Veterinarni Medicina, 51, 2006 (11): 497–511 Original Paper

Mycobacterial and equi in in the Czech Republic between the years 1996 and 2004: the causal factors and distribution of infections in the tissues

J.E. Shitaye1, I. Parmova2, L. Matlova1, L. Dvorska1, A. Horvathova1, V. Vrbas 1, I. Pavlik1

1Veterinary Research Institute, Brno, Czech Republic 2State Veterinary Diagnostic Institute, Prague, Czech Republic

ABSTRACT: Between 1996 and 2004, tissue samples from 3 630 slaughtered pigs were examined by gross exami- nation, microscopy after the Ziehl-Neelsen (ZN) staining of homogenised tissues for the detection of acid-fast rods (AFR) and by culture for the presence of mycobacteria and Rhodococcus equi: 1 781 head lymph nodes (ln), 1 123 mesenteric ln, 54 pulmonary ln, 32 inguinal ln, 562 non-identified ln and 78 samples of tissues from paren- chymatous organs (liver, spleen and kidneys). Tuberculous/tuberculoid lesions were not detected in 249 (6.9%) animals slaughtered due to a positive response to avian tuberculin. Various gross lesions were detected in 3 381 (93.1%) animals as follows: adenopathy in 150 (4.1%), tuberculous lesions with caseation in 2 026 (55.8%) and tuber- culous lesions with calcification in 1 205 (33.2%) of them. AFR were found in tissues from 2 047 (56.4%) animals: in 36 (14.5%) animals free from gross lesions, in 28 (18.7%) animals with adenopathy, in 801 (39.5%) animals with caseation and in 913 (75.8%) animals with calcified tuberculous lesions. Mycobacteria were isolated from the tissues of 289 (15.8%) of 1 852 animals without detected AFR and from the tissues of 1 290 (72.5%) of the 1 778 animals with detected AFR of various intensities. Of 1 579 mycobacterial isolates 1 493 (94.6%) were classified as M. avium complex (MAC) members: 469 (29.7%) M. a. avium (IS901+, serotypes 1, 2, and 3) and 891 (56.4%) M. a. hominissuis (IS901–) isolates of serotypes 4 (n = 1), 8 (n = 643), 9 (n = 74) and non-typed (n = 173). The other 52 (3.3%) iso- lates were members of other mycobacterial species: M. chelonae (n = 35), M. smegmatis (n = 4), M. xenopi (n = 3), M. terrae (n = 7), M. aurum (n = 1), M. scrofulaceum (n = 1), M. fortuitum (n = 1) and biochemically non-identified mycobacteria (n = 34). By examination of ZN stained homogenised tissues, AFR were detected significantly more frequently (P < 0.01) in samples from animals with caseated and/or calcified tuberculous lesions than in tissues from animals without tuberculous lesions. The detection rate of isolates from tissues with tuberculous lesions was likewise significantly higher (P < 0.01) than from tissues without tuberculous lesions.

Keywords: atypical mycobacteria; IS901; IS1245; PCR; serotyping; zoonosis; avian ; economic losses; bovine tuberculosis

Among domestic animals, pigs are particularly conditionally pathogenic mycobacteria (Morita et susceptible to mycobacterial infections caused es- al., 1994a,b; Thorel et al., 1997; Komijn et al., 1999; pecially by members of the tubercu- Offermann et al., 1999). The occurrence of tuber- losis complex (MTC; Thoen et al., 2006), M. avium culous/tuberculoid lesions in pigs brings consider- complex (MAC; Pavlik et al., 2003b, 2005a), and able economic losses (Berthelsen, 1974; Dey and

Supported by the Ministry of Agriculture of the Czech Republic (Grants No. MZE 0002716201 and No. NPV 1B53009) and Grant PathogenCombat (No. FOOD-CT-2005-007081, Brussels, EC).

497 Original Paper Veterinarni Medicina, 51, 2006 (11): 497–511

Parham, 1993; Sigurdardottir et al., 1994; Margolis morphism (RFLP) methods were used in the case of et al., 1994; Cvetnic et al., 1998). Twenty two to 24% the causative agent of avian tuberculosis (Dvorska of the total slaughter costs in the Czech Republic et al., 2003, Fischer et al., 2003, 2004a,b; Matlova consist of slaughtered pigs with detected tubercu- et al., 2004a,b; Dvorska et al., 2004), IS1245 RFLP lous/tuberculoid lesions (Pavlik et al., 2003b). The and Mycobacterial Interspersed Repetitive Unit causes of granuloma formation may also be other (MIRU) in the case of the causative agent of avian bacterial species e.g. Rhodococcus equi (Dvorska et mycobacteriosis (Fischer et al., 2004b; Dvorska et al., 1999; Flynn et al., 2001; Pavlik et al., 2005a). al., 2004; Matlova et al., 2004b, 2005; Romano et In the Czech Republic bovine tuberculosis was al., 2005). The introduced IS901 and IS1245 PCR put under control in 1968. In the following period, methods (Bartos et al., 2006) and biochemical tests incidence of bovine tuberculosis in and the according to Wayne and Kubica (1986) were per- other domestic animals including pigs was gradu- formed. These were used for identification of MAC ally decreasing (Pavlik et al., 1998, 2002a,d,e, 2005b; members during investigation of sources of myco- Pavlik, 2006). The last outbreak of bovine tuber- bacterial infections from pigs with kaolin feed as a culosis in cattle and domestic pigs was diagnosed supplement (Matlova et al., 2004a), peat (Matlova in 1995 and was caused by M. caprae (Pavlik et et al., 2003, 2004a, 2005), from the stable and the al., 2002a,b; Erler et al., 2004). Bovine tuberculo- external environment (Matlova et al., 2003). sis in free living animals and animals in captiv- Tuberculous/tuberculoid lesions, since the last ity was diagnosed occasionally, and no contact case of bovine tuberculosis diagnosed in cattle and with domestic animals occurred with registered domestic pigs in the Czech Republic in 1995 (Pavlik in any of the infected free living animals (Pavlik et et al., 2002b), were caused by MAC members (Pavlik al., 2002c,e; Machackova et al., 2003; Trcka et al., et al., 2003b, 2005a) and R. equi (Dvorska et al., 2006). Accordingly, the Czech Republic has been 1999; Pavlik et al., 2003b, 2005a), respectively. Of officially declared a bovine tuberculosis-free EU the MAC members, the causative agent of avian State (Anonymous, 2004; Pavlik, 2006). tuberculosis M. a. avium was predominantly iso- Besides gross examination and microscopy (and lated from tuberculous lesions in cattle (Pavlik et histology in some cases) after the Ziehl-Neelsen (ZN) al., 2002d, 2005a; Dvorska et al., 2004). In contrast, staining, culture examination for the presence of my- isolation of the causative agent of avian mycobac- cobacteria (Pavlik et al., 1998; Dvorska et al., 1999) was teriosis M. a. hominissuis from tuberculous lesions used for identification of pathogens causing tuberculous prevailed in pigs (Dvorska et al., 1999; Pavlik et al., lesions in domestic animals, above all in cattle and pigs 2000, 2003b, 2005a). in the Czech Republic. Mycobacterial isolates classi- From a veterinary aspect, the causative agent fied as MTC members have been routinely identified of avian tuberculosis M. a. avium (serotypes 1, 2, by biological experiments in animals (above all in guinea and 3), which is fully virulent to birds and caus- pigs), by biochemical tests (Wayne and Kubica, 1986) es , is a more consequential and since 1994 by Accu-Probes (Gen-Probe Inc., San pathogen (Yoder and Schaefer, 1971; Piening et Diego, California, USA). al., 1972; Pavlik et al., 2000; Dvorska et al., 2003, For identification of MAC members, the follow- 2004). In contrast, the other serotypes 4 to 28 were ing methods were used: biological experiments classified in the species formerly designated as in domestic fowl (Gallus domesticus; Pavlas and M. intracellulare (Wayne and Kubica, 1986), which Patlokova, 1977) and serotyping according to comprises subspecies M. a. hominissuis (Mijs et al., Wolinsky and Schaefer (1973) modified by Sussland 2002) with serotypes 4 to 6, 8 to 11 and 21 (Bartos and Hrdinova (1976). Since 1996 the IS901 PCR et al., 2006). These members formerly classified in method (Pavlik et al., 2000) that allows identifica- an intermediary group (Piening et al., 1972) only tion of the causative agent of avian tuberculosis cause tuberculous lesions at the site of inoculation M. a. avium of serotypes 1, 2, and 3. Introduction after intramuscular (Pavlik et al., 2000; of this method likewise allowed withdrawal of bio- Bartos et al., 2006). The members of the remain- logical experiments in domestic fowl from use for ing serotypes 7, 12 to 20 and 22 to 28 classified as the identification of the causative agent of avian species M. intracellulare are almost non-virulent to tuberculosis (Dvorska et al., 2006). birds (Bartos et al., 2006). These members were oc- For detailed identification of selected MAC iso- casionally isolated from pigs in the Czech Republic lates, IS901 Restriction Fragment Length Poly- (Pavlik et al., 2003b). 498 Veterinarni Medicina, 51, 2006 (11): 497–511 Original Paper

Among other subspecies classified in species Animals were classified in 4 groups according to M. avium (Thorel et al., 1990), which cannot be the results of gross examination (Table 3): serotyped and are not MAC members, M. a. para- PA 0 (without gross lesions). The vaccination tuberculosis was occasionally detected in domestic against avian tuberculosis/mycobacteriosis was pigs in Sweden (Pavlik et al., 1999) and in the USA undertaken to determine the actual status of the (Thoen et al., 1975; Songem et al., 1980), in wild boars prevalence of the disease in farms. (Sus scrofa) in the Czech Republic (Machackova et PA 1 (adenopathy, usually with concurrent swol- al., 2003; Trcka el al., 2006) and in Spain (Alvarez len lymph nodes as a consequence of effusion and et al., 2005). M. a. silvaticum, formerly designated congestion, pronounced trabeculae and developing as “wood pigeon” strains (mycobactin dependent lymphadenitis). isolates virulent to birds and isolated mainly from PA 2 (caseated tuberculous/tuberculoid lesions wild pigeons) has not been detected in pigs yet. visible by non-aided eye). The purpose of the present study was to analyse the PA 3 (calcified tuberculous/tuberculoid lesions reliability of respective diagnostic methods used in visible by non-aided eye). the investigation of sources of tuberculous/tubercu- Sample collection. Samples were collected im- loid lesions in domestic pigs and also to investigate mediately after slaughter into sterile polyethylene the distribution of respective mycobacterial species bags and were delivered to the laboratory fresh or in various tissues (above all MAC members) and frozen (–20°C). They were kept frozen for up to R. equi between the years 1996 and 2004. 6 weeks before laboratory analysis.

MATERIAL AND METHODS Detection of mycobacteria

Between the years 1996 and 2004, tissues from Microscopic examination. During the process- a total of 3 630 slaughtered pigs (only one tissue ing of samples for culture, imprint preparations from each pig was examined) were delivered to were made and stained according to the ZN method the National Referral Laboratory (NRL) in Prague. and at least 100 microscopic fields were examined The annual numbers of examined animals ranged for AFR in each sample. Based on the results ob- between 266 in 1998 and 922 in 1996. Among tained by microscopy, the pigs were classified into 3 552 lymph node (ln) samples from pigs the fol- four groups according to the amount of detected lowing were examined: head ln from 1 781 (50%) AFR (Table 3): animals, pulmonary ln from 54 (2.0%) animals, me- ZN 0 (AFR not detected) senteric ln from 1 123 (32%) animals, inguinal ln ZN I (AFR detected occasionally – up to 10 AFR from 32 (1%) animals and non-identified ln from per 100 microscopic fields) 562 (16%) animals (Tables 1 and 2). Among the ZN II (few AFR detected – up to 100 AFR per 100 micro- organ samples from 78 pigs, the following were ex- scopic fields) amined: liver from 53 (68%), spleen from 12 (15%), ZN III (frequently detected AFR – in each micro- lungs from 5 (6%), kidneys from 6 (8%) and pan- scopic field at least one AFR detected) creas from 2 (3%) animals (Table 2). Culture examination for mycobacteria. Appro- ximately 1 g of tissue was homogenised with sterile sea sand in a mortar. The homogenate was proc- Gross examination and sample collection essed with 1N HCl for 15 min and neutralised by 2N NaOH according to a previously described Gross examination. During meat inspection af- method (Fischer et al., 2000). One tissue sample ter slaughter, gross examination for the presence was examined from each animal; no more than one of tuberculous/tuberculoid lesions in respective isolate originated from each sample. lymph nodes was performed. Samples of tissues with tuberculous/tuberculoid lesions and lymph nodes from pigs with a positive response to the Identification of mycobacterial isolates tuberculin test with avian tuberculin AVITUBAL (Bioveta Ivanovice na Hane, Czech Republic; Pavlik Accu-Probes (Gen-Probe Inc., San Diego, Cali- et al., 2003b) were collected for laboratory testing. fornia, USA). Mycobacterial isolates were identified 499 Original Paper Veterinarni Medicina, 51, 2006 (11): 497–511 ) organs** Parenchymatous Parenchymatous = 2); ***in some animals 2); ***in some animals = n Rhodococcus equi and 2004 = 5), kidney (n = 6) and pancreas5), kidney= = ( (n n = 12), lung ( n Lymph nodes of Lymph = 53), spleen= ( n was diagnosed; + = positive and/or of mycobacteria (isolation results of examinations Rhodococcus equi head lung mesenterium inguinum non-identified isolations*** Total Rhodococcus equi isolation No. + % No. + % No. + % No. + % No. + % No. + % No. + % dual infection and with both mycobacteria Mycobacteria isolation Mycobacteria 199619971998 9221999 3352000 439 2662001 120 48 3762002 109 36 2902003 195 41 318 3252004 138 52 128 316 137Total 130 48 310 99 120Rhodococcus equi 40 40 43 183 490 3 630 138 38 174 39 1 579 31 190 85 45 184 9 44 67 39 39 186 46 5 66 1 781 203 5 39 52 3 306 647 36 7 2 77 56 28 5 84 36 1 40 38 4 2 365 28 7 2 54 33 188 29 7 0 96 40 19 7 0 52 83 35 126 6 0 351996 1 0 36 92 851997 37 2 86 1 1231998 14 93 60 43 68 601 9221999 0 76 3 335 43 682000 520 65 54 124 0 266 42 92001 146 56 0 33 0 46 376 792002 137 44 3 32 55 290 02003 0 260 49 52 318 0 64 213 3 3252004 200 69 1 0 128 3 316 210 0 102 208Total 0 69 3 310 0 99 6 194 62 64 99 3 66 183 0 48 490*each originated include liver ( organs sample from one animal; **parenchymatous 0 221 3 630 61 1 174 65 2 243 74 140 48 38 0 42 0 357 71 15 184 9 124 562 62 0 33 73 66 31 77 186 22 42 8 142 5 0 277 7 71 203 1 781 5 34 123 42 1 266 58 3 77 306 7 36 157 4 49 4 21 2 56 15 66 5 47 243 39 71 13 7 21 1 77 78 50 4 18 2 40 1 44 79 7 365 22 54 2 1 58 33 34 86 2 29 7 8 193 0 25 7 56 19 40 96 7 0 8 53 44 8 29 126 6 8 5 0 83 36 35 6 8 1 0 92 91 38 5 86 3 38 2 1 123 63 14 93 65 86 4 624 72 46 76 63 38 0 3 43 68 71 56 124 50 44 9 0 33 46 0 0 81 32 3 58 0 49 0 65 3 0 213 1 0 3 0 3 0 105 0 6 3 0 99 0 49 1 38 0 0 74 45 15 562 0 33 26 8 31 299 46 0 34 7 68 42 53 36 4 21 4 17 39 13 47 78 22 50 21 7 50 1 28 100 1 34 61 8 2 7 72 25 44 8 8 8 5 29 6 8 3 5 63 86 4 38 63 50 199619971998 9221999 3352000 81 2662001 26 3762002 9 28 2902003 8 65 11 3252004 318 62 17 316Total 128 78 21 310 73 and Mycobacteria 99 74 24 490 183 3 630 22 83 23 23 174 26 167 664 55 27 17 184 34 18 57 26 9 186 30 76 5 203 1 781 33 306 71 3 0 41 619 7 80 159 0 38 5 0 0 35 39 4 0 52 0 7 0 54 0 365 7 0 7 0 0 0 96 0 5 0 0 83 0 126 1 0 0 1 92 2 0 6 0 93 1 1 123 5 76 2 2 5 124 23 0 68 3 2 5 0 2 0 9 0 2 0 0 3 3 0 2 0 32 0 0 3 0 3 0 213 0 1 6 0 3 0 0 0 99 3 3 0 1 0 74 38 0 3 1 33 0 562 8 0 4 34 15 3 22 36 11 0 21 0 39 2 0 4 4 13 1 0 3 7 6 6 78 0 0 0 3 14 15 8 0 7 0 8 0 8 8 0 0 0 0 0 0 0 0 0 0 0 0 0 Table 1. Mycobacteria isolation from lymph nodes and parenchymatous organs from 3 630 slaughtered pigs* between from 3 630 slaughtered from organs lymph nodes isolation years 1. Mycobacteria the and parenchymatous 1996 Table Year

500 Veterinarni Medicina, 51, 2006 (11): 497–511 Original Paper by Accu-Probes using a MTC probe for the detec- tamination prior to the mycobacteria culture) and tion of all MTC species, the following probes were identified by biochemical tests (Goodfelow, 1986; used for the detection of MAC species: MAC probe Dvorska et al., 1999). for the detection of isolates of all 28 serotypes, MA probe for the detection of isolates of serotypes 1 to 6, 8 to 11, and 21, also the MI probe for the detec- Statistical assessment tion of isolates of the remaining serotypes 7, 12 to 20, and 22 to 28 (Bartos et al., 2006). The chi2-test (Stat Plus) was applied for the sta- PCR. Primers 5´-GCA ACG GTT GTT GCT tistical evaluation of results (Matouskova et al., TGA AA-3´ and 5´-TGA TAC GGC CGG AAT 1992). CGC GT-3´ were used for the detection of IS901 in all M. avium isolates (Kunze et al., 1992; Bartos et al., 2006). RESULTS Serotyping of MAC isolates. Most of the MAC isolates were examined by serotyping (Wolinsky Mycobacteria isolations and Schaefer, 1973), modified by Sussland and Hrdinova (1976). Examined tissue samples from different tis- Biochemical identification tests. Other my- sues. Mycobacteria were detected by culture in cobacterial species were identified biochemically tissues from 1 579 (44%) of 3 630 animals. Culture where negative results were obtained by the above positivity in respective years varied between 36% described methods (Wayne and Kubica, 1986). in 1997 and 52% in 1999. Culture positivity in 1999 Final identification. Based on these examina- was significantly increased (P < 0.01) in comparison tions, the isolates were identified as follows: with the years 1997, 1998, 2001, 2002 and 2004 MAA: M. a. avium: Accu-Probes MAC+, MA+, and (Table 1). MI–; IS901+; serotypes 1, 2 and 3 Examined tissue samples from lymph nodes MAH: M. a. hominissuis: Accu-Probes MAC+, (Table 1). The following positivity for mycobacteria MA+, and MI–; IS901–; serotypes 4–6, 8–11 was demonstrated in respective groups of lymph and 21 nodes: MI: M. intracellulare: Accu-Probes MAC+, MA–, (a) Mycobacteria were isolated from head ln and MI+; IS901–; serotypes 7, 11–20 and 22–28 samples from 647 (36%) of 1 781 animals. Culture MAC: includes MAA, MAH, and MI isolates positivity was significantly higher (P < 0.01) in 1999 Based on the previously described method (Pavlik than in 1997, 2002 and 2004. et al., 2003b; 2005a), the ratio MAA/MAH was cal- (b) Mycobacteria were isolated from lung ln sam- culated for the infection of respective organs. In ples from 19 (35%) of 54 animals. Due to a low the cases of mixed infections, the infection caused number of examined pigs (between 3 and 9 annu- by more consequential MAC members was taken ally) statistical evaluation was not possible. into consideration: in animals affected with mixed (c) Mycobacteria were isolated from ln samples infection caused by “MAA + MAH”, “ MAA + MI” from 601 (54%) of 1 123 animals. Culture positivity or “MAA + MAH + MI”, the MAA infection was was significantly higher (P < 0.01) in 1999 than in taken into consideration, in animals affected with 1996, 1997, 1998, 2001 and 2003. mixed infection caused by “MAH + MI”, t h e MAH (d) Mycobacteria were isolated from inguinal infection was taken into consideration (Table 2). ln samples from 1 (3%) of 32 pigs. Due to a low number (between 0 and 9 annually) of examined pigs statistical evaluation was not possible. Rhodococcus equi detection (e) Mycobacteria were isolated from ln samples of unknown origin from 277 (49%) of 562 animals. Prior to the decontamination of the homogenised Culture positivity was significantly higher (P < 0.01) lymph node samples were aseptically collected and in 2003 than in 1996, 1997, 1998 and 2001. cultured on blood agar supplemented with 5% of Culture positivity in mesenteric lymph nodes ram blood at 37°C for 48 hours. The R. equi iso- (54%) was significantly higher (P < 0.01) than in head lates were detected on blood agar or on solid egg (36%) and lung (35%) lymph nodes. Mycobacteria media for mycobacterial isolation (after the decon- were detected in only 3% of inguinal lymph nodes, 501 Original Paper Veterinarni Medicina, 51, 2006 (11): 497–511

MAC (78) total Accu Probe Probe Accu (2) pancreas hominissuis, (6) kidney subsp. ; serotypes 7, 11–20 and 22–28; (5) lung 901– ; IS MI+ (12) spleen and species; Uk = unknown tissues examined; which were Mycobacterium avium Mycobacterium = 7100 08 MA– , (53) liver MAH MAC+ Mycobacterium c total (3 552) Accu Probe Accu Uk (562) +, serotypes 1, 2 and 3; 901 (32) –, IS inguinal MI and (1 123) MA+ mesenteric mesenteric Mycobacterium intracellulare, Mycobacterium isolates +, = MI Examined lymph nodes (total No.) (total No.) organs Parenchymatous MI (54) lung MAC , and (for more details more (for see and Methods); Material chapter MAH , head (1 781) Accu Probe Accu MAH MAA and avium, 0.6 0.4 1.0 0.5 0 1.0 0.5 2.4 1.0 0 0 0 2.2 1 00 100 1 0000 00 030101 4 0000 101 7 00 0000 000 1 00 0000 300 3 00 0000 100 1 00 0000 00 40.320 00070.450 1 000010.110 00 30.200 10.100 10.11 MAA 25 0120000 00 6011320000 408170000 32150 171.141 53 18 3 17 0 14 52 1 0 0 0 0 1 4010210 01 1006231000 2470 38 12 0 19 0 5 36 1 1 0 0 0 2 34 2.2 25 0 2 0 7 34 0 0 0 0 0 0 35 2.2 27 0 4 0 4 35 0 0 0 0 0 0 782605700 00 640251730000 173822 subsp. –, serotypes 4–6, 8–11 and 21; a ratio of ratio 901 b complex includes complex MI IS + a b a MI–, MI MAH MI + + + and (No.) 547/983 171/429 10/11 209/394 1/0 132/138 523/972 22/9 2/2 0/0 0/0 0/0 24/11 c MAH MAA MAA+MAH MAA Mycobacterium avium Mycobacterium MA+ , MAH serotype 41 10 000 1 0000 00 serotype serotype 2 421 127 6 170 1 97 401 18 2 0 0 0 20 / = Subtotal Subtotal 1 493 94.6 586 19 589 1 264 1 459 30 4 0 0 0 34 species non-serotyped Mycobacterium avium Mycobacterium non-serotyped isolates; srtp 14 0120 02 31641 504142000 species mycobacterial 2. Distribution of different in tissues originating from 3 630 pigs Table isolates mycobacterial Identified SpeciesMAA serotype non-serotyped No. % srtp 6396505360 06 02 3709721100 6864329062560856376000 974260 0 00 000 0 0000 00 SubtotalMAH serotype serotype serotype 469 29.7 146 7 181 1 112 447 20 2 0 0 0 22 MAA MI Subtotal 891 56.4 399 8 357 0 119 883 SubtotalMAC MAA (ratio) 116 7.8 37 3 47 0 25 112 3 1 0 0 0 4 mixed or non-serotyped Mixed: a Subtotal 86Subtotal Total 5.4% 61 0 1 579 12 100 100 647 0 19 13 41.0 601 86 1.2 1 38.1 0 277 0.1 1 545 0 17.5 30 0 97.8 4 0 1.9 0.3 0 0 0 0 0 0 0 0 34 2.2 M. chelonae M. smegmatis M. terrae M. aurum M. xenopi M. scrofulaceum M. fortuitum M. MAC+ =

502 Veterinarni Medicina, 51, 2006 (11): 497–511 Original Paper which was significantly less (P < 0.01) than in the mals without gross lesions (animal group PA 0). other lymph nodes (Table 1). The differences were significant (P < 0.05) in two Examined parenchymatous organs (Table 1). groups: PA 2 and PA 1 (Table 3). Parenchymatous organs from 78 animals were examined and mycobacteria were isolated from 34 (44%) of them. Due to a low number of examined Gross examination and mycobacterial pigs (4 to 15 annually), statistical evaluation was species detection not possible. No significant differences were found between the detection rate of mycobacteria in pa- MTC members (Table 3). MTC members were renchymatous organs and in various lymph nodes. not detected in any of examined tissue samples dur- The detection rate of mycobacteria was significantly ing the whole study period. higher (P < 0.01) in parenchymatous organs in com- MAC members (Table 3). MAC members were parison with only inguinal lymph nodes. significantly (P < 0.01) most frequently detected in all four groups classified according to gross lesion incidence (PA 0 to 3). Among them, M. a. avium Gross examination and isolation was significantly (P < 0.01) more frequently isolated of mycobacteria from lymph nodes of animals without gross lesions (group PA 0) than from the remaining three groups Generalised or miliary forms of tuberculosis (PA 1, 2, and 3) with gross lesions. was not detected in any of 3 630 tested animals; Conditionally pathogenic mycobacteria (Table 3). tissues free from gross lesions (group PA 0) were Among conditionally pathogenic mycobacteria, found in 249 (7%) animals, tissues with adenopa- M. chelonae (35 isolates) was most frequently iso- thy (group PA 1) in 150 (4%) animals, tissues with lated. It was detected in 0.5 to 1.5% of animals from caseated tuberculous lesions (group PA 2) in 2 026 all four groups (PA 0 to PA 3). Among the other (56%) animals and tissues with calcified tubercu- conditionally pathogenic mycobacteria, species lous lesions (group PA 3) in 1 205 (33%) animals. M. smegmatis, M. xenopi, M. terrae, M. aurum, The highest detection rate of mycobacteria (57%) M. scrofulaceum and M. fortuitum were occasion- determined by culture was recorded in group PA 3. ally isolated. It was not possible to biochemically That was significantly higher (P < 0.01) than in identify 34 mycobacterial isolates that were nega- the remaining groups. The second highest culture tive by Accu-Probes and did not contain IS901; positivity was found in group PA 2 (39%). That was their occurrence was only occasional. significantly higher (P < 0.01) and (P < 0.05) in comparison with groups PA 0 and PA 1, respec- tively (Table 3). Microscopic detection of AFR and detection of mycobacterial species

Gross examination and AFR detection MAC members (Table 3). MAC members were detected in 16% of animals in the group without AFR AFR were found in ZN stained tissues from (ZN 0). Mycobacteria were detected in 69%, 83%, 36 (15%) animals of group PA 0, 28 (19%) animals and 74% of animals in groups ZN I, ZN II and ZN III, of group PA 1, 801 (40%) animals of group PA 2 and respectively. Differences between culture detection 913 (76%) animals of group PA 3. The difference of MAC in samples with AFR (animal groups ZN I, in frequencies of animals with AFR detected in the ZN II and ZN III) and the group without detected tissues were always significant (P < 0.01) between AFR (ZN 0) were significant (P < 0.01). all four groups from PA 0 to PA 3. No significant M. a. avium (Table 3). The detection rates of the differences were found between groups PA 0 and causative agent of avian tuberculosis M. a. avium PA 1. A rather high number of detected AFR (ani- were gradually increasing in respective groups of mal groups ZN II and ZN III) were significantly animals from 18% in group ZN 0 to 46% in group more frequently (P < 0.01) recorded in samples of ZN III; the differences were significant (P < 0.01). tissues with tuberculous lesions (animal groups PA Differences between groups ZN II and ZN III were 2 and 3) in comparison with samples from animals non-significant. A comparable gradual increase in showing adenopathy (animal group PA 1) and ani- the detection rates was recorded in the group of 503 Original Paper Veterinarni Medicina, 51, 2006 (11): 497–511 = MAA R. equi R. PCR and and PCR +% 901 /IS M. M. staining according staining according b species 6110 IS c itum –, serotypes 4–6, 8–11 and M. fortu- 901 IS isolates laceum MI–, M. scrofu- MI and and M. st one AFR detected); ally – up to 10 AFR perally – up to fields), 100 microscopic aurum MA+ , MAH (caseation) and 3 (calcification); (caseation) and 3 (calcification); , M. terrae MAC+ MAA M. xenopi : Accu-Probes matis M. smeg- lonae M. che- M. a. hominissuis = Mixed MAH probes negative biochemically, not identified mycobacterial isolates confirmed isolates not identified mycobacterial by ZN probes staining; negative biochemically, –, serotypes 7, 11–20 and 22–28. Mixed includes 901 IS MI+, +, serotypes 1, 2 and 3; M. intracellulare and 901 IS and MA– , MI–, MAA MAH MI MAC+ and M. avium MA+ +, complex, : Accu Probe : Accu MAC Animals M. avium No.+%+%+%+%+% : Accu-Probe : Accu-Probe b M. intracellulare ZN I0000000000 0 0 000 3I 0 0II 0 31185812675281 III0000000000000 0 0 5 0I 0 3II 60III 0 22 3 0 5 100 17 1 0 615I 77 1 3 4 156II 450 0 1 80III 5 73 137 100 0 29 88 106 2 0 24 30 0 45 50 10I 0 294 33 0 59II 17 65 2III 57 88 1 638 1 50 0 100 244 64 3 6 418 5 0 31 0 197 1 35 66 0I 2 81 0 142 0II 28 0 39 1 0 9 34III 81 1 306 90 0 0 53 6 41 0 250 410 903 0 15 0 60 104 0 69 62 54 0 341 0 0 0 1 53 265 83 0 0 1 46 13 29 0 131 0 0 74 0 46 1 0 1 0 38 559 0 0 21 0 62 0 12 0 0 194 1 0 0 46 0 57 0 7 6 2 0 0 23 0 0 0 1 2 1 3 0 50 0 0 2 0 0 1 0 0 51 0 0 0 1 0 0 1 4 0 7 0 0 0 0 0 0 0 2 0 1 0 3 0 0 1 0 0 0 0 0 0 0 1 2 1 0 0 0 1 0 0 0 0 7 0 1 1 1 1 31 0 0 0 0 0 0 5 0 5 0 0 1 2 0 6 0 40 0 0 0 0 0 0 4 0 0 1 0 0 1 0 0 8 3 0 0 17 0 4 0 3 4 0 0 0 16 2 0 50 4 0 0 4 12 0 3 1 2 = MI a xamination gross examination: 0 (no gross lesions were found), 1, 2 and 3 (tuberculoid/tuberculous lesions were found): 1 (adenopathy), 2 0 (no grossgross 1, 2 and 3 (tuberculoid/tuberculous lesions found), examination: were 1 (adenopathy), lesions found): were PA Table 3. Gross and laboratory (direct microscopy and culture) examinations of tissue samples from 3 630 pigs of tissue samples 3. Gross and laboratory examinations and culture) microscopy (direct Table E a II (few AFR detected 100 AFR per – up to detected III (frequently 100 microscopic fields), lea at AFR – in each field microscopic to Ziehl-Neelsento (ZN) detection the for of acid fast rods (AFR) was carried out: 0 (AFR not detected), I (AFR detected occasion 131141400 0 0 20 0 0 0 2133316154615460 0 0Subtotal1 0 249 0 1 0 54 7 3 22 122Subtotal2 30 23 150 56 0 19 45 20 7 1 225 30 37Subtotal 30 1893 14 1 15 2 026 14 31 0 61 1 793 19205687 27 39 10 0 60 1 Subtotal 292 0 176 57 64 30 to 0 22 0 0 44 249 1 34 455 15 36 1 0 1 852 57 6 0 389 11 289 0 11 1Total 57 25 0 16 0 1 55 0 22 4 1 52 3 630 50 94 18 0 5 1 1 579 1 1 115 5 44 3 22 0 22 40 469 1 1 0 25 1 30 0 1 9 3 891 0 0 2 1 7 56 2 1 0 0 0 58 17 2 1 4 3 4 1 0 1 5 0 1 116 1 29 0 0 3 1 0 2 0 2 1 8 0 35 0 1 3 0 0 2 1 0 4 6 1 0 0 5 0 14 5 3 9 564 21 1 0 46 602 6 0 7 30 12 1 1 45 0 4 1 0 1 24 14 601 8 1 33 34 664 18.3 Accu-Probes with M. a. avium 21;

504 Veterinarni Medicina, 51, 2006 (11): 497–511 Original Paper animals with caseated tuberculous lesions (PA 2) loid lesions (PA 2) that reached 46% in comparison to where the detection rate of M. a. avium isolates all the remaining groups of animals ZN I to ZN III. was gradually increasing with increasing numbers Comparable differences between ZN 0 and the three of detected AFR (groups ZN 0 to ZN III). groups of animals ZN I to ZN III were found for the M. a. hominissuis (Table 3). In contrast, the group of animals PA 3 (Table 3). causative agent of avian mycobacteriosis M. a. homi- nissuis was detected in all three groups of animals (ZN I to ZN III) with detected AFR. These ranged Detection of R. equi from different organs between 50 and 62% in comparison with ZN 0 without detected AFR where the positivity was 40% Lymph nodes (Table 1). R. equi was isolated from only; differences between this group and groups 619 (35%) of 1 781 animals whose head ln were ex- ZN I and ZN II were significant (P < 0.01). amined. Culture positivity detected between 2001 Of 1 579 mycobacterial isolates, 1 493 (94.6%) and 2004 was significantly higher (P < 0.01) than were the following MAC members: between 1996 and 1997. Different positivity for 469 (29.7%) M. a. avium (IS901+, serotypes 1, 2 R. equi was detected in respective groups of lymph and 3) nodes (Table 1). Culture positivity in head lymph 891 (56.4%) M. a. hominissuis (IS901–) of serotypes nodes (35%) was significantly higher (P < 0.01) in 4 (n = 1), 8 (n = 643), 9 (n = 74) and 173 non- comparison with all the other lymph nodes. serotyped isolates Parenchymatous organs (Table 1). R. equi was 17 (1.1%) M. intracellulare not isolated from any of parenchymatous organs The remaining 52 (3.3%) isolates were other my- from 78 animals. cobacterial species: M. chelonae (n = 35), M. sme- gmatis (n = 4), M. xenopi (n = 3), M. terrae n = 7), M. aurum (n = 1), M. scrofulaceum (n = 1), M. for- Mixed infection with mycobacteria and R. equi tuitum (n = 1) and biochemically non-identified mycobacteria (n = 34; Table 2). Mycobacteria and R. equi were detected together by culture in tissues from 2 243 (62%) of 3 630 animals. Culture positivity in respective years varied between R. equi isolation 44% in 1997 and 73% in 2004. Culture positivity was significantly lower (P < 0.01) in 1997 than in all the Examined tissue samples from different or- remaining years except for 1998 (Table 1). gans. R. equi was detected in tissues from 664 Culture positivity in head lymph nodes (71%) (18%) of 3 630 animals. In respective years, culture was significantly increased (P < 0.01) in compari- positivity ranged between 8% in 1997 and 34% in son with all the other ln. In contrast, the detection 2004. Culture positivity between 2001 and 2004 rate in inguinal lymph nodes was only 3%; that was was significantly higher (P < 0.01) than between significantly lower (P < 0.01) than in all the other 1996 and 1998 (Table 1). lymph nodes (Table 1). Gross examination and R. equi isolation. R. equi Mycobacteria and R. equi were together isolated was isolated from tissues of 8 (3%), 9 (6%), 602 (30%) from 34 (44%) of 78 animals whose parenchymatous and 45 (4%) animals from groups PA 0, PA 1, PA 2 organs were examined. That is significantly less and PA 3, respectively. The detection rate of R. equi (P < 0.01) than from head lymph nodes; however, it was the highest in group PA 2; it was significantly is significantly more (P < 0.01) than from inguinal higher (P < 0.01) in comparison with the remaining lymph nodes. three groups (Table 3). AFR detection and R. equi isolation. R. equi was isolated significantly more frequently (P < 0.01) from DISCUSSION tissues without detected AFR (group ZN 0; positivity 33%) in comparison to animals with detected AFR The prevalence of avian tuberculosis or mycobac- (groups ZN I, ZN II and ZN III: 4%, 3% and 2% posi- teriosis in pigs is assumed to be related to direct or tivity, respectively). R. equi was significantly more indirect exposure to infected animals, birds and/or frequently (P < 0.01) isolated from the animal group contaminated feed. In the Czech Republic since the ZN 0 without detected AFR with caseated/tubercu- early 1990s the epizootiological dynamics corre- 505 Original Paper Veterinarni Medicina, 51, 2006 (11): 497–511 sponding to the observation of tuberculous lesions Other isolated conditional pathogenic mycobac- in pigs is variable (Pavlik et al., 2003b). The main terial species such as M. chelonae, M. smegmatis, reasons were: M. terrae, M. xenopi, M. aurum, M. scrofulaceum (1) Transformation of the agricultural system con- and M. fortuitum may affect the health status of ani- nected with the impaired standard of animal mals, in particular, during unfavourable conditions hygiene and the frequent contact of domestic (Wayne and Kubica, 1986). M. a. avium was most pigs with game birds. commonly isolated from birds and cattle (Pavlik et (2) Application of different bedding materials. al., 2000, 2002d, 2005a,b; Dvorska et al., 2004), how- (3) Widely used peat as a feed supplement. ever, in our analysis of mycobacterial infection in Above all, using peat as a feed supplement for piglets pigs, it was found to share a considerable figure with in the end of 1998 and in 1999 resulted in a significant M. a. hominissuis in causing tuberculous lesions. increase (P < 0.01) in the prevalence of tuberculous The frequent detection of MAC members and lesions in slaughtered pigs in 1999 compared with the atypical mycobacteria from pigs implies that my- years 1997, 1998, 2001, 2002 and 2004. cobacterial infection was contracted from con- Our retrospective study in the years 1996 to 2004 taminated external and stable environments and showed that 1 579 (44%) and 664 (18%) of the exam- different contaminated feed supplements (Windsor ined pigs were found to be infected with the MAC et al., 1984; Gardner and Hird, 1989; Horvathova members and R. equi isolates respectively (Table 1). et al., 1997; Durrling et al., 1998; Pavlik et al., 1999, Both agents were predominantly found in the head 2000; Matlova et al., 2003, 2004a, 2005; Trckova et and mesenteric lymph nodes; it follows that the al., 2004). route of mycobacterial and R. equi infections of Furthermore, recent molecular studies showed pigs is the digestive tract, as mycobacteria pen- that can acquire the MAC infection from etrate there after ingestion of contaminated feed or pigs or both may be infected from a similar envi- bedding materials (Dvorska et al., 1999; Matlova et ronment (Komijn et al., 1999; Pavlik et al., 2000; al., 2003, 2004a,b). However, pigs themselves could Dvorska et al., 2002). This report and our result be the source of infection, as they are shedding bring to surface the importance of avian tuberculo- mycobacteria through faeces (Nasal et al., 1974; sis and mycobacteriosis in pigs in terms of economy Matlova et al., 2003). Despite this, contamination and epidemiology. of peat and bedding materials could have occurred Identification of MAC isolates predominantly during transportation and storage that resulted showed the presence of M. a. hominissuis isolates from contact with potentially infected free living of serotypes 8 and 9. This is evident that M. a. ho- birds and small terrestrial mammals (Fischer et al., minissuis species are the most common causes of 2000; Pavlik et al., 2000). tuberculous lesions particularly in pigs (Windsor et This long-term analysis showed a significant and al., 1984; Alfredsen and Skjerve, 1993; Nishimori et high prevalence of avian tuberculosis and mycobac- al., 1995; Dvorska et al., 1999; Komijn et al., 1999; teriosis in the pig farm industry. A wide use of saw- Pavlik et al., 2000, 2003b). Tuberculous lesions dust litter may also be an important risk factor, as it caused by M. a. avium and even R. equi have been allows multiplication of mycobacteria (Kazda, 2000). detected by other authors (Alfredsen and Skjerve, Hence, constant exposure of pigs to contaminated 1993; Dvorska et al., 1999). litter both before and after weaning may account No isolates of serotype 1 of M. a. avium and iso- for the high prevalence of mycobacterial lesions lates of serotype 6 of M. a. hominissuis have been (Rezinkov et al., 1971; Matlova et al., 2004b). isolated; this may be associated with the low viru- M. a. hominissuis (56.4%) and M. a. avium lence of a respective serotype to pigs or with their (29.7%) were the major dominant species, whilst absence in the environment and/or in host organ- M. intracellulare (1.1%) has been rarely detected isms. (Table 2); this may show that M. intracellulare is Soil contaminated with horse faeces is thought not virulent to pigs. However, it was identified to- to be the most common source for R. equi gether with both species. In comparison with the (Takai and Tsubaki, 1985; Prescott, 1991; Dvorska head and mesenteric lymph nodes, even with the et al., 1999). The population of horses in the Czech parenchymatous organs, the detection rate of MAC Republic has decreased (O.I.E., 1999). In contrast, isolates from the lung and inguinal lymph nodes however, R. equi was isolated from lymph nodes of was significantly (P < 0.01) lower. pigs with an increasing trend especially from the 506 Veterinarni Medicina, 51, 2006 (11): 497–511 Original Paper year 2002 to 2004. This may be due to the capac- Alvarez J., De Juan L., Briones V., Romero B., Aranaz A., ity of the R. equi to survive for a long time in the Fernandez-Garayzabal J.F., Mateos A. (2005): Myco- contaminated soil and other environmental sources bacterium avium subspecies in fallow and/or affiliating itself to other susceptible hosts. deer and in Spain. Veterinary Record, 156, For example, R. equi has also been isolated from 212–213. cattle, though it was rare compared to pigs (Dvorska Anonymous (2004): Commission Decision of 31st March et al., 1999; Pavlik et al., 2003a,b) and it was able 2004 amending Decisions 93/52/EEC, 2001/618/EC to cause formation of tuberculoid lesions. In both and 2003/467/EC as regards the status of acceding species (cattle and pigs), cervical lymphadenitis can countries with regard to brucellosis (B. melitensis), be caused by the host acquiring the pathogen from Aujeszky’s disease, enzootic bovine leukosis, bovine the soil (Quinn et al., 1994). brucellosis and tuberculosis and of France with regard Unlike the MAC isolates, R. equi species has not to Aujeszky’s disease (notified under document been detected in parenchymatous organs, in which number C (2004) 1094) (text with EEA relevance, R. equi may limit itself to the intestine, adjacent 2004/320/EC). Official Journal of the European Com- lymph nodes in pigs (Takai and Tsubaki, 1985; munities, 07.04.2004, L 102, 75. Prescott, 1991) and the head lymph nodes as the Bartos M., Hlozek P., Svastova P., Dvorska L., Bull T., predilection site. Matlova L., Parmova I., Kuhn I., Stubbs J., Moravkova Culture was found to be the reliable method for M., Kintr J., Beran V., Melicharek I., Ocepek M., Pav- making a definitive diagnosis in the examined sam- lik I. (2006): Identification of members of Mycobacte- ples. However, the acid fast staining (ZN) technique rium avium species by Accu-Probes, serotyping, and has also showed (48%) maximum agreement with single IS900, IS901, IS1245 and IS901– flanking region the culture (44%) which has also been significantly PCR with internal standards. Journal of Microbiologi- valuable. The tuberculous/tuberculoid lesions were cal Methods, 64, 333–345. observed in 93% of examined tissues which were Berthelsen J.D. (1974): Economics of the avian Tb prob- found to be less sensitive in the detection of the lem in swine. Journal of American Medical Associa- mycobacterial lesions. Development of granulomas tion, 164, 307–308. in animals is related to the reaction of the immune Cvetnic Z., Kovacic H., Ocepek M. (1998): Mycobacteria response of the host against infections, which can in the environment and in the feed of swine in Croatia even be of non mycobacterial origin (Takai, 1997). (in German). Wiener Tierarztliche Monatsschrift, 85, Hence, this finding strengthens our suggestion that 18–21. based on pathological lesions a presumptive diag- Dey B.P., Parham G.L. (1993): Incidence and economics nosis can be done, if not otherwise making a defini- of tuberculosis in swine slaughtered from 1976 to 1988. tive diagnosis remains troublesome. Journal of American Veterinary Medical Association, 203, 516–519. Durrling H., Ludewig F., Uhlemann J., Gericke R. 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Corresponding Author: Prof. MVDr. Ivo Pavlik, CSc., Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic Tel. +420 533 331 601, fax +420 541 211 229, e-mail: [email protected]

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