Optimization of procedures for isolation of mycobacteria from soil and water samples obtained in northern India

For isolation of environmental mycobacteria, a decontamination procedure has been standardized by which treatment with 3% sodium dodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slow growers, respectively) is followed by incubation with 2% cetrimide (5 and 15 min for fast- and slow-growing mycobacteria, respectively); this procedure was found to completely eliminate contamination with other organisms and resulted in the isolation of only mycobacteria.     

Optimization of Procedures for Isolation of Mycobacteria from Soil and Water Samples Obtained in Northern India

For isolation of environmental mycobacteria, a decontamination procedure has been standardized by which treatment with 3% sodium dodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slow growers, respectively) is followed by incubation with 2% cetrimide (5 and 15 min for fast- and slow-growing mycobacteria, respectively); this procedure was found to completely eliminate contamination with other organisms and resulted in the isolation of only mycobacteria.     

The division between fast- and slow-growing species corresponds to natural relationships among the mycobacteria.

Comparative 16S rRNA sequencing was used to infer the phylogenetic relationships among selected species of mycobacteria and related organisms. The phylogeny inferred reflects the traditional classification, with major branches of the phylogenetic tree in general correspondence to the four Runyon groups and with numerical classification analyses. All the mycobacterial species compared, with the exception of M. chitae, are closely related (average similarity values greater than 95%). The slow growers form a coherent line of descent, distinct from the rapid growers, within which the overt pathogens are clustered. The distant relationship between M. chitae and the remaining mycobacteria suggests that this organism is incorrectly classified with the mycobacteria. M. paratuberculosis 18 was indistinguishable from M. avium-M. intracellulare-M. scrofulaceum serovar 1 by this analysis.     

Environmental mycobacteria in Korea. I. Distribution of the organisms

Environmental mycobacteria in Korea have been investigated by examining 54 soil, 111 house dust, 63 well water, and 98 sewage samples collected from 123 randomly selected areas in Korea during the fourth nationwide tuberculosis prevalence survey in 1980. A variety of mycobacteria were isolated from 76% of soil, 67% of sewage, 43% of well water, and 7% of house dust samples. Some samples yielded more than one species; thus 56 strains were obtained from soil, 107 strains from sewage, 48 strains from well water and 8 strains from house dust. Mycobacterium fortuitum was the most common species of environmental mycobacteria in Korea and the species was distributed equally in all types of samples tested. The M. terrae complex was also one of the common species of environmental mycobacteria and it seemed to be more abundant in water samples than in soil. Scotochromogenic slow growers M. scrofulaceum and M. gordonae were common microbes in soil and water samples, although the latter was more frequently detected in water samples. Scotochromogenic rapid growers M. flavescens and M.phlei, and photochromogenic rapid grower M. vaccae were isolated more frequently from sewage or water samples than from soil. Nonphotochromogenic rapid growers M. chelonei (chelonae) and M. smegmatis were isolated mostly from sewage and the former was rarely found in soil and well water samples. The clinically important species M. avium-intracellulare complex was found less frequently in all types of test samples.     

Physiological and biochemical characteristics of fast-and slow-growing Rhizobium sp. pigeonpea (Cajanus cajan)

Thirty-two of 38 cultures isolated from the nodules of pigeonpea plants were rapid growers. The generation time of rapid growers varied from 0·71 to 1·31 h and slow growers from 7·7 to 9·9 h. Rapid growers oxidized pentoses, hexoses, polyhydric alcohols (except dulcitol), tricarboxylic acid cycle intermediates (except citrate) and disaccharides more rapidly than slow growers; disaccharides were not oxidized by any of the slow growers. The Embden-Meyerhof-Parnas and Entner-Doudoroff pathways and the tricarboxylic acid cycle were present in both rapid and slow growers, whereas the pentose-phosphate pathway was present only in rapid growers.     

Motivation and ability to walk for a food reward in fast- and slow-growing broilers to 12 weeks of age

Poor physical abilities of broilers may prevent them from performing behaviours for which they are motivated. The aim of this study was to measure the influence of physical ability and motivation on the performance of broilers in short physical tasks. We tested birds from a fast- and a slow-growing broiler strain in a runway to 12 weeks of age. To manipulate motivation, half of the birds of each strain was feed deprived for 3h and the other half for 24h before testing. Each bird was tested in a control and a slalom runway test once a week. With a similar motivation, slow growers had a shorter latency to start walking and walked faster through the runway than fast growers in both tests. In fast growers walking speed decreased faster with age than in slow growers. Slow growers vocalised more in both tests. In the slalom test, 24h deprived birds vocalised more than 3h deprived birds. Although the fast and slow growers have a different genetic background, the results indicated that motivation is the dominant determinative factor for walking in birds with a low body weight, while physical ability is the dominant determinative factor for walking in birds with a high body weight.     

Growth and mortality in a field population of Amphibolurus lizards exposed to seasonal cold and aridity

The demography of a discrete population of Amphibolurus ornatus lizards in Western Australia was studied over a period of four years. Large differences in the rate of growth of individuals from the same cohort were observed during each year of the study and each animal could be classified as maturing at one, two or three years of age. Fast- and slow growing individuals were shown to suffer differential mortality during seasonal stresses with fast growers being cold hardy and drought sensitive whereas slow-growers were cold sensitive but drought resistant. The potential longevity of fast- and slow-growing animals appears to be identical but the survival of animals in the field depends markedly upon the incidence of climatic extremes, slow growers tending to live longer than fast growers in the study area. Changes that were observed in the size and age structure of the breeding population during the period of study can be interpreted in terms of the weather conditions experienced by the population over this period. The significance of the variability in individual growth rates of Amphibolurus ornutus for the persistence of the study population is discussed.     

Studies on fast and slow growing Rhizobium spp. nodulating Cqjanus cajan and Cicer arietinum

Fast and slow growing Rhizobium spp. isolated from Cajanus cajan and Cicer arietinum were compared in terms of colony characteristics, utilisation of carbon sources, acid production, symbiotic effectiveness and nodulating competitiveness. Fast growing isolates from C. cajan and C. arietinum formed 3–6 mm diameter colonies on yeast-extract mannitol agar after 4 days and were unlike the slow growers which produced colonies of c. 1 mm diameter after 7–10 days at 28 °C. The fast growing Rhizobium spp. from C. cajan utilised a wider range of carbon sources than the slow growing isolates from this host. Fast and slow growing strains from C. arietinum were able to utilise most of the carbon sources tested suggesting that the slow growers possessed glycolytic pathways similar to those in other fast growing species of Rhizobium. In culture, slow growing isolates from C. cajan produced a near-neutral to alkaline reaction (pH 66·7-5) whereas the fast growers from this host and both fast and slow growing isolates from C. arietinum produced an acidic reaction (pH 4·4–5·6). These data are discussed in the context of Norris' (1965) evolutionary concept of the Leguminosae. Under glassshouse conditions, fast and slow growing strains isolated from C. cajan and C. arietinum were equally effective on their respective hosts. In competition with slow growing rhizobia, half of the fast growers formed more than 70% of the nodules on C. cajan grown in sand. In all but one instance similar results were obtained when plants were grown in soil. With C. arietinum grown in sand or soil, all fast growing isolates from this host formed more than 85% of the nodules in competition with slow growing strains.     

Differentiating Indigenous Soybean Bradyrhizobium and Rhizobium spp. of Indian Soils

Soybean is extensively cultivated worldwide and is the largest source of biologically fixed nitrogen among legumes. It is nodulated by both slow and fast growing rhizobia. Indigenous soybean rhizobia in Vertisols of central India were assessed for utilization of 35 carbon sources and intrinsic resistance to 19 antibiotics. There was greater utilization of trehalose and raffinose by fast growers (87 and 73 % by fast vs. 35 and 30 % by slow growers); but slow growers had higher ability to utilize glucosamine (75 % by slow vs. 33 % by fast growers). A larger proportion of slow growers were resistant to vancomycin, polymyxin-B and rifampicin (70, 65 and 55 %) compared to fast growers (13, 7 and 7 % each). Among the two 16S rRNA sequence types in the slow growers, those belonging to Bradyrhizobium spp. utilized glucosamine while those belonging to Rhizobium radiobacter did not. All the fast growers had 16S rRNA homology to R. radiobacter and majority could not utilize glucosamine. It is suggested that during initial isolations and screening of rhizobia in strain selection programmes, using carbon sources like glucosamine and antibiotics like vancomycin, polymyxin-B and rifampicin in the media may provide a simple way of distinguishing Bradyrhizobium strains from R. radiobacter among the slow growers.     

The influence of piscivory on life history traits of brown trout

Brown trout in Lake Femund migrated from the nursery streams mainly at 2 years old, but ranging between 1 and 8 years. Brown trout switched to piscivory from 3 years onwards, and a body length of 17·5 cm, according to back calculation from scales. Fast growers switched to piscivory at a younger age and smaller size than slow growers. The most slow-growing trout switched to fish feeding at 9 years old and a mean body length of 36 cm. The growth of the invertebrate feeders was almost rectilinear to c. 45 cm and 11 years of age. Switching to a fish diet induced increased growth rates. Age at sexual maturity increased with the age at which the fish became piscivorous. The invertebrate feeders matured at an age similar to that of the most fast growing piscivorous trout. The mortality rate of sub-adults and adults did not differ significantly between fish and invertebrate feeding trout. Longevity of piscivorous trout was estimated at 11 years and of invertebrate feeders at 10 years.     

Growth strategy of heterotrophic bacterial population along successional sequence on spoil of brown coal colliery substrate

The bacterial population of brown coal colliery spoil (Sokolov coal mining district, Czechia) was characterized by measuring viable bacterial biomass, the culturable to total cell ratio (C:T), colony-forming curve (CFC) analysis and species and/or biotype diversity. Bacterial representatives that differed in colony-forming growth (fast and/or slow growers) were used for growth-strategy investigation of heterotrophic bacteria. Spoil substrates from the surface (0–50 mm) and the mineral (100–150 mm) layers were sampled on 4 sites undergoing spontaneous succession corresponding to 1, 11, 21 and 43 years after deposition (initial, early, mid and late stages). The bacterial biomass of the surface layer increased during the initial and early stages with a maximum at mid stage and stabilized in the late stage while mineral layer biomass increased throughout the succession. The maxima of C:T ratios were at the early stage, minima at the late stage. Depending on the succession stage the C:T ratio was 1.5–2 times higher in the mineral than the surface layer of soil. An increase in the fraction of nonculturable bacteria was associated with the late succession stage. CFC analysis of the surface layer during a 3-d incubation revealed that the early-succession substrate contained more (75 %) rapidly colonizing bacteria (opportunists, r-strategists) than successively older substrates. The culturable bacterial community of the mineral layer maintained a high genera and species richness of fast growers along the succession line in contrast to the surface layer community, where there was a maximum in the abundance of fast growers in the early stage. There was a balanced distribution of Grampositive and Gram-negative representatives of fast growers in both layers. A markedly lower abundance of slow growers was observed in the mineral in contrast to the surface layer. Gram-positive species dominated the slow growers at the surface as well as in the mineral layers. The growth strategy of the heterotrophic bacterial population along four successional stages on spoil of brown coal colliery substrate in the surface layer displayed a trend indicative of a r-K continuum in contrast to the mineral layer, where an r-strategy persisted. This research was funded by theCzech Science Foundation grant no. 526/03/1259 and by theResearch Plan of the Institute of Soil Biology, Academy of Sciences of the Czech Republic, project no. AV 0Z 6066 0521.     

Optimized Method for Preparation of DNA from Pathogenic and Environmental Mycobacteria

Genomic studies on pathogenic and environmental mycobacteria are of growing interest for understanding of their evolution, distribution, adaptation, and host-pathogen interaction. Since most mycobacteria are slow growers, material from in vitro cultures is usually scarce. The robust mycobacterial cell wall hinders both experimental cell lysis and efficient DNA extraction. Here, we compare elements of several DNA preparation protocols and describe a method that is economical and practical and reliably yields large amounts—usually 10-fold increased compared to earlier protocols—of highly pure genomic DNA for sophisticated downstream applications. This method was optimized for cultures of a variety of pathogenic and environmental mycobacterial species and proven to be suitable for direct mycobacterial DNA extraction from infected insect specimens.     

Associations of the bovine major histocompatibility complex DRB3 (BoLA-DRB3) alleles with occurrence of disease and milk somatic cell score in Canadian dairy cattle

Potential associations were investigated between bovine leucocyte antigen (BoLA) alleles and occurrence of disease. Cows (Holstein n = 835; Jersey n = 66) were examined for polymorphisms of the second exon of the BoLA-DRB3 gene, using the polymerase chain reaction (PCR), followed by digestion of the amplified fragments with three restriction endonucleases. Disease occurrences were recorded for each cow throughout one lactation. Milk somatic cell count data were retrieved through the Dairy Herd Improvement records and converted to somatic cell score (SCS). There were no effects of BoLA alleles on SCS in Jersey cows, but BoLA-DRB3·2* 16 was significantly associated ( P ≤ 0·05) with lower SCS in Holsteins. Since the number of Jerseys was relatively small and prevalence of diseases in this population was low, health records of Jerseys were not analyzed further. BoLA associations with occurrence of disease in Holsteins were investigated using a log-linear model. There was a significant ( P ≤ 0·05) association between BoLA-DRB3.2* 23 and occurrence of severe mastitis, from which coliforms were the most commonly isolated bacteria. The BoLA allele *3 was associated with a lower risk of retained placenta ( P ≤ 0·05) and alleles *16 ( P ≤ 0·05) and *22 ( P ≤ 0·05) with a lower risk of cystic ovarian disease. Although more studies are required to confirm the present findings, it can be concluded that BoLA alleles may have potential usefulness as genetic markers of higher or lower risk of disease occurrence in cows.     

Prospecting Environmental Mycobacteria: Combined Molecular Approaches Reveal Unprecedented Diversity

Background

Environmental mycobacteria (EM) include species commonly found in various terrestrial and aquatic environments, encompassing animal and human pathogens in addition to saprophytes. Approximately 150 EM species can be separated into fast and slow growers based on sequence and copy number differences of their 16S rRNA genes. Cultivation methods are not appropriate for diversity studies; few studies have investigated EM diversity in soil despite their importance as potential reservoirs of pathogens and their hypothesized role in masking or blocking M. bovis BCG vaccine.

Methods

We report here the development, optimization and validation of molecular assays targeting the 16S rRNA gene to assess diversity and prevalence of fast and slow growing EM in representative soils from semi tropical and temperate areas. New primer sets were designed also to target uniquely slow growing mycobacteria and used with PCR-DGGE, tag-encoded Titanium amplicon pyrosequencing and quantitative PCR.

Results

PCR-DGGE and pyrosequencing provided a consensus of EM diversity; for example, a high abundance of pyrosequencing reads and DGGE bands corresponded to M. moriokaense, M. colombiense and M. riyadhense. As expected pyrosequencing provided more comprehensive information; additional prevalent species included M. chlorophenolicum, M. neglectum, M. gordonae, M. aemonae. Prevalence of the total Mycobacterium genus in the soil samples ranged from 2.3×10⁷ to 2.7×10⁸ gene targets g⁻¹; slow growers prevalence from 2.9×10⁵ to 1.2×10⁷ cells g⁻¹.

Conclusions

This combined molecular approach enabled an unprecedented qualitative and quantitative assessment of EM across soil samples. Good concordance was found between methods and the bioinformatics analysis was validated by random resampling. Sequences from most pathogenic groups associated with slow growth were identified in extenso in all soils tested with a specific assay, allowing to unmask them from the Mycobacterium whole genus, in which, as minority members, they would have remained undetected.     

Occurrence of Mycobacteria in Water Treatment Lines and in Water Distribution Systems

The frequency of recovery of atypical mycobacteria was estimated in two treatment plants providing drinking water to Paris, France, at some intermediate stages of treatment. The two plants use two different filtration processes, rapid and slow sand filtration. Our results suggest that slow sand filtration is more efficient for removing mycobacteria than rapid sand filtration. In addition, our results show that mycobacteria can colonize and grow on granular activated carbon and are able to enter distribution systems. We also investigated the frequency of recovery of mycobacteria in the water distribution system of Paris (outside buildings). The mycobacterial species isolated from the Paris drinking water distribution system are different from those isolated from the water leaving the treatment plants. Saprophytic mycobacteria (present in 41.3% of positive samples), potentially pathogenic mycobacteria (16.3%), and unidentifiable mycobacteria (54.8%) were isolated from 12 sites within the Paris water distribution system. Mycobacterium gordonae was preferentially recovered from treated surface water, whereas Mycobacterium nonchromogenicum was preferentially recovered from groundwater. No significant correlations were found among the presence of mycobacteria, the origin of water, and water temperature.     

Characterization of intermediate biotypes in atrazine-susceptible populations of Chenopodium polyspermum L. and Amaranthus bouchonii thell. in Hungary

Intermediate biotypes for atrazine herbicide resistance in Chenopodium polyspermum and Amaranthus bouchonii were characterized by a peculiar chlorophyll fluorescence induction curve. The intermediate biotypes were isolated from progenies of susceptible plants in maize grown in alternate years without atrazine. The lethal dose in seedling treatments was lower than that of the resistant plants but higher than for susceptible plants. Atrazine at 10 μM was near the I₅₀-value for in vivo nitrite reductase activity in both intermediate biotypes. The activity of nitrite reducttase in the intermediate biotypes was about 75% of that of susceptible biotypes. These characteristics of intermediate biotypes were maternally inherited in crosses.     

Identification of Aphis gossypii Glover (Hemiptera: Aphididae) Biotypes from Different Host Plants in North China

Background

The cotton-melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is a polyphagous species with a worldwide distribution and a variety of biotypes. North China is a traditional agricultural area with abundant winter and summer hosts of A. gossypii. While the life cycles of A. gossypii on different plants have been well studied, those of the biotypes of North China are still unclear.

Results

Host transfer experiments showed that A. gossypii from North China has two host-specialized biotypes: cotton and cucumber. Based on complete mitochondrial sequences, we identified a molecular marker with five single-nucleotide polymorphisms to distinguish the biotypes. Using this marker, a large-scale study of biotypes on primary winter and summer hosts was conducted. All A. gossypii collected from three primary hosts—hibiscus, pomegranate, and Chinese prickly ash—were cotton biotypes, with more cotton-melon aphids found on hibiscus than the other two species. In May, alate cotton and cucumber biotypes coexisted on cotton and cucumber seedlings, but each preferred its natal host. Both biotypes existed on zucchini, although the cucumber biotype was more numerous. Aphids on muskmelon were all cucumber biotypes, whereas most aphids on kidney bean were cotton biotypes. Aphids on seedlings of potato and cowpea belong to other species. In August, aphids on cotton and cucumber were the respective biotypes, with zucchini still hosting both biotypes as before. Thus, the biotypes had different fitnesses on different host plants.

Conclusions

Two host-specialized biotypes (cotton and cucumber) are present in North China. Hibiscus, pomegranate, and Chinese prickly ash can serve as winter hosts for the cotton biotype but not the cucumber biotype in North China. The fitnesses of the two host-specialized biotypes differ on various summer hosts. When alate aphids migrate to summer hosts, they cannot accurately land on the corresponding plant.     

Reproductive rates of Russian wheat aphid (Hemiptera: Aphididae) biotypes 1 and 2 on a susceptible and a resistant wheat at three temperature regimes

The reproductive rates of Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae), Biotype 1 (RWA 1) and Biotype 2 (RWA 2) were compared in the laboratory at three temperature regimes on a Russian wheat aphid resistant cultivar ('Prairie Red') and a susceptible cultivar ('TAM 107'). The objective of this study was to expose RWA 1 and RWA 2 to three temperature regimes and two levels of resistance to find whether there were reproductive differences that may occur within each biotype as well as differences in reproduction between biotypes. In addition, temperature effects of the Dn4 gene on biotype reproduction were noted. Differences in reproductive rates between the two biotypes seem to be driven by temperature. For both biotypes, longevity and reproductive rate parameters, except for intrinsic rate of increase, were lower at the 24-29 degree C temperature regime than the 13-18 degree C and 18-24 degree C temperature regimes. The intrinsic rate of increase was higher for both biotypes at the 18-24 degree C and 24-29 degree C temperature regimes than at the 13-18 degree C temperature regime. Reproductive rates between biotypes were similar at the two higher temperature regimes, but the fecundity for RWA 1 was less than RWA 2 at the 13-18 degree C temperature. The change in fecundity rates between RWA 1 and RWA 2 at lower temperatures could have ecological and geographical implications for RWA 2.     

Ultrastructural alterations to chloroplasts in triazine-resistant weed biotypes

Ultrastructural, morphometric and physiological techniques were used to determine the consistent chloroplast differences between triazine-resistant (R) and triazine-susceptible (S) biotypes of Amaranthus hybridus L., Chenopodium album L., and Brassica campestris L. All R biotypes had a larger proportion of the chloroplast volume as grana lamellae and a lower proportion of starch and stroma lamellae than S biotypes. In the R biotypes, a greater percentage of grana contain larger numbers of thylakoids per granum. A greater proportion of chlorophyll associated with the light-harvesting chlorophyll alb protein and a lower chlorophyll alb ratio, traits associated with an increase in grana lamellae, were noted in R biotypes. Chloroplasts of S biotypes could be modified to ultrastructural phenocopies of those in R biotypes by treatment with sublethal levels of the PSII inhibiting herbicides, bentazon, diuron, atrazine and prometon. Despite the structural similarities to R biotypes, the modified S biotypes were not resistant to atrazine as determined by fluorescence measurements. Thus, the structural alterations observed are apparently secondary effects of impaired photosynthetic electron transport in R biotypes, and are not the cause of triazine resistance.     

Mycorrhizae, crop growth, and crop phosphorus nutrition in maize-soybean rotations given various tillage treatments

Previously, tillage has been found to reduce early-season phosphorus (P) uptake from soil in continuous maize cropping systems. This reduced P uptake has often been associated with delayed colonization of roots by arbuscular mycorrhizal (AM) fungi. Our aim was to determine if similar responses occur in maize-soybean rotations, which are more typical of current farming in Ontario, Canada. Similar responses were expected because both are AM crops, and the mechanism by which tillage reduces P uptake is thought to be a negative impact on the development of effective mycorrhizae. Simultaneous field experiments with either maize-soybean-maize or soybean-maize-soybean rotations were conducted in 1992–4. Treatments imposed were no-till (NT), ridge-tillage (RT), and conventional tillage using a moldboard plow (MP). In 1993, early-season dry mass of maize was similar among treatments, but colonization of maize roots by AM fungi and P uptake of maize were stimulated by NT and RT, compared with MP. In 1994, early growth was more rapid overall than in 1993, but it was reduced in the NT and RT treatments compared with MP for reasons not related to P. For soybean, AM colonization in NT and RT systems was higher than with MP, but P uptake was unchanged. As was found for maize in 1994, early-season shoot dry mass of soybean was higher in the MP treatment than with NT, but both in 1993 and 1994. We conclude that colonization of both maize and soybean by AM fungi is susceptible to slower development in tilled systems, and that for maize, stimulation of P uptake under reduced tillage can occur in rotations with soybean just as easily as it does with continuous maize. Taken with other studies, the data here suggest that responses to tillage of colonization of roots by AM fungi and of P uptake could apply to many cropping systems. The slow early-season shoot growth seen in some years in response to reduced tillage is discussed.