Studies on histoplasmosis I. Comparative virulence of variant and parent strain Histoplasma capsulatum in Hamsters

Summary 1. A variant strain ofHistoplasma capsulatum was isolated from a secondary colony in cultures of tissue from hamsters infected with the parent strain ofHistoplasma. The variant strain was found to differ from the parent strain in colonial morphology, stability of the yeast phase in subculture, and in growth rate.2. Results of virulence testing with these two strains showed the parent strain to be somewhat more virulent than the variant strain as measured by survival time and slope of the death rate curve over a 19 day period.3. Nevertheless, in view of the more rapid growth rate of the variant and its probable antigenic variation from the parent strain, the development of such a variant is viewed as an increased hazard to the host, particularly in a longer term infection.4. The repeated isolation of a variant strain ofHistoplasma suggests that strain variation may play a significant role in the outcome of naturally acquired infections.This work was initiated at the State University of New York, Downstate Medical Center, Brooklyn, N. Y., and supported in part by funds ((#12-1081) from the National Fund for Medical Education, grant # 12-1054.     

Structural organization of cholera toxin gene and its expression in an environmental non-pathogenic strain ofVibrio cholerae

Non-pathogenic, environmental strain ofVibrio cholerae, ELTOR Ogawa EW6 carries a copy of the cholera toxin gene in its chromosome. Restriction enzyme digestion followed by Southern blot analysis revealed that the structure of the cholera toxin gene in this organism is different from that found in the virulent strains. The xbaI site which has been found to be conserved in the cholera toxin of the virulent strains examined so far, is absent here. Results of the RNA dot blot analysis indicated that the cholera toxin gene in EW6 is transcribed much less efficiently compared to the cholera toxin gene present in the virulent strainVibrio cholerae classical Inaba 569B.     

Über die weite Verbreitung lichenisierter Sippen vonDictyochloropsis und die systematische Stellung vonMyrmecia reticulata (Chlorophyta)

Catinaria grossa is lichenized withDictyochloropsis splendida var.gelatinosa, var. nova. When cultured isolated from the fungus the cells of this variety are covered individually by thick gelatinous envelopes. The phycobionts ofMegalospora gompholoma subsp.gompholoma andM. atrorubicans subsp.australis belong to a new variety ofD. symbiontica, i.e. var.pauciautosporica, which preferentially reproduces by zoo- and aplanospores. The phycobiont ofPseudocyphellaria aurata does not belong toMyrmecia reticulata as formerly thought, but toDictyochloropsis symbiontica. Specimens from one locality in Tenerife and from New Zealand are lichenized with a strain ofD. symbiontica var.symbiontica, those from another locality in Tenerife with a strain ofD. symbiontica var.pauciautosporica. These strains differ in certain characters from other lichenized strains of these varieties.

     

Changes in the levels of peroxidase and phenylalanine ammonia-lyase inBrassica napus cultivars showing variable resistance toLeptosphaeria maculans

Peroxidase and phenylalanine ammonia-lyase activities (PAL) were determined in leaves of healthy and inoculatedBrassica napus cultivars, showing differential disease reaction towards a virulent and a weakly virulent strain ofLeptosphaeria maculans, the black leg pathogen. Both enzymes showed increased activities as the result of inoculation, PAL activity increasing as early as 12 h after inoculation. The most significant increase in both peroxidase and PAL activity was observed when the moderately resistant cultivar, Cresor, was challenged with the weakly virulent strain. Highest activity of the two enzymes was detected 2 d after inoculation. Very low peroxidase activity was detected in both strains ofL. maculans, while no PAL activity was detectable in either of the strains. Cytochemical tests revealed increased peroxidase activity following inoculation, mainly in the epidermal and guard cells.     

Antigenic and deoxyribonucleic acid base composition of aMycoplasma and associated diphtheroids

Three diphtheroids isolated from cultures ofMycoplasma arthritidis (Campo strain) were shown to cross-react serologically with theMycoplasma. Stable L-forms prepared from these bacteria were shown to have still greater antigenic similarity to theMycoplasma. But the three diphtheroids isolated on three separate occasions were shown not to be identical antigenically. The buoyant densities of the DNA's of the L-forms were similar to those of the diphtheroids from which they were derived. The caesium chloride gradient method indicated a significant difference in nucleotide base composition between the purified deoxyribonucleic acid of the diphtheroids and of theMycoplasma.Junior Research Fellow supported by USPH training grant 5 ROI AI 00232.     

Intraspecific metabolic diversity among strains ofBurkholderia cepacia isolated from decayed onions,soils, and the clinical environment

A collection of 218 strains ofBurkholderia cepacia (including 18% strain replicates) was assembled from organic soils, decayed onions, and clinical sources. Each strain was characterized for virulence to onion, catabolic ability using the Biolog GN microtiter plate, and several other behaviors. Overall test reproducibility was estimated at 98%. The results obtained using the Biolog GN system corresponded well to those obtained using standard methods. Three coefficients of resemblance (Gower similarity, pattern difference, and Jaccard similarity) were calculated and clustered by the group-average method. The sorted matrices and phenograms, while giving evidence of an underlying phenetic structure to theB. cepacia nomenspecies, gave little evidence of sorting by broad source of isolation. Strains isolated from within fields or samples were frequently found to be similar, however, strains isolated from fields with similar cropping histories were not. The Gower-transformed centroids of ordained clusters were projected in a principal coordinate system and estimates of disjunction were calculated. Strains ofB. cepacia were shown to be non-uniformly distributed in taxonomic space. Strains isolated by serial dilution on onion slices formed a tight phenetic cluster which includes the type strain of the nomenspecies and that of a synonymous group (Pseudomonas multivorans); the strains in this phenon were generally virulent to onion and were partially differentiated from others by pectolytic behavior and by the production of diffusible pigment on King's medium A. Further characterization should better resolve the taxonomy of the nomenspecies.     

Deoxyribonucleic acid base composition of species ofKlebsiella,Azotobacter andBacillus

Methods of molecular taxonomy were used to study the genome or deoxyribonucleic acid (DNA) of different strains of five genera of nitrogen-fixing bacteria. The DNA base compositions of all strains ofKlebsiella pneumoniae (previously classified as strains ofAchromobacter sp.,Aerobacter aerogenes orK. pneumoniae) occupy a fairly narrow range of values from 56.7 to 62.5% G-C content. No significant difference was observed in the DNA base composition of bacteria which can fix molecular nitrogen and that of strains which do not fix nitrogen.Six strains of the speciesAzotobacter vinelandii and the one strain ofA. chroococcum tested possess similar DNA base composition. The strain ofAzotobacter agilis tested, however, had a DNA base composition different from these seven strains. The G-C content of the strains ofAzotomonas insolita falls within the broad range of thePseudomonas genus (55–70%), but the peritrichous flagellation of these strains eliminates them from this genus. Their classification still remains to be ascertained.Eleven strains of the speciesBacillus polymyxa andB. macerans formed two homogenous groups of organisms with different DNA base composition. The atypical strainB. polymyxa Hino is genetically similar to the strains of the speciesBacillus macerans and perhaps should be reclassified as such.Supported in part by National Science Foundation grant GB-483 and National Institutes of Health grants AI 01417 (11) and based on Ph.D. thesis of senior author (1967).     

Substrate specificities and electron paramagnetic resonance properties of benzylsuccinate synthases in anaerobic toluene and<Emphasis Type="Italic"> m</Emphasis>-xylene metabolism

The anaerobic degradation pathways of toluene and m-xylene are initiated by addition of a fumarate cosubstrate to the methyl group of the hydrocarbon, yielding (R)-benzylsuccinate and (3-methylbenzyl)succinate, respectively, as first intermediates. These reactions are catalyzed by a novel glycyl-radical enzyme, (R)-benzylsuccinate synthase. Substrate specificities of benzylsuccinate synthases were analyzed in Azoarcus sp. strain T and Thauera aromatica strain K172. The enzyme of Azoarcus sp. strain T converts toluene, but also all xylene and cresol isomers, to the corresponding succinate adducts, whereas the enzyme of T. aromatica is active with toluene and all cresols, but not with any xylene isomer. This corresponds to the capabilities of Azoarcus sp. strain T to grow on either toluene or m-xylene, and of T. aromatica to grow on toluene as sole hydrocarbon substrate. Thus, differences in the substrate spectra of the respective benzylsuccinate synthases of the two strains contribute to utilization of different aromatic hydrocarbons, although growth on different substrates also depends on additional determinants. We also provide direct evidence by electron paramagnetic resonance (EPR) spectroscopy that glycyl radical enzymes corresponding to substrate-induced benzylsuccinate synthases are specifically detectable in anoxically prepared extracts of toluene- or m-xylene-grown cells. The presence of the EPR signals and the determined amount of the radical are consistent with the respective benzylsuccinate synthase activities. The properties of the EPR signals are highly similar to those of the prototype glycyl radical enzyme pyruvate formate lyase, but differ slightly from previously reported parameters for partially purified benzylsuccinate synthase.     

Biological control of maize seed pathogenic fungi by use of actinomycetes

The effectiveness of twoStreptomyces spp. strains to controlpathogenic fungi was studied in stored maizegrain. The treatments included seeddisinfection and inoculation withStreptomyces spp. strains previously isolatedfrom maize rhizosphere. Actinomycete inoculumconsisted of filtered suspension and totalsuspension of fermentor-producedStreptomyces spp. strains biomass. Treatmentswith Streptomyces spp. strains aloneeffectively suppressed the development ofAspergillus spp., Curvularia lunata, andDrechslera maydis and significantly(p < 0,05) reduced the incidence ofFusarium subglutinans and Cephalosporiumacremonium. Among the inoculation treatments,nondisinfested seed inoculated with filteredsuspension was the only treatment that did notsuppress the development of Penicilliumspp. Maize seed inoculation with totalsuspension of strains was the most effectivetreatment to control the incidence of seedpathogenic fungi. The development of theDiplodia maydis was only suppressed by thecombination of seed disinfection andinoculation with total suspension of strains.Although, the strain DAUFPE 11470 showed thegreatest effectiveness for controlling thefungi pathogenic to seed, root and shootdevelopment was reduced by treatment with thisstrain.The results indicate thatStreptomyces spp. strains reduce the incidenceof seed pathogenic fungi and have potential asa biological control agent. However, an efficient methodof seed treatment with the biological controlagent must be developed before it can become anagricultural practice.     

Protection of apple against fire blight induced by an hrpL mutant of Erwinia amylovora

A regulatory hrpL non-virulent mutant of Erwinia amylovora is effective in controlling fire blight disease when inoculated on apple seedlings simultaneously with the pathogenic parental strain. Mechanisms involved in this protective effect were investigated. The use of two marker genes, uidA and lacZ, expressed in the hrpL mutant and the pathogenic strain, respectively, allowed to localize simultaneously the two inoculated strains in plant tissue. An anti-β-glucuronidase antibody was also used to detect the hrpL mutant. Both techniques indicated that the two strains localized mainly in separate areas of the leaf tissue. In addition, leaves infiltrated with the hrpL mutant exhibited a significant increase in peroxidase activity in contrast to a hrp secretion mutant known to be less effective in the protection. It is suggested that protection obtained with the hrpL mutant relies on the physical separation between the mutant and the parental strain after co-inoculation and the rapid and sustained activation of plant defense mechanisms in reactive tissue, i.e. not invaded by the virulent strain.     

Use of ELISA and GUS‐transformed strains to study competition between pathogenic and non‐pathogenic Fusarium oxysporum for root colonization

To characterize the ability of different strains of Fusarium oxysporum to colonize roots, and to analyze competition for root colonization between pathogenic and non‐pathogenic strains of F. oxysporum, it was necessary to develop specific labelling techniques for quantification of root colonization. Two methods were selected: the production of polyclonal antibodies, and the use of GUS‐transformed strains of F. oxysporum. The polyclonal antibodies recognized infected plants, and gave a minimum reaction with healthy plants, but were not specific for individual strains of F. oxysporum. These antibodies enabled total density of F. oxysporum to be assessed on roots, by ELISA. Metabolic activity of the root population of GUS‐marked strains was assessed by measuring the glucuronidase activity. Strains showed a diversity in their ability to colonize roots: patterns of root colonization were similar, but the intensity and the speed of colonization differed according to the plant—fungus combination used. Results demonstrated competition between the pathogenic and the non‐pathogenic strains for root colonization. In the presence of the non‐pathogenic strain Fo 47, the competition seems to be reciprocal, affecting both the pathogen and non‐pathogenic strain. Other non‐pathogenic strains reduced root colonization by the pathogenic strain, but some strains did not reduce the metabolic activity of the pathogen, suggesting that different mechanisms are involved in the interaction between pathogenic and non‐pathogenic F. oxysporum.     

Expression of antigens in virulent and avirulent Indian strains ofLeishmania donovani

Indo-Gen mediated surface labelling with¹²⁵I demonstrated differences in surface oriented antigens between virulent and virulent promastigote ofLeishniania donovani, In case of virulent strains, surface polypeptides with molecular masses of 63, 53, 42 and 38 kDa were found to be labelled with¹²⁵I whereas in the case of aviralent stains 68, 55, 50, 46, 42 and 33 Da, components were iodinated. Further studies by immunoblot assay using different subcellular fractions of virulent and avirulent parasites demonstrated that antibody raised against gp63 cross-reacted with the 63 and 60 kDa antigen of the virulent and avirulentLeishmania donovani strains of Indian origin respectively. It indicates that these two polypeptides are antigenically similar. When virulent and avirulent cells were grown in the presence of varying concentration of tunicarnycin and immunoblot with anti gp63, it was observed that with increasing concentration of tunicamycin the 63 kDa polypeptide of the virulent cells shifted to approximately 58–57 kDa and the 60 kDa polypoptide of the aviruleni cells shifted to 57 kDa. This suggests that glycosylation may play an important role in antigenic variation between virulent and avirulent parasites.     

Chromosome profile ofLeishmania donovani: Interstrain and interspecific variations

The genome ofLeishmania donovani AG83, a virulent strain causing kala-azar, was resolved into 29 chromosomal bands by pulsed field gel electrophoresis (pFGE) under standardized conditions. Comparison of the karyotype with those of other strains and species revealed variations. By Southern hybridization, specific genes were localized to individual chromosomes. Twenty-two copies ofβ-tubulin genes are located on band 27 (1.63 Mb); minor copies are present in band 16 (850 kb) and band 9 (650 kb). Aβ-tubulin related nontranscribed locus was isolated from a genomic library and shown to contain repetitive sequences hybridizing throughout the genome. Single chromosomes contain multicopy clusters of gp63 and rnini-exon-derived RNA genes, but interspecific variations were observed in each case. The results emphasize the importance of using a standard reference strain ofLeishmania donovani for coordinated genome mapping of this clinically important organism.     

Ectomycorrhizal fungal species and strains differ in their ability to produce free and conjugated polyamines

Production of free and conjugated polyamines by one strain of Laccaria proxima (Boud.) Maire, three strains (H, O, K) of Paxillus involutus (Batsch) Fr., and one strain of Pisolithus tinctorius was studied in vitro. Spermidine (Spd) was the main polyamine in the 4-week-old mycelium of all the fungi. It was mainly present in the free form, but it also occurred in conjugated forms. Paxillus involutus strain H released large amounts of free putrescine (Put), and the Pisolithus tinctorius released a compound probably related to cadaverine (Cad). On the other hand, these two fungi contained less conjugated polyamines than the other fungi. In addition to the amounts, the forms (perchloric acid soluble and insoluble) of conjugated polyamines in the mycelium varied between species and strains. L. proxima contained nearly as much insoluble conjugated Spd as free Spd, whereas Paxillus involutus strains O and K contained relatively large amounts of soluble conjugated Spd. The results suggest that ectomycorrhizal fungal species and strains differ in their ability and need to produce conjugated polyamines. The small amounts of soluble conjugated polyamines found in the culture filtrates indicate that some specific conjugated polyamines may be involved in polyamine translocation across the plasma membrane.     

Sexual fertility of fortyFusarium strains from the EuropeanFusarium sambucinum project

Fungus strains designated asFusarium sambucinum, F. torulosum, orFusarium sp. nov. were crossed withMAT1-1 andMAT1–2 tester strains ofGibberella pulicaris. Of the 40 field strains that were crossed with the tester strains, 13 strains produced fertile crosses and 27 strains did not produce fertile crosses. One strain designated asF. torulosum was fertile with a tester strain ofG. pulicaris, suggesting that this is an intraspecies cross and that the strain isG. pulicaris, and, consequently,F. sambucinum rather thanF. torulosum. The lack of fertile crosses between tester strains and 27 of the 40 field strains suggests that these strains are notG. pulicaris. Although the ability to form a fully fertile cross with a tester strain can determine the species of a fertile strain, it is more problematic to exclude a strain only because it is infertile.     

Immunologic reactions of rabbit anti-Mycoplasma arthritidis serum with in vitro cultivated rat synovial cells

Summary The pathogenesis of the intra-articular, arthritic-inflammatory reaction caused byMycoplasma arthritidis in susceptible rats and mice is poorly understood. To investigate this problem, synovial cells from normal Sprague-Dawley rats were cultured and studied in vitro. These cells continued to produce hyaluronic acid as measured by viscosity and chemical assays. Normal synovial cells were treated with rabbit serum specimens taken before and after immunization withM. arthritidis. Cytotoxicity assays indicated that the cells were killed in the presence of rabbit anti-M. arthritidis serum but not with preimmunization serum specimens. The anti-M. arthritidis serum was not cytotoxic to monolayer cultures of HEp-2, Vero, or L-cells. Antiserum produced in response toM. fermentans, M. hominis, andM. pulmonis did not produce a cytotoxic effect on the cultured synovial cells. From immunofluorescence studies it was demonstrated that the interactions occurred between the rabbit anti-M. arthritidis serum and synovial cell surface antigens. Extreme precautions were taken to prevent mycoplasmal contamination of rats and the synovial cells in culture. These observations would appear to support previous reports implicating mycoplasmas as biological triggering mechanisms of autoimmune reactions. This research was supported in part by funds from the National Science Foundation, Grant DPP72-05787, and the U.S. Veterans Administration.     

The effects of acetosyringone and pH on Agrobacterium-mediated transformation vary according to plant species

Summary Expiants of five plant species (Allium cepa, Antirrhinum majus, Brassica campestris. Glycine max, and Nicotiana tabacum) were co-cultivated with three Agrobacterium tumefaciens strains under different conditions to assess the effects of acetosyringone and medium pH on strain virulence. Tumours were incited on all dicotyledonous species by strains N2/73 and A281. The presence of acetosyringone during co-cultivation generally enhanced the virulence of these strains, most markedly N2/73 on A. majus and G. max, and A281 on G. max. Strain Ach5 was virulent only on N. tabacum in the absence of acetosyringone, which, when present, extended the host range to include A. majus. There was evidence to suggest that acetosyringone may suppress virulence in some strain/plant species interactions. Virulence was affected in some cases by medium pH, but there was no general effect across plant species.Abbreviations T-DNA DNA transferred to plant cells by Agrobacterium - BAP benzyl aminopurine - MS medium Murashige and Skoog (1962) medium     

Correlation ofLegionella pneumophila virulence with the presence of a plasmid

Legionella pneumophila is the causative agent of Legionellosis in man and considered an opportunistic intracellular Gram-negative bacterium that preferentially infects macrophages. The presence of a plasmid in these organisms was determined in cultures of the bacteria grown in vitro. A correlation was observed between the growth of virulent strains of theLegionella in murine macrophages and growth on standard buffered charcoal yeast extract agar supplemented with 0.1% -ketoglutarate (BCYE) agar medium rich in cysteine and widely used for growth of the bacteria in vitro. In contrast, the avirulent isolates of these strains grew well on supplemented Mueller-Hinton (SMH) agar utilized for differentiating virulent from avirulentLegionella. However, one virulent strain ofLegionella (the Iowa strain) was found to grow moderately well on the SMH agar. In addition, test strains ofLegionella that infect in vitro human monocytes were found to grow moderately well on the BCYE- agar, but did not grow on the SMH agar. Examination of these strains for plasmid DNA expression showed that extra chromosomal DNA-containing molecules were present in theL. pneumophila strains characterized as virulent for in vitro growth in macrophages. However, the avirulent strains that replicated in the human monocytes readily but only poorly in the permissive murine macrophages did not show evidence of similar plasmid DNA expression.     

Mink lung cells as a tool for detection ofMycoplasma hyorhinis contamination in cell cultures and virus stocks

Summary In an extensive host range study ofM. hyorhinis mink lung cells (MvlLu, ATCC CCL 64) were found to be the cells of choice for the propagation of this mycoplasm, which otherwise is often difficult to grow in a cell-free medium. Furthermore, rapid plaque assay and plaque purification procedures were developed forM. hyorhinis. The titer ofM. hyorhinis grew to 1×10⁷ to 1×10⁸ pfu/ml within three d postinoculation on mink lung cells. DNA restriction enzyme analysis of the genome ofM. hyorhinis was performed. Endonucleases Bst EII and Xho I are the most suitable enzymes for cleavingM. hyorhinis DNA into distinct fragment patterns. Thus, the use of the combination mink lung cells for mycoplasma growth with subsequent restriction enzyme analysis leads to an unamibiguous detection and identification toM. hyorhinis strains even in minute amounts.     

<Emphasis Type="Italic">Rhodococcus fascians</Emphasis>: Shoot Proliferation without Elevated Cytokinins?

In order to determine whether the disease symptoms caused by virulent strains of Rhodococcus fascians are due to increased cytokinin activity in infected tissues, germinating peas (Pisum sativum cv Novella) were inoculated with either a virulent strain or a nonvirulent strain of Rhodococcus fascians. The nonvirulent strain lacked both the ipt gene and the putative cytokinin oxidase/dehydrogenase homologue, fas5. Control peas were not inoculated. Twelve cytokinins were isolated from pea shoots 3, 6 and 9 days post-inoculation. Within 6 days of inoculation the levels of cytokinin free bases, ribosides, O-glucosides and nucleotides were decreased in shoots inoculated with the virulent strain, and were increased in shoots inoculated with the nonvirulent strain relative to the uninoculated control. The results are discussed with respect to the classic Skoog and Miller (1965) model of organogenesis and to the possible involvement of the plant cytokinin oxidase/dehydrogenase during infection by virulent strains of R. fascians.