JCM Accepts, published online ahead of print on 15 September 2010 J. Clin. Microbiol. doi:10.1128/jcm.01762-10 Copyright 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 2 Snapshot of Mycobacterium bovis and Mycobacterium caprae infections in livestock in a bovine tuberculosis low incidence scenario 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Filipa Matos, Mónica V. Cunha*, Ana Canto, Teresa Albuquerque, Alice Amado, and Ana Botelho INRB, I.P./LNIV- Laboratório Nacional de Investigação Veterinária Unidade de Sanidade Animal, Laboratório de Bacteriologia, Estrada de Benfica 701, 1549-011, Lisboa, Portugal. *Corresponding author. Mailing address: INRB, I.P./LNIV- Laboratório Nacional de Investigação Veterinária. Departamento de Bacteriologia, Estrada de Benfica 701, 1549-011, Lisboa, Portugal. Phone: +351-217115346; Fax: +351-217115336; E-mail: monica.cunha@lniv.min-agricultura.pt Keywords: Mycobacterium bovis, Mycobacterium caprae, Bovine Tuberculosis, Epidemiology, Spoligotyping Running title: M. bovis and M. caprae infections in livestock 1
23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Zoonotic bovine tuberculosis (btb), caused by Mycobacterium bovis and, more rarely, by Mycobacterium caprae, is an important disease of livestock and of Public Health concern (1). A low incidence of btb has been observed until now in Portugal, where a comprehensive eradication scheme is implemented. Epidemiological surveys carried out in several countries suggest that test and slaughter policies may reduce strain diversity favouring clonal expansion as a result of a bovine population bottleneck (2, 3). A remarkable exception to this observation was the first epidemiological analysis carried out in Portugal (4), which highlighted the apparent high genotype diversity of M. bovis, possibly due to the low prevalence rates and the absence of major epidemics favouring ongoing transmission of the same(s) strain(s). In the present work, we reassess and update the molecular epidemiology of btb in Portugal. The previous analysis, concerning the period 2002-2007 (4), was extended to 2009 by including additional 183 M. bovis and 10 M. caprae isolates from cattle, goat and sheep. Spoligotyping, based on the Direct Repeat (DR) region (5), identified 30 M. bovis patterns, including 11 profiles previously unrecognized in Portugal and seven new profiles that were deposited in the international database www.mbovis.org. Spoligotype SB0157 remains the single M. caprae pattern acknowledged in the territory. The epidemiological significance of the most relevant genotypes in relation to the temporal and geographical distribution was investigated globally for the period 2002-2009 [results from this study and (4)]. Consistent with previous findings, the high prevalence of SB0121 (25%) and SB0119 (13%), is confirmed. Remarkably, SB0119 and seven other profiles identified in Portugal differ from SB0121 in the absence of a single spacer (Table 1). Altogether, their cumulative prevalence in cattle is superior to 53%. Furthermore, they have also been detected in wildlife (4). MIRU-VNTR analysis based on eight previously validated 2
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 loci (6), distinguished 90 SB0121-related strains into 50 closely associated allelic profiles (data not shown), further suggesting their genetic relatedness. Taking this into consideration and the evolutionary trend of the DR region that is primarily by loss of single or multiple contiguous DVRs (7), our observations suggest the presence of a clonal complex. Supporting this hypothesis is also the recent detection of SB0121- like strains in the national territory in comparison to SB0121, the sole genotype identified until 2004. To our knowledge, three SB0121-related spoligotypes, SB1090, SB1093 and SB1095, have only been reported in Portugal. Geographical distribution analysis also shows that SB0121 and SB0119 have remarkably expanded throughout the territory, being isolated in roughly all btb affected districts, whilst most SB0121- related genotypes are geographically localized (Fig. 1). Our results support the notion that a country tends to have a dominant and locally evolved clone. Moreover, clonally related strains may emerge exhibiting biogeographical specificities, possibly in response to barriers for expansion or adaptation to new ecological niches and new host species. Further studies are needed to improve our understanding of the epidemiological significance of geographically important M. bovis strains which may define specific lineages with increased virulence and ability to escape tuberculin test and, consequently, slaughter. REFERENCES 1. Amanfu W. 2006. The situation of tuberculosis and tuberculosis control in animals of economic interest. Tuberculosis (Edinb) 86:330-335. 2. Costello, E., D. O'Grady, O. Flynn, R. O'Brien, M. Rogers, F. Quigley, J. Egan, and J. Griffin. 1999. Study of Restriction Fragment Length Polymorphism 3
73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 Analysis and Spoligotyping for epidemiological investigation of Mycobacterium bovis infection. J. Clin. Microbiol. 37:3217-3222. 3. Smith, N. H., K. Kremer, J. Inwald, J. Dale, J. R. Driscoll, S.V. Gordon, D. Van Soolingen, R. Glyn Hewinson, and J. Smith Maynard. 2006. Ecotypes of the Mycobacterium tuberculosis complex. J. Theoret. Biol. 239:220-225. 4. Duarte, E. L., M., Domingos, A. Amado, and A. Botelho. 2008. Spoligotype diversity of Mycobacterium bovis and Mycobacterium caprae animal isolates. Vet. Microbiol. 130:415 421. 5. Kamerbeek J., L. Schouls, A. Kolk, M. Van Agterveld, D. Van Soolingen, S. Kuijper, A. Bunschoten, H. Molhuizen, R. Shaw, M. Goyal, and J. Van Embden. 1997. Simultaneous detection and strain differentiation.of Mycobacterium tuberculosis for diagnosis and epidemiology. J. Clin. Microbiol. 35:907-914. 6. Duarte, E. L., M. Domingos, A. Amado, M. V. Cunha, and A. Botelho. 2010. MIRU-VNTR typing adds discriminatory value to groups of Mycobacterium bovis and Mycobacterium caprae strains defined by spoligotyping. Vet. Microbiol. 143:299-306. 7. Van Embden, J. D, T. Van Gorkom. K. Kremer, R. Jansen, B. A. van Der Zeijst, and L. M. Schouls. 2000. Genetic variation and evolutionary origin of the direct repeat locus of Mycobacterium tuberculosis complex bacteria. J. Bacteriol. 182:2393-2401. FIGURE LEGENDS Fig. 1. Geographical distribution across the districts of the six btb affected regions of Portugal mainland, during the 2002-2009 period [joint results from this study and (4)], of M. bovis spoligotype SB0121 ( ) and of strains with spoligotyping patterns that differ 4
98 99 100 101 102 103 from the previous by the absence of one spacer [strains SB0119 ( ), SB1090 ( ), SB1095 ( ), SB0295 ( ), SB122 (*), SB1064 ( ), SB0848 ( ), SB1093 ( )]. The geographical regions are indicated by the thick lines, while the thin lines indicate the districts. Legends: AL, Alentejo; BL, Beira Litoral; BI, Beira Interior; EDM, Entre Douro e Minho; TM, Trás-os-Montes; ERO, Extremadura, Ribatejo e Oeste; ALG, Algarve. 104 105 106 107 Downloaded from http://jcm.asm.org/ on April 16, 2018 by guest 5
Table 1. Patterns, hosts, prevalence and geographical distribution of M. bovis and M. caprae spoligotypes isolated in Portugal, during 2007-2009 (this study) and 2002-2009 [joint results from this study and (4)]. Spoligotypes Pattern Number of isolates Geographical 2007-2009 2002-2009 Regions Cattle Goat Sheep (%) (%) SB0121 47 24,35 116 (25.3) AL, BI, BL, EDM, TM, ERO SB0119 a 40 1 21,24 59 (12.9) AL, BI, EDM, TM, ERO SB0140 4 2,07 29 (6.33) AL, BI, BL, EDM, TM, ERO SB0886 1 0,52 28 (6.11) AL, EDM, ERO SB1090 a 8 4,15 21 (4.59) AL, BI, EDM, ERO SB1095 a 5 2,59 21 (4.59) AL, BI, BL, EDM, TM SB0295 a 3 1 2,07 20 (4.37) AL, BL, TM, ERO SB0157 c 5 5 5,18 19 (4.15) AL, BL, EDM, TM, ERO SB0124 - - 16 (3.49) EDM, TM, ERO SB1172 4 2,07 16 (3.49) AL, ERO SB1174 5 2,59 16 (3.49) AL, TM, ERO SB0120 9 4,66 12 (2.62) AL, EDM, ERO SB0265 4 2,07 10 (2.18) AL SB0134 d 8 4,15 8 (1.75) EDM, TM, ERO SB1264 d 6 3,11 6 (1.31) AL, EDM, TM SB1273 d 6 3,11 6 (1.31) AL, ERO SB1483 b 5 2,59 5 (1.09) AL, BI, EDM, ERO SB0848 a - - 4 (0.87) BI SB0856 d 4 2,07 4 (0.87) AL, EDM, TM, ERO SB1190 d 4 2,07 4 (0.87) AL, ERO SB0130 - - 3 (0.66) EDM, TM SB1167 - - 3 (0.66) AL, ERO SB1486 b 3 1,55 3 (0.66) TM SB1608 b 3 1,55 3 (0.66) AL, BL SB0122 a - - 2 (0.44) AL, BI
SB0822 d 2 1,04 2 (0.44) EDM, ERO SB0833 - - 2 (0.44) ERO SB1053 d 2 1,04 2 (0.44) ERO SB1173 - - 2 (0.44) BI SB1230 - - 2 (0.44) AL SB0849 d 1 0,52 1 (0.22) AL SB0127 d 1 0,52 1 (0.22) ERO SB0332 - - 1 (0.22) BL SB0334 - - 1 (0.22) AL SB0867 - - 1 (0.22) ERO SB1018 d 1 0,52 1 (0.22) AL SB1064 a,d 1 0,52 1 (0.22) BL SB1093 a - - 1 (0.22) BL SB1175 - - 1 (0.22) AL SB1191 - - 1 (0.22) BI SB1484 b 1 0,52 1 (0.22) BI SB1485 b 1 0,52 1 (0.22) EDM SB1487 b 1 0,52 1 (0.22) TM SB1607 b 1 0,52 1 (0.22) BI Total 186 6 1 100 458 (100) a Spoligotypes that differ from the pattern of SB0121 by the absence of one spacer each; b new spoligotypes (deposited in M. bovis database); c M. caprae isolates; d patterns isolated for the first time in Portugal; the geographical regions where the spoligotypes were isolated are indicated: AL, Alentejo; BL, Beira Litoral; BI, Beira Interior; EDM, Entre Douro e Minho; TM, Trás-os-Montes; ERO, Extremadura, Ribatejo e Oeste;
ERO EDM BL BI TM Downloaded from http://jcm.asm.org/ AL ALG Fig. 1. Geographical distribution across the districts of the six btb affected regions of Portugal mainland, during the 2002-2009 period [joint results from this study and (4)], of M. bovis spoligotype SB0121 ( ) and of strains with spoligotyping patterns that differ from the previous by the absence of one spacer [strains SB0119 ( ), SB1090 ( ), SB1095 ( ), SB0295 ( ), SB122 (*), SB1064 ( ), SB0848 ( ), SB1093 ( )]. The geographical regions are indicated by the thick lines, while the thin lines indicate the districts. Legends: AL, Alentejo; BL, Beira Litoral; BI, Beira Interior; EDM, Entre Douro e Minho; TM, Trás-os-Montes; ERO, Extremadura, Ribatejo e Oeste; ALG, Algarve. on April 16, 2018 by guest