Hominissuis Isolates Research Articles

Characterization of DNA Gyrase Activity and Elucidation of the Impact of Amino Acid Substitution in GyrA on Fluoroquinolone Resistance in Mycobacterium avium.

Mycobacterium avium, a member of the M. avium complex (MAC), is the major pathogen contributing to nontuberculous mycobacteria (NTM) infections worldwide. Fluoroquinolones (FQs) are recommended for the treatment of macrolide-resistant MACs. The association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA of M. avium is not yet clearly understood, as many FQ-resistant clinical M. avium isolates do not have such mutations. This study aimed to elucidate the role of amino acid substitution in the QRDR of M. avium GyrA in the development of FQ resistance. We found four clinical M. avium subsp. hominissuis isolates with Asp-to-Gly change at position 95 (Asp95Gly) and Asp95Tyr mutations in gyrA that were highly resistant to FQs and had 2- to 32-fold-higher MICs than the wild-type (WT) isolates. To clarify the contribution of amino acid substitutions to FQ resistance, we produced recombinant WT GyrA, GyrB, and four GyrA mutant proteins (Ala91Val, Asp95Ala, Asp95Gly, and Asp95Tyr) to elucidate their potential role in FQ resistance, using them to perform FQ-inhibited DNA supercoiling assays. While all the mutant GyrAs contributed to the higher (1.3- to 35.6-fold) FQ 50% inhibitory concentration (IC50) than the WT, Asp95Tyr was the most resistant mutant, with an IC50 15- to 35.6-higher than that of the WT, followed by the Asp95Gly mutant, with an IC50 12.5- to 17.6-fold higher than that of the WT, indicating that these amino acid substitutions significantly reduced the inhibitory activity of FQs. Our results showed that amino acid substitutions in the gyrA of M. avium contribute to FQ resistance. IMPORTANCE The emergence of fluoroquinolone (FQ) resistance has further compounded the control of emerging Mycobacterium avium-associated nontuberculous mycobacteria infections worldwide. For M. avium, the association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA is not yet clearly understood. Here, we report that four clinical M. avium isolates with a mutation in the QRDR of gyrA were highly resistant to FQs. We further clarified the impact of mutations in the QRDR of GyrA proteins by performing in vitro FQ-inhibited DNA supercoiling assays. These results confirmed that, like in Mycobacterium tuberculosis, mutations in the QRDR of gyrA also strongly contribute to FQ resistance in M. avium. Since many FQ-resistant M. avium isolates do have these mutations, the detailed molecular mechanism of FQ resistance in M. avium needs further exploration.

Genetic relatedness of Mycobacterium avium subsp. hominissuis isolates from bathrooms of healthy volunteers, rivers, and soils in Japan with human clinical isolates from different geographical areas

Japan reportedly has high incidence rate of nontuberculous mycobacterial lung disease (14.7 cases per 100,000 person in 2014). In Japan, the most common etiology is Mycobacterium avium subsp. hominissuis (MAH). MAH is a typical inhabitant of the environment, especially bathrooms, which are considered as a potential source of infection. To corroborate this hypothesis, we determined the detection rate of MAH in bathrooms of healthy volunteers by an ordinary culture method and we analyzed the genetic relatedness of these isolates with those from patients and other sources. We collected swabs of bathtub inlets, showerheads, bathroom drains, and shower water from 180 residences throughout Japan. The overall MAH detection rate was 16.1%, but the rate varied among regions: it was high in Kanto (9/34, 26.5%) and Kinki (9/33, 27.3%), but low in Kyushu (0/11, 0%), Tohoku (1/23, 4.3%), and Hokkaido (2/23, 8.7%). MAH was detected primarily in bathtub inlet samples (25 out of 170 residences). Variable numbers of tandem repeats (VNTR) analysis was used to examine the genetic relatedness of 57 MAH isolates from bathrooms of the healthy volunteers with human clinical isolates. A minimum spanning tree generated on the basis of the VNTR data indicated that isolates from the bathrooms of the healthy volunteers had a high degree of genetic relatedness with those from Japanese patients, bathrooms of patients, and river water, but not with those from Russian patients and Japanese pigs. These results showed that bathtub inlets in Japan provide an environmental niche for MAH and suggest that bathrooms are one of the important infection sources of MAH in Japan. Understanding country-specific lifestyle habits, such as bathing in Japan, as well as the genetic diversity of MAH, will help in elucidating the sources of this pathogen.

Metabolic phenotype of clinical and environmental Mycobacterium avium subsp. hominissuis isolates.

BackgroundMycobacterium avium subsp. hominissuis (MAH) is an emerging opportunistic human pathogen. It can cause pulmonary infections, lymphadenitis and disseminated infections in immuno-compromised patients. In addition, MAH is widespread in the environment, since it has been isolated from water, soil or dust. In recent years, knowledge on MAH at the molecular level has increased substantially. In contrast, knowledge of the MAH metabolic phenotypes remains limited.MethodsIn this study, for the first time we analyzed the metabolic substrate utilization of ten MAH isolates, five from a clinical source and five from an environmental source. We used BIOLOG Phenotype MicroarrayTM technology for the analysis. This technology permits the rapid and global analysis of metabolic phenotypes.ResultsThe ten MAH isolates tested showed different metabolic patterns pointing to high intra-species diversity. Our MAH isolates preferred to use fatty acids such as Tween, caproic, butyric and propionic acid as a carbon source, and L-cysteine as a nitrogen source. Environmental MAH isolates resulted in being more metabolically active than clinical isolates, since the former metabolized more strongly butyric acid (p = 0.0209) and propionic acid (p = 0.00307).DiscussionOur study provides new insight into the metabolism of MAH. Understanding how bacteria utilize substrates during infection might help the developing of strategies to fight such infections.

Molecular typing and genetic characterization of Mycobacterium avium subsp. hominissuis isolates from humans and swine in Japan.

Mycobacterium avium subsp. hominissuis (MAH) causes disease in both humans and swine; however, the genetic variations in MAH isolates are unclear. The aim of this study was to elucidate the genetic variations in MAH isolates from humans and swine in Japan. We analysed the 16S-23S rDNA internal transcribed spacer (ITS) sequence and variable number of tandem repeats (VNTRs) using the Mycobacterium avium tandem repeat loci, prevalence of ISMav6 and clarithromycin resistance for MAH isolates from patients with pulmonary MAC (pMAC) disease (n=69), and HIV-seropositive and blood culture-positive (HIV-MAC) patients (n=28) and swine (n=23). In the minimum spanning tree based on VNTR analysis, swine MAC isolates belonged to a cluster distinguishable from that of human pMAC isolates. Isolates from HIV-MAC were scattered throughout both clusters. The three major distinct sequevars, Mav-A, Mav-B and Mav-F, were determined according to 16S-23S rDNA ITS sequence analysis in addition to three new sequevars, Mav-Q, Mav-R and Mav-S. Mav-A and Mav-F comprised the majority of human pMAC strains; in contrast, Mav-B predominated in swine isolates. Distribution of ITS sequevars in the minimum spanning tree based on VNTR analysis showed similar clusters of isolates from different origins, i.e. human pMAC, HIV-MAC and swine. These results, together with ISMav6 possession and clarithromycin resistance, revealed the genetic diversity of MAH strains recovered from humans and swine. Molecular epidemiology and genetic characterization in the present study showed the distinctive genetic evolutionary lineage of MAH strains isolated from human pMAC diseases and swine.

A hypervariable genomic island identified in clinical and environmental Mycobacterium avium subsp. hominissuis isolates from Germany

Mycobacterium avium subsp. hominissuis (MAH) is an opportunistic human pathogen widespread in the environment. Genomic islands (GI)s represent a part of the accessory genome of bacteria and influence virulence, drug-resistance or fitness and trigger bacterial evolution. We previously identified a novel GI in four MAH genomes. Here, we further explored this GI in a larger collection of MAH isolates from Germany (n=41), including 20 clinical and 21 environmental isolates. Based on comparative whole genome analysis, we detected this GI in 39/41 (95.1%) isolates. Although all these GIs integrated in the same insertion hotspot, there is high variability in the genetic structure of this GI: eight different types of GI have been identified, designated A–H (sized 6.2–73.3kb). These GIs were arranged as single GI (23/41, 56.1%), combination of two different GIs (14/41, 34.1%) or combination of three different GIs (2/41, 4.9%) in the insertion hotspot. Moreover, two GI types shared more than 80% sequence identity with sequences of M. canettii, responsible for Tuberculosis. A total of 253 different genes were identified in all GIs, among which the previously documented virulence-related genes mmpL10 and mce. The diversity of the GI and the sequence similarity with other mycobacteria suggests cross-species transfer, involving also highly pathogenic species. Shuffling of potential virulence genes such as mmpL10 via this GI may create new pathogens that can cause future outbreaks.

Association of ISMav6 with the Pattern of Antibiotic Resistance in Korean Mycobacterium avium Clinical Isolates but No Relevance between Their Genotypes and Clinical Features.

The aim of this study was to genetically characterize clinical isolates from patients diagnosed with Mycobacterium avium lung disease and to investigate the clinical significance. Multi-locus sequencing analysis (MLSA) and pattern of insertion sequence analysis of M. avium isolates from 92 Korean patients revealed that all isolates were M. avium subspecies hominissuis. In hsp65 sequevar analysis, codes 2, 15, and 16 were most frequently found (88/92) with similar proportions among cases additionally two isolates belonging to code N2 and an unreported code were identified, respectively. In insertion element analysis, all isolates were IS1311 positive and IS900 negative. Four of the M. avium subsp. hominissuis isolates did not harbor IS1245 and 1 of the M. avium isolates intriguingly harbored DT1, which is thought to be a M. intracellulare-specific element. M. avium subsp. hominissuis harboring ISMav6 is prevalent in Korea. No significant association between clinical manifestation and treatment response has been found in patients with the hsp65 code type and ISMav6, indicating that no specific strain/genotype among M. avium subsp. hominissuis organisms was a major source of M. avium lung disease. Interestingly, the presence of ISMav6 was correlated with greater resistance to moxifloxacin. Conclusively, the genotype of Korean M. avium subsp. hominissuis isolates is not a disease determinant responsible for lung disease and specific virulent factors of M. avium subsp. hominissuis need to be investigated further.

Genetic diversity of clinical Mycobacterium avium subsp. hominissuis and Mycobacterium intracellulare isolates causing pulmonary diseases recovered from different geographical regions

Mycobacterium avium complex (MAC) infections are increasing annually in many countries. MAC strains are the most common nontuberculous mycobacterial pathogens isolated from respiratory samples and predominantly consist of two species, Mycobacterium avium and Mycobacterium intracellulare. The aim of this study was to analyze the molecular epidemiology and genetic backgrounds of clinical MAC isolates collected from The Netherlands, Germany, United States, Korea and Japan. Variable numbers of tandem repeats (VNTR) analysis was used to examine the genetic relatedness of clinical isolates of M. avium subsp. hominissuis (n=261) and M. intracellulare (n=116). Minimum spanning tree and unweighted pair group method using arithmetic averages analyses based on the VNTR data indicated that M. avium subsp. hominissuis isolates from Japan shared a high degree of genetic relatedness with Korean isolates, but not with isolates from Europe or the United States, whereas M. intracellulare isolates did not show any specific clustering by geographic origin. The findings from the present study indicate that strains of M. avium subsp. hominissuis, but not M. intracellulare, exhibit geographical differences in genetic diversity and imply that MAC strains may have different sources, routes of transmission and perhaps clinical manifestations.

Molecular characterization of Mycobacterium avium subspecies hominissuis isolated from humans, cattle and pigs in the Uganda cattle corridor using VNTR analysis

BackgroundMembers of the Mycobacterium avium complex (MAC) cause disease in both human and animals. Their ubiquitous nature makes them both successful microbes and difficult to source track. The precise characterization of MAC species is a fundamental step in epidemiological studies and evaluating of possible reservoirs. This study aimed at identifying and characterizing Mycobacterium avium subsp. hominissuis isolated from human, slaughter cattle and pigs in various parts of the Uganda cattle corridor (UCC) at two temporal points using variable number of tandem repeat (VNTR) analysis. MethodsA total of 46 M. avium isolates; 31 from 997 pigs, 12 from 43 humans biopsies and three from 61 cattle lesions were identified to subspecies level using IS1245 and IS901 PCR, thereafter characterized using VNTR. Twelve loci from two previously described VNTR methods were used and molecular results were analyzed and interpreted using Bionumerics 6.1. Principal findings37 of the isolates were identified as M. avium subsp. hominissuis and four as M. avium subsp. avium, while five could not be differentiated, possibly due to mixed infection. There was distinct clustering that coincides with the temporal and spatial differences of the isolates. The isolates from humans and cattle in the North Eastern parts of the UCC shared identical VNTR genotypes. The panel of loci gave an overall discriminatory power of 0.88. Some loci were absent in several isolates, probably reflecting differences in isolates from Uganda/Africa compared to isolates previously analyzed by these methods in Europe and Asia. ConclusionsThe findings indicate a molecular difference between M. avium subsp. hominissuis isolates from pigs in Mubende and cattle and human in the rest of the UCC. Although human and cattle shared VNTR genotypes in the North Eastern parts of the UCC, it is most likely a reflection of a shared environmental source.

Survival of three Mycobacterium avium subsp. hominissuis isolates in fish products after hot smoking and frying

SummaryFish mycobacteriosis should be considered as one possible means of transmission of mycobacterial infections to humans. In our study, we examined the survival of three field Mycobacterium avium subsp. hominissuis (MAH) isolates in fish meat during thermal processing technologies using culture and quantitative real‐time PCR (qPCR) methods. Minced carp meat mixture was artificially contaminated with known amount of MAH cells and survival and absolute numbers of MAH was monitored after hot smoking and frying. The viability of MAH was significantly lower after the frying process, whereas in response to hot smoking viable MAH cells could still be cultured even after 10 min at 70 °C. Significant differences in thermal stability were also observed among the three different MAH isolates. The human isolate was the most resistant, whereas the environmental isolate was the least resistant, which is probably due to host adaptability. In this work we confirmed that MAH can survive temperatures of 70 °C and thus can pose a risk to consumers.

Mycobacterium avium Subsp. avium and Subsp. hominissuis Give Different Cytokine Responses after in vitro Stimulation of Human Blood Mononuclear Cells

Background Mycobacterium avium is the principal etiologic agent of non-tuberculous lymphadenitis in children. It is also a known pathogen for birds and other animals. Genetic typing of M. avium isolates has led to a proposal to expand the set of subspecies to include M. avium subsp. hominissuis. Isolates associated with disease in humans belong to this subspecies.Methodology/Principal FindingsPeripheral blood mononuclear cells from six healthy blood donors were stimulated in vitro with ten isolates of M. avium avium and 11 isolates of M. avium hominissuis followed by multiplex bead array quantification of cytokines in supernatants. M. avium hominissuis isolates induced significantly more IL-10 and significantly less IL-12p70, TNF, IFN-γ and IL-17 when compared to M. avium avium isolates. All strains induced high levels of IL-17, but had very low levels of IL-12p70.Conclusion/SignificanceThe strong association between M. avium subsp. hominissuis and disease in humans and the clear differences in the human immune response to M. avium subsp. hominissuis compared to M. avium subsp. avium isolates, as demonstrated in this study, suggest that genetic differences between M. avium isolates play an important role in the pathogenicity in humans.

Diversity of Mycobacterium avium subsp. hominissuis mycobacteria causing lymphadenitis, France

The knowledge of Mycobacterium avium complex (MAC) genotypes responsible for lymphadenitis is limited. We retrospectively characterized all of the MAC isolates made in our laboratory in the last 18 years by sequence-based identification and genotyping, and compared the clinical and laboratory data for lymphadenitis-associated and non-lymphadenitis-associated MAC isolates. Of 67 MAC-infected patients, 25 lymphadenitis patients were significantly younger than 42 non-lymphadenitis patients, while the male/female ratio did not significantly differ between the two groups. Cervical topography found in 76.5% of lymphadenitis patients was significantly more frequent in non-immunocompromised patients (p=0.04). M. avium subsp. hominissuis was identified in 53 patients (24 lymphadenitis, 29 non-lymphadenitis), M. colombiense in six patients (five non-lymphadenitis, one lymphadenitis), M. intracellulare in four non-lymphadenitis patients, and M. chimaera in three non-lymphadenitis patients, while negative controls remained negative. M. hominissuis was significantly associated with lymphadenitis (p=0.03). M. hominissuis isolates yielded 15 genotypes in 29 non-lymphadenitis isolates (molecular diversity, 0.622) versus 11 genotypes in 24 lymphadenitis isolates (molecular diversity, 0.578), demonstrating a non-significant lower diversity of M. hominissuis isolates cultured from lymphadenitis. The genotypes did not correlate with the clinical features. These data suggest the presence of several environmental reservoirs for M. hominissuis causing lymphadenitis in France.

Mycobacterium chimaera pulmonary infection complicating cystic fibrosis: a case report

BackgroundMycobacterium chimaera is a recently described species within the Mycobacterium avium complex. Its pathogenicity in respiratory tract infection remains disputed. It has never been isolated during cystic fibrosis respiratory tract infection.Case presentationAn 11-year-old boy of Asian ethnicity who was born on Réunion Island presented to our hospital with cystic fibrosis after a decline in his respiratory function over the course of seven years. We found that the decline in his respiratory function was correlated with the persistent presence of a Mycobacterium avium complex organism further identified as M. chimaera.ConclusionUsing sequencing-based methods of identification, we observed that M. chimaera organisms contributed equally to respiratory tract infections in patients with cystic fibrosis when compared with M. avium subsp. hominissuis isolates. We believe that M. chimaera should be regarded as an emerging opportunistic respiratory pathogen in patients with cystic fibrosis, including young children, and that its detection warrants long-lasting appropriate anti-mycobacterial treatment to eradicate it.

MIRU–VNTR typing of Mycobacterium avium in animals and humans: Heterogeneity of Mycobacterium avium subsp. hominissuis versus homogeneity of Mycobacterium avium subsp. avium strains

Epidemiological studies on Mycobacterium avium are requisite for revealing infection sources and disease transmission. They are based upon genotyping methods like RFLP and MIRU–VNTR. In our study, MIRU–VNTR typing was applied to 121 previously RFLP typed M. avium field isolates to compare the discriminatory power of both methods. The applicability of MIRU–VNTR typing was studied for isolates from a limited geographic area, namely 41 M. avium subsp. avium and 80 M. avium subsp. hominissuis isolates. Among the former, exhibiting 12 IS 901 RFLP types, five MIRU–VNTR types were found with discriminatory index ( DI) of 0.716. Among the latter, exhibiting 56 IS 1245 RFLP types, 18 MIRU–VNTR types were found with DI of 0.866. Concomitant use of both methods increased DI to 0.981 and 0.995, respectively. MIRU–VNTR typing employing the selected markers provided discernible discrimination among M. avium subsp. hominissuis isolates, but more discriminative markers are needed for M. avium subsp. avium isolates.

IS1245 RFLP-based genotyping study of Mycobacterium avium subsp. hominissuis isolates from pigs and humans

Mycobacterium avium subsp. hominissuis, ubiquitous environmental mycobacterium, is an opportunistic pathogen that is regularly isolated from pigs and humans in Slovenia. Genetic diversity of 114 isolates from pigs (n = 57) and humans (n = 57), identified by means of bacteriology, DNA-RNA hybridization techniques, IS901 PCR and IS1245 PCR, was investigated in this study, using IS1245 restriction fragment length polymorphism (RFLP) analysis. Identical IS1245 RFLP profiles were found in isolates from pigs, isolates from humans and isolates from both origins. The proportion of clustered isolates varied as it depended on the similarity level (100% and 75%) chosen for the cluster limits. Using IS1245 RFLP, it was possible to detect monoclonal, polyclonal and recent infections and to monitor the genetic variability of the strains from individual patients. Our findings indicate the environment as the source of infection for both pigs and humans. The questions about the possibility of intra- and inter-species transmission of infection remain to be answered.

High genetic relatedness among Mycobacterium avium strains isolated from pigs and humans revealed by comparative IS 1245 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 IS 1245 RFLP analysis to evaluate the similarity of the isolates obtained from human and porcine samples. Nearly identical multicopy M. avium subsp. hominissuis IS 1245 RFLP fingerprints were obtained for isolates of porcine and human origin. IS 1245 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 IS 1245 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.

New probes used for IS1245 and IS1311 restriction fragment length polymorphism of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis isolates of human and animal origin in Norway

BackgroundMycobacterium avium is an environmental mycobacterium that can be divided into the subspecies avium, hominissuis, paratuberculosis and silvaticum. Some M. avium subspecies are opportunistic pathogens for animals and humans. They are ubiquitous in nature and can be isolated from natural sources of water, soil, plants and bedding material. Isolates of M. avium originating from humans (n = 37), pigs (n = 51) and wild birds (n = 10) in Norway were examined by IS1245 and IS1311 RFLP using new and specific probes and for the presence of IS901 and ISMpa1 by PCR. Analysis and generation of a dendrogram were performed with the software BioNumerics.ResultsIS1311 RFLP provided clear results that were easy to interpret, while IS1245 RFLP generated more complex patterns with a higher discriminatory power. The combination of the two methods gave additional discrimination between isolates. All avian isolates except one were M. avium subsp. avium with two copies of IS1311 and one copy of IS1245, while the isolates of human and porcine origin belonged to M. avium subsp.hominissuis. The isolates from human patients were distributed randomly among the clusters of porcine isolates. There were few identical isolates. However, one isolate from a human patient was identical to a porcine isolate. Regional differences were detected among the porcine isolates, while there was no clustering of human isolates according to type of clinical symptoms or geographical location of the patient's home addresses.ConclusionThe results demonstrate that a wide range of M. avium subsp.hominissuis are present in pigs and humans in Norway, and that some of these isolates are very similar. It remains to be determined whether humans are infected from pigs or if they are infected from common environmental sources.

Effect of feeding treated peat as a supplement to newborn piglets on the growth, health status and occurrence of conditionally pathogenic mycobacteria

The first purpose of this experiment was to investigate the effect of ad libitum feeding of peat as a supplement to piglets from the age of five days up 23 days of age on their growth performance and health status. The second purpose was to assess the risk of the occurrence of conditionally pathogenic mycobacteria (CPM) in peat treated with ionizing radiation (Group PI) or per acetic acid (Group PP) and fed as a supplement to piglets. In respective experimental periods (at the age of 4, 23, 41 and 67 days), no significant differences in the average body weight between control group (C) and experimental Groups PI and PP were detected. Levels of selected biochemical (total protein, albumin, glucose, cholesterol, Ca, P, Fe and I) and haematological (erythrocytes, leukocytes and immunoglobulin – Ig) parameters of the health status of the piglets from all three Groups C, PI and PP were comparable at the age of 41 and 67 days. Mycobacteria were detected by culture in one diet sample (Mycobacterium intracellulare), in all 10 peat samples (7 M. a. hominissuis isolates, 2 M. intracellulare isolates and 1 M. xenopi isolate) and in 4 samples of biofilm from the drinking water pipeline system in the stables (M. xenopi, M. a. hominissuis, M. gordonae and Mycobacterium sp., one isolate in each). In 15 slaughtered pigs (at 67 days of age), no gross lesions that would give evidence of tuberculosis were found either in lymph nodes or parenchymatous organs. In Group C, mycobacteria were detected in tissues from two piglets (Mycobacterium sp. and M. a. hominissuis), Group PI in four piglets (M. a. hominissuis) and in Group PP in all five piglets (Mycobacterium sp., M. a. hominissuis, M. terrae and M. intracellulare). High positivity for CPM in both types of treated peat caused disseminated infection of the digestive tract of piglets from Groups PI and PP. Based on these results, feeding peat treated with ionisation or per acetic acid may be viewed as risky.

Avian tuberculosis in naturally infected captive water birds of the Ardeideae and Threskiornithidae families studied by serotyping, IS 901 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 IS 901+ and IS 1245+) 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 IS 901 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 IS 901 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 IS 1245 RFLP “bird profile”. In 12 of 20 infected birds with MAA M.a. hominissuis isolates of serotypes 4, 8, 9 and genotype IS 901− and IS 1245+ 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.

INFECTIONS CAUSED BY MYCOBACTERIUM AVIUM SUBSPECIES AVIUM, HOMINISSUIS, AND PARATUBERCULOSIS IN FREE-RANGING RED DEER (CERVUS ELAPHUS HIPPELAPHUS) IN AUSTRIA, 2001–2004

Between 2001 and 2004, 14 Austrian free-ranging red deer (Cervus elaphus hippelaphus) infected by Mycobacterium avium species were observed. Eight of the cases were from different geographical regions, and six originated from the same hunting area. The affected animals had signs of diarrhea, severe weight loss, and emaciation. On post-mortem examination, lymphadenitis associated with grossly enlarged mesenteric lymph nodes as well as multiple caseous or purulent nodular lesions in the thickened wall of the intestines were present in all animals. In 10 cases M. avium subsp. avium and in four cases M. a. hominissuis were isolated. In three red deer, a mixed infection with M. a. hominissuis and M. a. paratuberculosis was evident. Typing of M. a. avium and M. a. hominissuis isolates was performed by polymerase chain reaction (PCR) detection of insertion sequence IS901 and the virulence-associated macrophage-induced gene (mig), inverted repeat (IR) typing (IS1245/IS1311), and random amplified polymorph DNA (RAPD) analysis. While all M. a. avium and M. a. hominissuis contained the mig gene, IS901 was detected only in M. a. avium. The prevalence of IS901-positive isolates correlated well with the geographic location of affected animals. The IS901-containing isolates were shown to be genotypically closely related, as they exhibit similar patterns in IR-typing and in RAPD analysis. In contrast, IS901-negative isolates (M. a. hominissuis) displayed distinct profiles in both molecular systems.

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 IS 1245 and IS 901 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. IS 1245-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 IS 901-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.