ALBERT

Notes: Two strains of Trichomonas vaginalis, JH162A, with low pathogenicity, and Balt 44, with high pathogenicity, as well as one highly pathogenic strain, KV-1, of Tritrichomonas foetus were studied by freeze-fracture electron microscopy.The protoplasmic faces (PFs) of the cell membranes of all three strains of both species had similar numbers of intramembranous particles (IMPs); however, the particles in the external faces (EFs) of these membranes were least abundant in Trichomonas vaginalis strain Balt 44 and most numerous in those of strain JH162A of this species. In Tritrichomonas foetus strain KV-1 the number of IMPs in the EF was close to but somewhat lower than that in the mild strain of the human urogenital trichomonad. In both species, the anterior, but not the recurrent, flagella had rosette-like formations, consisting of ∼9 to 12 IMPs on both the PFs and EFs. The numbers and distribution of the rosettes appeared to vary among different flagella and in different areas of individual flagella of a single organism belonging to either species. The freeze-fracture electron micrographs provided a more complete understanding of the fine structure of undulating membranes of Trichomonadinae, as represented by Trichomonas vaginalis, and of Tritrichomonadinae (the Tritrichomonas augusta-type), as exemplified by Tritrichomonas foetus, than was gained from previous transmission and scanning electron microscope studies. Typically three longitudinal rows of IMPs on the PF of the recurrent flagellum of Trichomonas vaginalis were noted in the area of attachment of this flagellum to the undulating membrane. The functional aspects of the various structures and differences between certain organelles revealed in the two trichomonad species by the freeze-fracture method are discussed.

Notes: SYNOPSIS. Several fine-structural changes were observed during formation of the so-called pseudocysts of Trichomitus batrachorum grown in an agar-free medium. Among these changes the most frequent were those of the undulating membrane complex. Internalization of the marginal lamella combined with disappearance of the fin-like, membranous dorsal fold occurred with the highest frequency. In many organisms these alterations were accompanied by internalization of the recurrent flagellum, which, however, remained external in other, presumably earlier stages of pseudocyst formation. In some of the internalized and even still external recurrent flagella the microtubules were in various states of disassembly. The often distended flagellar membrane enclosed large accumulations of filaments 5 nm in diameter. Internalization of the anterior flagella also was noted. Changes similar to those characteristic of the recurrent flagella were not seen, however, among the microtubules of the anterior flagella or among those of the pelta or the axostyle.

Notes: A relatively simple phenol extraction method, with EDTA as the nuclease inhibitor, is described for the isolation of purified, highly polymerized native DNA from Trichomonas vaginalis, Trichomonas gallinae, and Tritrichomonas foetus; it is applicable also to Tetrahymena pyriformis. RNase Tl, RNase A (Worthington's R), pronase, and α-amylase digestions constitute important steps in obtaining satisfactory yields of DNA. High degree of polymerization of the isolation product was estimated by hyperchromicity at O.D.260 after DNase treatment and by CsCl gradient analysis. The double-stranded condition of the DNA samples was estimated by the latter method and by denaturation with NaOH, and the molecular weight by sucrose gradient analysis. Purity of the samples was determined spectrophotometrically and by chemical analyses for protein and glycogen. DNA percent recovery was estimated by the diphenylamine reaction.

Notes: SYNOPSIS. Trichomitus batrachorum (Perty) shares with Trichomonadinae most of its fine-structural characteristics, especially those pertaining to the undulating membrane that consists of a high body fold, enclosing the loop-shaped, periodic marginal lamella, and of the recurrent flagellum applied to the fold. This flagellate has also certain structures, i.e. the costal base, comb-like organelle, and well developed marginal lamella, in common with Monocercomonas. It shares with Hypotrichomonas the costal base; large pelta; very fine fibers perpendicular to, and connecting the axostylar microtubules; structural details of the most proximal segment of the marginal lamella; and general relationships between dorsal body fold (poorly developed in Hypotrichomonas) and the recurrent flagellum. All these electronmicroscopic findings support the crucial position of Trichomitus in the evolutionary sequence Monocercomonas→Hypotrichomonas→Trichomitus→ Trichomonadinae suggested previously by lightmicroscopic observations. Further, Trichomitus shares with all Tritrichomonadinae the comb-like structure, not found either in Hypotrichomonas or Trichomonadinae; and it has in common with Tritrichomonas also the costa with Type A periodicity, being the only member of Trichomonadinae with this type of supporting organelle. It appears, therefore, that Trichomitus-type organisms represent an important link in the evolution of all Trichomonadidae from Monocercomonadidae.

Notes: SYNOPSIS. Antigenic constitution of 5 Trichomonas gallinae strains and substrains was analyzed by gel diffusion technics. Fresh isolates of the very virulent JB and of an avirulent SG strain as well as avirulent substrains JBC and SGC, derived from JB and SG respectively by prolonged in vitro cultivation, were used in the experiments. An originally avirulent AG strain that was attenuated still further and lost its infectivity for pigeons during many years of serial transfers in nonliving media also was analyzed.Two major groups of antigens, A and B, were differentiated on the basis of precipitin line patterns formed in gel diffusion reactions involving the 5 strains and substrains and antisera prepared in rabbits against each of these trichomonad stocks. Group A was subdivided further into subgroups [A] and (A). JB, JBC, AG, and SGC trichomonads appeared to share all or nearly all antigens of both these subgroups, but AG strain contained some unique [A] and (A) antigens in addition to those which it had in common with the remaining 4 strains and substrains.Group B antigens were divided into 5 subgroups, B1 to B5. The complete B1 antigenic complex was found in JB and JBC trichomonads and part of this complex was present also in SG strain and SGC substrain. In all instances, subgroup B1 antigens stimulated production of specific antibodies in rabbits and combined with these antibodies present in immune sera. The complete B2 antigenic complex was found only in JBC substrain. Some subgroup B2 antigens were present also in JB trichomonads. Very few of these, however, were capable of stimulating antibody production in rabbits. The more numerous B2 elements of JB strain that did not stimulate immunologic responses in rabbits, might be in the form of incomplete hapten-like antigens. All subgroup B2 antigens found in JB strain represented only a portion of the B2 complex associated with JBC substrain. Subgroup B2 was characteristic of SG and SGC trichomonads, the latter substrain differing from the parental SG strain in the levels of both B2 and B1 antigens; these differences, however, were purely quantitative. JB strain reacted with some of subgroup B3 antibodies present in SG and SGC antisera, but failed to stimulate antibody formation against any of these antigens in rabbits. The B3 elements of JB trichomonads might be incomplete antigens. AG strain was characterized by having B4 and B5 antigenic complexes. The very small part of subgroup B4, represented by a weak precipitin line in reactions between JB strain or JBC substrain and anti-AG serum, suggested the presence of some incomplete B4 antigens in these trichomonads.Irrespective of whether freshly isolated avirulent strains or substrains attenuated by prolonged in vitro cultivation are examined by gel diffusion, such organisms are found richer in subgroup B antigens than the fully virulent JB trichomonads. All the results suggest that there may be a direct relationship between antigenic constitution and virulence of T. gallinae strains.

Notes: SYNOPSIS. Tritrichomonas foetus shares many fine-structural features with the previously described genera of the subfamily Trichomonadinae. These include the arrangement and structure of the kinetosomes, of most rootlet filaments, including the sigmoid filaments of kinetosome #2, as well as those of the parabasal apparatus and of the pelta-axostyle complex. On the other hand, this species, and presumably all other Tritrichomonas augusta-type flagellates, differ from Trichomonadinae in certain important details. Among the features which T. foetus does not share with Trichomonadinae are the fine structure of the costa and of the undulating membrane, as well as several organelles not found in the latter subfamily. The costal base of Trichomonadinae is replaced in T. foetus and other Tritrichomonadinae by a comb-like structure, extending between the costa and the infrakinetosomal body. The suprakinetosomal body, connected to kinetosome #2 in the region of attachment of the sigmoid filaments, and the infrakinetosomal body, which appears to contribute to the proximal marginal lamella, are organelles evidently restricted to Tritrichomonadinae. The undulating membrane consists of 2 parts. The proximal part is a fold-like differentiation of the dorsal body surface, the dorsal part of which contains the proximal marginal lamella. The distal part of the undulating membrane, with no obvious physical connection to the fold, encloses the distal marginal lamella in its ventral, and the microtubules of the recurrent flagellum in its dorsal area. The organelle of T. foetus which by its size, certain structural characteristics, and relationship with the undulating membrane and some organelles, including the paracostal granules, is analogous to the costa of Trichomonadinae and of Trichomitopsis termopsidis (subfamily Tritrichomonadinae), conforms in the structure of its periodic cross-striations to that of the parabasal filaments of the latter organisms; its origin corresponds closely to that of parabasal filament 2 of Trichomonadinae.

Notes: SYNOPSIS. The nonpathogenic flagellate from the cecum of fowl, hitherto known as Histomonas wenrichi Lund, is an actively ameboid organism equipped with 4 flagella. Three of the flagella, subequal in length, are applied to one another for a short distance anterior to their origin in the kinetosomal complex and typically terminate in knob-like expansions; the 4th flagellum originates independently of the others and ends in a fine filament. A short, wide pelta is joined to the anterior part of the broad, spatulate axostylar capitulum applied to the left-ventral surface of the ellipsoidal, spheroidal, or ovoidal nucleus. Posterior to the nucleus the capitulum narrows into a slender axostylar trunk, which does not project beyond the body surface. The parabasal body, associated with a long, stout parabasal filament, is dorsal and to the right of the nucleus. On the basis of the flagellar number, the parasite is placed in a new genus Parahistomonas. A new subfamily, Protrichomonadinae, is created within the appropriately emended trichomonad family Monocercomonadidae for Parahistomonas wenrichi (Lund) comb. nov. as well as for Histomonas meleagridis (Smith) and Protrichomonas legeri (Alexeieff).

Notes: SYNOPSIS. Clones of Tritrichomonas foetus, referred to as strains KV-1, DK-2, and UT-1, which were judged by subcutaneous mouse assay to differ in pathogenicity, had different effects on trypsin-dispersed liver cell cultures prepared from 3 lines (Light Brown Leghorn, Massachusetts Brown, Massachusetts Low Growth) of chicks. The general pattern of parasite-cell interaction was similar in all cell cultures, but the intensity of certain responses of the cultures to trichomonads, reflected best in the levels of macrophage activity, depended on the line of chicks.The mild UT-1 strain was readily engulfed and digested by the macrophages, caused very little damage to the epithelial cells and fibroblasts, and had a minimal inhibitory effect on the division rate of the latter. Many abnormal changes were seen, however, in the cytoplasm and nucleil of fibroblasts and epithelial cells in cultures exposed to strains of high (KV-1) and intermediate (DK-2) pathogenicity or to cell-free filtrates of rich active cultures (henceforth referred to as filtrates) of these strains. The changes included retraction of cytoplasm which caused cell-free spaces to appear in the sheets of fibroblasts and around the epithelial islands; the islands became detached from the surrounding fibroblast sheets and tended to form multilayered cell mounds. The flagellates and filtrates of KV-1 strain greatly inhibited fibroblast division, and similar, but less pronounced, inhibitory effects were exerted by DK-2 strain and its filtrates. Trichomonads of all 3 strains did not attach themselves to fibroblasts and epithelial cells. Thus the abnormal changes in cultures infected with KV-1 and DK-2 parasites apparently were caused by some toxic substances produced by the trichomonads, which had to be responsible also for the identical changes in cultures exposed to filtrates of these strains. KV-1 and DK-2 strains inhibited the phagocytic activity of the macrophages in the early stages of infection, the former causing stronger and longer lasting inhibition. Trichomonads of KV-1 strain multiplied actively within the phagocytes which developed degenerative changes and ultimately burst, releasing healthy parasites into the medium. DK-2 flagellates, altho incapable of dividing inside the macrophages, usually were not digested and caused degeneration of the cells which contained them.