Journal of Microbiological Methods 64 (2006) 333–345 www.elsevier.com/locate/jmicmeth

Identification of members of 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. (n =961), Mycobacterium a. avium (n =677), Mycobacterium a. silvaticum (n =5), and Mycobacterium a. hominissuis (n =1566) were examined, and from Mycobacterium complex M. tuberculosis (n =2), (n =13), M. bovis BCG (n =4), and (n =10) were examined. From other mycobacterial species Mycobacterium intracellulare (n =60) and atypical mycobacteria (n =256) including , 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 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/+ 333–345 (2006) 64 Methods Microbiological of Journal / al. et Bartos M. 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 a Type 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). b One 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). c Some 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). d Strain used in the papers of Thorel and Desmettre (1982) and Thorel and Haagsma (1987). e Strains used in the paper of Thorel and Haagsma (1987). f Strain submitted as M. a. silvaticum by Finn Saxegaard (Central Veterinary Laboratory, Oslo, Norway) and used in the paper of Dvorska et al. (2003). g Provided by Sabine Ru¨sch-Gerdes (Reference Laboratory for Mycobacteria, Borstel, Germany). h Isolate described in the paper of Dvorska et al. (2003). i Provided by Hajime Saito and Haruaki Tomioka (National Institute for Research, Shimane Medical University, Tokyo, Japan). j Provided by Milan Slosarek (originally submitted from Werner B. Schaefer to Milan Kubin from National Institute of Public Health, Unit and Reference Laboratory for Mycobacterial Infections, The Czechoslovak National Collection of Type Cultures-CNCTC, Prague, the Czech Republic). k Provided by Dick Van Soolingen (National Institute of Public Health and Environmental Protection, Bilthoven, the Netherlands) and used as IWGMT strain in the paper of Ritacco et al. (1998). l Provided by Judy R. Stabel and Diana Whipple (United States Department of Agriculture, Ames, Iowa, USA). m Provided by Werner B. Schaefer (Division of Research, National Jewish Hospital and Research Centre, Denver, Colorado, now Colorado State University, Colorado, USA ) to Milan Pavlas (Veterinary Research Institute, Brno, the Czech Republic). n NJC (National Jewish Hospital and Research Centre), Denver, Colorado, now Colorado State University, Colorado, USA. o CDC (Centre for Disease Control), Atlanta, Georgia, USA. ATCC=American Type Culture Collection, Rockville, USA (ST18 originally described as M. a. paratuberculosis Strain 18). TMC=Trudeau Mycobacterial Culture Collection, Denver, Colorado, USA. IWGMT=International Working Group on Mycobacterial . 1 Cross reactions during the serotyping with minimally one of serotypes 1, 2, and 3 in the group of M. a. avium isolates and cross reactions of serotypes 4–6, 8–11 and 21 under the group of M. a. hominissuis isolates. 2 Not tested. 3 Some field

isolates were mixture of M. a. avium and M. a. hominissuis (more details see in the Table 3) and/or in the paper of Dvorska et al. (2004). 335 336 M. Bartos et al. / Journal of Microbiological Methods 64 (2006) 333–345 al., 1989; Pavlik et al., 1999; Bull et al., 2000; Iko- The primary purpose of this study was to develop nomopoulos et al., 2004), IS901/902 for M. a. avium and validate a set of specific PCR with internal stan- and M. a. silvaticum (Kunze et al., 1991, 1992; Moss dards for IS900,IS901,IS1245 and FR300, and to et al., 1992; Pavlik et al., 2000c; Svastova et al., 2002; test their presence in a defined strain collection com- Dvorska et al., 2003), IS1245 for the causative agent prising M. avium species and other closely related of mycobacteriosis (Guerrero et al., 1995; Van Soo- Mycobacterial species. This system was then used to lingen et al., 1998), and FR300, typical of some together with other routine procedures (serotyping and members of M. avium species (Kunze et al., 1991; examination using Accu-Probes) for identification of Bartos et al., 2001). pathogenic mycobacteria.

Table 2 Mycobacterium intracellulare collection strains and field isolates Collection serotype strains Field isolatesd Total no. Accu-Probesf IS* and FR300 Serotype Strain designation (synonyms) and origin No. MAC MA MI 7 Maureya, 158 Leonardb, 157 Mantenb, 42 6 + À + À Thrift-S VIIc 12 73Howell-Hatchera, Wood Duckb, P42-Howell 40 4 + À + À (ATCC 35762, TMC 1405)a,b,c, 6845b 13 Chance-Leplera, Chance-Chance (ATCC 35769, 40 4 + À + TMC 1466)a,b, ATCC 25122 (Borstel 5509)b, Lynn-Chancec 14 Edgar Boone (ATCC 35761, TMC 1403)b, 20 2 + À + À P 39 Boonea,b,c 15 Dent-Simpsona,c, Dent-Denta, Dent 50 5 + À + À (ATCC 35848, TMC 1473)b, Simpsonb, ATCC 23435b 16 Yandle-Yandlea, Yandle-Gisellea, Yandle 40 4 + À + À (ATCC 13950, P 44)b, ATCC 15987b 17 Wilson P54 (ATCC 35763, TMC 1411)a,b,c 10 1 + À + À 18 Altmann-Melnick (ATCC 35770)a,c, 30 3 + À + À O’Connor bb, 2219 Altmanna,b 19 Darden McAnamya,c, Darden 9666a, 40 4 + À + À Darden (ATCC 35772, TMC 1469)b, W-552b 20 Arnold Armstronga, TMC 1419 40 4 + À + À (ATCC 35764)b, AT 545 Findleyb, Newberny-Arnoldc 22 5154 O’connora,b, 10409a,b 20 2 + À + À 23 CDC 1214a,b, CDC 841Bb, 23393a,b, 12645a,b 40 4 + ÀÀÀ 24 2154b 10 1 + À +ÀÀ 25 72-888a,b, 1195a,b 20 2 + À + À 26 Hillbery 1244a,b, Cox 1994b, Mackenzie 30 3 + À +ÀÀ 2233a,b 27 Harrisona,b, Lane 3081a,b 20 2 + ÀÀÀ 28 153081a, Matthews 9055a,b 20 2 + À +ÀÀ Field isolates with cross-reactions and autoagglutinations Nt 7 7 + À + À Total 51 9 60 aProvided by Sabine Ru¨sch-Gerdes (Reference Laboratory for Mycobacteria, Borstel, Germany). bProvided by Hajime Saito and Haruaki Tomioka (National Institute for Leprosy Research, Shimane Medical University, Tokyo, Japan). cProvided by Werner B. Schaefer (Division of Research, National Jewish Hospital and Research Centre, Denver, Colorado, now Colorado State University, Colorado, USA ) to Milan Pavlas (Veterinary Research Institute, Brno, the Czech Republic). dField isolated originated from pigs (n =3), birds (n =1), cattle (n =2), and other sources (n =3). ePCR method used for the detection of IS900,IS901,IS1245, and FR300. fAccu-Probe Inc. (San Diego, California, USA). CDC=Centre for Disease Control, Atlanta, Georgia, USA. ATCC=American Type Culture Collection, Rockville, USA. TMC=Trudeau Mycobacterial Culture Collection, Denver, Colorado, USA. Nt=not tested. M. Bartos et al. / Journal of Microbiological Methods 64 (2006) 333–345 337

2. Materials and methods Other mycobacterial species. Reference collection strains, 213, and field isolates of MTC (M. tuberculo- 2.1. Mycobacterial strains and isolates sis, M. bovis, M. bovis BCG, and M. caprae) and other 15 mycobacterial species were examined (Table M. avium and M. intracellulare. A total of 85 4). Isolates were obtained from OIE Reference labo- collection strains of M. avium species [M. a. paratu- ratory for paratuberculosis in the Veterinary Research berculosis (n =6),M. a. silvaticum (n =5), M. a. avium Institute, The Methodical Centre for mycobacterial (n =10), M. a. hominissuis (n =13)] and M. intracellu- infections in animals of the State Veterinary Admin- lare (n =51) were examined (Tables 1 and 2). The istration of the Czech Republic and three reference 3175 field isolates of M. avium species shown in laboratories for mycobacterial infections of animals Table 1 originated from various hosts: birds (n =73), and humans in the Czech Republic, Slovakia and pigs (n =1658), cattle (n =965), and from hosts includ- Slovenia. ing HIV positive and negative human patients, small Growth conditions of mycobacterial collection terrestrial mammals (n =156), and the environment strains and field isolates. M. a. paratuberculosis (n =323). All 1177 mycobacterial isolates of subspe- (n =6) and M. a. silvaticum (n =5) collection strains cies of M. a. avium and M. a. hominissuis from cattle and field isolates (n =953) in which Mycobactin J and pigs were obtained from Reference laboratory for dependency was expected, were subcultured on Her- mycobacterial infections in Prague were examined rold’s Egg Yolk Medium (HEYM; Whipple et al., over the period from 1996 to 2001 (Table 3). 1991)at378C for up to 36 weeks and identified by

Table 3 Identification of pigs and cattle isolates from the Czech Republic from Reference Laboratory for Mycobacterial Infections in Animals (Prague, the Czech Republic) Host/ Total PCR IS901 (FR300) PCRb FR300 a b year no. IS1245 Positive 1 2 3 2/3 2/4 2/6 2/9 3/8 3/9 Pos. 4 8 9 10 Ag.c Pigs 1996 439 439 199 (12) 6 146 (0) 24 (0) 9 (1) 0 (0) 1 (1) 0 (0) 12 (9) 1 (1) 240 10 170 48 1 11 1997 161 161 74 (8) 0 67 (1) 1 (1) 0 (0) 0 (0) 0 (0) 1 (1) 5 (5) 0 (0) 87 0 76 7 0 4 1998 167 167 50 (6) 0 43 (0) 1 (0) 0 (0) 0 (0) 0 (0) 0 (0) 6 (6) 0 (0) 117 0 100 11 0 6 1999 149 149 45 (7) 0 45 (7) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 104 0 83 11 0 10 2000 98 98 37 (0) 0 37 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 61 1 39 11 0 10 2001 81 81 16 (0) 0 16 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 65 0 53 4 0 8 Total 1095 1095 421 (33) 6 354 (8) 26 (1) 9 (1) 0 (0) 1 (1) 1 (1) 23 (20) 1 (1) 674 11 521 92 1 49

Cattle 1996 34 34 28 (5) 0 23 (3) 4 (1) 0 (0) 1 (1) 0 (0) 0 (0) 0 (0) 0 (0) 6 2 2 0 0 2 1997 17 17 15 (0) 0 15 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2 0 1 1 0 0 1998 10 10 10 (0) 0 10 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 0 0 0 0 0 1999 11 11 11 (2) 0 11 (2) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 0 0 0 0 0 2000 6 6 5 (0) 0 5 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 0 0 0 0 1 2001 4 4 4 (0) 0 4 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 0 0 0 0 0 Total 82 82 73 (7) 0 68 (5) 4 (1) 0 (0) 1 (1) 0 (0) 0 (0) 0 (0) 0 (0) 9 2 3 1 0 3

Pigs and cattle Total 1177 1177 494 (40) 6 422 (13) 30 (2) 9 (1) 1 (1) 1 (1) 1 (1) 23 (20) 1 (1) 683 13 524 93 1 52 a All isolates were positive with all three Accu-Probes (Accu-Probe Inc., San Diego, California, USA): (i) MAC probe for the detection of all 28 serotypes of M. avium complex, (ii) MA probe for the detection of serotypes 1, 2, and 3 of M. a. avium and serotypes 4–6, 8–11, and 21 of M. a. hominissuis and (iii) MI probe for the detection of serotypes 7, 12–20 and 21 of M. intracellulare. b Instead of FR1700 bp the PCR product of 300 bp (FR300 bp) was detected, which documented the mixed infection of M. a. avium and M. a. hominissuis. c Agg. Auto-agglutination (not possible to identify isolates by serotyping) or cross reactions under different serotypes of M. a. hominissuis. 338 M. Bartos et al. / Journal of Microbiological Methods 64 (2006) 333–345

Mycobactin J dependency test: subculture on three Serotyping of M. avium complex isolates. Serotyp- slants with HEYM with Mycobactin J and one slant ing of MAC isolates was carried out according to with HEYM without Mycobactin J (Pavlik et al., Wolinsky and Schaeffer (1973), modified by Su¨ssland 2000b). The rest of all collection strains and field and Hrdinova (1976). isolates (n =3765) were subcultured on Lo¨wenstein– Biochemical identification. Field isolates non-de- Jensen medium and serum medium according to Sula pendent on Mycobactin J, not belonging to M. a. (Kubin et al., 1986)at378C for up to 12 weeks. avium and M. a. hominissuis, after the IS901 and

Table 4 Collection strains and field isolates others than Mycobacterium avium and M. intracellulare Species Origin No. IS* and FR300 IS 1245 Accu-Probes** MTC MAC MI M. tuberculosis Strain H37Rv, NCTC 74161 1 ÀÀ+ ÀÀ Cattle2 1 ÀÀ+ ÀÀ M. bovis ATCC 192103,4 1 ÀÀ+ ÀÀ Capybara5 1 ÀÀ+ ÀÀ Cattle5 10 ÀÀ+ ÀÀ Wild red deer5 1 ÀÀ+ ÀÀ M. bovis BCG Vaccine strain5 1 ÀÀ+ ÀÀ Patients—children5 3 ÀÀ+ ÀÀ M. caprae Cattle5,6 8 ÀÀ+ ÀÀ Farmed red deer5 1 ÀÀ+ ÀÀ Patient5 1 ÀÀ+ ÀÀ M. gastri ATCC 157543 1 ÀÀÀÀÀ Wild bird7,8 1 ÀÀÀÀÀ M. phlei NCTC 81511 1 ÀÀÀÀÀ Wild bird7,8 1 ÀÀÀÀÀ Pig farms7,9 6 ÀÀÀÀÀ M. smegmatis NCTC 102651 1 ÀÀÀÀÀ Pig farms7,10 3 ÀÀÀÀÀ M. malmoense NCTC 112981 1 ÀÀÀÀÀ Patient7,11 1 ÀÀÀÀÀ M. fortuitum NCTC 103941 1 ÀÀÀÀÀ Pig farms7,12 37 À + ÀÀÀ Wild bird7,13 3 À + ÀÀÀ Invertebrates7,14 3 À + ÀÀÀ Small terrestrial mammal7,15 1 À + ÀÀÀ M. diernhoferi Pig farms7,16 3 ÀÀÀÀÀ M. chelonae Pig farms7,17 8 ÀÀÀÀÀ Wild bird7,18 1 ÀÀÀÀÀ Invertebrates7,19 8 ÀÀÀÀÀ M. flavescens Pig farms7,20 14 ÀÀÀÀÀ M. gordonae Pig farms7,21 63 ÀÀÀÀÀ Wild bird7,22 1 ÀÀÀÀÀ M. scrofulaceum Pig farms7,23 5 ÀÀÀÀÀ Invertebrates7,24 1 ÀÀÀÀÀ Wild bird7,25 1 ÀÀÀÀÀ M. szulgai Pig farms7,26 1 ÀÀÀÀÀ M. triviale Pig farms7,27 3 ÀÀÀÀÀ M. terrae Pig farms7,28 11 ÀÀÀÀÀ M. ulcerans Wild bird7,29 1 ÀÀÀÀÀ M. xenopi Pig farms7,30 2 ÀÀÀÀÀ Total 213 M. Bartos et al. / Journal of Microbiological Methods 64 (2006) 333–345 339

IS1245 PCR, were identified by biochemical methods and 72 8C for 4 min, using a Thermocycler (model MJ (Wayne and Kubica, 1986). Research PTC200, USA). After the last amplification cycle, the samples were incubated further at 72 8C for 2.2. PCR analysis 3 min for complete elongation of the final PCR pro- ducts. Amplicons were then purified by QIAQuick DNA isolation. A loopful of a single bacterial PCR purification kit (QIAGEN, Germany) according colony was taken from a Lo¨wenstein–Jensen medium to the manufacturer’s instructions. The resulting mole- and resuspended in 50 Al distilled water in a screw- cules were then cloned into T-vector pCR2.1 using cap micro-centrifuge tube. The samples were boiled TA-Cloning Kit (Invitrogen, USA) and recombinant for 20 min prior to being centrifuged for 5 min/14000 plasmids isolated by the bmini-scale isolation systemQ Âg to settle cell debris. Two microliters of superna- described by Sambrook and Russell (2001). tant, containing the genomic DNA, was used for PCR Amplification. DNA isolated from single bacterial amplification. colony was amplified with the primers based on in- Construction of internal standard molecules. Inter- sertion sequences IS900,IS901,IS1245, and FR300 nal standard molecules corresponding to IS901, PCR amplification. PCR was carried out on DNA IS900,IS1245, and FR300 were produced by PCR isolated from single bacterial colony in a final volume using a RAPD segment of the alfalfa genome (Acces- of 20 Al consisting of 2 Al of DNA supernatant con- sion No. AF222048) as target. The 40mer primers taining approximately 10 ng of genomic DNA, 10 contained sequences specific for alfalfa genome seg- pmol of each primer and 17.8 Al Taq PCR Master ment and sequences specific for IS901,IS900, Mix (QIAGEN, Germany). Amplification for inser- IS1245, and FR300 fragments as 5V overhangs. tion sequence IS901 of M. a. avium, resulted in a These were designed to provide amplicon standards specific 1108 bp PCR product. For the amplification of 591 bp for IS900, 1 336 bp for IS901, 583 bp for of IS901 flanking region (FR300) the primers were as IS1245, and 488 bp for FR300. The amplicons were described previously (Kunze et al., 1992) resulting in amplified by a preliminary incubation at 94 8C for 3 a specific 300 bp product. Primers specific for IS1245 min then 33 cycles of 94 8C for 1 min, 66 8C for 45 s, were as described by Guerrero et al. (1995) resulting

Notes to Table 4: *IS=IS900 and IS901. 1NCTC; National Collection of Type Cultures, Colindale, UK. 2Isolate obtained from Dr. Zeljko Cvetnic from Croatian Veterinary Institute, Zagreb, Croatia. 3ATCC; American Type Culture Collection, Rockville, USA. 4Strain was obtained by Dr. Milan Slosarek from The Czech National Collection of Type Cultures (CNCTC), where it was designated as My 310/87. 5Described previously (Pavlik et al., 2002b). 6Described previously (Pavlik et al., 2002a). 7Described previously (Matlova et al., 2003). 8Isolate from starling (Sturnus vulgaris). 9Isolates from environment: bedding (n =2), biofilm from the pipeline (n =2), and from the stable scrapings (n =2). 10Isolates from environment: bedding (n =1), biofilm from the pipeline (n =1), and from the stable scrapings (n =1). 11Isolate obtained from Dr. Manca Zolnir-Dovc from University Clinic of Respiratory and Allergic Diseases, Golnik, Slovenia. 12Isolates from environment: peat (n =3), feed concentrates (n =2), bedding (n =5), biofilm from the pipeline (n =12), pig faeces (n =2), stable scrapings (n =2), dust and spider webs (n =2), water from the pipeline (n=9). 13One isolate from starling (Sturnus vulgaris) and two isolates from house sparrow (Passer domesticus). 14Four isolates from fly imagoes: Musca spp. (n =2), Scatophagidae (n =1), Stomoxys calcitrans (n =1); one isolate from larvae and one isolate from pupa of Musca spp., described previously by Fischer et al. (2001). 15One isolate from common vole (Microtus arvalis) described previously by Fischer et al. (2000). 16Isolates from environment: biofilm from the pipeline (n =1), pig faeces (n =1), and water from the pipeline (n =1). 17Isolates from environment: peat (n =1), bedding (n =1), biofilm from the pipeline (n =3), pig faeces (n =1), and water from the pipeline (n =2). 18One isolate from white wagtail (Motacila alba). 19Eight isolates from larvae of Musca spp. (n =1) and Eristalis tenax (n =7). 20Isolates from environment: peat (n =1), feed concentrates (n =1), biofilm from the pipeline (n =7), stable scrapings (n =1), and water from the pipeline (n =4). 21Isolates from environment: peat (n =2), feed concentrates (n =1), biofilm from the pipeline (n =36), stable scrapings (n =1), pig faeces (n =1), stable scrapings (n =1), and water from the pipeline (n =22). 22One isolate from white wagtail (Motacila alba). 23Isolates from environment: biofilm from the pipeline (n =3), pig faeces (n =1), and water from the pipeline (n =1). 24One isolate from earthworms (Lumbricus rubellus)as described previously (Fischer et al., 2003). 25One isolate from house sparrow (Passer domesticus). 26One isolate from biofilm from the pipeline. 27Isolates from environment: feed concentrates (n =2) and water from the pipeline (n =1). 28Isolates from environment: peat (n =2), feed concentrates (n =1), bedding (n =1), biofilm from the pipeline (n =2), stable scrappings (n =2), water from the pipeline (n =2) and soil and moos (n =1). 29One isolate from starling (Sturnus vulgaris). 30Isolates from environment: peat (n =1) and dust in spider webs(n =1). 340 M. Bartos et al. / Journal of Microbiological Methods 64 (2006) 333–345 in a 427 bp specific product. Detection of IS900 used the following primers designed by GeneBase software (Applied Maths, Belgium) IS900-P3N 5V-GGG TGT GGC GTT TTC CTT CG-3V and IS900-P4N 5V-TCC TGG GCG CTG AGT TCC TC-3V. A positive con- trol, 250 fg DNA from M. a. avium isolates of geno- type IS901+ and IS1245+, M. a. paratuberculosis IS900+, and a negative control, 2 Al sterile water, were included in each PCR. Amplification conditions were as described above.

2.3. Accu-probe test

The collection of MTC and MAC strains and M. a. avium and M. a. hominissuis field isolates from Ref- erence Laboratory for Mycobacterial Infections in Animals (Prague; the Czech Republic) were tested with the MTC, MAC, MA, and MI Accu-Probes (Accu-Probe Inc., San Diego, California, USA) Fig. 1. The example of real sample analysis. M =100 bp ladder. Line 1=internal standard ISIS901 (1336 bp) with DNA IS901 +(1108 according to the manufacturer’s instructions. bp). Line 2=DNA IS901 +(1108 bp) only. Line 3=internal standard ISIS901 (1336 bp) only. Line 4=internal standard ISIS901 (1336 bp) with inhibiting sample. Line 5=inhibiting sample only. 3. Results fication of M. avium genomic DNA (competitive ef- Sample analysis with the internal standard mole- fect) was determined by comparing amplification cules. The internal standard was co-amplified in the under varying concentrations of purified genomic same tube with the same primers as the target DNA. DNA or crude bacterial lysates (data not shown). Absence of the internal standard amplicon indicated The PCR reaction was completely inhibited by chro- inhibition of the amplification. Triplicate reactions for mosomal DNA in concentrations more then 50 ng per each sample included; one reaction with bacterial su- reaction. When the concentration of internal standard pernatant of lysate only, a second including the molecules was 108 molecules per reaction the ampli- corresponding internal standard, and a third with a con corresponding to internal standard was not ob- mixture of both. Absence or decreasing of the amount served until the target DNA concentration was of the internal standard amplicon was interpreted as decreased to 50 pg per reaction. The inhibiting effect inhibition (Fig. 1). The size differences between ampli- of bacterial lysate was observed when bacterial cells cons obtained after amplification by specific primer were greater than 5Â103 colony forming units (CFU) sets from IS900 (258 bp), IS901 (1108 bp), IS1245 per reaction for ISIS900,5Â103 CFU per reaction for (427 bp), and FR300 (300 bp), and from corresponding ISIS901,2Â103 CFU per reaction for ISIS1245, and internal standard molecules ISIS900 (591 bp), ISIS901 2Â105 CFU per reaction for ISFR300. (1336 bp), ISIS1245 (583 bp), and ISFR300 (488 bp), Test of the system. The equivalent concentration respectively, allowed easy discrimination. for each internal standard was carefully tested so the Sensitivity of reactions and amount of internal system could be used for proper analysis of real standards in the PCR. The internal standard amplifica- samples. Aliquots of the correctly diluted internal tions reliably generated amplicons visible on an aga- standards were kept at À20 8C. In the system rose gel when 101 to 103 molecules were used and no designed in this paper the internal standards compete bacterial DNA was added (data not shown). The with real samples; the sensitivity of our detection amount of internal standard molecules that could be system was 1Â101 CFU per reaction for ISIS900, used in the PCR without affecting the specific ampli- 5Â103 CFU per reaction for ISIS901,1Â102 CFU M. Bartos et al. / Journal of Microbiological Methods 64 (2006) 333–345 341 per reaction for ISIS1245, and 1Â103 CFU per strains and 9 field samples of M. intracellulare were reaction for ISFR300, then the amount of negative for each of the genomic markers tested by corresponding internal standard molecules added to PCR (Table 2). amplification was 200 to 300 copies. M. tuberculosis complex members. All 29 collec- Examination of collection strains and field isolates tion strains and field isolates of MTC were positive by of M. avium species. All 34 collection strains of M. MTC Accu-Probe, negative by MAC, MA, and MI avium were examined by aliquots of the correctly Accu-Probes and were negative for each of the ge- diluted internal standards and by MTC, MAC, MA nomic markers tested (Table 4). and MI Accu-Probes. Other collection strains and field isolates. All of M. a. paratuberculosis. In all 3 Mycobactin J 184 collection strains and field isolates of species dependent and 3 Mycobactin J non-dependent (EII, other from M. avium, M. intracellulare and species 316F, and ATCC 19068) collection strains of M. a. that are not members of MTC were of genotype paratuberculosis, presence of IS900 and FR300 only IS6110À,IS901À,IS1245À, and FR300À (except was detected. All 955 Mycobactin J dependent field for the collection strain and 44 field isolates of M. isolates of M. a. paratuberculosis obtained mostly fortuitum, which gave positive reactions with IS1245 from cattle, outdoor environment in the farms and PCR). No reaction with MTC, MAC, MA, and MI other hosts were of identical genotype IS900+, Accu-Probes (Table 4) was detected either. IS901À, IS1245À, and FR300+(Table 1). Mixed infection with M. a. avium and M. a. homi- M. a. silvaticum. Five M. a. silvaticum strains nissuis isolates. In 40 of 494 M. a. avium isolates (Mycobactin J dependant in primary culture but from cattle and pigs the genotypic profile was IS901+, Mycobactin J non-dependent in subsequent subcul- IS1245+, and FR300+ suggesting a mixed infection. tures) contained the genotypic profile IS900À, Serotype, cross-reactivity was detected in 25 of these IS901+, IS1245+, and FR300+ (Table 1). isolates, most frequently showing mixed infection M. a. avium. Ten strains of M. a. avium serotypes 1 between one serotype of M. a. avium and one serotype to 3 were of genotype IS901+, IS1245+, and of M. a. hominissuis (Table 3). FR300À (Table 1). All 667 isolates of M. a. avium False negativity analysis. The numbers of inhibi- serotypes 1 to 3 primarily obtained from external tions detected in performed analysis are collected in environment, birds, pigs, cattle and other hosts also Table 5. Reaction was considered as inhibited if no showed this genotypic profile. However, FR300 amplicon, both from specific insertion element and (Table 3, see below, in the chapter Mixed infection) internal standard, was observed. Partial inhibitions was detected in only 40 of these isolates. All the were not included. For individual M. avium subspe- isolates were positive by MAC and MA and negative cies the following frequencies of total inhibition were by MTC and MI Accu-Probes (Table 3). observed: 4.5% for MAP, 0.0% for MAS, 0.7% for M. a. hominissuis. All 13 strains of M. a. homi- MAA and 2.4% for MAH. A total of 57 from 60 nissuis serotypes 4 to 6, 8 to 11 and 21 were of collection strains of M. intracellulare, 29 collection genotype IS901À, IS1245+, and FR300+. All of strains and field isolates of MTC, and 248 from 256 1553 isolates of M. a. hominissuis serotypes 4 to 6, 8 to 11 and 21 obtained from external environment, pigs and other hosts (humans, cattle, birds and others) Table 5 were of genotype IS901À, IS1245+, and FR300+ Analysis of inhibition during PCR (Table 1). All 683 M. a. hominissuis isolates were Subspecies No. of Inhibition Percentage of positive by MAC and MA and negative by MTC and isolates inhibition (%) MI Accu-Probes (Table 3). MAP 961 43 4.5 M. intracellulare. All 51 collection strains of M. MAS 5 0 0.0 MAA 677 5 0.7 intracellulare serotypes 7 (n =6), 12 to 20 (n =38), MAH 1566 38 2.4 and 22 to 28 (n =16) were positive by the test MAC M. intracellulare 60 3 5.0 Accu-Probe with four strains (each of serotype 7) M. tuberculosis complex 29 0 0.0 being positive using MI Accu-Probe. All 51 tested Other mycobacteria 256 8 3.1 342 M. Bartos et al. / Journal of Microbiological Methods 64 (2006) 333–345 collection strains and field isolates of species other samples before amplification. For this reason the di- from M. avium and MTC were of genotype IS6110À, lution of bacterial lysates or DNA samples is advis- IS901À,IS900À,IS1245À, and FR300À. In all these able to perform PCR analysis on important samples. analysis the positive signals from the internal stan- The developed sets of internal standards were test- dards for IS901,IS1245, and FR300 were identified ed using a collection of reference strains including M. which enabled to exclude false negativity. A total of 3 avium, M. intracellulare, MTC, and other mycobac- from 60 (5.0%) isolates of M. intracellulare, and terial species. Although the frequency of inhibitions 8 from 256 (3.1%) isolates of other species was was not more than 5.0% for individual subspecies, it false negative in PCR. By repeated PCR analysis was concluded that using the internal standard mole- after dilution samples 10 and 100 times the confirma- cules for coamplification could be a useful tool in tion of correct genotypes was done. screening material, especially in avoiding false-nega- tive results. The number of inhibited reactions was significantly higher for MAP than for other M. avium 4. Discussion subspecies. The reason for this could be done by more complex bacterial cell wall for this subspecies, by The PCR is a powerful tool in diagnosing myco- different growth media or by different stage of bacte- bacterial infection (Hawkey, 1994). However, reduced rial growth. In the tested reference strains of M. a. sensitivity has been observed when amplification avium and M. a. silvaticum identical genotype techniques have been used for analysis of clinical IS900À,IS901+, IS1245+, and FR300À (Table 1) samples. The presence of inhibitors, inefficient extrac- and likewise identical positive reaction in the tests by tion of mycobacterial DNA, and low number of bac- MAC and MA Accu-Probes was always obtained. teria present in the sample can cause the low The internal standardised PCR system was used for sensitivity. The culture is considered the definitive routine identification of field mycobacterial isolates test for the confirmation of mycobacterial infection; obtained from cattle, pigs, birds, humans and envi- however this may take weeks or months to achieve ronmental samples (Tables 1, 2 and 3). The results (Ayele et al., 2001). The use of PCR for reliable and using this system of identification were completely fast detection in primary cultures or in subcultures consistent with identifications obtained by Accu- reduces the time for the identification of isolates. To Probes. In addition, the use of species specific overcome the problem with inhibitors some investi- IS901,IS1245,andFR300 molecular markers gators use complex DNA extraction protocols. The allowed detection of mixed mycobacterial infection. use of an internal control in PCR is essential for One limitation of this system is its inability to reliable results (DeWit et al., 1993) therefore we reveal mixed infection caused by M. a. avium and have constructed internal control molecules to check M. a. silvaticum. It can be only discriminated by the efficiency and sensitivity of amplification. cloning of the isolate upon subculture to individual A set of internal standards for IS900,IS901, colonies and their RFLP analysis using IS1245 probe IS1245, and FR300 was developed with the size of (Dvorska et al., 2003). the internal standard amplicons designed to be larger The use of the system with internal standard for the than that of the sample PCR product to ensure an detection of insertion sequence IS1245 confirms that efficient competition of amplification between stan- this element is not present in the members of M. a. dard and genomic target sample. The positive signals paratuberculosis. Although literature refers to IS1245 from internal standard molecules enabled to exclude if occurrence in this subspecies (Guerrero et al., 1995; false negative results were obtained (Ballagi-Pordany Ritacco et al., 1998; Keller et al., 2002), other authors and Belak, 1996). When bacterial cells were present at disprove it (Collins et al., 1997). The cause of the high concentration in the sample internal standards discrepancy may be 85% sequence homology of were also not amplified to a detectable level attribut- IS1245 and IS1311, which is present in M. a. para- able to competition. The same competition was ob- tuberculosis (Roiz et al., 1995). served in pure chromosomal DNA. However The PCR systems with internal standard that pre- inhibition could be removed by sufficient dilution of cludes wrong interpretation because of PCR system M. Bartos et al. / Journal of Microbiological Methods 64 (2006) 333–345 343 inhibition with well selected primers, which can dis- Ayele, W.Y., Machackova, M., Pavlik, I., 2001. The transmission criminate between closely related sequences IS1245 and impact of paratuberculosis infection in domestic and wild ruminants. Vet. Med.-Czech 46, 205–224. and IS1311, help correct interpretation. However, fur- Ayele, W.Y., Svastova, P., Roubal, P., Bartos, M., Pavlik, I., 2005. ther, particularly whole genome studies will be neces- Mycobacterium avium subspecies paratuberculosis cultured sary to come to the solution of the problem concerning from locally and commercially pasteurized cow’s milk in the IS1245 presence in M. a. paratuberculosis. Addition- Czech republic. Appl. Environ. Microbiol. 71, 1210–1214. ally, the element IS1245 and similar sequences were Ballagi-Pordany, A., Belak, S., 1996. The use of mimics as internal standards to avoid false negatives in diagnostic PCR. Mol. Cell. identified in another mycobacterial species (Keller et Probes 10, 159–164. al., 2002). Its presence was detected in all 44 tested Bartos, M., Svastova, P., Dvorska, L., Weston, R.T., Pavlik, I., 2001. isolates of M. fortuitum in the present study. Mycobacterium avium insertion element hot spot flanking re- An identical genotype (IS901À,IS1245À, and gion FR300. Unpublished. Access Number AF369936. FR300À) was detected for the tested isolates of M. Bull, T., Hermon-Taylor, J., Pavlik, I., El-Zaatari, F., Tizard, M., 2000. Characterisation of IS900 loci in Mycobacterium avium intracellulare and isolates of atypical mycobacteria. subsp. paratuberculosis and development of multiplex PCR Positive reactions obtained with MAC and MI Accu- typing. Microbiology 146, 2185–2197. Probes and negative reactions obtained with MA and Collins, D.M., Cavaignac, S., De Lisle, G.W., 1997. Use of four MTC Accu-Probes allow identification of this myco- DNA insertion sequences to characterize strains of the Myco- bacterial species (Table 4). bacterium avium complex isolated from animals. Mol. Cell. Probes 11, 373–380. DeWit, D., Wooton, M., Allan, B., Steyn, L., 1993. Simple method for production of internal control DNA for Mycobacterium Acknowledgements tuberculosis polymerase chain reaction assays. J. Clin. Micro- biol. 31, 2204–2207. We wish to thank all laboratories and colleagues Dvorska, L., Havelkova, M., Bartos, M., Bartl, J., Pavlik, I., 1999. Insertion sequences of mycobacteria and their use in the study of mentioned in (Tables 1, 2 and 4) for providing us with epidemiology of mycobacterial infections (in Czech). Vet. Med.- collection strains and field isolates. Dr. Wuhib Yayo Czech 44, 233–251. Ayele, Mrs. Zdenka Rozsypalova, Jana Srytrova and Dvorska, L., Bartos, M., Martin, G., Erler, W., Pavlik, I., 2001. Marcela Fisakova are gratefully acknowledged for Strategies for differentiation, identification and typing of med- their theoretical and technical help. The work was ically important species of mycobacteria by molecular methods. Vet. Med.-Czech 46, 309–328. supported by the grants Nos. QD1191 and MZE Dvorska, L., Bull, T.J., Bartos, M., Matlova, L., Svastova, P., 0002716201 from Ministry of Agriculture, Czech Re- Weston, R.T., Kintr, J., Parmova, I., Van Soolingen, D., Pavlik, public and grant No. 524/03/1532 from the Grant I., 2003. A standardised restriction fragment length polymor- Agency of the Czech Republic. phism (RFLP) method for typing Mycobacterium avium isolates links IS901 with virulence for birds. J. Microbiol. Methods 55, 11–27. Fischer, O., Matlova, L., Bartl, J., Dvorska, L., Melicharek, I., References Pavlik, I., 2000. Findings of mycobacteria in insectivores and small rodents. Folia Microbiol. 45, 147–152. Amemori, T., Matlova, L., Fischer, O.A., Ayele, W.Y., Machackova, Fischer, O., Matlova, L., Dvorska, L., Svastova, P., Bartl, J., Meli- M., Gopfert, E., Pavlik, I., 2004. Distribution of Mycobacterium charek, I., Weston, R.T., Pavlik, I., 2001. 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