Sialic acid-specific lectin from Tritrichomonas foetus.

A novel sialic acid-specific lectin (TFL) was isolated from Tritrichomonas foetus culture supernatant and purified by erythrocyte adsorption followed by fetuin-agarose affinity chromatography. According to gel filtration TFL is a protein of 728 kDa, different from the two sialidases of 853 and 254 kDa, secreted by T. foetus into the medium. The lectin is formed by multimeric complexes of 66 kDa subunit according to SDS-PAGE under reducing conditions. TFL is glycosylated with 4.2% of carbohydrates, half of which is represented by glucose. The lectin reacts equally with N-acetyl and N-glycolyl neuraminic acid, free, in alpha2,3- or alpha2,6-linkage. TFL has 7-fold weaker affinity to alpha2,8-linked N-acetylneuraminic acid (Neu5Ac) in colominic acid. Horse erythrocytes containing 4-O-acetyl Neu5Ac are agglutinated equally as compared to the human cells. TFL affinity to 9-O-acetyl Neu5Ac is 4-fold weaker as documented by hemagglutination inhibition with de-O-acetylated bovine submaxillary mucin, and ovine submaxillary mucin. A panel of mono- and oligosaccharides other than Neu5Ac do not inhibit TFL activity at 200 mM. The lectin does not require bivalent cations for activity, shows optimal reactivity at neutral pH and is stable at 4 degrees C. Anti-TFL antibodies identify membrane positivity on T. foetus, suggesting that the lectin functions in adhesion of the parasites. These findings, together with good stability and immunogenicity, make TFL a prospective candidate for further studies, especially in searching for efficient diagnostics and prevention of bovine trichomoniasis.

Purification and basic characteristics of sialidase from Tritrichomonas mobilensis.

Sialidase, produced by Tritrichomonas mobilensis, a colonic parasite of squirrel monkeys, was purified by adsorption on human RBCs in ice-cold culture supernatant followed by release in PBS at 37 degrees C. The enzyme was purified by size-exclusion chromatography of RBC-eluted material giving a single peak with sialidase activity of molecular weight approx. 630 kDa, representing a quadrimer of the complex of three subunits. SDS-PAGE under reducing conditions showed three bands of 56, 61, and 66 kDa, identical with the molecular weight of the three subunits of T. mobilensis sialic acid-specific lectin (Babál et al., 1994). The enzyme treatment changed the agglutination of human RBCs by other lectins: created agglutinability with galactose-specific peanut agglutinin, but did not change agglutination with alpha 2,6 sialic acid linkage-specific Sambucus nigra lectin and linkage nonspecific TML. A 4-fold decrease of the agglutination with alpha 2,3 sialic acid linkage-specific Maackia amurensis lectin was observed. Histochemistry of kidney glomeruli after T. mobilensis sialidase treatment showed peanut agglutinin positivity on membranes of podocytes indicating selectivity for alpha 2,3 linked sialic acid. The sialidase did not hydrolyze colominic acid and was inhibited by 2,3-dehydro-2-deoxy-NeuAc. Divalent cations were not required for activity. The enzyme activity was optimal at pH 6.5-7 with RBCs as substrate and could be stored for 1 year at 4 degrees C.

Purification and characterization of a sialic acid-specific lectin from Tritrichomonas mobilensis.

New sialic acid-specific lectin has been isolated from culture supernatant of the protozoan Tritrichomonas mobilensis. It was purified by adsorption by erythrocytes or bovine submaxillary gland mucin (BSM)-Sepharose affinity chromatography. The T. mobilensis lectin (TML) does not require bivalent cations for activity and agglutinates all human erythrocytes. The lectin forms multimeric complexes with molecular mass 556 and 491 kDa as determined by size-exclusion chromatography. SDS/PAGE under reducing conditions disclosed a large band of 343 kDa and three bands of 246, 265 and 286 kDa which, after denaturation with urea, were split into three subunits of 56, 61 and 66 kDa; under non-reducing conditions there were two bands, of 360 and 260 kDa. Western blots performed with anti-TML monoclonal antibodies revealed bands identical with those in the silver-stained gels, suggesting homogeneity of the BSM -Sepharose-purified lectin. TML is a highly glycosylated protein with approx. 8% of N-linked glycosides found by protein-N-glycanase F treatment; the total amount of saccharides revealed by chemical deglycosylation was 20%. Haemagglutination-inhibition studies documented exclusive specificity for sialic acid (NeuAc). Both (alpha 2-->6)- and (alpha 2-->3)-linked and free NeuAc were eight times more potent inhibitors than N-glycolylneuraminic acid. The lectin does not require O-acetyl groups on NeuAc for recognition. A spectrum of mono- and oligo-saccharides other than sialic acid had no inhibitory effect at 200 mM. Anti-TML monoclonal antibodies strongly inhibited the lectin activity. TML was stable at temperatures below 4 degrees C and lyophilized with 3% (w/w) glycerol.

Contact-independent cytotoxicity of Trichomonas vaginalis.

OBJECTIVE: To test the dependency of haemolytic and cytocidal manifestations of pathogenicity of Trichomonas vaginalis on direct contact between the target cells and the organism. TEST ORGANISM: T vaginalis strain Baltimore 42. DESIGN: Haemolysis in the presence of live T vaginalis and of its filter-sterilised metabolic products was compared. The dependence of haemolytic and cytocidal effects on retention of low pH of metabolic products of the organism was demonstrated by parallel titrations of sterile filtrates in normal saline and in phosphate buffered saline (PBS) pH 7.0. RESULTS: Near complete lysis was obtained when erythrocytes mixed with T vaginalis were incubated for 1 h at 37 degrees C in saline containing 1% glucose. The same degree of haemolysis was present in filter-sterilised glucose-saline in which the organism was incubated (1 h/37 degrees C) before erythrocytes were added and incubated under the same conditions as in the mixture with the organism. The degree of haemolysis in filtrates was dependent on retention of low pH (below 5.0) of the suspending fluid in which the organism alone was incubated. Dilution of filtrates in PBS, as opposed to normal saline, abolished or diminished the haemolytic effect. Presence of glucose (energy source) in the saline during incubation of the organism had a pronounced enhancing effect. The production of haemolytic metabolites was temperature dependent, whereas the haemolytic process per se was not. The effect was not an exclusive property of T vaginalis since it was also demonstrated with other trichomonads. The same filtrates applied to tissue culture exerted cytocidal effect strikingly similar to that observed in the haemolysis experiments. CONCLUSION: Neither haemolytic nor cytocidal effect of T vaginalis was contact-dependent.

Acquisition and retention of viruses by Trichomonas vaginalis.

Recently described occurrence of virus-like particles (VLP) in some strains of Trichomonas vaginalis suggests the possibility that the pathogenic significance of this organism may be broadened by its potential for viral transmission. Inasmuch as neither the source nor the host range of the VLP are known, any hazard which they may present for man cannot be estimated. A model has been established for the study of acquisition of known human viruses by T vaginalis. Tissue cultures were infected with two reoviruses and a fresh isolate of genital herpes simplex virus (HSV). A squirrel monkey reovirus was also included in the study. T vaginalis was inoculated into the virus cultures three days later. The progress of virus acquisition by the trichomonads was monitored by transmission electron microscopy and by culture. Virus-containing cell fragments were engulfed by trichomonads and internalised in vacuoles. After digestion of cellular debris only virus particle aggregates were retained. Viable reoviruses were recovered from the trichomonads for nine days, and HSV for six days. The results suggest the possibility of transmission of at least some viruses by T vaginalis.

Adherence and surface properties of Tritrichomonas mobilensis, an intestinal parasite of the squirrel monkey.

Adherence properties of the potentially enteropathogenic Tritrichomonas mobilensis were studied in vitro. Axenically cultivated trichomonads readily attached to isolated intestinal epithelial cells and mucus of the squirrel monkey. The kinetics and nature of T. mobilensis cytadherence were microscopically evaluated in cell-suspension assay using Chinese hamster ovary (CHO) cells and in microplate hemagglutination assay with human erythrocytes. Adherence of the parasites to target cells was concentration- and time-dependent; it was inhibited by sialic acid (N-acetylneuraminic or N-glycolylneuraminic acid) and sialyllactose. Neither trypsinization of the flagellates nor their exposure to low temperature (4 degrees C) affected their cytadherence capacities. The data indicate the presence of adhesin(s) with lectin properties on T. mobilensis. Agglutination of live protozoa by animal and plant lectins with various carbohydrate-binding specificities as well as the occurrence of an electron-dense cell coat on plasma membrane suggest marked glycosylation of the parasite surface.

Basic properties of Tritrichomonas mobilensis hemagglutinin.

Tritrichomonas mobilensis is a recently described enteric protozoon of squirrel monkeys. An earlier report identified one of the metabolic products of this organism as a lectinlike hemagglutinin. Its further properties were determined in this study. Culture supernatants of T. mobilensis FP4190 were concentrated by ultrafiltration through a membrane with 100,000-molecular-weight cutoff. High titers of agglutinin against human erythrocytes were obtained. Incubation at 70 degrees C for 15 min resulted in complete inactivation. Exposure to 56 degrees C for 30 min was without effect, and only partial loss of activity was obtained during incubation for up to 18 h. Maintenance at pH 4 to 9 for 4 h at room temperature had no deleterious effect. Apparent degradation of the hemagglutinin was achieved by 18 h of contact with proteinase K, but trypsin and collagenase were essentially ineffective. Papain increased the sensitivity of the test. In the presence of this enzyme hemagglutinin was demonstrated also in cultures of Tritrichomonas foetus and Tritrichomonas augusta but not in those of Pentatrichomonas hominis or Trichomonas vaginalis.

Neurovirulence in an experimental focal herpes encephalitis: relationship to observed seizures.

An animal model of focal herpes simplex encephalitis was used to study several strains of type-1 herpes simplex virus. Rabbits were inoculated in the olfactory bulb by a standardized technique. Virus strains resulting in mortality of greater than 70% produced seizures of 3 types, and all animals that seized became moribund or died. In contrast, a virus strain resulting in a 20% mortality produced no seizures. Administration of 60 mg phenobarbital intramuscularly daily reduced mortality significantly in animals given the epileptogenic viruses. Cultures from temporal and frontal lobes showed viral growth more frequently than did cultures of other brain areas. Microscopic examination of routine and immunoperoxidase-stained brain sections confirmed the focal nature of the infection. Clinical syndromes such as seizures arising from viral brain disease may influence mortality in animal model systems.

Experimental genital trichomoniasis in the squirrel monkey (Saimiri sciureus).

The squirrel monkey (Saimiri sciureus) has been proposed as a model for urogenital trichomoniasis in man, but has not been accepted as such because of the purported presence of naturally occurring vaginal trichomonads in this animal. The study published here shows that these are easily eradicated organisms of intestinal origin, which eliminates the potential confusion created by them. In addition, our experiments have shown that the hormonal status of primates seems to be a determinant in successfully establishing experimental trichomoniasis. This experimental infection recapitulates the clinical observations sufficiently to warrant the use of this model for studies of vaginal trichomoniasis.

Detection of hemagglutinins in cultures of squirrel monkey intestinal trichomonads.

Intestinal trichomonads are very common inhabitants of captive squirrel monkeys. In evaluating the potential pathogenicity of these organisms, we encountered hitherto unknown hemagglutinins in their culture fluids. The cytopathic effect associated with a number of the isolates resembled that caused by vacuolating viruses. We have ruled out conventional viruses as the cause of the cytopathic effect and as the source of the hemagglutinin. The agglutinin has some of the basic characteristics of lectins. Parallel experiments demonstrated agglutination of erythrocytes from squirrel monkeys, humans, dogs, cats, guinea pigs, and horses, with the first two types being the most sensitive. Relatively less agglutination was seen with rat erythrocytes. Agglutination of sheep, rabbit, chicken, and bovine erythrocytes was virtually absent.

Tritrichomonas mobilensis n. sp. (Zoomastigophorea: Trichomonadida) from the Bolivian squirrel monkey Saimiri boliviensis boliviensis.

A trichomonad flagellate, Tritrichomonas mobilensis n. sp., is described from the large intestine of the squirrel monkey, Saimiri boliviensis boliviensis. The organism has a lanceolate body 7-10.5 micrometers in length; a well developed undulating membrane; a stout, tubular axostyle with periaxostylar rings that terminate in a cone-shaped segment projecting from the posterior end of the cell; and a moderately wide costa. The anterior flagella are about as long as the body, and the recurrent flagellum is of the acroneme type. All its characteristics suggest that the new species belongs in the Tritrichomonas augusta type of the subfamily Tritrichomonadinae.

Growth and cytopathogenicity of Trichomonas vaginalis in tissue cultures.

The primary purpose of this study was to identify the mammalian tissue cultures which were most suitable for investigations of the cytopathogenicity of Trichomonas vaginalis. A recently isolated strain of the organism was inoculated into 15 different tissue cultures which were maintained in an appropriately modified growth medium. Proliferation of the protozoon was accompanied by the progressive disintegration of cell culture monolayers. Initial focal lesions consisting of detached cells and an accumulation of trichomonads gradually enlarged until the entire monolayer was disrupted. When judged by the size of the inoculum required to obtain this effect, differences among the tissue cultures were noted. An inoculum of approximately 10(3) viable trichomonads was sufficient to completely disrupt monolayers of HeLa 229, HeLa, McCoy, HEp-2, and RK-13 cells. To obtain a comparable effect with other cells, 10- to 100-fold higher levels of inoculum were required. Polyethylene glycol concentrates from culture filtrates contained a cell-detaching factor (CDF) which caused detachment and clumping of susceptible cells. Freshly seeded cells in growth medium containing CDF failed to form a monolayer. Aggregates of cells maintained for up to 1 week in the presence of CDF remained viable and formed a monolayer after being washed and suspended in normal growth medium. The activity of the CDF was not lost during 1 week of contact with the cells. The CDF may contribute to the pathogenicity mechanisms of T. vaginalis.

Detection and cultivation of intestinal trichomonads of squirrel monkeys (Saimiri sciureus).

Routine examinations of fecal samples from squirrel monkeys suggested that intestinal trichomonads might be common inhabitants of these animals. In pursuit of these observations, microscopic examination of fecal suspensions and cultures have demonstrated a 100% incidence of trichomonads in 30 arbitrarily selected animals from a colony of more than 300 housed in groups of ten. The most prominent species was Pentatrichomonas hominis. A not yet fully characterized tritrichomonad was also found on several occasions. The main obstacle in establishing individual strains in culture was the presence of bacterial and fungal flora in the samples. Nevertheless, abundant cultures were obtained from 28 animals by inoculation of fecal suspensions into tissue cultures with appropriately formulated medium and high concentration of antibiotics. In several unattended cultures maintained at room temperature, the flagellates retained motility for at least 4 months. This long survival may explain the widespread occurrence of the parasites within a confined animal community.

Viral replication in human ovarian cell culture.

A method is presented for the preparation and maintenance of human ovarian cell cultures. Sections of normal ovaries removed at surgery were minced, trypsinized, and seeded as cell cultures grown in minimal essential media at 37 C. Long-term, low-passage cultures were grown in quantities sufficient to permit viral studies. Ovarian cells, of passage 23, were challenged with 57 known viruses. Of these viruses, 52 (especially viruses of the picornavirus and adenovirus groups) produced typical cytopathic effects. The significance of these studies is discussed.

Further studies on the influence of steroids on viral infection in mice.

Certain steroids have been reported to enhance experimental viral infections, whereas others have little or no effect. Interference with the interferon system has been suggested as a possible mechanism for the viral infection-enhancing (VIE) activity of hormones. In the present study, steroids (prednisolone, progesterone, testosterone) which had no effect on MM virus infection demonstrated VIE activity against Mayaro virus infection. It is suggested that the VIE activity of a hormone may be dependent on the viral agent used for challenge. Data are also presented which suggest that, in addition to interference with the interferon system, the VIE activity of steroid hormones may be the result of at least two other actions: (i) alteration of cell membranes; (ii) stimulation of initial viral replication.

Tilorone hydrochloride: lack of correlation between interferon induction and viral protection.

The protection of mice against MM virus infection and the induction of circulating interferon by tilorone hydrochloride were determined. Whereas protection was evident with doses of 0.15 and 1.5 mg/kg, interferon was not detected with doses lower than 150 mg/kg. Protection was apparently not dependent on interferon induction.

Effect of interferon inducers and interferon on bacterial infections.

The effect of interferon inducers and exogenous L-cell interferon on the infection of mice by Pasteurella tularensis or Diplococcus pneumoniae was investigated. The results indicate that the degree of protection is dependent on the type of inducer used. A variety of defense mechanisms with limited nonspecific activity appear to be involved.