Genetic characterization of German Mycobacterium avium strains isolated from different hosts and specimens by multilocus sequence typing.

Infections caused by Mycobacterium avium and its subspecies are reported as emerging disease in many countries worldwide. In our study we applied the multilocus sequence typing technology to 98 German M. avium strains originating from different hosts and specimens to examine the degree of the genetic diversity. By MLST, 80% of strains were identified as subspecies 'M. avium hominissuis', and 20% as subspecies M. avium avium/M. avium silvaticum. Distinctly different MLST profiles were identified for both subspecies. Based on the analysis of 4 and 5 loci, 87 and 106 SNPs and 1 codon deletion could be detected, respectively, resulting in 40 different strain profiles. Twelve out of these have recently been described for strains coming from different countries, yet in our study, additional new strain profiles (n=28) were found. The high degree of diversity within 'M. avium subsp. hominissuis' as well as the relatedness of human, porcine and environmental strains could be confirmed by IS1245 RFLP fingerprinting. The detection of ISMav6 and hsp65 code 15 in one adult patient strain being positive for IS901, but displaying 'M. avium subsp. hominissuis' MLST profile revealed that PCR for detection of IS901 is not a definitive proof of M. avium subsp. avium/M. avium subsp. silvaticum.

Facts, myths and hypotheses on the zoonotic nature of Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis (Johne's disease [JD]), a chronic granulomatous enteritis in ruminants. JD is one of the most widespread bacterial diseases of domestic animals with significant economic impact. The histopathological picture of JD resembles that of Crohn's disease (CD), a human chronic inflammatory bowel disease of still unresolved aetiology. An aetiological relevance of MAP for CD has been proposed. This and the ambiguity of other published epidemiological findings raise the question whether MAP represents a zoonotic agent. In this review, we will discuss evidence that MAP has zoonotic capacity.

Molecular Diversity of Mycobacterium avium subsp. paratuberculosis in Four Dairy Goat Herds from Thuringia (Germany).

This study investigated the intra- and inter-herd diversity of Mycobacterium avium subsp. paratuberculosis (MAP) isolates from four goat herds in Thuringia (Germany) that were affected by paratuberculosis for several years. The main focus was on the characterization and distribution of genotypes among animals and the environment of goat herd 1. This study included 196 isolates from the feces of 121 infected goats, various tissues from 13 clinically diseased goats, 29 environmental samples from herd 1, and additionally, 22 isolates of different origin from herds 2 to 4. The isolates, sampled between 2018 and 2022, were genotyped using short-sequence-repeat (SSR) analysis, mycobacterial-interspersed repetitive units-variable-number tandem repeat (MIRU-VNTR) analysis, and a single nucleotide polymorphism (SNP)-based assay for phylogenetic grouping. All the isolates belonged to the MAP-C group. In herd 1, one predominant genotype was determined, while two other genotypes were identified very rarely and only in fecal and environmental samples. One of three further genotypes was found in each of herds 2 to 4. The assignment of genotypes to different phylogenetic clades suggested six different infection strains. The results indicated no epidemiological links between the examined herds. Based on the current MAP genotyping data from Germany, possible sources of infection are MAP-contaminated barns previously used by infected cattle and the purchase of sub-clinically infected goats.

Suspicion of Mycobacterium avium subsp. paratuberculosis transmission between cattle and wild-living red deer (Cervus elaphus) by multitarget genotyping.

Multitarget genotyping of the etiologic agent Mycobacterium avium subsp. paratuberculosis is necessary for epidemiological tracing of paratuberculosis (Johne's disease). The study was undertaken to assess the informative value of different typing techniques and individual genome markers by investigation of M. avium subsp. paratuberculosis transmission between wild-living red deer and farmed cattle with known shared habitats. Fifty-three M. avium subsp. paratuberculosis type II isolates were differentiated by short sequence repeat analysis (SSR; 4 loci), mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis (MIRU-VNTR; 8 loci), and restriction fragment length polymorphism analysis based on IS900 (IS900-RFLP) using BstEII and PstI digestion. Isolates originated from free-living red deer (Cervus elaphus) from Eifel National Park (n = 13), six cattle herds living in the area of this park (n = 23), and five cattle herds without any contact with these red deer (n = 17). Data based on individual herds and genotypes verified that SSR G2 repeats did not exhibit sufficient stability for epidemiological studies. Two common SSR profiles (without G2 repeats), nine MIRU-VNTR patterns, and nine IS900-RFLP patterns were detected, resulting in 17 genotypes when combined. A high genetic variability was found for red deer and cattle isolates within and outside Eifel National Park, but it was revealed only by combination of different typing techniques. Results imply that within this restricted area, wild-living and farmed animals maintain a reservoir for specific M. avium subsp. paratuberculosis genotypes. No host relation of genotypes was obtained. Results suggested that four genotypes had been transmitted between and within species and that one genotype had been transmitted between cattle herds only. Use of multitarget genotyping for M. avium subsp. paratuberculosis type II strains and sufficiently stable genetic markers is essential for reliable interpretations of epidemiological studies on paratuberculosis.

Mycobacteriosis in Various Pet and Wild Birds from Germany: Pathological Findings, Coinfections, and Characterization of Causative Mycobacteria.

A total of 50 birds diagnosed with mycobacteriosis were examined for pathomorphological lesions, coinfections, and causative agents. Mycobacterial species were identified and isolates differentiated using multilocus sequence typing (MLST) and mycobacterial interspersed repetitive-unit variable-number of tandem-repeat (MIRU-VNTR) analysis. Possible associations between mycobacterial species, pathomorphological findings, coinfections, bird orders, and husbandry conditions were evaluated statistically. Mycobacteria were isolated from 34 birds (13 of 22 Psittaciformes, 12 of 18 Passeriformes, five of six Columbiformes, and four other orders) belonging to 26 species in total. Mycobacterium genavense (Mg) was cultured from 15 birds, Mycobacterium avium subsp. avium (Maa) from 20 birds, and Mycobacterium avium subsp. hominissuis (Mah) from three birds; hence, four birds had mixed infections. About equal numbers of psittacines and passerines were infected with Ma and Mg. The genetic diversity differed; Mg isolates belonged to one MLST type, Maa to six, and Mah to three combined genotypes. Several coinfections were detected; viruses and/or endoparasites affected 44%, fungi 38%, and bacteria 29% of the birds. Pathological findings and mycobacteriosis-affected organs were independent of coinfections. Overall, gross pathological findings were more often seen in mycobacteriosis caused by Ma (95%) compared with Mg (66%). Organ distribution of mycobacteriosis was independent of the mycobacterial species. Pathomorphological changes were seen in the small intestine of 71% and the lung of 65% of the birds, suggesting oral or pulmonal ingestion of mycobacteria. There were no associations between mycobacterial species and bird orders or bird husbandry conditions. Not only Mg, but also Maa and Mah, were clearly identified as primary cause of mycobacteriosis in pet birds. IMPORTANCE In this study, the causative agents and confounding factors of mycobacteriosis in a set of pet and some wild birds from Germany were examined. Not only Mycobacterium genavense, but also M. avium subsp. avium and M. avium subsp. hominissuis, contributed to mycobacteriosis in these birds. Various coinfections did not affect the manifestation of mycobacteriosis. Due to different gross necropsy findings, however, a different pathogenicity of the two species was assumed. New strains of M. avium subsp. hominissuis originating from birds were identified and characterized, which is important for epidemiological studies and for understanding the zoonotic role of this pathogen, as the subsp. hominissuis represents an increasing public health concern. The study provides some evidence of correlation between M. avium subsp. avium genotypes and virulence which will have to be confirmed by broader studies.

Differential proteome analysis of Mycobacterium avium subsp. paratuberculosis grown in vitro and isolated from cases of clinical Johne's disease.

Bovine Johne's disease (paratuberculosis), caused by Mycobacterium avium subspecies paratuberculosis, poses a significant economic problem to the beef and dairy industry worldwide. Despite its relevance, however, pathogenesis of Johne's disease is still only partially resolved. Since mycobacterial membrane proteins expressed during infection are likely to play an important role in pathogenesis, membrane-enriched fractions, namely mucosa-derived membranes (MDM) and culture-derived membranes (CDM), of M. avium subsp. paratuberculosis from three cows with clinical paratuberculosis were investigated. An initial analysis by 2D difference gel electrophoresis (2D DIGE) and MALDI-TOF-MS analysis revealed four differentially expressed proteins with only one predicted membrane protein. Due to this limited outcome, membrane preparations were subjected to a tube-gel trypsin digestion and investigated by using nanoflow-liquid-chromatography-coupled tandem MS. Based on this approach a total of 212 proteins were detected in MDM including 32 proteins of bovine origin; 275 proteins were detected in CDM; 59 % of MDM and CDM proteins were predicted to be membrane-associated. A total of 130 of the proteins were detected in both MDM and CDM and 48 predicted membrane proteins were detected in MDM from at least two cows. Four of these proteins were not detected in CDM, implying differential expression in the host. All membrane-associated proteins, especially the four identified as being differentially expressed, might be relevant targets for further analyses into the pathogenesis of bovine paratuberculosis.

Clinical outcome and diagnostic methods of atypical mycobacteriosis due to Mycobacterium avium ssp. hominissuis in a group of captive lowland tapirs (Tapirus terrestris).

Tapirs seem particularly susceptible to mycobacterial infections, especially to tuberculosis caused by M. tuberculosis or M. bovis. In this case series, we report an infection with the non-tuberculous mycobacteria (NTM) species M. avium ssp. hominissuis (MAH) in a group of four (2.2) captive lowland tapirs (Tapirus terrestris). Two female tapirs showed mild respiratory signs such as coughing and mucous sputum production for several years, one juvenile male tapir had to be euthanized due to severe dyspnoea, and the adult male only showed mild respiratory signs in 2010. Post-mortem histopathology of the euthanized animal revealed a chronic bronchopneumonia, and MAH was detected via culture. Subsequently, the three remaining tapirs were tested further: serologically, the tapirs had high antibody titres against M. avium, but they showed no reaction in the comparative skin test (TST). At several time points, the animals were tested for the presence of mycobacteria in different sample matrices including sputum samples, pooled faecal samples as well as swabs from the tapir enclosure to identify potential environmental niches of the pathogen. Moreover, animals were directly sampled using nasal swabs, endoscopic broncho-alveolar (BAL) and gastric lavages. MAH was detected by culture in the sputum samples, in the BAL of the breeding pair, as well as in the swimming pool water and walls, and in swabs taken from the tapir's sleeping beds. We conclude that the TST is not a useful diagnostic tool to detect MAC infections in tapirs, whereas antibody ELISA and culture from BAL appear more sensitive.

Detection of Mycobacterium avium ssp. paratuberculosis in Cultures From Fecal and Tissue Samples Using VOC Analysis and Machine Learning Tools.

Analysis of volatile organic compounds (VOCs) is a novel approach to accelerate bacterial culture diagnostics of Mycobacterium avium subsp. paratuberculosis (MAP). In the present study, cultures of fecal and tissue samples from MAP-infected and non-suspect dairy cattle and goats were explored to elucidate the effects of sample matrix and of animal species on VOC emissions during bacterial cultivation and to identify early markers for bacterial growth. The samples were processed following standard laboratory procedures, culture tubes were incubated for different time periods. Headspace volume of the tubes was sampled by needle trap-micro-extraction, and analyzed by gas chromatography-mass spectrometry. Analysis of MAP-specific VOC emissions considered potential characteristic VOC patterns. To address variation of the patterns, a flexible and robust machine learning workflow was set up, based on random forest classifiers, and comprising three steps: variable selection, parameter optimization, and classification. Only a few substances originated either from a certain matrix or could be assigned to one animal species. These additional emissions were not considered informative by the variable selection procedure. Classification accuracy of MAP-positive and negative cultures of bovine feces was 0.98 and of caprine feces 0.88, respectively. Six compounds indicating MAP presence were selected in all four settings (cattle vs. goat, feces vs. tissue): 2-Methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, heptanal, isoprene, and 2-heptanone. Classification accuracies for MAP growth-scores ranged from 0.82 for goat tissue to 0.89 for cattle feces. Misclassification occurred predominantly between related scores. Seventeen compounds indicating MAP growth were selected in all four settings, including the 6 compounds indicating MAP presence. The concentration levels of 2,3,5-trimethylfuran, 2-pentylfuran, 1-propanol, and 1-hexanol were indicative for MAP cultures before visible growth was apparent. Thus, very accurate classification of the VOC samples was achieved and the potential of VOC analysis to detect bacterial growth before colonies become visible was confirmed. These results indicate that diagnosis of paratuberculosis can be optimized by monitoring VOC emissions of bacterial cultures. Further validation studies are needed to increase the robustness of indicative VOC patterns for early MAP growth as a pre-requisite for the development of VOC-based diagnostic analysis systems.

Complete Genome Sequence of Ovine Mycobacterium avium subsp. paratuberculosis Strain JIII-386 (MAP-S/type III) and Its Comparison to MAP-S/type I, MAP-C, and M. avium Complex Genomes.

Mycobacterium avium (M. a.) subsp. paratuberculosis (MAP) is a worldwide-distributed obligate pathogen in ruminants causing Johne's disease. Due to a lack of complete subtype III genome sequences, there is not yet conclusive information about genetic differences between strains of cattle (MAP-C, type II) and sheep (MAP-S) type, and especially between MAP-S subtypes I, and III. Here we present the complete, circular genome of MAP-S/type III strain JIII-386 (DE) closed by Nanopore-technology and its comparison with MAP-S/type I closed genome of strain Telford (AUS), MAP-S/type III draft genome of strain S397 (U.S.), twelve closed MAP-C strains, and eight closed M.-a.-complex-strains. Structural comparative alignments revealed clearly the mosaic nature of MAP, emphasized differences between the subtypes and the higher diversity of MAP-S genomes. The comparison of various genomic elements including transposases and genomic islands provide new insights in MAP genomics. MAP type specific phenotypic features may be attributed to genes of known large sequence polymorphisms (LSPSs) regions I-IV and deletions #1 and #2, confirmed here, but could also result from identified frameshifts or interruptions of various virulence-associated genes (e.g., mbtC in MAP-S). Comprehensive core and pan genome analysis uncovered unique genes (e.g., cytochromes) and genes probably acquired by horizontal gene transfer in different MAP-types and subtypes, but also emphasized the highly conserved and close relationship, and the complex evolution of M.-a.-strains.

High genetic diversity among Mycobacterium avium subsp. paratuberculosis strains from German cattle herds shown by combination of IS900 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive unit-variable-number tandem-repeat typing.

Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease and is endemic to the national cattle herds of many countries. Because of the very low level of genetic heterogeneity of this organism, it is difficult to select a workable procedure for strain differentiation at a resolution sufficient to investigate epidemiological links between herds or different ruminant species and the suggested zoonotic potential of M. avium subsp. paratuberculosis for Crohn's disease. Analysis of restriction fragment length polymorphisms (RFLPs) based on the insertion element IS900 (IS900 RFLP) with four restriction enzymes and 10 markers of specific mycobacterial interspersed repetitive units (MIRUs) and variable-number tandem repeats (VNTRs) was applied to 71 bovine M. avium subsp. paratuberculosis isolates originating from 14 herds from different regions in Germany. Among these isolates, all of which belonged to the M. avium subsp. paratuberculosis type II group, 17 genotypes were detected by IS900 RFLP and consisted of a combination of seven BstEII, eight PstI, nine PvuII, and four BamHI restriction patterns. Novel RFLP types were found. The diversity of the M. avium subsp. paratuberculosis isolates inside the herds was different depending on the frequency of animal purchase. The results of typing by IS900 RFLP and MIRU-VNTR analyses were not associated. Fifteen MIRU-VNTR patterns were identified with a discriminatory index of 0.905. The most common BstEII-based IS900 RFLP type, type C1 (72%), was subdivided into 14 types by MIRU-VNTR analysis. A combination of fingerprinting and PCR-based techniques resulted in 24 M. avium subsp. paratuberculosis genotypes and achieved a discriminatory index of 0.997. By using only BstEII and PstI digestion together with typing by MIRU-VNTR analysis, a discriminatory index of 0.993 was achieved. This is high enough to support epidemiological studies on a national as well as a global scale.

Comparison of 13 single-round and nested PCR assays targeting IS900, ISMav2, f57 and locus 255 for detection of Mycobacterium avium subsp. paratuberculosis.

For molecular biological detection of Mycobacterium avium subsp. paratuberculosis (MAP), PCR methods with primers targeting different regions specific for MAP are used worldwide. However, some uncertainties exist concerning the specificity of certain target regions and the sensitivity. To identify the methods which are best suited for diagnostics, 8 single-round and 5 nested PCR systems including 12 different primer pairs based on IS900 (9 x), ISMav2 (1x), f57 (1x), and locus 255 (1x) sequences were compared regarding their analytical sensitivity and specificity under similar PCR conditions. Reference strains and field isolates of 17 Mycobacterium species and subspecies, 16 different non-mycobacterial bovine pathogens and commensals were included in this study. Single-round PCR resulted in a detection limit of 100 fg to 1 pg, and nested PCR in 10 fg or below. Depending on the specific primer sequences targeting IS900, false positive results occurred with one of the five single-round and two of the four nested PCR systems. This also applied to the single-round PCR based on ISMav2 and the nested PCR based on f57. A high number of non-specific products were primarily detected for the single-round PCR assay based on ISMav2, but also for a single-round PCR targeting the IS900 and the locus 255. In conclusion, stringent selection of IS900-specific primers ensures that IS900 remains a favourite target sequence for amplification of MAP specific loci. The studied PCR systems based on f57, and locus 255 can also be recommended. Revision of ISMav2 primers is necessary. Single-round PCR systems are very reliable. Nested PCR assays were occasionally disturbed by contaminations, thus bearing a risk for routine diagnostics.

Evaluation of associations between genotypes of Mycobacterium avium subsp. paratuberculsis and presence of intestinal lesions characteristic of paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis affecting ruminants worldwide. Depending on the MAP-Type (MAP-C or MAP-S, cattle or sheep type), strains differ in virulence and host preference. There is not yet any strong evidence indicating that individual field strains of the same MAP-subgroup exhibit differences in virulence. The aim of this study was to evaluate a potential association between the genotype of individual field strains belonging to the MAP-C group and the presence of macroscopic intestinal lesions characteristic of paratuberculosis in the infected animals. 88 MAP-C isolates were sampled from clinically healthy cows at slaughter. Cows were grouped as A (n=46) with, and B (n=42) without macroscopic intestinal lesions. Sampled cows from both the A and B groups came from different farms and had a similar age distribution. MAP isolates were characterized by MIRU-VNTR and IS900-RFLP analysis. Resulting genotypes were examined for an association with the presence of macroscopic intestinal lesions characteristic of paratuberculosis. MAP isolates from groups A and B exhibited similar strain diversity: 20 and 18 combined genotypes, altogether 32 genotypes. Six of these genotypes were detected in both groups. Although no association was found between individual combined genotypes and presence of macroscopic intestinal lesions, IS900-RFLP-(BstEII)-Type-C1 (the most common type worldwide) was found more often in group A (p<0.01). The data give only weak indication for the existence of differences in virulence among MAP-cattle type isolates. Differences in the development and severity of lesions may rather depend on unknown host factors or inoculation dose. Virulence properties of IS900-RFLP-(BstEII)-Type-C1 isolates should be examined in more detail.

Complete Genome Sequence of JII-1961, a Bovine Mycobacterium avium subsp. paratuberculosis Field Isolate from Germany.

Mycobacterium avium subsp. paratuberculosis causes Johne's disease in ruminants and was also detected in nonruminant species, including human beings, and in milk products. We announce here the 4.829-Mb complete genome sequence of the cattle-type strain JII-1961 from Germany, which is very similar to cattle-type strains recovered from different continents.

Comprehensive insights in the Mycobacterium avium subsp. paratuberculosis genome using new WGS data of sheep strain JIII-386 from Germany.

Mycobacterium avium (M. a.) subsp. paratuberculosis (MAP) - the etiologic agent of Johne's disease - affects cattle, sheep and other ruminants worldwide. To decipher phenotypic differences among sheep and cattle strains (belonging to MAP-S [Type-I/III] respectively MAP-C [Type-II]) comparative genome analysis needs data from diverse isolates originating from different geographic regions of the world. The current study presents the so far best assembled genome of a MAP-S-strain: sheep isolate JIII-386 from Germany. One newly sequenced cattle isolate (JII-1961, Germany), four published MAP strains of MAP-C and MAP-S from U.S. and Australia and M. a. subsp. hominissuis (MAH) strain 104 were used for assembly improvement and comparisons. All genomes were annotated by BacProt and results compared with NCBI annotation. Corresponding protein-coding sequences (CDSs) were detected, but also CDSs that were exclusively determined either by NCBI or BacProt. A new Shine-Dalgarno sequence motif (5'AGCTGG3') was extracted. Novel CDSs including PE-PGRS family protein genes and about 80 non-coding RNAs exhibiting high sequence conservation are presented. Previously found genetic differences between MAP-types are partially revised. Four out of ten assumed MAP-S-specific large sequence polymorphism regions (LSPSs) are still present in MAP-C strains; new LSPSs were identified. Independently of the regional origin of the strains, the number of individual CDSs and single nucleotide variants confirm the strong similarity of MAP-C strains and show higher diversity among MAP-S strains. This study gives ambiguous results regarding the hypothesis that MAP-S is the evolutionary intermediate between MAH and MAP-C, but it clearly shows a higher similarity of MAP to MAH than to M. intracellulare.

Stability of genotyping target sequences of Mycobacterium avium subsp. paratuberculosis upon cultivation on different media, in vitro- and in vivo passage, and natural infection.

Mycobacterium (M.) avium subsp. paratuberculosis - the causative agent of paratuberculosis (Johne's disease) - affects domestic and wild ruminants worldwide. Recently, different typing techniques have been combined to provide sufficient discriminatory power for the differentiation of isolates and for epidemiological studies. In order to challenge the reliability of this approach the stability of different M. avium subsp. paratuberculosis genotypes determined after primary isolation was investigated after sub-cultivation on six different media (A), twelve in vitro passages (B), or a singular in vivo passage (C). In addition, different isolates from a single animal or herd were investigated (D). Sub-cultures of type- and reference strains, re-isolated inoculation strain after in vivo passage, and 23 field isolates were genotyped by mycobacterial interspersed repetitive unit-variable-number of tandem-repeat (MIRU-VNTR)-, short-sequence-repeat (SSR)-, and IS900-based restriction-fragment length-polymorphism (IS900-RFLP)-analyses and compared with initial genotypes. MIRU-VNTR-alleles (at loci 292, X3, 25, 47, 7, and 32) were stable after in vitro cultivations and after animal passage. Results of SSR analysis at Locus 1 with 7G nucleotides and at Loci 8 and 9 (tri-nucleotides) were also stable. At Locus 2 9G repeats changed into 10G after goat passage. After in vitro subculture (A+B) but not after animal passage (C) IS900-RFLP-typing revealed changes of BstEII-patterns for 3 of 23 strains (including ATCC 19698). Multiple isolates from individual animals or from a single cattle herd with natural infection (D) which exhibited identical IS900-RFLP- and MIRU-VNTR- genotypes, showed different G repeat numbers at SSR locus 2. This implies strand slippage events during chromosomal duplication of bacteria in the course of bacterial spreading within hosts and herds. Consequently, SSR-Locus 2 is not suitable as genome marker for epidemiological studies.

[Paratuberculosis in a miniature donkey (Equus asinus f. asinus)]. / Paratuberkulose bei einem Zwergesel (Equus asinus f. asinus).

Paratuberculosis is mainly an infectious disease of ruminants with worldwide distribution. Infection occurs in early stages of life. Other animal species beyond ruminants are rarely affected, however, experimental and natural infections are possible. A case of paratuberculosis in a miniature donkey (Equus asinus f. asinus) with typical clinical and pathomorphological changes is reported here. Lesions were mainly observed in the intestine. Causative for the profuse diarrhoea with emaciation was massive diffuse granulomatous enteritis involving large quantities of acid-fast organism mainly in macrophages. Granulomatous inflammation with acid-fast bacilli again in macrophages to a lesser degree could be detected in the liver. Mycobacterium avium subsp. paratuberculosis (MAP) was isolated from intestinal contents after an incubation period of four weeks. MAP-specific DNA (IS900 and f57) was detected by polymerase chain reaction in culture material. Additionally MAP-isolates were characterized by multi-target genotyping (MIRU-VNTR- and MLSSR-typing). Isolates belonged to the Type II group and exhibited a unique genotype different from other MAP strains in Germany. The donkey originated from a donkey breeding farm in France with intensive free ranging cattle in the neighbourhood and could have been infected there. Donkeys should be considered as paratuberculosis-susceptible animals in exceptional cases and as possible reservoirs or disseminators of infection.

Divergent cytokine responses of macrophages to Mycobacterium avium subsp. paratuberculosis strains of Types II and III in a standardized in vitro model.

Based on epidemiological and clinical observations, different strains of Mycobacterium avium subsp. paratuberculosis (MAP) are suspected to significantly differ in their virulence for ruminants. In the pathogenesis of paratuberculosis, macrophages represent the principal target cell for MAP. In order to judge the ability of different MAP-genotypes to modulate macrophage responses, the cytokine responses of the monocyte cell line THP-1 were studied after challenge with three different MAP strains under standardized conditions. The bovine field isolate J1961 (major Type II) and the ovine field isolate JIII-86 (Type III) were compared with the laboratory adapted reference strain ATCC 19698 (Type II). Strains were shown by three different typing methods (IS900-RFLP-, MIRU-VNTR-, and SSR-analysis) to substantially differ in several genotypic features. Macrophage function was assessed by quantifying mRNA of the cytokines TNF-α, IL-1ß, and IL-10 by quantitative RT-PCR. Secreted TNF-α protein was measured by a cytotoxicity test, IL-1ß and IL-10 using ELISA tests. The three MAP strains of various genotypes differ in their effect on human macrophages depending on challenge dose and infection time. These differences concerned both the mRNA level and secreted protein amounts of proinflammatory cytokines TNF-α, IL-1ß and anti-inflammatory cytokine IL-10. Type III strain produced less IL-10 and IL-1ß mRNA and protein but more TNF-α protein at 2h than the Type II strains. In summary, our results support the hypothesis that strain characteristics might have relevance for the host response towards MAP and, consequently, for the pathogenesis of paratuberculosis.

Unique genotypes of Mycobacterium avium subsp. paratuberculosis strains of Type III.

Mycobacterium avium subsp. paratuberculosis (MAP) strains with two new IS900 restriction fragment length polymorphism (RFLP) BstEII types intermediate suspected to belong to the MAP Type III group were isolated from migrating sheep in Germany. Such strains have only been sporadically identified in a few studies. For a better understanding of the genomic diversity of MAP with regard to specific host associations, geographic origin, and the discussed classification into Type I, Type II and Type III, these isolates were further characterized. Using IS900-RFLP, the isolates showed unique fingerprint patterns after BstEII-, PstI-, PvuII- and BamHI-digestion which had not been published before. Additionally, using gyrB-PCR-restriction endonuclease analysis (PCR/REA) and mycobacterial interspersed repetitive unit (MIRU)-PCR, the two strains showed differences to known patterns of the Type I as well as the Type II group. Unique genotypes were also obtained with multilocus short sequence repeat (MLSSR) sequencing and MIRU-variable-number tandem-repeat (VNTR) typing. As expected, genomic profiles identical to the Type I and different from the Type II group were detected by IS1311-PCR/REA, IS1311 sequencing as well as by Large Sequence Polymorphism analysis (LSP(A) 8, 17, 20, 4-II, and 18). In addition to distinct growth characteristics, the unique genotypes of the studied sheep strains support their affiliation to the assumed third group within the MAP subspecies and suggest the existence of different genotypes within this Type III group. The results could serve as further evidence that Type I and Type III groups are more closely related to each other than to the bovine Type II group.

Cloning, biochemical properties, and distribution of mycobacterial haloalkane dehalogenases.

Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M. bovis 5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M. africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45 degrees C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47 degrees C and DmbB is 57 degrees C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.