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.     

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.     

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.     

Serologic survey for antibodies against Mycobacterium a vium subsp. paratuberculosis in free-ranging cervids from Norway

Affinity between protein-G and immunoglobulins from red deer (Cervus elaphus), moose (Alces alces), roe deer (Capreolus capreolus), and reindeer (Rangifer tarandus tarandus) was tested in a competition binding assay. Sera from red deer, reindeer, and moose inhibited the assay less than sera from cattle (less affinity), whereas sera from roe deer showed a slightly higher affinity to protein-G than did sera from cattle. The conclusion was made that protein-G could be used instead of anti-species antibodies for these cervid species, where the aim of the screening was to look for exposure or lack of exposure to mycobacteria in the tested populations. Serologic screening of 1,373 free-ranging cervids for antibodies against Mycobacterium avium subsp. paratuberculosis was conducted. All sera were tested by a protein-G-based antigen-absorbed enzyme-linked immunosorbent assay (ELISA). Seropositive moose (10/537; 1.9%), red deer (14/371; 3.8%), roe deer (6/49; 12.2%), and semidomesticated reindeer (11/325; 3.4%) were found, whereas wild reindeer (n = 91) were seronegative. In addition, the red deer sera were tested with a commercial ELISA, by which two animals tested positive and nine were suspicious of having M. avium subsp. paratuberculosis antibodies. Tissue samples and feces from 10 moose originating from a population with a clustering of seropositive animals were investigated by histology and bacteriology with negative results. Paratuberculosis has never been diagnosed in free-ranging or farmed cervid species in Norway. Thus, further studies are indicated to prove that the present findings reflect an infection with M. avium subsp. paratuberculosis.     

The relationship of temperature to desiccation and starvation tolerance of the Mycobacterium avium complex

Mycobacterium avium grew in media at 14-37 degrees C, and persisted at 4 degrees C and 42 degrees C. The bacteria lost approximately 90% viability after 3 months in reverse-osmosis deionized water at 4-37 degrees C. Cooler temperatures lowered the death rate. Death rates also decreased after a 5- to 10-day starvation adaptation period. Alterations of the steady-state levels of different mycolic acid classes, presumably to facilitate thermoadaptation, were found. Following desiccation, M. avium lost viability at a constant rate (half-life of 2.3 days). This implies that bacilli contaminating dry medical surfaces would persist for short periods of time. The remarkable stress survival exhibited by M. avium further suggests persistence in a range of environmental and clinical settings.     

Culture- and Quantitative IS900 Real-Time PCR-Based Analysis of the Persistence of Mycobacterium avium subsp. paratuberculosis in a Controlled Dairy Cow Farm Environment

The aim of this study was to monitor the persistence of Mycobacterium avium subsp. paratuberculosis in environmental samples taken from a Holstein farm with a long history of clinical paratuberculosis. A herd of 606 head was eradicated, and mechanical cleaning and disinfection with chloramine B with ammonium (4%) was carried out on the farm; in the surrounding areas (on the field and field midden) lime was applied. Environmental samples were collected before and over a period of 24 months after destocking. Only one sample out of 48 (2%) examined on the farm (originating from a waste pit and collected before destocking) was positive for M. avium subsp. paratuberculosis by cultivation on solid medium (Herrold's egg yolk medium). The results using real-time quantitative PCR (qPCR) showed that a total of 81% of environmental samples with an average mean M. avium subsp. paratuberculosis cell number of 3.09 × 10(3) were positive for M. avium subsp. paratuberculosis before destocking compared to 43% with an average mean M. avium subsp. paratuberculosis cell number of 5.86 × 10(2) after 24 months. M. avium subsp. paratuberculosis-positive samples were detected in the cattle barn as well as in the calf barn and surrounding areas. M. avium subsp. paratuberculosis was detected from different matrices: floor and instrument scrapings, sediment, or scraping from watering troughs, waste pits, and cobwebs. M. avium subsp. paratuberculosis DNA was also detected in soil and plants collected on the field midden and the field 24 months after destocking. Although the proportion of positive samples decreased from 64% to 23% over time, the numbers of M. avium subsp. paratuberculosis cells were comparable.     

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.     

Molecular detection of Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in soil

PCR primers specific for the Mycobacterium tuberculosis complex were used to detect the presence of Mycobacterium bovis BCG (Pasteur) in soil microcosms and Mycobacterium bovis in environmental samples taken from a farm in Ireland with a history of bovine tuberculosis. M. bovis genes were detected in soil at 4 and 21 months after possible contamination. Gene levels were found in the range of 1 x 10(3) to 3.6 x 10(3) gene copies g of soil(-1), depending on the sampling area. Areas around badger setts had the highest levels of detectable genes and were shown to have the highest levels of gene persistence. M. bovis-specific 16S rRNA sequences were detected, providing evidence of the presence of viable cells in Irish soils. Studies of DNA turnover in soil microcosms proved that dead cells of M. bovis BCG did not persist beyond 10 days. Further microcosm experiments revealed that M. bovis BCG survival was optimal at 37 degrees C with moist soil (-20 kPa; 30% [vol/wt]). This study provides clear evidence that M. bovis can persist in the farm environment outside of its hosts and that climatic factors influence survival rates.     

Fatty acid oxidation and the beta-oxidation complex in Mycobacterium leprae and two axenically cultivable mycobacteria that are pathogens

Intact, non-growing Mycobacterium leprae, M. avium and M. microti oxidized a wide range of 1-14C-labelled fatty acids (C8 to C24) to 14CO2. Laurate (C12) was oxidized most rapidly, and its oxidation by M. leprae was inhibited by the antileprosy agents Dapsone, clofazamine and rifampicin. Key enzymes of beta-oxidation were detected in extracts from all three mycobacteria. All these activities (both in intact mycobacteria and the enzymes) were stimulated in M. avium grown in Dubos medium plus palmitate but activities in M. microti or M. avium grown either in Dubos medium with added liposomes or triolein, or in vivo were similar to those detected in the same strain grown in Dubos medium alone. M. avium could be grown in medium in which 95% of its fatty acyl elongase activity is acetyl-CoA dependent. In this medium growing M. avium organisms oxidized [1-14C]palmitate to 14CO2 but simultaneously elongated palmitate to C24 acids and even longer. Acetyl-CoA-dependent elongase activity is similar but clearly not identical to reversed beta-oxidation, but the exact point(s) of difference have not yet been identified.     

Modified enrichment-serology procedure for detection of salmonellae in soy products.

A modified enrichment-serology (MES) procedure was used to reduce the time necessary for salmonella analysis. Naturally contaminated samples of soy products were preenriched in 1% proteose peptone for 6 h at 37 degrees C followed by inoculation into tetrathionate broth for 18 h at 37 degrees C. Two drops of the tetrathionate sample were inoculated into M broth. After incubation at 37 degrees C for 6 h, 0.85 ml of the mixture was formolized, and 0.1 ml of polyvalent H antiserum was added. After incubation in water bath at 50 degrees C for 1 h, the appearance of a typical floccular flagellar precipitate was observed in tubes positive for salmonellae. Over 3,000 samples were subjected to standard biochemical and serological procedures, and the results were compared with those of the MES method with a 96.7% correlation. Eleven of the samples (0.3%) were false-negative with the MES procedure, and 3% were false-negative with the U.S. Food and Drug Administration Bacteriological Analytical Manual procedure. The 3% negative samples by this latter procedure were subsequently found to be positive by the MES procedure. The MES procedure reduced the time required for salmonella analysis from 4 days to 32 h.     

The isolation of Mycobacterium avium complex from soil, water, and dusts

Previously, it was difficult to isolate the Mycobacterium avium complex from soil, water, and dusts, because rapidly growing mycobacteria always overgrew slowly growing ones. We used Ogawa egg medium containing both ethambutol and ofloxacin, which inhibit the nonpathogenic slowly growing mycobacteria and most rapidly growing mycobacteria, respectively, as an aid to screen for pathogenic slowly growing mycobacteria; we could thereby isolate a number of the M. avium complex and M. scrofulaceum strains from soil, water, and dusts in this country.     

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.     

Mycobacterial screening of Czech red deer (Cervus elaphus) populations in overwintering sites, 2004-2006

One isolate of Mycobacterium avium subsp. paratuberculosis was detected in 2,212 fecal samples of wild deer assembled in overwintering sites (OwS). Neither M. bovis nor M. a. subsp. avium was found. Therefore, congregating deer in OwSs does not automatically lead to the amplification of these pathogens among animals in OWSs.     

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.     

Mycobacterium avium subspecies paratuberculosis cultured from locally and commercially pasteurized cow's milk in the Czech Republic

Between November 2002 and April 2003, 244 bottles and cartons of commercially pasteurized cow's milk were obtained at random from retail outlets throughout the Czech Republic. During the same period, samples of raw milk and of milk that was subsequently subjected to a minimum of 71.7 degrees C for 15 s in a local pasteurization unit were also obtained from two dairy herds, designated herds A and B, with low and high levels, respectively, of subclinical Mycobacterium avium subsp. paratuberculosis infection, and from one herd, herd C, without infection. Infection in individual cows in each herd was tested by fecal culturing. Milk samples were brought to the Veterinary Research Institute in Brno, Czech Republic, processed, inoculated onto Herrold's egg yolk slants, and incubated for 32 weeks. Colonies were characterized by morphology, Ziehl-Neelsen staining, mycobactin J dependency, and IS900 PCR results. M. avium subsp. paratuberculosis was cultured from 4 of 244 units (1.6%) of commercially pasteurized retail milk. M. avium subsp. paratuberculosis was also cultured from 2 of 100 (2%) cartons of locally pasteurized milk derived from infected herds A and B and from 0 of 100 cartons of milk from uninfected herd C. Raw milk from 1 of 10 (10%) fecal culture-positive cows in herd A and from 13 of 66 (19.7%) fecal culture-positive cows in herd B was culture positive for M. avium subsp. paratuberculosis. These findings confirm that M. avium subsp. paratuberculosis is present in raw milk from subclinically infected dairy cows. The culture of M. avium subsp. paratuberculosis in the Czech Republic from retail milk that had been pasteurized locally or commercially to the required national and European Union standards is in agreement with similar research on milk destined for consumers in the United Kingdom and the United States and shows that humans are being exposed to this chronic enteric pathogen by this route.     

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.     

Comparison of methods for processing drinking water samples for the isolation of Mycobacterium avium and Mycobacterium intracellulare

Several protocols for isolation of mycobacteria from water exist, but there is no established standard method. This study compared methods of processing potable water samples for the isolation of Mycobacterium avium and Mycobacterium intracellulare using spiked sterilized water and tap water decontaminated using 0.005% cetylpyridinium chloride (CPC). Samples were concentrated by centrifugation or filtration and inoculated onto Middlebrook 7H10 and 7H11 plates and Lowenstein-Jensen slants and into mycobacterial growth indicator tubes with or without polymyxin, azlocillin, nalidixic acid, trimethoprim, and amphotericin B. The solid media were incubated at 32 degrees C, at 35 degrees C, and at 35 degrees C with CO(2) and read weekly. The results suggest that filtration of water for the isolation of mycobacteria is a more sensitive method for concentration than centrifugation. The addition of sodium thiosulfate may not be necessary and may reduce the yield. Middlebrook M7H10 and 7H11 were equally sensitive culture media. CPC decontamination, while effective for reducing growth of contaminants, also significantly reduces mycobacterial numbers. There was no difference at 3 weeks between the different incubation temperatures.     

Effect of chemical decontamination and refrigerated storage on the isolation of Mycobacterium avium subsp. paratuberculosis from heat-treated milk

AIMS: To assess the impact of chemical decontamination and refrigerated storage before culture on the recovery of Mycobacterium avium subsp. paratuberculosis from heat-treated milk. METHODS AND RESULTS: Five-millilitre samples of ultra heat-treated (UHT) milk spiked with Myco. paratuberculosis NCTC 8578, B4 or 806R (ca 10(6) CFU ml(-1)) were heated at 63 degrees C for 20 or 30 min by submersion in a water bath. Heat-treated milk (0.5 ml) was cultured immediately into BACTEC 12B medium or refrigerated at 4 degrees C for 48 h before culture. Milk samples that received a 20-min heat treatment were also subjected to decontamination with 0.75% cetylpyridinium chloride (CPC) for 5 h at room temperature before inoculation into BACTEC 12B medium when tested immediately and after 48 h at 4 degrees C. BACTEC vials were monitored for evidence of growth over an 18-week incubation period at 37 degrees C. CPC decontamination resulted in a significant reduction in the number of culture-positive milk samples recovered immediately after heating (P < 0.05) and after refrigerated storage for 48 h (P < 0.01). Refrigerated storage for 48 h before testing did not have any significant effect, beneficial or detrimental, on Myco. paratuberculosis recovery rates. CONCLUSIONS: CPC decontamination applied to milk immediately or 48 h after heating will adversely affect the recovery of viable Myco. paratuberculosis, possibly leading to nonrecovery of the organism although viable cells are present in the original milk sample. SIGNIFICANCE AND IMPACT OF THE STUDY: Published pasteurization studies in which milk samples were decontaminated before culture will have underestimated the survival capability of Myco. paratuberculosis after high-temperature, short-time pasteurization. CPC decontamination should not be applied to pasteurized milk in future studies.     

Disinfectant effects of hot water, ultraviolet light, silver ions and chlorine on strains of Legionella and nontuberculous mycobacteria

The disinfectant effects on Legionella and nontuberculous mycobacteria of hot water, ultraviolet light, silver ions and chlorine, were evaluated. The bacterial strains Legionella pneumophila ATCC33152 and Mycobacterium avium ATCC25291 and strains of L. pneumophila and M. avium which had been isolated from a 24 h bath, were examined for their resistance to treatments. All strains were killed within 3 min on exposure to hot water at 70 degrees C and exposure to ultraviolet light at 90 mW.s/cm2. The strains of L. pneumophila tested were killed within 6 h on exposure to a solution of silver ions at 50 micrograms/l. The number of viable cells of strains of M. avium fell from 10(5) CFU/ml to 10(3) CFU/ml after exposure to an aqueous solution of silver ions at 100 micrograms/l for 24 h. Chlorine effectively killed strains of Legionella which were exposed to an aqueous solution of chlorine at 2 mg/l within 3 min, but strains of Mycobacterium survived exposure to chlorine at 4 mg/l for more than 60 min.