Irmgard Moser

Characterization of Mycobacterium caprae Isolates from Europe by Mycobacterial Interspersed Repetitive Unit Genotyping

ABSTRACT Mycobacterium caprae, a recently defined member of the Mycobacterium tuberculosis complex, causes tuberculosis among animals and, to a limited extent, in humans in several European countries. To characterize M. caprae in comparison with other Mycobacterium tuberculosis complex members and to evaluate genotyping methods for this species, we analyzed 232 M. caprae isolates by mycobacterial interspersed repetitive unit (MIRU) genotyping and by spoligotyping. The isolates originated from 128 distinct epidemiological settings in 10 countries, spanning a period of 25 years. We found 78 different MIRU patterns (53 unique types and 25 clusters with group sizes from 2 to 9) but only 17 spoligotypes, giving Hunter-Gaston discriminatory indices of 0.941 (MIRU typing) and 0.665 (spoligotyping). For a subset of 103 M. caprae isolates derived from outbreaks or endemic foci, MIRU genotyping and IS6110 restriction fragment length polymorphism were compared and shown to provide similar results. MIRU loci 4, 26, and 31 were most discriminant in M. caprae, followed by loci 10 and 16, a combination which is different than those reported to discriminate M. bovis best. M. caprae MIRU patterns together with published data were used for phylogenetic inference analysis employing the neighbor-joining method. M. caprae isolates were grouped together, closely related to the branches of classical M. bovis, M. pinnipedii, M. microti, and ancestral M. tuberculosis, but apart from modern M. tuberculosis. The analysis did not reflect geographic patterns indicative of origin or spread of M. caprae. Altogether, our data confirm M. caprae as a distinct phylogenetic lineage within the Mycobacterium tuberculosis complex.

Genomic Heterogeneity and O-Antigenic Diversity of Campylobacter upsaliensis and Campylobacter helveticus Strains Isolated from Dogs and Cats in Germany

ABSTRACT A serotyping scheme based on heat-stable surface antigens was established for 101 Campylobacter upsaliensis and 10Campylobacter helveticus strains isolated from 261 dogs and 46 cats of different ages originating from two geographically distinct regions in Germany. The prevalence of C. upsaliensis varied between 27.8% in juvenile dogs (<12 months of age) and 55.4% in adult dogs (P < 0.05). Of the cats, 19.6% harbored C. upsaliensis, whereas 21.7% carried C. helveticus. Of the C. upsaliensis isolates from both host species, 93.1% belonged to five different serogroups, two of them being prevalent at rates of 47.5 and 27.7%, with different frequencies in both regions. Six (54.6%) of the C. helveticus isolates also belonged to serotypes found among C. upsaliensis strains, whereas five (45.4%) possessed an O antigen unique for C. helveticus. In contrast, a considerable degree of genomic diversity of the isolates was assessed by macrorestriction analyses with the endonucleases SmaI and XhoI, using pulsed-field gel electrophoresis as well as enterobacterial repetitive intergenic consensus sequence PCR (ERIC PCR). Restriction with SmaI pointed towards the existence of clonal groups associated to some extent with serotypes, while restriction withXhoI disintegrated these groups to smaller noncoherent subgroups. Analysis of ERIC PCR profiles did not exhibit any associations with serotypes. In conclusion these data demonstrate the genomic heterogeneity among C. upsaliensis strains and indicate that the combination of SmaI restriction with serotyping is a useful tool to investigate the expansion of clonal groups of C. upsaliensis.

Rapid Spoligotyping of Mycobacterium tuberculosis Complex Bacteria by Use of a Microarray System with Automatic Data Processing and Assignment

ABSTRACT Membrane-based spoligotyping has been converted to DNA microarray format to qualify it for high-throughput testing. We have shown the assays validity and suitability for direct typing from tissue and detecting new spoligotypes. Advantages of the microarray methodology include rapidity, ease of operation, automatic data processing, and affordability.

Bovine tuberculosis and brucellosis in cattle and African buffalo in the Limpopo National Park, Mozambique.

Bovine tuberculosis (BTB) and brucellosis are prevalent in buffaloes of the Kruger National Park (KNP, South Africa). Both diseases were considered to have no or a very low prevalence in wildlife and livestock in and around the Limpopo National Park (LNP, Mozambique). The same applies for tuberculosis in Gonarezhou National Park (GNP, Zimbabwe), but just recently, BTB was detected in buffaloes in the GNP and fears arose that the disease might also spread to the LNP as a result of the partial removal of the fences between the three parks to form the Great Limpopo Transfrontier Park. To assess the status of both diseases in and around LNP, 62 buffaloes were tested for bovine tuberculosis (BTB) and bovine brucellosis. The percentage of positive BTB reactors in buffalo was 8.06% using BovidTB Stat-Pak® and 0% with BOVIGAM® IFN-γ test and IDEXX ELISA. The brucellosis seroprevalence in buffalo was found to be 17.72% and 27.42% using Rose Bengal Test (RBT) and ELISA, respectively. In addition, 2445 cattle in and around the LNP were examined for BTB using the single intradermal cervical comparative tuberculin test (SICCT), and an apparent prevalence of 0.98% was found with no significant difference inside (0.5%) and outside (1.3%) the park. This is the first published report on the presence of positive reactors to BTB and bovine brucellosis in buffalo and cattle in and outside the LNP. Monitoring the wildlife-livestock-human interface of zoonotic high-impact diseases such as BTB and brucellosis is of outmost importance for the successful implementation and management of any transfrontier park that aims to improve the livelihoods of the local communities.

Outbreak of reproductive disorders and mycobacteriosis in swine associated with a single strain of Mycobacterium avium subspecies hominissuis

In a breeding and fattening pig farm an increasing number of cases of abortion and generalized mycobacteriosis at slaughter occurred. Pathological findings compatible with mycobacteriosis, acid-fast organisms in tissues, and isolation of mycobacteria from tissue samples including fetuses, lungs and reproductive organs from sows, genital swabs, mesenteric lymph nodes, and from a sperm sample revealed the cause of the disease. Bacterial cultures were identified as Mycobacterium avium subsp. hominissuis using IS901-/IS1245-specific PCR. Genotyping of selected isolates from animals as well as from their environment by MIRU-VNTR analysis showed that the herd was infected with one single outbreak strain. The same genotype was also isolated from pigs of two other farms which showed comparable symptoms and were in direct contact with the index farm as well as from their environment. Immunological host responses detected by tuberculin skin test and ELISA gave positive results at herd level only. Despite the detection of other potential pathogens mycobacteria were regarded as the causative agent of the reproductive disorders. To our knowledge this is the first report of an epidemic mycobacterial infection in a pig holding associated with reproductive disorders, which could be attributed to one single virulent strain, and the first report of detection of M. avium subsp. hominissuis in pig sperm.

High-Resolution Genotyping of Campylobacter upsaliensis Strains Originating from Three Continents

ABSTRACT Ninety-six Campylobacter upsaliensis strains that originated from Australia, Canada, and Europe (Germany) and that were isolated from humans, dogs, and cats were serotyped for their heat-stable surface antigens. All of them were genotyped by enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) profiling, and 83 strains were genotyped by macrorestriction analysis with the endonuclease XhoI. Eighty-four percent of the strains belonged to five different serotypes (serotypes OI, OII, OIII, OIV, and OVI), with the proportions of strains in each serotype being comparable among the groups of strains from all three continents. Two serotypes, OIII and OIV, were prevalent at rates of 35 to 40%. Serotypes OI, OII, and OVI were detected at rates of 1.5 to 15%. Between 10 and 17.7% of the strains did not react with the available antisera. Analysis of the ERIC-PCR profiles revealed two distinct genotypic clusters, which represented the German and the non-European strains, respectively. XhoI macrorestriction yielded two genotypic clusters; one of them contained 80.2% of the German strains and 34.6% of the non-European strains, and the second cluster consisted of 65.4% of the non-European strains and 19.8% of the German strains. Fourteen strains from all three continents were analyzed for their 16S rRNA gene sequences. Only two minor variations were detected in four of the strains. In conclusion, C. upsaliensis has undergone diverging processes of genome arrangement on different continents during evolution without segregating into different subspecies.

Molecular characterization of Mycobacterium tuberculosis isolates from Tanga, Tanzania: First insight of MIRU-VNTR and microarray-based spoligotyping in a high burden country.

Molecular typing of Mycobacterium tuberculosis(MTB) has greatly enhanced the understanding of the population structure of MTB isolates and epidemiology of tuberculosis (TB). To characterize prevalent genotypes of MTB, microarrays‑based spoligotyping and mycobacterial interspersed repetitive unit‑variable number of tandem repeats (MIRU‑VNTR) were applied on 80 isolates collected from primary health care facilities in Tanga, North‑eastern Tanzania. A total of 18 distinct spoligotypes were identified. The lineages by order of their predominance were EAI and CAS families (26.25%, 21 isolates each), LAM family and T super‑family (10%, 8 isolates each), MANU family (3.75%, 3 isolates), Beijing family (2.5%, 2 isolates) and S family (1.25%, 1 isolate). Overall, sixteen (20%) strains could not be allocated to any lineage according to the SITVIT_WEB database. The allelic diversity (h) for specific MIRU‑VNTR loci showed a considerable variation ranging from 0.826 of VNTR locus 3192 to 0.141 of VNTR locus 2059. The allelic diversity for 11 loci (VNTR 3192, 2996, 2165, 960, 4052, 424, 4156, 2531, 1644, 802 and 3690) exceeded 0.6, indicating highly discriminatory power. Seven loci (VNTR 2163b, 2401, 1955, 577, 4348, 2687 and 580) showed moderate discrimination (0.3 ≤ h ≥ 0.6), and three loci (VNTR3007, 154 and 2059) were less polymorphic. The present study suggests that the TB cases in Tanga might be caused by a diverse array of MTB strain families that may be indicative of a cosmopolitan population with frequent migration and travel. Microarray‑based spoligotyping and MIRU‑VNTR could be reliable tools in detecting different MTB genotypes in high burden settings.

Mycobacterial Infections in Free-living Cervids in Germany (2002–2006)

We examined 1,022 free-living roe deer, red deer, and fallow deer for mycobacteria in Germany, 2002–2006. Retropharyngeal lymph nodes and other tissues were processed for culture and isolates were identified with the use of polymerase chain reaction and DNA sequencing. Mycobacteria were found in 18.3% of deer, with Mycobacterium avium in 14.8%. Other atypical mycobacteria were detected in 5.3%. Members of the Mycobacterium tuberculosis complex were not detected.

Concurrent infection with Mycobacterium avium subsp. hominissuis and Giardia duodenalis in a chinchilla (Chinchilla lanigera f. dom.).

A 3-year-old, female chinchilla (Chinchilla lanigera f. dom.) suffered from prolonged vaginal discharge. Sonographically, multiple nodules were detected in the uterus, and the lung showed a diffuse radiodensity. Ovario-hysterectomy was performed and histology of the uterus revealed a severe multifocal pyogranulomatous metritis with myriads of acid-fast rod-shaped bacilli. Microbiological culture of formalin-fixed uterine tissue and a native vaginal swab resulted in the growth of mycobacteria that were identified as Mycobacterium (M.) avium subsp. hominissuis. The animal was euthanized and pathomorphological examination revealed severe multifocal granulomatous inflammation of lung, mediastinal and mesenteric lymph nodes, intestine, pancreas and kidneys. In addition, an infection of the small intestine with Giardia duodenalis was confirmed immunohistochemically. This is the first report describing a concurrent infection with M. avium subsp. hominissuis and Giardia duodenalis in a chinchilla. Both pathogens represent a potential health risk especially for young or immunosuppressed persons, in particular if infected animals show unspecific clinical symptoms.

Mycobacterium avium subspecies hominissuis infection in a dog from Germany with multifocal alopecia, exfoliative dermatitis, hypercalcaemia and subsequent sebaceous atrophy

Mycobacterium avium subspecies hominissuis belongs to the M avium complex and infections with this agent are reported rarely in the dog. A nine-year-old intact female Elo dog was presented with widespread hypotrichosis, erythema and exfoliation. Other clinical signs were polyuria/polydipsia and severe weight loss. No other animals or humans in the household showed any dermatological abnormalities. Multiple skin biopsies identified a pyogranulomatous dermatitis but no causative agent could be detected. Skin tissue culture and PCR revealed M avium subspecies hominissuis. Hypercalcaemia (ionised calcium) was initially present. All other serum biochemistry parameters, chest radiographs, abdominal ultrasound, parathyroid hormone and parathyroid hormone-related protein were within the reference interval. Subsequent to therapy with rifampicin, pradofloxacin and oral fatty acids the pyogranulomatous dermatitis resolved and mycobacteria could not be detected but sebaceous glands were lost histopathologically. This is the first report describing a dog infected with M avium subspecies hominissuis successfully treated with antibiotic therapy and with no zoonotic transmission.