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Mycobacterium Avium Species by Accu-Probes, Serotyping, and Single IS900,IS901, IS1245 and IS901-Flanking Region PCR with Internal Standards

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Mycobacterium Avium Species by Accu-Probes, Serotyping, and Single IS900,IS901, IS1245 and IS901-Flanking Region PCR with Internal Standards Journal of Microbiological Methods 64 (2006) 333–345 www.elsevier.com/locate/jmicmeth Identification of members of Mycobacterium avium species by Accu-Probes, serotyping, and single IS900,IS901, IS1245 and IS901-flanking region PCR with internal standards Milan Bartos a,*, Pavel Hlozek a, Petra Svastova a, Lenka Dvorska a, Tim Bull b, Ludmila Matlova a, Ilona Parmova a,c, Isolde Kuhn a, Janine Stubbs a, Monika Moravkova a, Jaromir Kintr a, Vladimir Beran a, Ivan Melicharek d, Matjaz Ocepek e, Ivo Pavlik a aVeterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic bSt. George’s Hospital, Medical School, Cranmer Terrace, London, United Kingdom cState Veterinary Diagnostic Institute, Sidlistni 24, 165 03 Prague-Lysolaje, Czech Republic dState Veterinary Diagnostic Institute, Akademicka 3, 949 01 Nitra, Slovakia eVeterinary Faculty, University of Ljubljana, Gerbiceva 60, 61 000 Ljulbljana, Slovenia Received 5 January 2005; received in revised form 11 May 2005; accepted 24 May 2005 Available online 2 August 2005 Abstract From Mycobacterium avium species Mycobacterium avium subsp. paratuberculosis (n =961), Mycobacterium a. avium (n =677), Mycobacterium a. silvaticum (n =5), and Mycobacterium a. hominissuis (n =1566) were examined, and from Mycobacterium tuberculosis complex M. tuberculosis (n =2), Mycobacterium bovis (n =13), M. bovis BCG (n =4), and Mycobacterium caprae (n =10) were examined. From other mycobacterial species Mycobacterium intracellulare (n =60) and atypical mycobacteria (n =256) including Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium scroful- aceum, Mycobacterium gastri and other species of conditionally pathogenic mycobacteria were analysed. The internal standard molecules corresponding to insertion sequences IS900,IS901,IS1245, and flanking region (FR300)ofIS901 were produced by PCR of alfalfa genome segment and inserted into plasmid vector. The resulting recombinant plasmid molecules were used as internal standards in coamplification with a total of 4729 mycobacterial collection strains and field isolates between 1996 and 2003. The size differences between amplicons obtained from IS900 (258 bp), IS901 (1108 bp), IS1245 (427 bp), and FR300 (300 bp) and from corresponding internal standard molecules ISIS900 (591 bp), ISIS901 (1 336 bp), ISIS1245 (583 bp), and IS901 flanking region of 300 bp ISFR300 (488 bp), respectively, allowed easy discrimination. The internal amplicons were visible by naked aye on agarose gel when 101,103,102, and 102 molecules for ISIS900, ISIS901, ISIS1245, and ISFR300 were * Corresponding author. Tel.: +420 5 3333 1615; fax: +420 5 4121 1229. E-mail address: [email protected] (M. Bartos). URL: http://www.vri.cz (M. Bartos). 0167-7012/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2005.05.009 334 M. Bartos et al. / Journal of Microbiological Methods 64 (2006) 333–345 used in the PCR, respectively, when no bacterial DNA was added to the reaction. The system was tested to define the amount of internal standards that could be used in the PCR without affecting the amplification of the specific segment. Non-specific amplifications were observed in M. fortuitum with IS1245 PCR and mixed infections with M. a. avium and M. a. hominissuis from pigs and cattle were found. PCR results of typing were compared with serotyping and Accu-Probes analyses in selected field isolates. D 2005 Elsevier B.V. All rights reserved. Keywords: Avian tuberculosis; Bovine tuberculosis; Crohn’s disease; Human tuberculosis; Johne’s disease; Mycobacteriosis 1. Introduction of M. avium species over the two past decades (Pavlik et al., 2000b; Dvorska et al., 2001; Svastova Mycobacterial infections of farm animals in Cen- et al., 2002). tral Europe cause considerable economic losses Commercially available radioactively labelled despite programmes attempting to eradicate bovine probes 125I and 32P Gen-Probe (Gen-Probe Inc., San tuberculosis (Pavlik et al., 1998, 2002b, 2003). Diego, California, USA) and a non-radioactive Accu- Since 1990s, members of Mycobacterium avium Probe series (San Diego, California, USA) kits for species have been frequently isolated from infected rapid diagnosis of mycobacteria were methods initial- cattle (Pavlik et al., 2002b), domestic pigs (Pavlik ly used for rapid identification of MAC and MTC et al., 2000c, 2003; Matlova et al., 2005), wild isolates. MAC probe was suitable for identification birds (Pavlik et al., 2000c), and wild ruminants of isolates of all 28 serotypes. (Pavlik et al., 2000a; Machackova et al., 2003, However subsequent experience with these sys- 2004a,b). tems has shown that MAC and MA probes positively All subspecies of M. avium species are pathogens reacted with some isolates of M. a. paratuberculosis causing serious disease. Mycobacterium avium and the MA probe does not allow differentiation of subsp. paratuberculosis particularly causes paratu- serotypes 1, 2, and 3 of subspecies M. a. avium, berculosis in ruminants, affecting digestive tract from serotypes 4 to 6, 8 to 11 and 21 of subspecies (Ayele et al., 2001; Amemori et al., 2004), reproduc- M. a. hominissuis (Saito et al., 1989, 1990; Thoresen tive organs or mammary gland (Ayele et al., 2005). and Saxegaard, 1991). Therefore, in veterinary labo- Mycobacterium a. avium (serotypes 1, 2 and 3) and ratories in the Czech Republic MA probe was Mycobacterium a. silvaticum primarily cause avian replaced in the mid-1990s with the IS901 PCR tuberculosis in birds (Pavlik et al., 2000c; Thorel et system for identification of serotypes 1, 2, and 3 al., 2001; Dvorska et al., 2003). Mycobacterium a. of M. a. avium which are fully virulent for birds hominissuis (serotypes 4 to 6, 8 to 11, and 21) (Pavlik et al., 2000c); that method has been used predominates in external environment and causes since 1996 in all accredited laboratories of the Czech mycobacteriosis in animals (Mijs et al., 2002; Republic (according to ISO/IEC 17025) and autho- Matlova et al., 2005) and in humans. Mycobacterium rized veterinary laboratories of the State Veterinary intracellulare (remaining serotypes 7, 12 to 20, and Administration of the Czech Republic (Anonymous, 22 to 28) is the remaining member among 28 MAC 1996, 1997a). Similarly, IS900 PCR method has serotypes and has been occasionally isolated from been used for identification of the causative agent the animals in the Czech Republic (Pavlik et al., of paratuberculosis in those laboratories since 1997 2002, 2003; Matlova et al., 2003). (Anonymous, 1997b). Culture detection of these pathogens using various The introduction of PCR and RFLP methods into solid and liquid culture media is the most common diagnostic laboratories for mycobacterial infections diagnostic method of these diseases (Kubin et al., was possible due to the discovery of specific insertion 1986; Pavlik et al., 2000b). However, molecular– sequences (IS) for each significant mycobacterial biological methods have been frequently employed pathogen in animals (Dvorska et al., 1999). These for identification and differentiation of the members include IS900 for M. a. paratuberculosis (Green et Table 1 Examined collection strains and field isolates of Mycobacterium avium: M. a. paratuberculosis (MAP), M. a. silvaticum (MAS), M. a. avium (MAA) and M. a. hominissuis (MAH) Collection and serotype strains of M. avium species Field isolates Total collection strains and field isolates examined by PCR Subsp. (serotype) Designation and origin of collection strains No. Environment Birds Pigs Cattle Others No. No. IS900 IS901 IS1245 FR300 MAP ATCC 19068, Linda, Ben, Dominic, EII, 316Fa 6750 2b 755 123c 955 961 + ÀÀ + MAS 6861d, 5329e, 3135e,Ti94f, T93/94f 50 0 000 05À ++ À MAA (1) 2827, 14141-139, ATCC 35716h, TMC 715g 4 5 28 10 3 0 46 50 À ++ À/+3 (2) 6194i,j, B-92i 2 15 37 452 15 5 524 526 À ++ À/+3 (3) 6195 (I0WGMT 26)I,k, 19.88g, 128 Germanyi, 41 1 41424953À ++ À/+3 ST18 (ATCC 12227)l 1 2 3 (Cross reactions) Nt 3 4 35 4 2 48 48 À ++ À/+ M. Bartos et al. / Journal of Microbiological Methods 64 (2006) 333–345 Subtotal (MAA) 10 24 70 538 26 9 667 677 À ++ À MAH (4) 13528-1079 (IWGTM 62)i,k 11 0 14101617ÀÀ++ (5) 3259-685m 11 0 000 12ÀÀ++ (6) 41258g 14 0 26223435ÀÀ++ (8) Davis 13628, Davis 14658-1686g, SJB#2 3 179 2 878 157 9 1225 1228 ÀÀ++ (IWGTM 29)i,k (9) Watson12306g 1 35 1 137 24 7 204 205 ÀÀ++ (10) IIIa1062-1695g, IIIa1602-1695g,i, TMC1461i,n 30 0 100 14ÀÀ++ (11) 14186-1424 (ATCC 35766, TMC 1462)g,i,n 11 0 000 12ÀÀ++ (21) T77g,i,,o, 5154 O’Connorg,i,n 21 0 200 35ÀÀ++ (Cross reactions)1 Nt2 2 0 60 0 6 68 68 ÀÀ++ Subtotal (MAH) 13 224 3 1118 184 24 1553 1566 ÀÀ++ Total No. of M. avium 34 323 73 1658 965 156 3175 3209 strains and field isolates aType strain ATCC 19068 (Merkal, 1979) and collection Strains Linda ATCC 43015, Ben (ATCC 43544) and Dominic (ATCC 43545) provided by R. J. Chiodini, Rehoboth, Mass., and vaccine Mycobactin J non-dependent Strains EII and 316F were kindly provided by John Hermon-Taylor, John Ford and Tim Bull (University of London, St George’s Hospital, UK). bOne isolate from wild boar (Sus scrofa) described in previous study (Machackova et al., 2003) and one isolate from domestic pig (Sus scrofa f. domestica) kindly provided by Goran Bo¨lske and Stina Englund (National Veterinary Institute, Uppsala, Sweden). cSome isolates described in papers dealing with non-vertebrates as potential vectors of causal agent of paratuberculosis (Fischer et al., 2001, 2003, 2004a,b; Machackova et al., 2004a,b). dStrain used in the papers of Thorel and Desmettre (1982) and Thorel and Haagsma (1987). eStrains used in the paper of Thorel and Haagsma (1987). fStrain submitted as M. a. silvaticum by Finn Saxegaard (Central Veterinary Laboratory, Oslo, Norway) and used in the paper of Dvorska et al. (2003). gProvided by Sabine Ru¨sch-Gerdes (Reference Laboratory for Mycobacteria, Borstel, Germany).
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