4):373-387.
• [19]Singh SV, Sohal JS, Singh PK, Singh AV: Genotype profiles of Mycobacterium avium subspecies paratuberculosis isolates recovered from animals, commercial milk, and human beings in North India. Int J Infect Dis 2009, 13(5):e221-e227.
• [20]Sevilla I, Li L, Amonsin A, Garrido J, Geijo M, Kapur V, Juste R: Comparative analysis of Mycobacterium avium subsp. paratuberculosis isolates from cattle, sheep and goats by short sequence repeat and pulsed-field gel electrophoresis typing. BMC Microbiol 2008, 8(1):204. BioMed Central Full Text
• [21]Harris NB, Payeur JB, Kapur V, Sreevatsan S: Short-sequence-repeat analysis of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium isolates collected from animals throughout the United States reveals both stability of loci and extensive diversity. J Clin Microbiol 2006, 44(8):2970-2973.
• [22]Motiwala AS, Amonsin A, Strother M, Manning EJ, Kapur V, Sreevatsan S: Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild animal species. J Clin Microbiol 2004, 42:1703-1712.
• [23]Motiwala AS, Strother M, Theus NE, Stich RW, Byrum B, Shulaw WP, Kapur V, Sreevatsan S: Rapid detection and typing of strains of Mycobacterium avium subsp. paratuberculosis from broth cultures. J Clin Microbiol 2005, 43:2111-2117.
• [24]Möbius P, Luyven G, Hotzel H, Köhler H: High Genetic Diversity among Mycobacterium avium subsp. paratuberculosis Strains from German Cattle Herds Shown by Combination of IS900 Restriction Fragment Length Polymorphism Analysis and Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem-Repeat Typing. J Clin Microbiol 2008, 46(3):972-981.
• [25]Moravkova M, Hlozek P, Beran V, Pavlik I, Preziuso S, Cuteri V, Bartos M: Strategy for the detection and differentiation of Mycobacterium avium species in isolates and heavily infected tissues. Res Vet Sci 2008, 85(2):257-264.
• [26]Zhang Z, Yang X, Meng L, Liu F, Shen C, Yang W: Enhanced amplification of GC-rich DNA with two organic reagents. Biotechniques 2009, 47(3):775-779.
• [27]Whipple DL, Callihan DR, Jarnagin JL: Cultivation of Mycobacterium Paratuberculosis from bovine fecal specimens and a suggested standardized procedure. J Vet Diagn Invest 1991, J3:368-373.
• [28]van Soolingen D, Hermans PW, de Haas PE, Soll DR, van Embden JD: Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbiol 1991, 29(11):2578-2586.
• [29]Poland: Prediction of melting transitions of dsRNA, DNA or RNA/DNA hybrids. http://www.biophys.uni-duesseldorf.de/local/POLAND/poland.html webciteaccessed on 4/3/2010
• [30]Steger G: Thermal denaturation of double-stranded nucleic acids: prediction of temperatures critical for gradient gel electrophoresis and polymerase chain reaction. Nucleic Acids Res 1994, 22(14):2760-2768.
• [31]Whittington R, Marsh I, Choy E, Cousins D: Polymorphism in IS1311, an insertion sequence common to Mycobacterium avium and Mycobacterium avium subsp. paratuberculosis, can be used to distinguish between and within these species. Mol Cell Probes 1999, 12:349-358.
• [32]Vary PH, Andersen PR, Green E, Hermon-Tailor J: McFadden: Use of highly specific DNA probes and polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne’s disease. J Clin Microbiol 1990, 28(5):933-937.
• [33]Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol 2011, 28(10):2931-2939.