Morphology of Pseudomonas aeruginosa phages from the sputum of cystic fibrosis patients and from the phage typing set. An electron microscopy study.

Sixteen phage suspensions isolated from the sputum of sixteen cystic fibrosis patients and five phages from the present phage typing set were studied by electron microscopy. All sputum samples contained at least one type of bacteriophage (range: 1-4) which could be classified by the morphology and dimensions of the virion. All phages isolated from sputum as well as the four typing phages were tailed phages. The clinical phages belonged either to the Myoviridae, Siphoviridae or Podoviridae family. The four typing phages belonged to the Myoviridae family. The detection of the presence of tailed phages in sputum further supports the prevailing theory that phages play a role in the phenotypical change of Pseudomonas aeruginosa during the chronic lung infection of cystic fibrosis patients, since only tailed phages are known to be temporate and thus mediate transduction and conversion.

Echinostoma caproni in mice: ultrastructural studies on the formation of immune complexes on the surface of an intestinal trematode.

The binding of mouse antibodies to the surface antigens of juvenile and 7 and 28 day old Echinostoma caproni was examined by transmission electron microscopy of thin sections of parasites, which were treated with antibodies in a double sandwich technique with ferritin-conjugated antibody. The surface of freshly recovered mature adult parasites was covered with an irregular but often rather intensive mouse antibody containing matrix, which probably represents a layer of mouse antibody/parasite antigen complexes. The complexes were lost after in vitro culturing of the parasites for 24 h, but incubation of the in vitro-maintained antibody-negative adult parasites with immune mouse serum led to reformation of a similar but less intensive cover with immune complexes. Juvenile and young stages of E. caproni, which had never been exposed to host antibodies, obtained a layer of immune complexes on their surface after incubation with immune mouse serum in vitro. In both young and mature parasites, the antibody-antigen complexes were observed to be rather loosely attached to the outer surface of the parasites, where the antigens probably constitute a part of the irregular glycocalyx of the organisms. It may also be that the antigens are present as isolated excretion along the surface of the parasites. Several sections indicated that the parasite surface antigens may be present in the tegument in vesicles which fuse with the outer membrane of the parasite whereby their contents are released to the exterior.

Morphological heterogeneity among spirochetes isolated from cases of swine dysentery.

Pathogenic and non-pathogenic spirochetes isolated from the intestines of pigs were examined by electron microscopy using the negative staining and ultrathin sectioning techniques. Morphological differences were observed among cells of different strains. The cells differed in length as well as in width and in the number of flagella inserted at each end. In addition, the cells from different strains also varied in their resistance to the action of the detergents, Teepol and sodium deoxycholate. Three of the strains studied contained weakly haemolytic spirochetes, two of which differed markedly in their morphology from the cells of the other strains. These spirochetes had fewer flagella inserted at each end than those from other isolates and showed a distinct lattice-like substructure covering the ends of the cells. The spirochetes examined were found to be morphologically more similar to those of the genus Borrelia than to those of the genus Treponema but were clearly different from the cells of both of these genera. The taxonomic implications of the observations are discussed in brief.

The effect of cholesterol on the morphology and reactivity of the mixture of lipids used in syphilis serology.

The effect of increasing amounts of cholesterol on the morphology of the liposomes constituting the VDRL-antigen was studied. The morphological parameters examined were the shape of the lipoidal particles and especially the number of lamellae on each particle in the various mixtures of lipids studied. Cholesterol in the presence of cardiolipin and lecithin is observed as rhomboid crystals, indicating that the majority of the cholesterol is located exterior to the lamellar membranes of lecithin and cardiolipin. It is shown that the effect of cholesterol is to reduce the number of individual lamellae per liposome, mainly by mechanically dispersing the cardiolipin and lecithin on the surface of the cholesterol crystals. It is suggested that cholesterol has no effect on the structure of the epitopes which react with antibodies in sera from patients with syphilis, but that, as a result of the mechanical dispersion of cardiolipin and lecithin, it creates liposomes with more accessible epitopes.

Echinostoma caproni in mice: shedding of antigens from the surface of an intestinal trematode.

The surface antigens, which induce a serum antibody response during infection of mice with the intestinal trematode Echinostoma caproni, were examined. It was demonstrated that antigens are shed from the surface of juvenile and 4-week old adult E. caproni during in vitro culture. SDS-PAGE and Western blot analysis of in vitro shed and detergent solubilized surface antigens indicated that the four major antigens released from the surface of adult parasites had molecular masses of approximately 26,000, 66,000, 75,000 and 88,000. A modified ELISA technique showed the in vitro turn-over rate of the surface antigens to be very high, with a half-life of 8-15 min in both juvenile and adult E. caproni trematodes. Transmission electron microscopy of the surface of adult parasites revealed a highly active secreting tegument which was densely packed with membrane-bound vesicles, reflecting the high rate of shedding of the surface antigens. An attempt to immunize mice with detergent solubilized adult surface antigens failed to induce resistance to infection with metacercariae of E. caproni.

Ultrastructural localization of capsules, cell wall polysaccharide, cell wall proteins, and F antigen in pneumococci.

The localization of pneumococcal capsular and cell wall antigens was examined by immunoelectron microscopy. C polysaccharide (C-Ps), a common component of all pneumococci, was uniformly distributed on both the inside and outside of the cell walls. The thickness of the C-Ps varied with the strain. Encapsulated strains were covered by varied amounts of capsular polysaccharide concealing the C-Ps of the bacteria so as to render it inaccessible to anti-C-Ps antibodies. In addition to C-Ps, protein antigens were demonstrable on the surface of nonencapsulated pneumococci. The proteins were not masked by the C-Ps layer. An extra layer on the cell walls was conspicuous on electron micrographs of both rough and encapsulated pneumococci. The nature of this extra layer has not been disclosed. F antigen, another common antigen of pneumococci, was uniformly distributed on the surface of the plasma membranes. During the course of the experimental work a reproducible method of gold labeling immunoglobulins was developed.

An adhesive protein capsule of Escherichia coli.

The nature of the adhesive capacity of three hemagglutinating Escherichia coli strains that had earlier been described as nonfimbriated was studied. The strains that were isolated from human disease adhered to human buccal and urinary tract epithelial cells, an adhesion that was not inhibited by D-mannose. By crossed immunoelectrophoresis it was shown that the three strains produced a common antigen, Z1, developed after growth at 37 degrees C but not 18 degrees C. One of the strains produced an additional antigen, Z2, of almost the same electrophoretic mobility in crossed immunoelectrophoresis. A mutant of this strain deficient of its polysaccharide K antigen had maintained the adhesive capacity, indicating that the K antigen was not responsible for adhesion. A further mutant of the acapsular mutant produced a strongly reduced amount of the Z antigens and had lost the ability to adhere. The Z1 (and Z2?) antigens were therefore deemed to be responsible for adhesion. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracts of cells of the three strains, a heavy Coomassie-blue stained line was seen, indicating the presence of a protein subunit of molecular weight slightly above 14,400. By immunoblotting with absorbed antiserum, it was shown that this protein was the same as that detected by crossed immunoelectrophoresis. Protease from Streptomyces griseus, but not trypsin, digested the protein. Heating to 100 degrees C did not affect it. By immunoelectron microscopy of embedded and sectioned bacteria that had first been treated with specific antisera and ferritin-labeled antirabbit immunoglobulin, the protein adhesin-antibody complex was found to surround the bacteria as a heavy capsule. After negative staining with uranylacetate (pH approximately 4), the capsule appeared as a mesh of very fine filaments. The possible role of this capsule in the pathogenesis of disease is discussed.

Intestinal spirochetosis: morphological characterization and cultivation of the spirochete Brachyspira aalborgi gen. nov., sp. nov.

The ultrastructure of spirochetes obtained from rectal biopsies of patients with intestinal spirochetosis was studied by means of negative staining and ultrathin sectioning. The cells were sigmoidal with tapered ends, 2 to 6 microns long, with a wavelength of 2 microns. Four flagella were inserted at each end of the cells. The maximal cell width was about 0.2 microns. The spirochetes were cultured on tryptose soy blood agar plates. They were anaerobic and grew, although very slowly, at 37 to 38.5 degrees C in an atmosphere of 5% CO2-95% H2. Two types of colonies could be distinguished. The growth characteristics and the morphology of the isolated spirochetes differ from those of previously isolated spirochetal strains. Consequently, it is proposed that the present strains constitute a new genus, Brachyspira, of the family Treponemataceae. The type species is Brachyspira aalborgi, the type strain of which is 513A (NCTC 11492).

Scanning electron microscopy of the oocyst and sporocyst of Toxoplasma gondii.

Oocysts and sporocysts of T. gondii were examined with the scanning electron microscope. The oocysts were slightly ellipsoidal in shape with a smooth outline. The sporocysts were also ellipsoidal with a smooth surface on which there were a number of raised ridges. The ridges represent the junctions between the four plates comprising the sporocyst wall. From the distribution of the ridges it was concluded that the wall consisted of two upper and two lower plates which were at an angle to each other and which interlocked along the mid-line suture. The specific orientation of the plates would provide a structure extremely resistant to mechanical disruption. During excystation, an infolding of the surface was observed along the suture lines between the plates of the sporocyst wall.

O, K, H and fimbrial antigens in Escherichia coli serotypes associated with pyelonephritis and cystitis.

O:K:H serotype determination of 267 E. coli strains from patients with pyelonephritis or cystitis led us to tentatively consider some O:K:H types, e.g. O6:K2:H1 as pyelonephritis and e.g. O6:K13:H1 as cystitis associated types. By means of crossed immunoelectrophoresis (CIE) several strains of such special O:K:H serotypes were examined for presence of the previously described fimbrial antigens, F7 and F8. Three new F antigens were designated F10, F11 and F12. The fimbrial structures of these antigens were demonstrated by immunoelectron microscopy (IEM). Based on the haemagglutinating (HA) capacity of the strains it was possible to separate differently fimbriated organisms of the same strain. The bacteria which agglutinate human erythrocytes could be absorbed out of the mixture when the erythrocytes were removed by sedimentation. When the bacteria in the supernatant no longer agglutinated human erythrocytes they were regarded as HA absorbed and were compared with non-HA absorbed bacteria for their ability to attach to epithelial cells from the urinary tract. Hence, it was concluded that F7, F8 and F10 caused haemagglutination and were responsible for the attachment to urinary epithelial cells, while the fimbriae antigenically termed pseudotype 1 did not induce haemagglutination and played little or no role for the attachment to this kind of epithelial cells.

Leptospira parva sp.npv.: some morphological and biological characters.

Two lines of a spirochete were isolated as contaminants in a culture of a leptospire and from a bottle of uninoculated medium. Studies on morphological and biological characters led to the conclusion that these lines represent a new species of the genes Leptospira, Leptospira parva sp. nov. The morphology of the cells was rather similar to that of previously examined leptospires, but the cells were shorter and had a shorter wavelength (more tightly wound). Furthermore, the surface layer of cells of L. parva formed numerous small blebs with no apparent substructure when detached during preparation for negative staining, while the surface layer of previously studied leptospires formed cross-striated tubules under similar conditions. L. parva shows biological characteristics of both parasitic and saprophytic leptospires. The deoxyribonucleic acid base composition of cells of this organism differed from that found in leptospires, as well as from that found in cells of Leptonema illini.

Isolation of a heat-stable antigen from Treponema Reiter, using an immunoadsorbent with antibodies from syphilitic patients.

It was attempted to isolate antigens from Treponema Reiter, relevant to syphilis serology, by immunoadsorption with patients' antibodies coupled to CNBr--Sepharose 4B. One antigen was desorbed by 2 M KSCN in 0.05 M Tris--barbital buffer, pH 8.6. The recovery was 3% and 7% in two experiments. A small amount of human antibodies in the isolate was removed on an immunoadsorbent column with insolubilized rabbit antibodies against normal human serum proteins. The antigen thus obtained was immunologically pure when analysed by crossed immunoelectrophoresis. By electron microscopy of immunoprecipitates and by tandem crossed immunoelectrophoresis it was shown that the antigen differed from the flagellar antigen of T. Reiter, but was identical to antigen d previously described in T. Reiter. Antigen d could also be isolated from supernatants of T. Reiter cultures. The d antigen was not denatured at pH 2.8, by 8 M urea or by 3 M KSCN, and it resisted heating to 100 degrees C for 30 min. No protein could be detected in a concentrated preparation, and the antigen might be a polysaccharide. Antigen d is probably present in the sorbent used in the fluorescence treponemal antibody absorption (FTA-ABS) test and may constitute the active substance of this reagent.

Ultrastructure of the tail stump sperm defect in the bull.

A characteristic major sperm defect, with about 60% of the sperm cells showing a rudimentary tail stump, was found in two sterile Danish bulls of imported origin. The condition seldom occurs, but seems to be widespread and has been observed in several breeds of cattle and in different animal species, including man. Electron microscopy of ejaculated sperm and testicular tissue from one of the bulls has shown that a dysfunction of the centriolar apparatus during spermiogenesis is likely to be the cause of the defect. In ejaculated defective sperm the proximal centriole is often found connected with the normally transitory organelle, the centriolar adjunct. In the defective spermids the distal centriole has lost the faculty to form axonemal fibers, so that no tail formation can take place.

Ultrastructural observations on microgametogenesis and the structure of the microgamete of Isospora felis.

The endogenous forms of Isospora felis were observed within the epithelial cells of the small intestine of the cat. They were situated within a parasitophorous vacuole which was limited by a multimembranous wall. The ultrastructural features of microgametogenesis were studied at 8 days post-infection. The initial phase of microgamont development consisted of cytoplasmic growth accompanied by a number of nuclear divisions. The gamont was enclosed by a pellicle and its surface area was greatly increased by deep invaginations. In the later stages of development the numerous nuclei were situated close to the pellicle. Each nucleus has peripherally condensed chromatin. Formation of the microgametes occurred as protrusions from the microgamont surface. Two basal bodies, the dense portion of a nucleus and a mitochondrion entered each protrusion. The microgametes matured while still attached to the gamont from which they finally budded off into the parasitophorous vacuole leaving a large residual cytoplasmic mass. The mature microgamete was found to consist of an elogate nucleus which overlaps with a mitochondrion towards the anterior end of the organism. The anterior portion contains a dense perforatorium and two basal bodies with attached flagella. In addition a number of microtubules (5-9) were found to run longitudinally from the basal body region.

Ultrastructural observations on macrogametogenesis and the structure of the macrogamete of Isospora felis.

The ultrastructural changes occurring during macrogametogenes of Isospora felis were studied in the small intestine of the cat at 8 and 9 days post-infection. The parasites were situated in parasitophorous vacuoles within the host epithelial cells. The early macrogamont could be identified by its ellipsoidal shape and by the presence of a large nucleus with a distinct nucleolus. Throughout the development of the macrogamont the organism remained limited by a pellicle. At an early stage of macrogametogenesis the formation of the wall-forming bodies of types I and II (WFB I and WFB II) was initiated simultaneously. The formation of the WFB I appeared to involve the rough endoplasmic reticulum (rER) and the Golgi bodies, whereas the WFB II developed within cisternae of the rER. Early in the developmental process vacuoles were observed which budded off from the nucleus and formed multimembranous vacuoles. During gametogenesis both lipid globules and polysaccharide granules developed within the cytoplasm. The mature macrogamete was spherical in appearance and was limited by a pellicle. It possessed a large nucleus with a nucleolus and the cytoplasm was filled with numerous WFB I, WFB II, lipid globules and polysaccharide granules. A few multimembranous vacuoles were also present together with canaliculi, mitochondria, Golgi bodies and some rER.

Comparison of Escherichia coli fimbrial antigen F7 with type 1 fimbriae.

Two Escherichia coli O6:K2:H1 strains, C1212 and C1214, isolated from urinary tract infections, were compared for their capacity to adhere to various cells. After growth on solid medium, only C1212 bacteria agglutinate human erythrocytes and attach to urinary epithelial cells. Both of these reactions are mannose resistant. In contrast, C1214 bacteria cause a mannose-sensitive agglutination of guinea pig erythrocytes, show a mannose-sensitive attachment to buccal epithelial cells, and attach to urinary mucus. Immunoelectron microscopy revealed that C1214 bacteria possess type 1 fimbriae (mannose sensitive), which are not present in C1212 bacteria when this strain is grown on solid medium. The fimbriae of C1212 (mannose resistant) were also demonstrated by immunoelectron microscopy. We call these fimbriae demonstrated in C1212 the E. coli F7 antigen. Urinary mucus, and probably mucous material elsewhere, may function as a trap for Enterobacteriaceae with type 1 fimbriae by the specific adherence of such bacteria. We consider this a nonimmune resistance mechanism against disease caused by Enterobacteriaceae.

An ultrastructural study on the excystation of the sporozoites of Toxoplasma gondii.

The ultrastructural changes which occur during the in vitro excystation of the sporozoites of Toxoplasma gondii were examined. The excystation was carried out at 37 degrees C on suspensions of oocysts which had been ground and then treated with an excysting medium containing 0.25% trypsin and 0.75% sodium taurocholate in phosphate buffered saline, pH 7.3. It was found that sporocysts within intact oocysts were unaffected while sporocysts exposed to the medium ruptured. The sporocyst wall consisted of two layers and during excystation the four plates which form the inner layer started to curl inward. At the same time changes were seen at the specialized junctions between these plates. When the junctions finally break, the plates separate. The outer layer of the sporocyst wall is then ruptured at points directly above where the plates were joined. Each of the four portions of the sporocyst wall curled inward to form a tightly wound whorl. The sporozoites can escape through the openings created between the portions of the sporocyst wall.

Electron microscopy of a filamentous, segmented bacterium attached to the small intestine of mice from a laboratory animal colony in Denmark.

A filamentous, segmented bacterium was observed in the small intestine of the SSC:AH stock of mice from the Statens Seruminstitut (Denmark) animal colony but was absent in golden hamsters and guinea pigs from the same colony. The bacterium is attached to the epithelial cell by a special segment (holdfast) and causes specific changes in the epithelial cell at the site of attachment. Once attached the bacterium appears to undergo a complex life cycle which involves the development of a long filament divided into a number of segments within which holdfasts or spores are formed. This organism is morphologically identical to a bacterium found in mice and rats in the USA, but this is the first report of such an infection in Europe.