Reedbed structure and habitat preference of reed passerines during the post-breeding period

Post-breeding habitats of nine passerine species were studied in the permanently flooded reed beds of ponds near the village Pusté úlany (SW Slovakia) in 2002. Structural features of reed beds and the abundance of all birds studied were sampled at two sites and eight study plots. The reed bed structure and abundance of four passerines differed considerably between the individual study plots. The variations in vegetation structure were also caused by winter reed burning in the SE part of study area. Stems in burnt reed beds were smaller and thinner than unburnt. Abundance of the reed warbler and to a lesser extent sedge warbler was higher in tall reed beds, while the bearded tit preferred thick reed stems. Position of reed beds along the shore — open water gradient was an important factor in terms of abundance at the study plots in the case of the reed bunting. This species showed a strong preference for the reed bed edge with open water. Horizontal distribution of other species seems to be random, however, the birds with the exception of Savi’s warbler preferred the upper stratum of reed bed in vertical distribution. Our field data corroborate with those found in experimental studies concerning with ecomorphological characteristics of birds. Some inconsistencies, however, could also be caused by other factors (e.g. foraging preferences). Comparison of breeding and post-breeding habitat requirements of the studied bird species confirmed a more or less random distribution of the majority of species in the post-breeding period. Some variation was found in the cases of only the reed warbler and bearded tit.     

Phytoremediation of selenium using subsurface-flow constructed wetland

The potential of two plant species, Phragmites australis (common reed) and Typha latifolia (cattail), in the phytoremediation process of selenium (Se) was studied in subsurface-flow constructed wetland (SSF). Se was supplemented continuously at a concentration of 100 microg Se L(-1) in the inlet of the cultivation beds of the SSF. Water samples collected from the outlet of the Phragmites bed after 1, 3, 6, 9, and 12 wk of treatments showed that Se content was under detectable limits. Water samples collected from the Typha bed at the same five periods showed that Se concentrations in the outlet were 55, 47, 65, 76, and 25 microg/L, respectively. The results of bioaccumulation in the biomass of both species after 12 wk of treatment indicated that Typha plants accumulated Se mainly in fine roots. Phragmites accumulated Se mainly in leaves and rhizomes, and moderate levels were found in stems and fine organic materials. The results indicate that common reed is a very good species for Se phytoextraction and phytostabilization (immobilization) and that cattail is only a phytostabilization species. The use of common reed and cattail for Se phytoremediation in a SSF system and in constructed wetland models are discussed.     

Response to mowing of coastal brackish meadow plant communities along an elevational gradient

Over a six year period, 1979–1985, eight stands of till then ungrazed and unmown brackish meadow plant communities were treated by mowing once a year (August), in order to detect the response of the vegetation. The stands were distributed along a local elevation gradient, which was found to influence the composition of the vegetation through a complex-gradient, composed by unidirectional gradients in inundation, in ground water level and in Na as per cent of Ca+Mg+Na+K.
The mowing was carried out within 5 ×5 m treatment plots and the effect was indicated by quantitative analysis of the vegetation. A natural succession progressing at the locality, most distinctly manifested by expansion of Phragmites australis , was traced during the study period by means of unmown control plots.
The mowing caused increase in species diversity in all stands, mainly due to increase in evenness among the species.
The resistance of the stands to the mowing was found to differ in relation to elevation and to the initial composition of the vegetation. In upper geolittoral Festuca rubra stands the resistance was high and the mowing did not affect presence or colonisation of Phragmites . In upper-middle Festuca rubra stands resistance was lower, mainly indicated by suppression of Phragmites . Low elevation Scirpus maritimus reed beds on waterlogged soil was rather resistant, but four additional years of mowing led to a change into Agrostis stolonifera-Puccinellia maritima meadow. The resistance of low elevation Phragmites reed bed was low, as the mowing within a few years changed the reed bed into Agrostis stolonifera meadow.
The results were discussed in relation to the use of mowing as a nature conservation management tool in salt meadows.     

Putative in vitro expressed gene fragments unique to Mycobacterium avium subspecies paratuberculosis

By a suppression subtractive hybridization based method, nine novel Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) fragments of between 318 and 596 bp have been identified and characterized. Database search revealed little or no similarity with other mycobacteria. The uniqueness and diagnostic potential of seven of these fragments in relation to M. paratuberculosis closest relative Mycobacterium avium subsp. avium (M. avium) was confirmed by species-specific PCR and Southern blot. Furthermore, RT-PCR indicated that eight of the nine fragments originate from areas of the genome that are expressed in vitro.     

Identification and semiquantitation of Mycobacterium avium using a competitive PCR method.

A competitive PCR method with standard DNA (MIMIC) was developed for the rapid detection and semiquantitation of Mycobacterium avium (M. avium) using primers specific for the alpha antigen sequence of the bacteria. DNA from both M. avium and Mycobacterium marinum was amplified by polymerase chain reaction (PCR), but only M. avium could be detected by subsequent blotting confirmation with a probe specific for the bacteria. With the PCR and subsequent dot blot hybridization, as little as 10 fg of the M. avium DNA could be detected, equivalent to about 2 cells of the mycobacteria. In addition, we could distinguish 10(5) CFUs of M. avium from 10(4) CFUs or less by competitive PCR using a MIMIC. The present competitive PCR test enabled rapid identification and semiquantitation of M. avium, and could be used clinically to monitor disease severity and response to treatment of human M. avium disease.     

Characterization of the intracellular survival of Mycobacterium avium ssp. paratuberculosis: phagosomal pH and fusogenicity in J774 macrophages compared with other mycobacteria

The phagosomes containing viable pathogenic mycobacteria, such as Mycobacterium ( M .) tuberculosis and Mycobacterium avium ssp. avium ( M. avium ), are known to be limited in their ability to both acidify and fuse with late (but not early) endocytic organelles. Here, we analysed the pH and fusogenicity of phagosomes containing M. avium ssp. paratuberculosis ( M. ptb ), the causative agent of paratuberculosis in ruminants. Using the murine J774 macrophage cell line, we compared viable and heat-killed M. ptb and, in addition, viable or dead M. avium , as well as two non-pathogenic mycobacteria, Mycobacterium smegmatis and Mycobacterium gordonae . Electron microscopic analysis revealed that M. ptb persisted intracellularly in phagosomes for up to 15 days. The phagosomes containing live M. ptb and M. avium were significantly reduced in their ability to acquire some markers for the endocytic pathway, such as internalized calcein, BSA–gold or the membrane protein Lamp 2. However, they were almost completely accessible to 70 kDa fluorescein isothiocyanate (FITC)–dextran and Lamp 1. Overall, the phagosomes containing dead pathogenic mycobacteria behaved similarly to the ones containing live non-pathogenic mycobacteria in all experiments. Using FITC–dextran in a novel fluorescence-activated cell sorting (FACS)-based method, we could also show that the bulk of endocytic compartments, including phagosomes, were only very mildly acidified to ≈ pH 6.3 over at least 72 h in J774 cells infected with live M. ptb and M. avium . In contrast, J774 cells treated with heat-killed M. ptb or BSA-coated latex beads showed substantial acidification of the phagosome/endocytic compartments to a pH value of ≈ 5.2. After infection with M. smegmatis and M. gordonae , acidification was initially (1–5 h after infection) inhibited, but increased after longer infection to levels similar to those with dead mycobacteria.     

Comparison of the (CCG)4-based PCR and MIRU-VNTR for molecular typing of Mycobacterium avium strains

Mycobacterium avium, a member of the group of non-tuberculous mycobacteria, is most often responsible for the serious diseases in humans and is frequently isolated from NTM-caused pulmonary events. In this connection the epidemiological aspect is also of great importance. Here we present a useful genetic assay that uses (CCG)(4)-based PCR for genotyping M. avium. After applying this test to 33 strains of M. avium, we found a discriminatory index of 0.979. The accuracy of this analysis was supported by a reasonable reproducibility of 95.1%. These results were compared with the Mycobacterial Intergenic Repeat Unit-Variable Number Tandem Repeats (MIRU-VNTR) typing scheme which had slightly lower discriminatory index of 0.945 however, the method was able to cluster different strains compared to CCG-PCR. Taking into account high discriminatory index and reproducibility, this test scheme has the potential as a screening tool in the investigation of M. avium infections, especially if combined with MIRU-VNTR.     

Comparison of three DNA extraction methods for Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium subsp. avium

Aims:  To compare three methods for DNA extraction from Mycobacterium bovis , Mycobacterium tuberculosis and Mycobacterium avium subsp. avium .
Methods and Results:  The DNA was extracted from mycobacterial cultures using enzymatic extraction, combined bead beating and enzymatic extraction and cetyltrimethylammonium bromide (CTAB) extraction. The yield and quality of DNA were compared by spectrophotometry, agarose gel electrophoresis, restriction endonuclease analysis and PCR. The combined bead beating and enzymatic extraction method yielded more DNA. However, that method produced some sheared DNA, visible either by agarose gel electrophoresis or by restriction endonuclease analysis. All methods were appropriate for PCR amplification of a 123 bp fragment of IS 6110 in M. bovis and M. tuberculosis , and of a 1700 bp fragment of FR300 region in M. avium avium .
Conclusions:  Combined bead beating and enzymatic extraction method was the most efficient and easy method for extracting DNA from bacteria of the M. tuberculosis complex.
Significance and Impact of the Study:  The results reveal important differences among the DNA extraction methods for mycobacteria, which are relevant for the success of further downstream molecular analysis.     

Nymphs of the Oriental cockroach (Blatta orientalis) as passive vectors of causal agents of avian tuberculosis and paratuberculosis

The potential transmission of the causal agent of paratuberculosis Mycobacterium avium ssp. paratuberculosis and avian tuberculosis Mycobacterium avium ssp. avium (Actinomycetales: Mycobacteriaceae) by nymphs of the Oriental cockroach Blatta orientalis L. (Blattodea: Blattidae) was investigated by oral infection with mycobacterial suspensions and examination of their droppings and bodies. Both the subspecies of M. avium were isolated from droppings at 3 days post-infection and M. a. avium was found in homogenized bodies at 10 days post-infection. The identity of M. a. avium and M. a. paratuberculosis isolates was demonstrated by Restriction Fragment Length Polymorphism (RFLP) analysis. The M. a. avium isolate used as the inoculum and the isolates from the bodies and droppings of the nymphs were shown to be virulent in chickens. The results show that orally infected nymphs of B. orientalis can harbour and shed viable and virulent mycobacteria. This hazard should be considered in the implementation of control measures against mycobacterial infections of animals and humans, which should include destruction of all developmental stages of cockroaches and prevention of their access to materials that can be contaminated by mycobacteria.     

Mycobacterial ecology of the Rio Grande

This is the first study to characterize the environmental conditions which contribute to the presence and proliferation of environmental mycobacteria in a major freshwater river. Over 20 different species of environmental mycobacteria were isolated, including the pathogenic M. avium and M. kansasii. Species of the rapidly growing M. fortuitum complex were the most commonly isolated mycobacteria, and one-third of all isolates were not identified at the species level, even by 16S sequencing. PCR restriction analysis of the hsp65 gene was more accurate and rapid than biochemical tests and as accurate as yet less expensive than 16S sequencing, showing great promise as a new tool for species identification of environmentally isolated mycobacteria. Total environmental mycobacteria counts positively correlated with coliform and Escherichia coli counts and negatively correlated with chemical toxicity and water temperature. Environmental mycobacteria can survive in the alkaline conditions of the river despite previous reports that especially acidic conditions favor their presence. A representative river isolate (M. fortuitum) survived better than E. coli O157:H7 at pHs below 7 and above 8 in nutrient broth. The river strain also retained viability at 8 ppm of free chlorine, while E. coli was eliminated at 2 ppm and above. Thus, in vitro studies support environmental observations that a variety of extreme conditions favor the hardy environmental mycobacteria.     

Nutrient removal from on-site domestic wastewater in horizontal subsurface flow reed beds in Ireland

In order to investigate the nutrient (namely nitrogen and phosphorus) removal efficiency and the governing internal dynamics of the most widely used wetland type, the horizontal subsurface flow reed bed, in receiving domestic septic tank and secondary effluent in a temperate climate such as Ireland, two systems were designed, constructed and rigorously monitored for a period of over 2 years. Nitrogen removal, as expected, was found to be poor across both reed beds, with only 29% removal of TN across the secondary treatment bed and 41% removal across the tertiary treatment bed, with little distinctive seasonal change. A ¹⁵N stable isotope tracer study revealed, in line with the results from the chemical analysis, that nitrogen kinetics in the secondary treatment bed were dominated by continuous plant litter decomposition and mineralisation processes converting stored org-N to NH₄-N indefinitely. Similar analysis on the tertiary treatment bed indicated that only limited denitrification of the oxidized forms of N was occurring in the anoxic environment of the bed, while NH₄-N and org-N were merely changing form on a cyclic basis. Removal of PO₄-P from the secondary and tertiary treatment beds was equally poor at rates of 45% and 22%, respectively. While at their maximum growth in the third year of operation, the total phosphorus in the stems and roots of the Phragmites australis in the secondary treatment bed equated to only 10% of the total P removed over the duration of the bed's operation. In the tertiary treatment bed, more seasonal variability was recorded with intermittent negative removal found during winter periods. This was somewhat more reflected in the P-uptake study for this bed with the roots and stems of the Typha and Iris containing phosphorus, which accounted for 31% of the overall mass removed.     

Methods used to investigate a possible environmental source of Mycobacterium avium-intracellulare-scrofulaceum (MAIS) infection in farmed deer

Currently used methods for the culture of mycobacteria from contaminated material were found to be unsatisfactory in an investigation of a possible environmental source of Mycobacterium avium-intracellulare-scrofulaceum (MAIS) infection in a New Zealand deer farm. Different combinations of established procedures were investigated using soil spiked with a laboratory strain of M. avium. The most successful combination involved mixing the soil in nutrient broth (pH 8.0) containing Tween 80, incubating at 37 degrees C for 1 h to germinate sporing contaminants, treatment for 24 h with 1% cetylpyridinium chloride, followed by washing and culture on Lowenstein-Jensen slopes and incubation at 37 degrees C and 42 degrees C in approximately 5% CO2 atmosphere. This procedure allowed good recovery of M. avium while successfully inhibiting saprophytic mycobacteria and other soil organisms, and was chosen to process the deer farm samples. No mycobacteria resembling the deer strains were found in these samples.     

Relationships between Mycobacterium isolates from patients with pulmonary mycobacterial infection and potting soils

High numbers of mycobacteria, including known pathogenic species such as Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium chelonae, were recovered from aerosols produced by pouring commercial potting soil products and potting soil samples provided by patients with pulmonary mycobacterial infections. The dominant mycobacteria in the soil samples corresponded to the dominant species implicated clinically. Profiles of large restriction fragments obtained by pulsed-field gel electrophoresis demonstrated a closely related pair of M. avium isolates recovered from a patient and from that patient's own potting soil. Thus, potting soils are potential sources of infection by environmental mycobacteria. Use of dust-excluding masks should be considered during potting or other activities that generate aerosol with soil.     

Identification of mycobacteria in peat moss processing plants: application of molecular biology approaches

Peat moss processing plant workers are exposed to high concentrations of bioaerosols. Although mycobacteria have been cultured from peat moss, no study has examined the workers' exposure to mycobacterial bioaerosols. We evaluated the presence of mycobacteria in air samples from peat moss processing plants using molecular biology approaches (cloning-sequencing and polymerase chain reaction (PCR)) and the workers exposure using immunoglobulin G (IgG) complexes to mycobacteria. In addition, species detected in air samples and in peat moss were compared. Two peat moss processing plants were chosen among 14 previously studied. A total of 49 clones were sequenced. Real-time PCR was also performed on the same air samples to evaluate the airborne concentration of mycobacteria and estimate exposure levels. Several Mycobacterium species were present in the air samples (M. malmoense, M. smegmatis, M. graceum, M. bohemicum, and M. interjectum). Mycobacterium avium was recovered by culture in peat moss but not in the air using the molecular approach. Total airborne Mycobacterium concentration was estimated at 8.2 x 10(8)/m3. Workers had IgG against the mycobacterial mix and M. avium, suggesting significant exposure. The findings from air samples, supported by IgG measurements, demonstrate that peat moss processing plant workers are exposed to mycobacteria in addition to other biological agents.     

Methods used to investigate a possible environmental source of Mycobacterium avium-intracellulare-scrofulaceum (MAIS) infection in farmed deer

Currently used methods for the culture of mycobacteria from contaminated material were found to be unsatisfactory in an investigation of a possible environmental source of Mycobacterium avium-intracellulare-scrofulaceum (MAIS) infection in a New Zealand deer farm. Different combinations of established procedures were investigated using soil spiked with a laboratory strain of M. avium.
The most successful combination involved mixing the soil in nutrient broth (pH 8mD0) containing Tween 80, incubating at 37°C for 1 h to germinate sporing contaminants, treatment for 24 h with 1% cetylpyridinium chloride, followed by washing and culture on Lowenstein-Jensen slopes and incubation at 37°C and 42°C in approximately 5% CO₂ atmosphere. This procedure allowed good recovery of M. avium while successfully inhibiting saprophytic mycobacteria and other soil organisms, and was chosen to process the deer farm samples. No mycobacteria resembling the deer strains were found in these samples.     

Various stages in the life cycle of syrphid flies (Eristalis tenax; diptera: Syrphidae) as potential mechanical vectors of pathogens causing mycobacterial infections in pig herds

We defined the role of the syrphid fly Eristalis tenax in the survival and transmission of mycobacteria in pigs. The conditionally pathogenic mycobacterial (CPM) species Mycobacterium chelonae was isolated from 10 % of liquid dung samples, and both M. chelonae and another CPM species M. fortuitum were isolated from 7 (78 %) of the examined E. tenax larvae collected from the same location. Mycobacteriosis of the lymph nodes of pigs from 3 infected farms was caused by M. avium subsp. avium, M. avium subsp. hominissuis, and M. fortuitum. M. avium subsp. avium and M. avium subsp. hominissuis of identical genotype and serotypes and M. fortuitum were isolated from 7 (1.9 %) larvae, 2 (7.4 %) puparia, and one (1.6 %) imago. The count of colony forming units isolated from larval skin covering (pouch) was higher (p < or = 0.01) than that isolated from the internal organs of larvae. These results showed the potential for E. tenax larvae to spread mycobacteria throughout pig herds and the surrounding environment.     

Factors influencing numbers of Mycobacterium avium, Mycobacterium intracellulare, and other Mycobacteria in drinking water distribution systems

Eight water distribution systems were sampled over an 18-month period (528 water and 55 biofilm samples) to measure the frequency of recovery and number of mycobacteria, particularly Mycobacterium avium and Mycobacterium intracellulare, in raw source waters before and after treatment and within the distribution system. The systems were chosen to assess the influence of source water, treatment, and assimilable organic carbon levels on mycobacterial numbers. Overall, mycobacterial recovery from the systems was low (15% of samples). Numbers of mycobacteria ranged from 10 to 700,000 CFU liter(-1). The number of M. avium in raw waters was correlated with turbidity. Water treatment substantially reduced the number of mycobacteria in raw waters by 2 to 4 log units. Mycobacterial numbers were substantially higher in the distribution system samples (average, 25,000-fold) than in those collected immediately downstream from the treatment facilities, indicating that mycobacteria grow in the distribution system. The increase in mycobacterial numbers was correlated with assimilable organic carbon and biodegradable organic carbon levels (r(2) = 0.65, P = 0.03). Although M. intracellulare was seldom recovered from water samples, it was frequently recovered (six of eight systems) in high numbers from biofilms (average, 600 CFU/cm(2)). Evidently, the ecological niches of M. avium and M. intracellulare are distinct.     

Nontuberculous mycobacteria isolated from pulmonary specimens between 2004 and 2009: causative agent or not?

Nontuberculous mycobacteria were identified from 45891 samples of 19553 patients with a prediagnosis of pulmonary tuberculosis between November 2004 and January 2009. Among 10041 (21.9%) culture positive samples, 208 (2.1%) pulmonary samples recovered from 77 individual patients were differentiated as mycobacteria other than tuberculosis (MOTT). Proportion of mycobacteria evaluated as causative agent for clinical infection were found as 0.16% (n = 31), mostly M. avium complex, M. abscessus and M. kansasii. Additionally, M. fortuitum-peregrinum complex, M. simiae, M. szulgai / intermedium and M. scrofulaceum were found as causative agent in 2, 2, 2 and 1 patient, respectively. Identification of infections caused by environmental or opportunistic pathogen mycobacteria is required in rapid and accurate diagnosis, infection control and treatment planning of infections caused by M. tuberculosis complex and/or MOTT.     

Replication and long-term persistence of bovine and human strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga

Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.