Studies on Trichomonads. I. The Metabolism of Tritrichomonas foetus and Trichomonads from the Nasal Cavity and Gecum of Swine

SUMMARY. The metabolism of Tritrichomonas foetus (strain, BP-1) and trichomonads from the nasal cavity and cecum of swine was studied manometrically under similar experimental conditions. At pH 6.4, quantitative and qualitative differences were observed. The cecal (probably T. suis ) and nasal trichomonad used glucose, galactose, fructose, mannose, lactose, sucrose, raffinose, and trehalose. T. foetus used all except lactose and raffinose. All three were inhibited by iodoacetate and arsenite. T. foetus and the nasal form were significantly inhibited by fluoride and 8-hydroxyquinoline, whereas the cecal trichomonad was not. At varied pH, all failed to oxidize Krebs' cycle intermediates. The amounts of oxygen consumed by T. foetus and the nasal trichomonad in the presence of lactate and pyruvate were at levels similar to those with disaccharides; the cecal trichomonad was indifferent toward both substances. Anaerobically, lactate and pyruvate increased the evolution of gas by all three trichomonads. Aerobic acid formation was demonstrated for all three forms. Anaerobically, metabolic CO₂ and gas(es) that were not absorbed by KOH were evolved by all three. Pure oxygen was inhibitory to glucose utilization and stimulatory to the endogenous respiration of all trichomonads; the nasal form was affected the least.
The writer believes that the cecal trichomonad is different from T. foetus and the nasal trichomonad of swine. The relationship between the nasal trichomonad and T. foetus remains in doubt.     

Purification of cysteine proteinases from trichomonads using bacitracin-sepharose

Abstract Bacitracin affinity chromatography has been used to purify proteinases of the parasitic protozoon Tritrichomonas foetus . It proved superior to other affinity chromatography methods we have tested for the purification of trichomonad proteinases and should prove a useful procedure for purifying cysteine proteines from these parasites and other parasitic protozoa. The main cysteine proteinases of T. foetus were purified over 100-fold to be free from the majority of other cell proteins. About 90 μg of protein containing 1.56-fold more proteinase activity than was detectable in the original cell lysate was obtained from 10⁹ cells (7.2 mg protein). SDS-PAGE revealed that the eluate contained two main Coomassie blue-staining bands. N-terminal amino acid sequence analysis of these proteins confirmed that one of them was a cysteine proteinase with unusuall features. Cysteine proteinases were also purified from cell lysates of Trichomonas vaginalis and a N-terminal sequence determined. This is the first amino acid sequence information that has been obtained for trichomonad cysteine proteinases. The method was also used to purify proteinases from the medium of T. foetus cultures. Some selectivity in binding of the proteinases to the affinity column was found.     

Entry and intracellular location of Mycoplasma hominis in Trichomonas vaginalis

The parasite Trichomonas vaginalis causes one of the most common non-viral sexually transmitted infections in humans. The coexistence of different sexually transmitted diseases in the same individual is very common, such as vaginal infections by T. vaginalis in association with Mycoplasma fermentans or Mycoplasma hominis. However, the consequences and behavior of mycoplasma during trichomonad infections are virtually unknown. This study was undertaken to elucidate whether mycoplasmas enter and leave trichomonad cells and if so how. M. hominis was analyzed in different trichomonad isolates and the process of internalization and the pathway within the parasite was studied. Parasites naturally and experimentally infected with mycoplasmas were used and transmission electron microscopy, cytochemistry and PCR analyses were performed. The results show that: (1) M. hominis enters T. vaginalis cells by endocytosis; (2) some mycoplasmas use a terminal polar tip as anchor to the trichomonad plasma membrane; (3) some trichomonad isolates are able to digest mycoplasmas, mainly when the trichomonads are experimentally infected; (4) some fresh virulent isolates are able to maintain mycoplasmas as cohabitants in the cell’s interior; (5) some mycoplasmas are able to escape from the vacuole to the trichomonad cytosol, and trichomonad plasma membrane budding suggested that mycoplasmas could leave the parasite cell.     

Studies on Trichomonads. II. The Metabolism of a Trichomonas batrachorum-type Flagellate from the Cecum of Swine

SYNOPSIS. Certain aspects of the metabolism of a Trichomonas batrachorum -type flagellate from the cecum of swine were studied. This trichomonad (1) oxidized glucose, mannose, maltose, sucrose, and inulin, (2) was incapable of oxidizing Krebs' cycle intermediates, (3) possessed peroxide-splitting capabilities, (4) was inhibited by only iodoacetate and arsenite, and (5) formed acid(s) aerobically. Although there was no effect on oxygen uptake, pyruvate and lactate increased the anaerobic evolution of gas(es). In addition to CO₂, other gas, not absorbed by KOH, was produced anaerobically.
Compared with other porcine trichomonads, the metabolism of this small trichomonad resembles most closely that of the large cecal trichomonad, T. suis. However, the smaller trichomonad had a generally lower respiratory rate, a slightly lower optimal pH, and failed to oxidize fructose, galactose, lactose, raffinose, and trehalose.     

Comparable growth of Tritrichomonas mobilensis in two commercially available culture media

The investigation reported herein was undertaken to determine which medium is more practical for the axenic laboratory culture of trichomonads. The growth of Tritrichomonas mobilensis was monitored in 2 different types of commercially available growth media. Although Roswell Park Memorial Institute (RPMI) 1640 medium is typically used as a mammalian cell culture medium, it was found to support the growth of trichomonads as well as the American Type Culture Collection (ATCC) medium 745 under similar conditions. Environmental variables, such as temperature and pH, known to affect the success of trichomonad cultures were controlled. The mean generation times (MGTs) of T. mobilensis in the log phase of growth were 5.1 and 4.9 hr for RPMI 1640 and ATCC medium 745, respectively. A stationary phase of zero growth was reached more quickly in the ATCC medium 745 cultures, and in both media a phase of rapid attrition followed this period of static growth. In assessing the practicality of the media, total cell amplification, as well as factors such as cost, ease of preparation, and storage capacity, were considered.     

The proteins secreted by Trichomonas vaginalis and vaginal epithelial cell response to secreted and episomally expressed AP65

We showed recently that contact of human vaginal epithelial cells (VECs) by Trichomonas vaginalis and incubation with trichomonad proteins in conditioned medium induced expression of VEC genes. We performed 2-D SDS-PAGE followed by MALDI-TOF to identify the major secreted proteins. Based on protein abundance and separation of spots in 2-D gels, 32 major secreted proteins were examined, which gave 19 proteins with accession numbers. These proteins included known secreted cysteine proteinases. In addition, other secreted proteins were enzymes of carbohydrate metabolism, adhesin protein AP65, heat shock proteins, thioredoxin reductase and coronins. We confirmed that the secreted trichomonad proteins induced expression of VEC genes, including interleukin 8 (IL-8), COX-2 and fibronectin. Purified AP65 added to VECs had a pronounced effect only on IL-8 gene expression, which was inhibited in the presence of 12G4 monoclonal antibody to AP65. Moreover, AP65 expressed episomally within epithelial cells was found to enhance the expression of IL-8 and COX-2. This may be the first report of analysis of the secreted proteins of T. vaginalis and of the host epithelial cell response to these proteins and to the prominent adhesin AP65.     

Electron microscope observations on the interaction of Mycoplasma fermentans with Trichomonas vaginalis

Cultures of Trichomonas vaginalis were found to be contaminated with Mycoplasma fermentans. By means of electron microscopy the interaction between the prokaryotic organisms and the trichomonads was examined. Cells of M. fermentans were observed in the medium; some of them were attached to the surface of the trichomonads and others were observed in membrane-bounded vacuoles of trichomonads. They were also present in the ground substance of the cytoplasm. The mycoplasmas divided by binary fission like other prokaryotes. The most obvious change occurring in the infected trichomonad cells was an increase in number of vacuoles containing mycoplasmas.     

Functional capacities of lungs and thorax in beagles after prolonged residence at 3,100 m

Functional capacities of the lungs and thorax in beagles taken to high altitude as adults for 33 mo or in beagles raised from puppies at high altitude were compared with functional capacities in corresponding sets of beagles kept simultaneously at sea level. Comparisons were made after reacclimatization to sea level. Lung volumes, airway pressures, esophageal pressures, CO diffusing capacities (DLCO), pulmonary blood flow, and lung tissue volume (Vt) were measured by a rebreathing technique at inspired volumes ranging from 15 to 90 ml/kg. In beagles raised from puppies we measured anatomical distribution of intrathoracic air and tissue using X-ray computed tomography at transpulmonary pressures of 20 cm H2O. Lung and thoracic distensibility, DLCO, and Vt were not different between beagles that had been kept at high altitude for 33 mo as adults and control subjects kept simultaneously at sea level. Lung distensibility, DLCO, and Vt were significantly greater in beagles raised at high altitude than control subjects raised simultaneously at sea level. Thoracic distensibility was not increased in beagles raised at high altitude; the larger lung volume was accommodated by a lower diaphragm, not a larger rib cage.     

Trichomonas vaginalis: in vitro survival in swimming pool water samples

In this work it is shown that Trichomonas vaginalis remains viable and infective in swimming pool water samples for several hours. After survival, trichomonad cytotoxicity was tested on primary cultures of epithelial cells. It demonstrates that the some trichomonad strains are able to survive in water pools and survival time is dependent on the degree of strain infectivity and also if it is a long term cultured or fresh isolate.     

Plasma concentrations of thyroid hormones in dogs: influence of sampling hour, breed and age

Thyroxine (T4) and triiodothyronine (T3) plasma concentrations have been determined during 24-hr sampling periods in six mongrels (age 12-36 months), six beagles (age 35-37 months), three labradors (age 3.5 months) and three beagles (age 5 months). The mean T4 levels of the labradors were significantly lower than the values found for mongrels or older beagles (P less than 0.05), whereas T3 was higher in the 5 month old beagles compared to the mongrels (P less than 0.001), young beagles (P less than 0.05) or labradors (P less than 0.01). Circadian and ultradian rhythmicities have been evaluated by cosinor and Fourier analysis. Mongrels and older beagles did have a 12-hr rhythmicity in plasma T4 (P less than 0.05), whereas 5 month old beagles had a circadian one (P less than 0.01). A 12-hr rhythmicity was also found for T3 in the older Beagles (P less than 0.05). However, Fourier analysis indicated that the daily variation in T4 and T3 plasma levels was inadequately mathematically described by single sinusoidal rhythm and that more harmonic components are to be taken into account. The obtained data during a 24-hr period indicate that T4 and T3 concentrations in plasma may vary according to breed, age and sampling hour.     

Profile of hematological values in hybrid hairless dogs

The purpose of this paper is to describe some fundamental physiological data in F 1 hybrids bred from a Mexican hairless dog and beagle cross. These F 1 hybrids numbered 5 hairless dogs and 12 haired dogs. The hematological profile of these offspring was assessed via an automated cell counter and compared with those of healthy beagles. In hairless dogs, red blood cell count, hemoglobin concentration and packed cell volume tended to be higher than in beagles. White cell distribution curves in hairless dogs and beagles yielded a single peak, while in haired dogs one or two peaks were present. Red blood cell and platelet distribution curves revealed few differences among the 3 kinds of dogs.     

Phylogenetic position of the trichomonad parasite of turkeys, Histomonas meleagridis (Smith) Tyzzer, inferred from small subunit rRNA sequence.

The phylogenetic position of the trichomonad, Histomonas meleagridis was determined by analysis of small subunit rRNAs. Molecular trees including all identified parabasalid sequences available in data bases were inferred by distance, parsimony, and likelihood methods. All reveal a close relationship between H. meleagridis, and Dientamoeba fragilis. Moreover, small subunit rRNAs of both amoeboid species have a reduced G + C content and increased chain length relative to other parabasalids. Finally, the rRNA genes from H. meleagridis and D. fragilis share a recent common ancestor with Tritrichomonasfoetus, which exhibits a more developed cytoskeleton. This indicates that Histomonas and Dientamoeba secondarily lost most of the typical trichomonad cytoskeletal structures and hence, do not represent primitive morphologies. A global phylogeny of parabasalids revealed significant discrepancies with morphology-based classifications, such as the polyphyly of most of the parabasalid families and classes included in our study.     

Trichomonad infection in endemic and introduced columbids in the Seychelles

Island endemic avifaunas face many threats, including the now well-documented impacts of pathogens. The impacts of pathogens on the endemic Seychelles avifauna, however, have been little studied. The protozoan parasite Trichomonas gallinae has been shown to reduce survival and reproductive success of the endemic Pink Pigeon Columba mayeri on the nearby island of Mauritius. I investigated trichomonad infection prevalence and pathogenicity in endemic Seychelles Blue Pigeons, Alectroenas pulcherrima, and two introduced species of columbid, the Madagascar Turtle-dove, Streptopelia picturata, and the Barred Ground Dove, Geopelia striata, on the Seychelles island of Mahé during September-October 2007. I asked whether: 1) trichomonad infections occur in these species; 2) prevalence varies among species; and 3) birds show any signs of pathogenicity consistent with tricho-monosis. I use the results to assess the potential threat of this pathogen to A. pulcherrima. All three species were infected with trichomonads, and the overall prevalence was 27.5%. Alectroenas pulcherrima had higher prevalence (47.1%) than the two introduced species combined (24.3%). No infected individuals showed any signs of disease. These findings suggest that trichomonad parasites should be considered as a potential disease threat to the A. pulcherrima population.     

Ultrastructure of Ditrichomonas honigbergii N. G., N. Sp. (Parabasalia) and Its Relationship to Amitochondrial Protists

ABSTRACT. Ditrichomonas honigbergii n. g., n. sp. is a small trichomonad flagellate that has three emergent flagella arising from four basal bodies, a parabasal apparatus (single dictyosome with associated striated flagellar rootlets), a microtubular axostyle, a short undulating membrane, and hydrogenosomes. Cultures of D. honigbergii were isolated from the sediments of a freshwater lake and there is no known metazoan host. Cells form walled cysts with internalized flagella and go through all phases of the life cycle (excystment, binary division, encystment) without any perturbations to the culture medium. Ditrichornonas honigbergii is capable of ingesting and digesting bacteria by phagocytosis. These facts suggest that D. honigbergii may be a free-living inhabitant of oxygen-reduced environments. The structure of D. honigbergii is similar to that of retortamonads and the relationship of trichomonads to other amitochondrial protists is discussed.     

Orcadian Variation in Human Stature

Thyroxine (T₄) and triiodothyronine (T₃) plasma concentrations have been determined during 24-hr sampling periods in six mongrels (age 12-36 months), six beagles (age 35-37 months), three labradors (age 3.5 months) and three beagles (age 5 months). The mean T₄ levels of the labradors were significantly lower than the values found for mongrels or older beagles (P < 0.05), whereas T₃ was higher in the 5 month old beagles compared to the mongrels (P < 0.001), young beagles (P < 0.05) or labradors (P < 0.01).

Circadian and ultradian rhythmicities have been evaluated by cosinor and Fourier analysis. Mongrels and older beagles did have a 12-hr rhythmicity in plasma T₄ (P < 0.05), whereas 5 month old beagles had a circadian one (P < 0.01). A 12-hr rhythmicity was also found for T₃ in the older Beagles (P < 0.05). However, Fourier analysis indicated that the daily variation in T₄ and T₃ plasma levels was inadequately mathematically described by single sinusoidal rhythm and that more harmonic components are to be taken into account.

The obtained data during a 24-hr period indicate that T₄ and T₃ concentrations in plasma may vary according to breed, age and sampling hour.     

Isolation of Campylobacter jejuni from experimental dogs and monkeys in Japan

Isolation of Campylobacter jejuni (C. jejuni) from experimental dogs and monkeys was undertaken. C. jejuni was detected from 14.7% of the fecal samples obtained from beagles in a production colony, whereas 32% of newly imported beagles harbored the organisms. C. jejuni was isolated from 25% of the young and 3.9% of adult dogs in an animal center. The organisms were isolated from newly imported cynomolgus and rhesus monkeys at high frequencies (49.2% and 38.8%, respectively). Almost all of the strains isolated were highly sensitive to erythromycin, chloramphenicol and gentamicin.     

Location of the cell-binding domain of CP65, a 65kDa cysteine proteinase involved in Trichomonas vaginalis cytotoxicity

The cysteine proteinase (CP) of 65 kDa, CP65, binds to the surface of HeLa cells and is involved in Trichomonas vaginalis cellular damage. To identify and locate the CP65 cellular-binding domain, we enriched the CP65 protein band by ammonium sulfate fractionation and ion-exchange chromatography and the N-terminal sequence was obtained. A 618 bp gene fragment was obtained by PCR using genomic DNA as template and primers derived from the N-terminal sequence of CP65 and the Asn papain-catalytic conserved region. This gene fragment encodes for 206 amino acid (aa) residues corresponding to the N-terminal region of a mature CP with 67–76% identity to the reported trichomonad cathepsin-L-like CPs. This gene fragment was expressed in a bacterial system for antibody production and functional analysis. Antibodies against the native trichomonad CP65 recognized the recombinant protein, referred to as rCP65, confirming its relationship with the CP65 gene. The rCP65 protein was bound to the surface of HeLa cells and competed with the native CP65 for binding. Antibodies to the rCP65 (-rCP65) reacted with the trichomonad CP65 located on the parasite surface, and inhibited trichomonal cytotoxicity in a concentration-dependent manner. These data strongly suggest that this gene fragment encodes for the putative cell-binding domain (CBD) of CP65 located at its N-terminal region.     

Effect of chronic cigarette smoke exposure on pulmonary vasomotion in beagle dogs

To study the effect of chronic cigarette smoke exposure on the resistive properties of the pulmonary vasculature, left lower lobes from 12 control beagles and 6 beagles who had smoked cigarettes (50 cigarettes/wk for 40 wk) were perfused in situ to measure the vascular pressure-flow relationship and the resistance of the three vascular segments with the arterial and venous occlusion technique. In control subjects the vascular resistance in the arterial, middle, and venous segments was 23, 36, and 41% of the total, respectively. The segmental distribution of vascular resistance was not significantly different in the cigarette smoke-exposed dogs, despite the fact that the absolute values were 30-40% less than that of the control group. The longitudinal distribution of resistance among the three vascular segments and their response to drugs were different in beagles than was previously found in mongrels. In all beagles the veins were considerably more reactive than arteries. Vasoconstriction with serotonin (5-HT) prostaglandin F2 alpha (PGF2 alpha), norepinephrine, histamine, and methacholine (M) infusion occurred predominantly in the veins. The effect of PGF2 alpha and 5-HT was totally different than that previously observed in mongrels in which the constriction was predominantly in the arteries. Chronic cigarette smoking reduced the basal pulmonary vascular resistance and attenuated the venoconstrictor response to 5-HT and M but potentiated the hypoxic pressor response of the microvessels.