High genetic relatedness among Mycobacterium avium strains isolated from pigs and humans revealed by comparative IS1245 RFLP analysis

Members of the Mycobacterium avium complex cause pig mycobacteriosis and opportunistic human infections. Infections due to environmental mycobacteria are increasing in both industrial and developing countries. Mycobacterium-infected pig carcasses can pass for human consumption due to the poor specificity of meat control by visual detection at the slaughter houses. The genetic relatedness of porcine and human MAC isolates in Finland has been unknown. M. avium isolates isolated from pig organs (n=16) and clinical samples (n=13) were compared by IS1245 RFLP analysis to evaluate the similarity of the isolates obtained from human and porcine samples. Nearly identical multicopy M. avium subsp. hominissuis IS1245 RFLP fingerprints were obtained for isolates of porcine and human origin. IS1245 RFLP patterns of 38% of the porcine and human M. a. hominissuis isolates were >90% similar. The RFLP patterns of two porcine and two human isolates showed >95% similarity. The high similarity of the IS1245 RFLP patterns of the human and porcine M. a. hominissuis isolates indicates close genetic relatedness, suggesting that M. a. hominissuis is transmitted between pigs and humans, or that pigs and humans share common environmental sources of infection. Porcine and human isolates with RFLP patterns differing by only one or two bands were found, which shows that the same M. a. hominissuis strains may infect both humans and pigs.     

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.     

Strategy for the detection and differentiation of Mycobacterium avium species in isolates and heavily infected tissues

The members of Mycobacterium avium species, comprising M. avium subsp. paratuberculosis, M. a. hominissuis, M. a. avium, M. a. silvaticum, are currently the most prevalent opportunistic pathogenic mycobacteria causing mycobacterial infection in animals and humans. The ability to distinguish between these subspecies is of relevance for proper diagnosis and control programmes of the diseases. The aim of this study was to design a fast and specific PCR strategy for the detection and differentiation of M. avium subspecies from the solid plate cultures for use in routine veterinary diagnosis. We have developed a multiplex PCR based on IS900, IS901, IS1245 and the dnaJ gene. This method allows the detection of M. a. paratuberculosis, M. a. hominissuis and M. a. avium/M. a. silvaticum in one PCR reaction and theoretically enables mixed infections of M. a. paratuberculosis and M. a. avium or M. a. paratuberculosis and M. a. hominissuis to be revealed. The sensitivity of this multiplex PCR is 10(3)CFU for each bacterial strain in one PCR reaction, which also enabled the use of this test directly for DNA isolated from the tissue of the heavily infected sheep.     

Impact of sawdust and wood shavings in bedding on pig tuberculous lesions in lymph nodes, and IS1245 RFLP analysis of Mycobacterium avium subsp. hominissuis of serotypes 6 and 8 isolated from pigs and environment

Among 25,027 slaughter pigs raised in two farms, tuberculous lesions were detected in the lymph nodes of 898 (3.6%) of them. Tuberculous lesions were most commonly found in the mesenteric (601; 2.4%) and head (451; 1.8%) lymph nodes. Mycobacteria were isolated from 49 of 120 randomly selected mesenteric, head and bronchial lymph nodes with diagnosed tuberculosis originating from both farms. Forty six Mycobacterium avium subsp. hominissuis, one M. chelonae and two M. fortuitum isolates were found in the lymph nodes of pigs. No statistically significant difference was detected between farms A and B for isolation rates of mycobacteria from the lymph nodes of pigs and their species composition. To investigate the source of the pigs' infections, culture examinations of 117 samples from the external environment were performed. Mycobacteria were isolated from 25 samples from the external environment (21.4%). Mycobacterial isolates were also detected in eleven (91.7%) and two (16.7%) of 12 used sawdust and 12 of non-used (fresh) sawdust samples, respectively. None of 12 wood shavings was culture-positive. Twelve of 13 sawdust isolates were classified as M. a. hominissuis of serotypes 6 and 8 and genotype IS901- and IS1245+; the remaining isolate was classified as species M. fortuitum. Other conditionally pathogenic mycobacteria were only isolated from 12 of the remaining 81 samples from the external environment (excluding bedding). A total of eight isolates (two pig and six sawdust samples originating from farms A and B) were examined by IS1245 restriction fragment length polymorphism (IS1245 RFLP) analysis. These isolates produced five distinct IS1245 RFLP types with more than 20 bands. Based on identical IS1245 RFLP types of one pig isolate and two isolates of used sawdust from farm A, we have concluded that contaminated sawdust was the source of mycobacterial infection for pigs in our study.     

Comparative macrorestriction and RFLP analysis of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis isolates from man, pig, and cattle

In Germany, tuberculous lesions in slaughtered pigs due to infection with members of the Mycobacterium avium complex are increasingly reported. Contaminated food originating from pig or other livestock is discussed as potential source of human infection. M. avium isolates from man (n=45), pig (n=29), and cattle (n=13) were characterised by restriction fragment length polymorphism (RFLP) with respect to insertion sequences IS1245 and IS901 as well as by XbaI-based pulsed-field gel electrophoresis (PFGE) and the results were compared by computer cluster correlation analysis, to determine potential sources of infection in man. By PCR, 55% of animal isolates was identified as M. avium subsp. avium, and 45% as M. a. hominissuis. All human isolates belonged to M. a. hominissuis. IS1245-RFLP and PFGE resulted in two distinct main groupings reflecting the two subspecies, and dividing the isolates into several subgroups. Animal isolates of M. a. hominissuis were widely distributed within the subgroups of human isolates. M. a. avium isolates, further discriminated by IS901-RFLP, formed host-associated subgroups for animals. Comparison of RFLP patterns with those of PFGE resulted in different subgroups as well as different pairs of isolates with high similarities. Only two isolates exhibited identical patterns by both methods. In general, results of both methods support the possibility that M. a. hominissuis isolates from livestock represent a source of infection for man, probably by common environmental reservoirs. There was no evidence of human infections caused by M. a. avium in Germany.     

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.     

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.     

Avian tuberculosis in naturally infected captive water birds of the Ardeideae and Threskiornithidae families studied by serotyping, IS901 RFLP typing, and virulence for poultry

Avian tuberculosis was detected in one flock of 38 water birds of the families Ardeideae (n = 20) and Threskiornithidae (n = 18). Mycobacterium avium subsp. avium (MAA, serotype 1, genotype IS901+ and IS1245+) was more often (p = 0.01) detected in tissue and/or faecal samples in 18 (90.0%) birds form the Ardeideae family: little egret (Egretta garzetta), buff-backed heron (Bubulcus ibis), great white egret (Egretta alba), and bittern (Botaurus stellaris) in comparison to two (11.1%) birds from the Threskiornithidae family: sacred ibis (Threskiornis aethiopicus). Avian tuberculosis was not diagnosed in spoonbills (Platalea leucorodia). Tuberculous lesions were found in nine birds. MAA isolates of IS901 RFLP type F-C3 were present in all of the 20 infected birds and in all environmental isolates. A mixed infection with the MAA isolates of three RFLP types F-C3 (tissue isolate), G-C3, and T-C3 (faecal isolates) was found in one sacred ibis. All 20 tissue isolates of IS901 RFLP type F-C3 from 20 birds and 8 environmental MAA isolates were fully virulent in pullets, whilst the isolates of RFLP types G-C3 and T-C3 were non-virulent in pullets. All of the tested MAA isolates had the same IS1245 RFLP "bird profile". In 12 of 20 infected birds with MAA M.a. hominissuis isolates of serotypes 4, 8, 9 and genotype IS901- and IS1245+ were detected and in 8 other birds mycobacteria not belonging to the M. avium complex were found. The presence of MAA in the environment may be a source for further spread of the causal agent of avian tuberculosis among other groups of animals in zoological gardens, farm animals, and also among their keepers.     

Outbreak of Mycobacterium avium subsp. avium infection in one flock of domestic pigeons

An outbreak of Mycobacterium avium subsp. avium infection was diagnosed in one breed of domestic pigeons (Columba livia f. domestica) in the Czech Republic. Nodular granulomatous lesions were found in 42 (9.7%) pigeons of the 435 examined; histopathologic examination of livers with gross lesions of mycobacteriosis from 15 randomly selected pigeons revealed granulomatous inflammation typical for avian mycobacteriosis in all samples. Direct Ziehl-Neelsen (ZN) microscopy and conventional culture were performed for a total of 117 liver samples (42 pigeons with nodular lesions, 55 randomly selected pigeons without nodular lesions, and 20 randomly selected squabs). Acid-fast bacilli were observed in 19 (16.2%), and conventional culture yielded growth of M. a. avium in 40 (34.2%) liver samples. A triplex quantitative real-time PCR assay based on the IS901 detection system was performed successfully in 115 liver samples and revealed M. a. avium in 63 (54.8%) of them. Mycobacterium a. avium was also detected in two squabs. Eight domestic rabbits (Oryctolagus cuniculus f. domestica) living in the breeding facility were also examined. Pyogranulomatous lesions were only found in one adult male rabbit. At necropsy, both direct ZN microscopy and culture gave negative results for mycobacteria in all examined rabbit tissues. Mycobacterium a. avium was diagnosed in a liver sample of one juvenile rabbit using triplex qPCR, suggesting that M. a. avium infection can occur as early as juvenile animals.     

IS900/ERIC-PCR as a tool to distinguish Mycobacterium avium subsp. paratuberculosis from closely related mycobacteria

There is an increasing demand for fast and reliable methods to distinguish Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) from closely related mycobacteria and also a need for rapid strain specific typing of clinical isolates for epidemiological reasons. In the present study, the potential of rep-PCR as a fingerprinting method for M. paratuberculosis was assessed and compared to conventional RFLP. A PCR assay was designed and optimised to obtain reproducible fingerprints of mycobacterial DNA with primers targeting the enterobacterial intergenic consensus (ERIC) sequence and the M. paratuberculosis specific insertion sequence IS900. Reproducible fingerprints were obtained with 60 strains of M. paratuberculosis, 16 strains of M. avium subsp. avium, 3 strains of M. intracellulare, and 11 other mycobacterial strains. A species-specific band pattern that was clearly distinguishable from that of other mycobacteria was obtained with M. paratuberculosis. The rep-PCR did not detect any differences among M. paratuberculosis strains of different RFLP types, and was therefore not considered as an alternative fingerprinting method. However, the species-specific band pattern make IS900/ERIC-PCR a suitable alternative for distinguishing M. paratuberculosis from other mycobacteria, especially in cases of IS900 PCR positive mycobacteria. The fingerprinting method reported was fast and easy to perform, and produced highly reproducible results.     

Apa antigen of Mycobacterium avium subsp. paratuberculosis as a target for species-specific immunodetection of the bacteria in infected tissues of cattle with paratuberculosis

Comparative genomics of Mycobacterium spp. have revealed conservative genes and respective proteins differently expressed in mycobacteria that could be used as targets for the species-specific immunodiagnostics. The alanine and proline-rich antigen Apa is a mycobacterial protein that present significant variability in primary sequence length and composition between members of M. avium and M. tuberculosis complexes. In this study, the recombinant Apa protein encoded by the MAP1569/ModD gene of M. avium subsp. paratuberculosis (Map) was used to generate a panel of monoclonal antibodies which were shown to recognize the most important veterinary pathogens of the M. avium complex, specifically Map and M. avium subsp. hominissuis, and which did not cross-react with M. bovis or M. tuberculosis. The produced antibodies were demonstrated to be a useful tool for the species-specific immunofluorescence or immunohistochemical detection of Map in experimentally infected cell cultures or intestinal tissues from cattle with bovine paratuberculosis and, additionally, they may be employed for the discrimination of pathogenic M. avium subspecies via Western blotting.     

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.     

A novel IS element, ISMpa1, in Mycobacterium avium subsp. paratuberculosis

A novel insertion element belonging to the IS110 family was identified in Mycobacterium avium subsp. paratuberculosis. The IS element, ISMpa1, is 1500 bp and has one ORF encoding a putative transposase. Three copies of ISMpa1 were identified in the M. avium subsp. paratuberculosis genome. The element had inserted into the 3' end of the highly conserved mycobacterial genes prrB and a homologue of M. tuberculosis Rv1593c, and between a putative cytochrome p450 oxygenase and a putative hydrolase. The IS element was present in all (n = 11) M. avium subsp. paratuberculosis strains but not detected in most other mycobacterial species examined, including 10 M. avium subsp. avium isolates of human, avian and porcine origin. However two porcine isolates of M. avium subsp. avium and the reference strain IWGMT49 did harbour ISMpa1. These three strains belong to a previously described subgroup of M. avium subsp. avium based on IS1245 restriction fragment length polymorphism (RFLP) pattern and serovars. All of the M. avium subsp. paratuberculosis strains examined had an identical RFLP pattern when probed with sequences corresponding to the 5' end of ISMpa1, whereas a different pattern was seen in the positive M. avium subsp. avium strains. This novel IS element might be a useful tool in strain classification of M. avium subsp. avium and also for the identification of M. avium subsp. paratuberculosis when used in combination with IS900.     

Isolation and characterisation of Mycobacterium avium and Rhodococcus equi from granulomatous lesions of swine lymph nodes in Slovenia

Granulomatous lesions in bovine and especially swine lymph nodes are still frequently observed during routine veterinary meat inspections even though Mycobacterium bovis infections are no longer detected in domestic animals in Slovenia. Different lymph nodes of pigs (n = 260) were investigated using classical bacteriological and molecular methods. Mycobacterium avium alone was isolated in 47.3% of pigs and in mixed infection with Rhodococcus equi in 3.9% of pigs. R. equi alone was isolated in 27.3% and in mixed infection with mycobacteria other than M. avium in 1.5% of pigs. A total of 133 M. avium isolates were typed using the IS1245, IS901 and FR300 PCR. Almost two thirds (60.9%) of isolates belonged to M. avium hominissuis (IS901-, IS1245+ genotype), 33.8% of isolates belonged to M. avium avium (IS901+, IS1245+ genotype) and 5.3% of isolates remained non-typed. Fifty out of 85 R. equi isolates were tested for the virulence-associated antigens (VapA and VapB). Nearly two thirds (60.0%) were positive for VapB while all the other isolates were VapA- and VapB-negative.     

Polymerase chain reaction identification of Mycobacterium avium in formalin-fixed, paraffin-embedded animal tissues.

A PCR procedure previously developed for identification of Mycobacterium bovis in formalin-fixed tissues was used to identify mycobacteria of the M. avium complex. Tissues were examined from 100 culture-positive cases of M. avium complex infection, including 86 in which the subspecies was not identified and 14 that had been identified as M. avium subsp. paratuberculosis. Each sample was tested with 5 primer sets, 16S ribosomal RNA (rRNA), IS900, IS901, IS1245, and a heat shock protein (hspX), that detect 1 or both M. avium subspecies. The success rate of PCR detection varied with the primers used and the animal species tested. Among the 86 cases with no M. avium subspecies designation, primers for the 16S rRNA gene were clearly the most efficient because they produced amplicons from all samples that reacted with any other primer set. The overall detection rate in this group of samples was 71%: highest in avian tissues (89%) followed by swine (72%) and ruminants (57%) None of the avian or swine tissues reacted with primers for IS900 or hspX, which identify M. a. paratuberculosis. In contrast, 7 of the 12 ruminant samples that were 16S rRNA positive reacted with 1 or both of these primers. All of the 14 cases shown by culture to be M. a. paratuberculosis infections were positive with IS900 primers, whereas only 11 were positive for 16S rRNA. These results indicate that 16S rRNA primers are the most useful for PCR identification of M. avium in formalin-fixed tissues of nonruminant species. However, IS900 primers should also be used when ruminant tissues are examined because these primers provide the greatest sensitivity for detection of M. a. paratuberculosis infections.     

PCR-based typing of Mycobacterium avium isolates in an epidemic among farmed lesser white-fronted geese (Anser erythropus)

Mycobacterium avium is an important veterinary pathogen causing avian tuberculosis in birds. The aim of the study was to evaluate the genetic relatedness in M. avium isolates from deep tissues of farmed lesser white-fronted geese with avian tuberculosis and in samples from the farm environment. The strains were analyzed by two PCR-based typing methods, inverted repeat (IR) typing and random amplified polymorphic DNA (RAPD) analysis. The primers for the inverted repeats of the insertion sequences IS1245 and IS1311 were used in IR typing, and the RAPD analysis was performed with six primers. Seven of the nine avian strains yielded an identical pattern in the IR typing, but they could be divided into two groups in the RAPD analysis. The remaining two bird isolates had an identical IR pattern (IR cluster II) which they shared with two environmental isolates. However, the RAPD analysis revealed that these environmental isolates had a RAPD pattern (RAPD cluster VI) distinct and different from either of the bird isolates (RAPD clusters II and IV). In all, four M. avium strains were verified as being inducers of avian tuberculosis in birds, and all were distinct from the three environmental strains identified. Thus, the results did not confirm the preliminary idea that a single strain had caused the epidemic. The polymorphism among M. avium strains highlighted the great biodiversity among an M. avium population even in a limited environmental setting during a short time span, and indicated the high susceptibility to avian tuberculosis of lesser white-fronted geese.     

贵阳市规模化猪场猪繁殖与呼吸综合征血清学调查研究

试验应用ELISA检测方法对贵阳市3个未免疫猪繁殖与呼吸综合征（PRRS）猪场和9个免疫猪场的后备母猪、生产母猪和5～10周龄断奶仔猪3个猪群进行猪繁殖与呼吸综合征病毒（PRRSV）抗体检测。结果表明：贵阳市3个未免疫猪场中2个有PRRSV感染,猪群抗体阳性率在0％-10％之间,平均阳性率为5％;9个免疫猪场猪群PRRSV抗体阳性率在65％～90％之间,平均阳性率为82．80％,未免疫猪场与免疫猪场猪群抗体平均阳性率相比,差异极显著（P〈0．01）。     

Molecular epidemiology of Mycobacterium avium subsp. avium and Mycobacterium intracellulare in captive birds

Mycobacterium avium subsp. avium and Mycobacterium intracellulare are primary causes of mycobacteriosis in captive birds throughout the world, but little is known about how they are transmitted. To define the local epidemiology of infection, we strain-typed 70 M. avium subsp. avium and 15 M. intracellulare culture isolates obtained over a 4-year period from captive birds. Typing was performed using randomly amplified polymorphic DNA (RAPD) PCR, amplified fragment length polymorphic (AFLP) fragment analyses, and for a subset of isolates, DNA sequencing of a segment of the 16S-23S rRNA internal transcribed spacer region. Six strain clusters comprising 43 M. avium subsp. avium, isolates were identified; 42 isolates had unique typing patterns, including all M. intracellulare isolates. Phylo-geographical analyses using RAPD and AFLP fingerprints and animal confinement histories showed no correlation between housing of infected birds and mycobacterial strain-type, except for two animals. The diversity of M. avium subsp. avium and M. intracellulare isolates and minimal evidence for bird-to-bird transmission suggest that environmental reservoirs may be important sources of infection in captivity.     

Description of the infection status in a Norwegian cattle herd naturally infected by Mycobacterium avium subsp. paratuberculosis

The Norwegian surveillance and control programme for paratuberculosis revealed 8 seroreactors in a single dairy cattle herd that had no clinical signs of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) infection. Paratuberculosis had been a clinical problem in goats several years previously in this herd. All 45 cattle were culled and a thorough investigation of the infection status was conducted by the use of interferon-gamma (IFN-gamma) immunoassay, measurement of antibodies, and pathological and bacteriological examination. In the IFN-gamma immunoassay, 9 animals gave positive results, and 13 were weakly positive, while 19 animals were negative. In the serological test, 10 animals showed positive reactions, and 5 were doubtful, while 30 animals gave negative reactions. There appeared to be a weak trend toward younger animals having raised IFN-gamma and older animals having raised serological tests. Histopathological lesions compatible with paratuberculosis were diagnosed in 4 animals aged between 4 and 9 years. Three of these animals had positive serological reaction and one animal gave also positive results in the IFN-gamma immunoassay. Infection was confirmed by isolation of M. a. paratuberculosis from 2 of these 4 animals. One single bacterial isolate examined by restriction fragment length polymorphism (RFLP) had the same profile, B-C1, as a strain that had been isolated from a goat at the same farm several years previously. Despite many animals being positive in one or both of the immunological tests, indicative of a heavily infected herd, none of the animals showed clinical signs and only one cow was shown to be shedding bacteria. A cross-reaction with other mycobacteria might have caused some of the immunoreactions in these animals. It is also possible that the Norwegian red cattle breed is resistant to clinical infection with M. a. paratuberculosis.     

Comparison of Variable-Number Tandem-Repeat Markers typing and IS1245 Restriction Fragment Length Polymorphism fingerprinting of Mycobacterium avium subsp. hominissuis from human and porcine origins

Background

Animal mycobacterioses are regarded as a potential zoonotic risk and cause economical losses world wide. M. avium subsp. hominissuis is a slow-growing subspecies found in mycobacterial infected humans and pigs and therefore rapid and discriminatory typing methods are needed for epidemiological studies. The genetic similarity of M. avium subsp. hominissuis from human and porcine origins using two different typing methods have not been studied earlier. The objective of this study was to compare the IS1245 RFLP pattern and MIRU-VNTR typing to study the genetic relatedness of M. avium strains isolated from slaughter pigs and humans in Finland with regard to public health aspects.

Methods

A novel PCR-based genotyping method, variable number tandem repeat (VNTR) typing of eight mycobacterial interspersed repetitive units (MIRUs), was evaluated for its ability to characterize Finnish Mycobacterium avium subsp. hominissuis strains isolated from pigs (n = 16) and humans (n = 13) and the results were compared with those obtained by the conventional IS1245 RFLP method.

Results

The MIRU-VNTR results showed a discriminatory index (DI) of 0,92 and the IS1245 RFLP resulted in DI 0,98. The combined DI for both methods was 0,98. The MIRU-VNTR test has the advantages of being simple, reproducible, non-subjective, which makes it suitable for large-scale screening of M. avium strains.

Conclusions

Both typing methods demonstrated a high degree of similarity between the strains of human and porcine origin. The parallel application of the methods adds epidemiological value to the comparison of the strains and their origins. The present approach and results support the hypothesis that there is a common source of M. avium subsp. hominissuis infection for pigs and humans or alternatively one species may be the infective source to the other.