Distribution of mycobacteria in clinically healthy ornamental fish and their aquarium environment.

Some mycobacterial species (particularly Mycobacterium marinum) found in aquarium environments may cause chronic diseases in fish and cutaneous infections in humans, the so-called 'fish tank granuloma'. The presence and distribution of mycobacterial species in clinically healthy aquarium fish and their environment has not been adequately explored. The present study analysed the occurrence of mycobacteria in a decorative aquarium (Brno, South Moravia) and in five aquaria of a professional fish breeder (Bohumin, North Moravia). After Ziehl-Neelsen staining, acid-fast rods (AFR) were observed in six (14.3%) and mycobacteria were detected by culture in 18 (42.9%) of 42 tissue samples from 19 fish. Sixty-five samples of the aqueous environment from all six aquaria were examined; AFR were found in 16 (24.6%) and mycobacteria were detected by culture in 49 (75.4%) samples. Forty-one (70.7%) of 58 selected mycobacterial isolates were identified biochemically as follows: M. fortuitum, M. flavescens, M. chelonae, M. gordonae, M. terrae, M. triviale, M. diernhoferi, M. celatum, M. kansasii and M. intracellulare. The clinically important species for humans and fish, M. marinum, was not detected. Mycobacterium kansasii was isolated from one sample of the aquarium environment from North Moravia, which is a region of the Czech Republic with endemic incidence of M. kansasii in water. The incidence of other conditionally pathogenic mycobacterial species in healthy fish and in all investigated constituents of the aquarium environment including snails and crustaceans used for fish feeding, was quite high. Accordingly, mycobacterial species from aquarium environments may serve as a possible source of infection for both aquarium fish and immunodeficient fish handlers.

Various stages in the life cycle of syrphid flies (Eristalis tenax; Diptera: Syrphidae) as potential mechanical vectors of pathogens causing mycobacterial infections in pig herds.

We defined the role of the syrphid fly Eristalis tenax in the survival and transmission of mycobacteria in pigs. The conditionally pathogenic mycobacterial (CPM) species Mycobacterium chelonae was isolated from 10 % of liquid dung samples, and both M. chelonae and another CPM species M. fortuitum were isolated from 7 (78 %) of the examined E. tenax larvae collected from the same location. Mycobacteriosis of the lymph nodes of pigs from 3 infected farms was caused by M. avium subsp. avium, M. avium subsp. hominissuis, and M. fortuitum. M. avium subsp. avium and M. avium subsp. hominissuis of identical genotype and serotypes and M. fortuitum were isolated from 7 (1.9 %) larvae, 2 (7.4 %) puparia, and one (1.6 %) imago. The count of colony forming units isolated from larval skin covering (pouch) was higher (p < or = 0.01) than that isolated from the internal organs of larvae. These results showed the potential for E. tenax larvae to spread mycobacteria throughout pig herds and the surrounding environment.

Potential risk of Mycobacterium avium subspecies paratuberculosis spread by syrphid flies in infected cattle farms.

The syrphid Eristalis tenax Linnaeus (Diptera: Syrphidae) may be found in and around dung storage pits at cattle farms at various developmental stages of their life cycle. The purpose of this study was to investigate the occurrence of Mycobacterium avium ssp. paratuberculosis in 1044 E. tenax samples at various developmental stages, as well as fresh and stored dung originating from nine cattle farms. Mycobacterium fortuitum was isolated from one (1.5%) larva from the vicinity of three paratuberculosis-free herds of cattle. Mycobacterium a. paratuberculosis was isolated from 111 (21.4%) of E. tenax larvae collected from two of seven farms known to be infected with the causal agent of paratuberculosis. Mycobacteria were not isolated from any of the 340 pupae, 41 adults of 78 samples of exoskeletal exuviae. Mycobacterium a. paratuberculosis isolates from E. tenax larvae were of the IS900 restriction fragment length polymorphism (RFLP) type B-C1, identical to that detected in faecal samples from cattle herds infected with paratuberculosis. Larvae artificially infected with mycobacteria of IS900 RFLP type B-C9 did not contain statistically more CFU of identical IS900 RFLP type B-C9 in the intestinal tract and internal organs than on the body surface. These results show that M. a. paratuberculosis can survive in the intestinal tract and internal organs of E. tenax.

IS900 restriction fragment length polymorphism (RFLP) analysis of Mycobacterium avium subsp. paratuberculosis isolates from goats and cattle in Norway.

In Norway, paratuberculosis has been frequently diagnosed in goats, while cattle have been almost free of the infection. This difference in prevalence between goats and cattle has led to speculations about the existence of a Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) isolate that is non-pathogenic for cattle. There is little information available on genotypic variation of M. a. paratuberculosis isolated from animals in Norway. In the present study, genotypic information on 51 isolates from goats and four isolates from cattle in Norway was obtained by use of IS900 restriction fragment length polymorphism (RFLP) analysis. All isolates from cattle and 84% of the isolates from goats had the same RFLP pattern (B-C1). Five RFLP patterns not previously detected were found. No genotypic variation that could explain a difference in host origin was found between the isolates from cattle and the majority of the Norwegian goat isolates. This lack of difference indicates that the most common M. a. paratuberculosis isolates in Norway may infect both cattle and goats.

Paratuberculosis and avian tuberculosis infections in one red deer farm studied by IS900 and IS901 RFLP analysis.

As the attempt to eradicate paratuberculosis in one red deer (Cervus elaphus) farm failed, all 167 red deer of different age groups were slaughtered and examined by culture for mycobacteria, and the farm was closed down. Spleen and hepatic lymph nodes, mediastinal lymph node, ileocecal lymph node, and ileum were collected from each animal and examined (a total of 835 organs). Neither tuberculosis lesions nor pathognomic signs of paratuberculosis were detected. Among all microscopically negative for mycobacteria organs, Mycobacterium avium subsp. paratuberculosis alone was isolated from 165 organs, M. a. avium alone from 41 organs, and both pathogens from four organs. M. a. paratuberculosis alone was detected in 71 red deer, M. a. avium alone in 13 red deer and both pathogens in 18 red deer. Using standardised RFLP methods, three IS900 RFLP types B-C1, B-C16, and B-C32 were identified among 40 M. a. paratuberculosis isolates and four IS901 RFLP types N-B1, N-B3, N-B4, and P-B3 among 17 M. a. avium isolates.

Distribution of Mycobacterium avium subsp. paratuberculosis in organs of naturally infected bull-calves and breeding bulls.

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, has particular importance in cattle due to the resulting chronic diarrhoea, weight loss, decreased production, infertility and eventual death. While faecal oral route of infection is generally recognised, reports about semen-derived infection are rare. The objective of this work was to assess whether M.a. paratuberculosis may disseminate from the gastrointestinal tract to reproductive organs, and compare this event between naturally infected bull-calves and breeding bulls. Ten bull-calves, aged 6-28 weeks and four breeding bulls were tested by serology, faecal and tissue culture, IS900 PCR and RFLP. In seven bull-calves M.a. paratuberculosis was isolated predominantly from mesenteric lymph nodes (75%); isolates from mucosa of the intestine constituted 25%. In three breeding bulls, M.a. paratuberculosis was isolated both from intestinal mucosa and mesenteric lymph nodes. Head and mediastinal lymph nodes, liver, spleen and semen of bull no. 1 (Holstein-Friesian); testes and epididymis of bull no. 2 (Piemonte); testes, epididymides and seminal vesicle of bull No. 3 (Hereford); and seminal vesicle of bull No. 4 (Simmental) tested positive by culture. Hot-start PCR revealed M.a. paratuberculosis in semen, seminal vesicle and intestinal tissue where culture isolation was difficult. Isolates from bull-calves and breeding bulls were of RFLP types B-C9 and B-C1, respectively. Bull-calves born in infected herd can be sources of infection when later used for natural mating or artificial insemination. Sub-clinically infected bulls release M.a. paratuberculosis into semen, consequently infecting the uterine environment of cows.

Beetles as possible vectors of infections caused by Mycobacterium avium species.

Mycobacteria were not isolated from any of 229 beetle imagoes of 29 species originating from 14 distinct localities in the Czech and Slovak Republics: 186 imagoes (34 samples) and 43 imagoes (12 samples) from the wild and herds with paratuberculosis infected ruminants, respectively. From 75 environmental samples taken from barns with infected ruminants, Mycobacterium avium subsp. paratuberculosis was isolated from five scrapings of the floors in barns and a feed processing room. From bran and peat taken from pig farms, M. a. hominissuis was diagnosed in 13% of 72 samples and in 69% of 70 samples, respectively. M. a. avium was isolated from 2 (2.9%) and atypical mycobacteria from 12 (17.1%) peat samples. In the respective experiments, larvae of Tenebrio molitor Linnaeus and Zophobas atratus Fabricius were infected in vitro with isolates of M. a. paratuberculosis of IS900 RFLP type B-C1 and M. a. avium of IS901 RFLP type F-C3. T. molitor larvae were also infected with M. a. hominissuis by naturally contaminated bran and peat. M. a. paratuberculosis and M. a. avium were diagnosed in larvae of both species on days 1 to 3 post infection (p.i.). M. a. hominissuis was isolated from T. molitor larvae fed by bran on days 4 to 9 p.i. and from imagoes on day 35 p.i. and from larvae fed by peat on days 4 to 14 p.i. RFLP types of all the isolates identified before infection and after isolation from larvae were identical. Thus, beetles could mechanically transmit mycobacteria, this hazard should be considered for both the implementation of control measures and feeding captive animals with larvae.

Paratuberculosis in farmed and free-living wild ruminants in the Czech Republic (1999-2001).

Due to the occurrence of the infection of Mycobacterium avium subspecies paratuberculosis among domestic ruminants and the rapid development of farmed deer industry and the market of cloven-hoofed game we have carried surveys of paratuberculosis, beginning in 1997, in the most common four species of wild ruminants in the Czech Republic [Pavlik et al., Vet. Microbiol. 77 (2000) 231-251]. From 1999 the prevalence of paratuberculosis has been slightly reduced in all three types of husbandry of wild ruminants. Nevertheless paratuberculosis has been diagnosed in wild ruminants in three districts, in four game parks and in five farms. M. a. paratuberculosis was isolated from 128 (5.3%) out of 2,403 wild ruminants of four animal species: 106 red deer, 2 roe deer, 4 fallow deer and 16 mouflons. In red deer farms, the highest number of clinical paratuberculosis cases was in yearling deer. RFLP type B-C1 of M. a. paratuberculosis predominated during the second period (1999-2001) in all types of husbandry with no relationship to wild ruminant species. New "cattle" RFLP types B-C5 and B-C16 of M. a. paratuberculosis were described in infected farmed red deer and one "intermediate" RFLP type R-I4 in fallow deer from one game park. The survival of M. a. paratuberculosis was found to be 4 months during winter in the pasture after destocking of all cattle infected with paratuberculosis. We found that non-vertebrates, wild ruminants or non-ruminant wildlife can be vectors and potentially become a risk factor in the spread of M. a. paratuberculosis infection.

Blowflies Calliphora vicina and Lucilia sericata as passive vectors of Mycobacterium avium subsp. avium, M. a. paratuberculosis and M. a. hominissuis.

Mycobacterium avium subsp. paratuberculosis (Actinomycetales: Mycobacteriaceae) isolates of identical restriction fragment length polymorphism (RFLP) type B-C1 were isolated from: intestinal mucosa of two cows showing clinical signs of paratuberculosis, a specimen of the blowfly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) captured while perched on these cattle intestines in a waste container at the site of the slaughter, and the blowflies C. vicina and Lucilia caesar Linnaeus captured the next day at the same site when no infected cattle with paratuberculosis were slaughtered. Subsequently, second-stage larvae of the blowflies C. vicina and Lucilia sericata (Meigen) were experimentally infected by feeding them liver from hens with avian tuberculosis caused by M. a. avium (serotype 1, genotype IS901+ and IS1245+) and small cuts of pork meat contaminated with M. a. hominissuis (serotype 8, genotype IS901- and IS1245+). Mycobacterium a. avium of identical serotype, genotype and RFLP type F-C3 was isolated from C. vicina larvae on days 4 and 11 post infection (p.i.) and from L. sericata larvae on day 4 p.i. Identical RFLP type B-C1 of M. a. paratuberculosis was isolated from adult C. vicina fed with artificially contaminated saccharose solution on day 2 p. i. Investigation of M. a. paratuberculosis distribution inside the adult C. vicina showed that the majority of Colony Forming Units (CFU) were isolated from the abdomen and head, fewer from the thorax and wings and none from the legs. Larvae and adults may participate in spreading causal agents of mycobacterial infections and this fact should be considered during sanitation of infected herds and in slaughterhouses when materials from animals affected by mycobacterial infections are processed.

Study of Mycobacterium avium complex strains isolated from cattle in the Czech Republic between 1996 and 2000.

This study surveys 2,593,348 cattle slaughtered between 1996 and 2000, and further investigates 571 (0.02%) animals found to have tuberculous lesions. Culture of 346 randomly selected tissue samples from animals younger (n = 215) and older (n = 131) than 2 years, isolated mycobacteria from 91 animals (26.3%). These included 74 Mycobacterium avium subsp. avium isolates of IS901+ and IS1245+ genotype and serotype 2, 13M. avium subsp. hominissuis isolates of IS901- and IS1245+ genotype and serotypes 8 (n = 7) and 4 (n = 6), two M. chelonae, one M. avium subsp. paratuberculosis (RFLP type B-C1), and one M. terrae. Culture of mesenteric lymph node samples obtained 66 isolates of M. avium complex (MAC) and four isolates of other mycobacterial species. M. bovis was significantly absent from all samples. Mycobacteria were more frequently (P = 0.01) isolated from tissues of animals under 2 years (34.4%) than animals over 2 years (13.0%). IS901 and IS1245 RFLP methods were used to type 17 randomly selected MAC isolates, virulent after intramuscular inoculation of pullets, from 17 different cattle herds. These revealed 11 distinct IS901 RFLP types and three IS1245 RFLP profiles. Polyclonal infection of individual animals was detected by IS901/IS1245 typing in 2 of the 17 selected isolates.

Nymphs of the Oriental cockroach (Blatta orientalis) as passive vectors of causal agents of avian tuberculosis and paratuberculosis.

The potential transmission of the causal agent of paratuberculosis Mycobacterium avium ssp. paratuberculosis and avian tuberculosis Mycobacterium avium ssp. avium (Actinomycetales: Mycobacteriaceae) by nymphs of the Oriental cockroach Blatta orientalis L. (Blattodea: Blattidae) was investigated by oral infection with mycobacterial suspensions and examination of their droppings and bodies. Both the subspecies of M. avium were isolated from droppings at 3 days post-infection and M. a. avium was found in homogenized bodies at 10 days post-infection. The identity of M. a. avium and M. a. paratuberculosis isolates was demonstrated by Restriction Fragment Length Polymorphism (RFLP) analysis. The M. a. avium isolate used as the inoculum and the isolates from the bodies and droppings of the nymphs were shown to be virulent in chickens. The results show that orally infected nymphs of B. orientalis can harbour and shed viable and virulent mycobacteria. This hazard should be considered in the implementation of control measures against mycobacterial infections of animals and humans, which should include destruction of all developmental stages of cockroaches and prevention of their access to materials that can be contaminated by mycobacteria.

Earthworms (Oligochaeta, Lumbricidae) and mycobacteria.

The objective of the study was to define the role of earthworms in the survival of mycobacteria in animal populations. In 13 sampling sites mycobacteria were detected in 53 (5.5%) samples of faeces and parenchymatous tissues from animals, in 25 (7.3%) environmental and in nine (8.2%) earthworm samples. In cattle and goat farms affected by Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) of IS900 restriction fragment length polymorphism (RFLP) type B-C1 was isolated from 37 (4.6%) faecal samples, three (1.4%) environmental and one (3.1%) earthworm sample. Investigations of aviaries affected by avian tuberculosis detected M. avium of genotype IS901+ and IS1245+ in six (7.9%) bird's faecal and in four (4.4%) environmental samples. M. avium (genotype IS901- and IS1245+) was detected in four (4.4%) and M. abscessus in one (1.1%) environmental sample. M. avium of genotype IS901- and IS1245+ and M. gastri were isolated from three (6.4%) earthworm samples. In pig farm with mycobacteriosis M. avium of genotype IS901- and IS1245+ was detected in five (20.0%) faecal samples from pigs and in four (12.9%) environmental samples. M. scrofulaceum was isolated in one (4.6%) sample of Lumbricus rubellus. In laboratory experiments identical RFLP types of M. paratuberculosis were isolated from bodies and faeces of earthworms 1-2 days after the last contact with the faeces contaminated with the same RFLP type of M. paratuberculosis. The results suggest that earthworms may become vectors of mycobacteria.

Diptera as vectors of mycobacterial infections in cattle and pigs.

Mycobacteria were isolated from 14 (4.5%) of 314 samples, containing 7791 adult Diptera, which were collected in the Czech Republic and Slovakia in 1997-2000. These flies were collected from three cattle herds with paratuberculosis, two pig herds with mycobacterial infections and one farm that kept both cattle and pigs and that did not have problems of mycobacterial infections. Mycobacterium intracellulare was isolated from Eristalis tenax Linnaeus (Diptera: Syrphidae) captured from a pig herd. Mycobacterium avium ssp. avium (serotype 8) was isolated from flies of the genera Drosophila Fallen (Diptera: Drosophilidae) and Musca Linnaeus (Diptera: Muscidae) originating from a pig herd. Mycobacterium spp. were isolated from Musca spp. and Mycobacterium fortuitum was isolated from dung flies of the genus Scatophaga Meigen (Diptera: Scatophagidae), Musca spp. and Stomoxys calcitrans Linnaeus (Diptera: Muscidae) captured in the same herd. Mycobacterium scrofulaceum was isolated from S. calcitrans from the farm with both cattle and pigs. Mycobacterium avium ssp. paratuberculosis was isolated from Scatophaga spp. collected from pastures grazed by one of the cattle herds and from Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) and Lucilia caesar Linnaeus (Diptera: Calliphoridae) captured in a slaughterhouse, where cattle infected with paratuberculosis were slaughtered. Mycobacterium phlei was isolated from flies of the genus Lucilia captured at a waste bin. These data indicate that mycobacteria may be spread by adult flies that have been in contact with material contaminated with these pathogens.

Epidemiology of paratuberculosis in wild ruminants studied by restriction fragment length polymorphism in the Czech Republic during the period 1995-1998.

In two studies carried out during the period 1995-1998, paratuberculosis was diagnosed in domestic and wild ruminants in the Czech Republic. The isolated Mycobacterium avium subspecies paratuberculosis strains were analysed by standardised restriction fragment length polymorphism (RFLP) [Pavlik, I., Horvathova, A., Dvorska, L., Bartl, J., Svastova, P., du Maine, R., Rychlik, I., 1999. J. Microbiol. Methods 38, 155-167]. In December 1992, 19 late pregnant Charolais heifers were imported to the Czech Republic from Hungary (original import from France to Hungary). One 11-month-old heifer roamed in the wild in a range of approximately 15-20km for 7 months from November 1993 to May 1994. Upon capture, the animal showed clinical signs of paratuberculosis (emaciation and diarrhoea). Seven other animals from the same herd were infected with the identical RFLP type B-C1 of M. paratuberculosis. During the period 1995-1996, samples were taken and examined from the small intestine and corresponding lymph nodes of 84 wild ruminants: 19 red deers (Cervus elaphus) and 65 roe-deers (Capreolus capreolus). These wild ruminants originated from 44 different locations within the same district from as the infected escaped heifer. Five M. paratuberculosis strains were isolated: one strain of RFLP type B-C1 from a stag and three strains of RFLP type B-C1 and one strain of RFLP type B-C9 from roe-deer. The three wild ruminants (one stag and two roe-deer) infected with the same RFLP type B-C1 were detected in the same area as the heifer, suggesting that this was the likely infection source. However, the infection source of the roe-deer infected with strain of RFLP type B-C9 was obviously different, and the stags that escaped from the farm were purchased from an area infected with this RFLP type. In the second study carried out during 1997-1998 in the whole Czech Republic (divided into 76 districts), 718 wild ruminants were examined from 90% of the districts. M. paratuberculosis was isolated from 25 (3.5%) animals from the wild, from farms and from game parks: 7.1% of 132 red deers, 1.5% of 336 roe-deers, 3.9% of 178 fallow deers (Dama dama), and 4.2% of 48 moufflons (Ovis musimon). This study discovered three RFLP types (B-C1, D-C12 and M-C16). A surprising finding was that of M. paratuberculosis (RFLP type B-C1) infection in roe-deer and a fallow deer in their natural habitat. The infection source was determined to have originated from two imported Holstein and Limousine cattle herds infected with the same strain. In the case of a mother and daughter roe-deer infected with RFLP type M-C16 and a fallow deer infected with RFLP type D-C12, all roaming in their natural habitat, the infection source was not discovered. The highest incidence of clinically ill wild ruminants was found in farmed red deer, and no relationship was found between the RFLP type or ruminant species and clinical status of animal.

Parallel faecal and organ Mycobacterium avium subsp. paratuberculosis culture of different productivity types of cattle.

Faecal (at least 3 months before slaughtering) and organ examinations were carried out in 611 animals (497 dairy, 69 dual-purpose and 44 beef cattle) originating from eight paratuberculosis infected cattle herds. The diagnosis in cattle was established by routine intestinal culture (ileum and the adjacent lymph nodes) after slaughter. In selected 132 animals, post-mortem intensive culture was performed on tissue samples collected from the gastrointestinal tract (duodenum, jejunum, ileum, ileocecal valve, caecum, rectum) and the corresponding lymph nodes, submandibular, retropharyngeal, tracheobronchial, liver and supramammary lymph nodes, kidney, liver and spleen. In 251 (41.1%) of all 611 animals, Mycobacterium avium subspecies paratuberculosis could be isolated from the faeces; in 164 (65.7%) out of 251 shedding animals the infection was detected in the ileum and adjacent lymph nodes. The detection of M. paratuberculosis by routine intestinal culture of faecal culture positive animals varied from 46.0% in animals shedding 1 CFU (colony forming unit), to 94.7% in massive shedders. On the contrary, M. paratuberculosis was detected by routine intestinal culture in 92 (25.5%) of the 360 faecal culture negative animals. Shedding animals had significantly higher (P<0.01) number of organisms in their organs than non-shedding animals. During the intensive tissue cultivation from selected 132 animals, 72 (54.5%) of them were positive. For the negative animals, no significant difference was found between the detection rate in organs examined after slaughter with routine and intensive method. However, in the subgroup of tissue culture positive animals a highly significant difference (P<0.01) was found by intensive examination (83.0%) compared with the routine examination (60.4%). Out of 72 tissue culture positive animals 73.6% of them harboured M. paratuberculosis in the gastrointestinal tract, 16.7% in the gastrointestinal tract and the parenchymatous organs, tracheobronchial and mandibular lymph nodes. The rest of the 9.7% of the infection was detected in the lymph nodes of head and lungs. Our study concerning the distribution of M. paratuberculosis by intensive examinations revealed a minimum effect of breed and production type on localisation of the agent. Thus, the results suggest that in case of an active infection, M. paratuberculosis can be localised in different organs of animals irrespective of their breed or production type.

Relationship between IS901 in the Mycobacterium avium complex strains isolated from birds, animals, humans, and the environment and virulence for poultry.

A total of 738 strains of Mycobacterium avium complex (MAC) were examined in biological experiments on poultry by use of PCR methods with primers for detection of the insertion sequence IS901. Serotype strains of MAC from all known 28 serotypes were examined. Further strains were isolated from human immunodeficiency virus (HIV)-negative and HIV-positive patients, 6 animal species, 17 bird species, and the environment. Of 165 strains virulent for poultry, characterized by generalized tuberculosis, 164 strains contained IS901, a result which is statistically highly significant (P, 0.01). The remaining 573 strains were nonvirulent; however, IS901 was present in 24 strains. From among 20 strains of serotypes 1, 2, and 3, IS901 was found in 15 strains, only 5 of which were virulent for poultry. The remaining 111 strains, of serotypes 4 to 28, were nonvirulent and did not incorporate IS901. None of the 152 strains isolated from humans was virulent for poultry, including 12 strains which were IS901 positive.

Standardisation of restriction fragment length polymorphism analysis for Mycobacterium avium subspecies paratuberculosis.

DNA from 1008 strains of Mycobacterium avium subspecies paratuberculosis, digested by restriction endonucleases PstI and BstEII, was hybridised with a standard IS900 probe prepared by PCR and labelled non-radioactively by ECL. DNA fingerprints were scanned by CCD camera and analysed using the software Gel Compar (Applied Maths, Kortrijk, Belgium). Thirteen restriction fragment length polymorphism (RFLP) (PstI) types were detected, which where designated as A, B, C, D, E, F, G, H, I, J, K, L and M in accordance with the study of Pavlik et al. (1995) [Pavlik, I., Bejckova, L., Pavlas, M., Rozsypalova, V., Koskova, S., 1995. Characterization by restriction endonuclease analysis and DNA hybridization using IS900 of bovine, ovine, caprine and human dependent strains of Mycobacterium paratuberculosis isolated in various localities. Vet. Microbiol. 45, 311-318]. Twenty RFLP (BstEII) types were detected and designated as C1-3, C5, C7-20, S1 and I1 in accordance with the study by Collins et al. 1990 [Collins, D.M., Gabric, D.M., de Lisle, G.W., 1990. Identification of two groups of Mycobacterium paratuberculosis strains by restriction endonuclease analysis and DNA hybridization. J. Clin. Microbiol. 28, 1591-1596]. A combination of both RFLP (PstI) and RFLP (BstEII) results revealed a total of 28 different RFLP types. All the RFLP types and detailed protocols are available at Intemet web site WWW...: http:/ /www.vri.cz/wwwrflptext.htm.