Molecular epidemiology of Types I/III strains of Mycobacterium avium subspecies paratuberculosis isolated from goats and cattle

Molecular characterization of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) isolates classifies them into three groups: cattle or Type II, sheep or Type I, and intermediate or Type III. To avoid problems associated with characterization of extremely slow growth strains, PCR-based techniques that divide the M. a. paratuberculosis strains in two main groups (cattle or Type II, and sheep or Types I/III) can be performed. The objectives of this study were to characterize the M. a. paratuberculosis isolates identified by different PCR-based tests (IS1311-PCR and restriction endonuclease analysis, PCR test based on a DNA sequence difference, and a PCR aimed at three Type I-specific loci), and to determine the clinical and epidemiological implications of Types I/III M. a. paratuberculosis strains in livestock. One hundred and fifty-eight M. a. paratuberculosis strains from domestic ruminants were analyzed. One hundred and six M. a. paratuberculosis isolates (61 from goats and 45 from cattle) were classified as Type II strains; and 52 (29 from cows, 20 from goats, and three from sheep) were included in the Types I/III. The Types I/III M. a. paratuberculosis strains were associated to Spanish native breeds. The majority of these animals had not been in direct or indirect contact with sheep flocks infected with M. a. paratuberculosis. This fact should be taken into account when implementing paratuberculosis control programs.     

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.     

Specificity of absorbed ELISA and agar gel immuno-diffusion tests for paratuberculosis in goats with observations about use of these tests in infected goats

OBJECTIVE: To determine the specificity of serological tests that are currently used in veterinary diagnostic laboratories in Australia for detection of Mycobacterium avium subsp paratuberculosis infection in goats. DESIGN: A laboratory study. PROCEDURE: Four tests were studied, comprising AGID with M. a. paratuberculosis antigen derived from cattle isolates of caprine or bovine origin, the EMAI caprine Johne's disease absorbed ELISA and the CSL PARACHEK Johne's absorbed EIA. The specificities of AGID and ELISA for paratuberculosis (Johne's disease) were estimated after examining a panel of 1000 serum samples collected from goats in Western Australia, a region free of paratuberculosis. In addition a comparison was made of test performance in a small number of paratuberculous goats from New South Wales using sera from two archival collections. RESULTS: The specificity of the AGID tests was 100% while the specificities of the two absorbed ELISA were 99.7 to 99.8% at appropriate positive-negative cut-offs. Based on testing the small sample of sera from infected goats, the absorbed ELISA tests detected about twice as many goats with Johne's disease as the AGID. Each test detected paratuberculous animals regardless of whether infection was caused by cattle or sheep strains of M. a. paratuberculosis. CONCLUSIONS: Both ELISA and AGID tests for paratuberculosis have high specificity and can be used in a market assurance program without risk of generating large proportions of false positive test results. However, the results suggested the ELISA is more sensitive for detection of infected goats and should be used in preference to the AGID. The two formats of ELISA evaluated in this study have similar characteristics and could be used in paratuberculosis control programs for the goat industries, but further data on sensitivity would increase confidence in their application.     

Molecular analysis of field strains of Mycoplasma capricolum subspecies capripneumoniae and Mycoplasma mycoides subspecies mycoides, small colony type isolated from goats in Tanzania.

A molecular analysis of strains of Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae) and Mycoplasma mycoides subsp. mycoides, small colony type (M. mycoides SC) isolated from goats was performed using the amplified fragment length polymorphism (AFLP) and pulsed-field gel electrophoresis (PFGE) fingerprinting techniques. Among the 11 field strains of M. capripneumoniae from Tanzanian goats, two AFLP patterns were demonstrated, with 10 of the strains showing indistinguishable patterns. Five Kenyan strains of M. capripneumoniae produced three AFLP patterns, with two of them being indistinguishable from the 10 identical Tanzanian and one Ugandan strain (M74/93) isolated from sheep. The AFLP pattern of the type strain (F38(T)) was identical to two Kenyan strains (Baringo and G183/82). On PFGE analysis, all the examined M. capripneumoniae strains exhibited identical PFGE profiles.Five field strains of M. mycoides SC isolated from goats displayed identical AFLP patterns except for one strain which differed from others at only one position. The AFLP pattern of the type strain of M. mycoides SC (PG1(T)) was different from the field strains. The five field strains of M. mycoides SC produced identical PFGE profiles, which were, however, different from the type strain. The AFLP and PFGE profiles of M. mycoides SC strains from goats were identical to those of six strains isolated from cattle affected with contagious bovine pleuropneumonia (CBPP) in the same areas. The results of this study suggest a close epidemiological linkage between strains of M. capripneumoniae and between M. mycoides SC type, respectively, isolated from goats in Tanzania.     

Molecular characterisation of avian Pasteurella multocida isolates from Australia and Vietnam by REP-PCR and PFGE

A total of 95 isolates of Pasteurella multocida were analysed by pulsed field gel electrophoresis (PFGE) using the enzyme ApaI, including 73 avian isolates from Australia and 22 from Vietnam. The majority of field isolates were capsular Type A, with the predominant somatic serovars of 1, 3, 4 and 3,4. Twenty-one distinct profiles were evident among the Australian isolates, with only 3 profiles observed among the 22 P. multocida strains isolated from Vietnam. Within the Australian isolates, related and unrelated outbreaks could be identified by PFGE. These results correlated well with previously published studies, with greater discrimination shown by PFGE. Repetitive extragenic palindromic sequence PCR (REP-PCR) analysis of representative isolates from PFGE classifications yielded 21 profiles, with most of the subgroups in accordance with PFGE analysis. While REP-PCR was shown to be less discriminating than PFGE, the epidemiological relatedness of strains compared favourably between the techniques. Thus, the ease and rapidity of REP-PCR while maintaining a high level of differentiation, supports the use of REP-PCR as a competent alternative to the more labour-intensive PFGE system for strain identification and epidemiological studies of avian P. multocida.     

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.     

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.     

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.     

Molecular epidemiology of contagious bovine pleuropneumonia in Tanzania based on amplified fragment length polymorphism and pulsed-field gel electrophoresis analysis

The genetic diversity of 60 field strains of Mycoplasma mycoides ssp. mycoides, small colony type (M. mycoides SC), comprising 56 isolates from cattle in Tanzania, one from Kenya, two from Botswana and one from Portugal, as well as the type (PG1T) and vaccine (T1-SR49) strains, was investigated. The strains were analyzed for variations in the EcoRI and Csp6I restriction sites in the genomic DNA using the amplified fragment length polymorphism (AFLP) technique, and variations in the BamHI restriction sites using pulsed-field gel electrophoresis (PFGE). Six AFLP types were detected among the analysed strains. The AFLP profiles of the type and vaccine strains were indistinguishable from each other. Indistinguishable AFLP profiles were found for 55 Tanzanian field strains, one of them isolated in 1990 and the other 54 isolated in 1998/1999, although one strain isolated in 1999 showed a different profile. Strains from different countries revealed different AFLP profiles. Six PFGE types were detected among the analysed strains, with all the 56 Tanzanian field strains displaying indistinguishable PFGE profiles. Strains from different countries revealed different PFGE profiles, and so did the type and vaccine strains. The strong genomic homogeneity among M. mycoides SC strains associated with outbreaks of contagious bovine pleuropneumonia in different regions of Tanzania suggests that the outbreaks of the disease in the 1990-99 period might have been caused by a single epidemic clone. Moreover, this study has demonstrated that AFLP and PFGE are potential tools for molecular epidemiological studies of M. mycoides SC infections.     

Distribution and hybridization patterns of the insertion element IS900 in clinical isolates of mycobacterium paratuberculosis

Reference strains and 31 clinical isolates of M. paratuberculosis, mainly from goats, were analysed for restriction fragment length polymorphism (RFLP). Restriction digests of bacterial DNA were hybridized with a repetitive insertion sequence, IS900, to obtain banding patterns for comparison of strains. Twenty-five of the 31 field-strains hybridized with IS900, and five hybridization patterns were identified. It was not possible to identify specific patterns for goat strains of M. paratuberculosis. Four hybridization patterns were similar, whereas the fifth pattern of a sheep strain diverged considerably in position and number of band. Six goat strains failed to hybridize with IS900, and the absence of IS900 was verified by the polymerase chain reaction and hybridization with an oligonucleotide probe. The six IS900-negative goat strains had diverging phenotypic properties, and the identification of these strains is discussed. The present study shows that M. paratuberculosis strains infecting goats are genetically similar to cattle strains and that IS900 is a specific genetic element for identification of M. paratuberculosis.     

Pooled faecal culture for the detection of Mycobacterium avium subsp paratuberculosis in goats

OBJECTIVE: To evaluate pooled faecal culture for herd diagnosis of caprine Johne's disease and relate these findings to faecal shedding rates of Mycobacterium avium subsp paratuberculosis (Map). DESIGN: Radiometric broth culture was applied to several pooling dilutions, and shedding rates were estimated from a regression equation based on bacterial growth rates and known processing losses during radiometric culture. PROCEDURE: Sixteen faecal samples from goats naturally infected with sheep (n = 3) or cattle (n = 13) strains of Map, were diluted in normal goat faeces from 1 in 5 to 1 in 50. Cultures were confirmed by IS900 polymerase chain reaction and restriction endonuclease analysis, and mycobactin dependency. The numbers of viable Map in the culture inocula were determined by endpoint titration (most probable number) of nine samples and related to a cumulative growth index. RESULTS: A pooling dilution of 1 in 25 with an incubation period of 10 weeks detected 13 of 16 culture positive goats, all shedding > or = 2 x 10(4) Map per gram of faeces. Two samples containing very low numbers of Map (< 2 x 10(3)/g) were only culture positive from undiluted faeces. Thirteen of 16 goats were considered to be shedding low to moderate concentrations of Map (< 2 x 10(5)/g faeces). CONCLUSIONS: These data support a pooling dilution of 1 in 25 for application of pooled faecal culture as a diagnostic tool in caprine Johne's disease control. A test based on this dilution would reduce laboratory costs of whole herd testing in goats by approximately 40% relative to serology and 75 to 90% relative to individual faecal culture.     

Development and application of quantitative polymerase chain reaction assay based on the ABI 7700 system (TaqMan) for detection and quantification of Mycobacterium avium subsp. paratuberculosis.

Numerous reports have described diagnostic methods based on the polymerase chain reaction (PCR) used to detect Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease. The result of conventional PCR tests has been only qualitative, either positive or negative; it does not present any quantitative information about the number of the agents in the specimen. A quantitative PCR method (IS900 TaqMan) was developed to measure the number of M. a. paratuberculosis organisms present in field and clinical samples. The sensitivity of IS900 TaqMan was 1 colony-forming unit (CFU) for M. a. paratuberculosis ATCC 19698. The specificity of the method was determined by testing 14 mycobacterial species (M. abscessus, M. asiaticum, M. avium subsp. avium, M. bovis, M. fortuitum subsp. fortuitum, M. intracellulare, M. kansasii, M. marinum, M. phlei, M. scrofulaceum, M. simiae, M. smegmatis, M. terrae, and M. ulcerans) and 9 nonmycobacterial species (Borrelia burgdorferi, Chlamydia psittaci, Ehrlichia canis, E. equi, E. risticii, Escherichia coli, E. coli O157:H7, Streptococcus equi, and S. zooepidemicus). Even at high cell numbers (10(5) CFU/reaction), most of the organisms tested negative for the IS900 insertion element except M. marinum and M. scrofulaceum. This finding for M. scrofulaceum was consistent with previous reports that several M. scrofulaceum-like isolates were positive for IS900. Those isolates had 71-79% homology with M. a. paratuberculosis in the region of IS900. When used in conjunction with the new liquid medium-based ESP culture system II for bovine clinical fecal samples, IS900 TaqMan confirmed that the ESP II-positive samples contained 10(5)-10(6) CFU/ml of M. a. paratuberculosis. All of the 222 ESP II-positive and acid-fast bacilli-positive samples tested in this study were positive by IS900 TaqMan. IS900 TaqMan was also useful in the study of growth characteristics of 3 groups of M. a. paratuberculosis strains in bovine fecal samples from 3 shedding levels (heavy, medium, and low) based on cell numbers measured by Herrold egg yolk (HEY) agar culture. When cultured in ESP medium, M. a. paratuberculosis reached 10(5)-10(6) CFU/ml within 2 weeks for heavy shedders, 3-4 weeks for medium, and 6-8 weeks for low shedders. No significant growth was observed after up to 5 weeks of incubation for some of low shedders. No or extremely slow growth characteristic of low shedders might be a possible explanation for frequent false-negative results by HEY. The detection time was dependent on the inoculum size and the growth rate of M. a. paratuberculosis. Generation times were inversely proportional to the shedding level: 1-2 days for medium and heavy shedders and >4 days for low shedders. IS900 TaqMan could be a useful tool for determining viable cell counts by measuring changes in cell numbers over the incubation period.     

Molecular Epidemiology of Contagious Bovine Pleuropneumonia in Tanzania Based on Amplified Fragment Length Polymorphism and Pulsed‐field Gel Electrophoresis Analysis

The genetic diversity of 60 field strains of Mycoplasma mycoides ssp. mycoides, small colony type (M. mycoides SC), comprising 56 isolates from cattle in Tanzania, one from Kenya, two from Botswana and one from Portugal, as well as the type (PG1^T) and vaccine (T₁‐SR49) strains, was investigated. The strains were analysed for variations in the EcoRI and Csp6I restriction sites in the genomic DNA using the amplified fragment length polymorphism (AFLP) technique, and variations in the BamHI restriction sites using pulsed‐field gel electrophoresis (PFGE). Six AFLP types were detected among the analysed strains. The AFLP profiles of the type and vaccine strains were indistinguishable from each other. Indistinguishable AFLP profiles were found for 55 Tanzanian field strains, one of them isolated in 1990 and the other 54 isolated in 1998/1999, although one strain isolated in 1999 showed a different profile. Strains from different countries revealed different AFLP profiles. Six PFGE types were detected among the analysed strains, with all the 56 Tanzanian field strains displaying indistinguishable PFGE profiles. Strains from different countries revealed different PFGE profiles, and so did the type and vaccine strains. The strong genomic homogeneity among M. mycoides SC strains associated with outbreaks of contagious bovine pleuropneumonia in different regions of Tanzania suggests that the outbreaks of the disease in the 1990–99 period might have been caused by a single epidemic clone. Moreover, this study has demonstrated that AFLP and PFGE are potential tools for molecular epidemiological studies of M. mycoides SC infections.     

Proteome-determined type-specific proteins of Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (M. a. paratuberculosis) is a pathogen of ruminants, causing paratuberculosis (characterized by severe emaciation). The disease is endemic in many countries including the UK and places a severe economic burden on the global livestock industry. Two types of M. a. paratuberculosis can be classified by pulsed-field electrophoresis (I/III and II), which are phenotypically distinct and appear to have different host preferences. Proteomes of Type I and Type II M. a. paratuberculosis were analyzed by 2-D gel electrophoresis to determine if any significant differences existed between the subtypes. Seven different strains of Type I and 18 strains of Type II were analyzed and compared to detect type-specific differences. These 'type-specific' differences existed regardless of growth phase and were also exhibited in cells isolated directly from pathogenic lesions. Twenty-three spots predominated on the Type I profile, from which 17 proteins were identified. Twenty-one spots predominated on the Type II profile, from which 16 proteins were identified. None of the proteins identified as differentially represented on the profiles of Type I or Type II corresponded to open reading frames of the defining genomic regions as previously described for the Type I (sheep) and Type II (cattle). Sequence polymorphisms existing in Type I and II strains were identified in some open reading frames or regulatory regions of genes that correspond to proteins expressed in a type-specific fashion. The consequence of these is discussed in relation to protein expression and their impact on the type phenotype is discussed.     

Bacteriological investigations about paratuberculosis in dairy herds in Switzerland

In Switzerland clinical bovine paratuberculosis is registered sporadically with on average seven outbreaks per year. Our present studies are aimed to investigate the prevalence of Mycobacterium avium ssp. paratuberculosis (MAP)-infections in the Swiss cattle population and, therefore methods to culture MAP from bovine feces as well as a commercially available ELISA to detect MAP-specific antibodies are evaluated by using fecal samples and blood sera from herds with cases of clinical paratuberculosis. A series of molecular methods i.e. PCR-coupled RFLP analysis of the IS1311-insertion element of M. avium, PCR-coupled RFLP-analysis of the mycobacterial rpoB-gene, and DNA analysis of the mycobacterial 16S rRNA gene are used to identify mycobacterial isolates grown from bovine feces. Up to now, MAP was detected by culture in 12 of 155 (7.7%) animals from herds with paratuberculosis. A rather striking result is the finding of atypical mycobacteria in feces of 75 cattle (48.3%). Among these isolates, M. avium ssp. avium, M. thermoresistibile, and M. hassiacum/M. buckleii have been identified so far.     

Molecular genotyping of multinational ovine and caprine Corynebacterium pseudotuberculosis isolates using pulsed-field gel electrophoresis

Caseous lymphadenitis (CLA) is a chronic, suppurative disease, with a worldwide distribution, caused by Corynebacterium pseudotuberculosis. The clinical manifestation of CLA is known to vary between different countries, and has been postulated to be due to differences in the strains present in these countries. Forty-two sheep and goat isolates of C. pseudotuberculosis from Australia, Canada, Eire, The Netherlands and Northern Ireland were characterized by pulsed-field gel electrophoresis (PFGE), biotyping, antimicrobial susceptibility, and production of phospholipase D. The PFGE-determined genotypes of this multicentric collection were then compared with representative ovine and caprine isolates from a previously published panel of PFGE profiles of United Kingdom isolates. Digestion with SfiI generated 16-18 bands in the 48.5 and 290 kb range, and differentiated four distinct pulsotypes amongst the 36 ovine and 6 caprine strains which displayed remarkable homogeneity. Based on these results, it would appear that the genome of C. pseudotuberculosis is highly conserved, irrespective of the country of strain origin.     

Detection by immunomagnetic PCR of Mycobacterium avium subsp. paratuberculosis in milk from dairy goats in Norway

Milk samples from 340 individual goats in 34 dairy herds throughout Norway were examined for Mycobacterium avium subsp. paratuberculosis (M.a. paratuberculosis) by culture and immunomagnetic separation combined with PCR (IMS-PCR). The samples included three categories; (A) vaccinated dairy goats in herds with paratuberculosis; (B) vaccinated dairy goats in herds with no history of paratuberculosis; (C) unvaccinated goats in herds with no history of paratuberculosis.Viable M.a. paratuberculosis were not detected by culture in any sample, but 24 samples (7.1%) tested positive by IMS-PCR when the PCR products were visualised by dot blot hybridisation. PCR products from five milk samples originating from five different herds were sequenced; all showed 99% homology with the IS900 sequence from M.a. paratuberculosis.M.a. paratuberculosis were detected in all sampled categories. The percentage of IMS-PCR positive samples from herds where paratuberculosis had previously been reported was significantly lower than from herds where the infection had never been diagnosed (3.3 and 9.1%, respectively, P=0.048). Similar proportions of milk samples from vaccinated and non-vaccinated goats tested positive for the presence of M.a. paratuberculosis. Vaccinated goats older than 4 years tested positive more often than vaccinated animals less than 2 years old. Samples collected in May tested significantly more often positive than milk sampled during February-March (13.8 and 2.9%, respectively, P=0.001). This study showed that raw goats' milk in Norway might be contaminated with M.a. paratuberculosis.     

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.     

Restriction endonuclease analysis of various strains of Mycobacterium paratuberculosis isolated from cattle

Deoxyribonucleic acid (DNA) preparations from 3 reference strains of Mycobacterium paratuberculosis and from 23 isolates of M paratuberculosis obtained from cattle in New Zealand were characterized by restriction endonuclease analysis, using the enzymes BstE II, Pvu II, and Bcl I. Patterns of DNA fragments for strain 18 (one of the reference strains) differed markedly from patterns of other strains, indicating genetic differences between strain 18 and the other strains of M paratuberculosis evaluated. The other 2 reference strains (TMC 1613 and Weybridge strain 316) and all but 1 of the isolates from cattle had identical patterns with the 3 enzymes. These 2 reference strains differed from each other in their dependence on exogenous mycobactin, but this was not reflected in their restriction patterns. The single variant isolate from cattle had patterns identical to those of the other isolates, using Pvu II and Bcl I, and had only 1 fragment line difference with BstE II. Although close genetic homogeneity of cattle strains of M paratuberculosis prevented development of a typing system on the basis of restriction endonuclease analysis, the results provided a basis for genomic comparison with other closely related organisms.