Journal: Microbiology Spectrum

Article Title: Prolonged survival of venereal Tritrichomonas foetus parasite in the gastrointestinal tract, bovine fecal extract, and water

doi: 10.1128/spectrum.00429-23

Figure Lengend Snippet: Orally administered T. foetus survive through the bovine gastrointestinal tract and are discharged by feces. ( A ) Timeline, experimental design, and sample collection to evaluate T. foetus survival through the bovine gastrointestinal tract. ( B ) A representative optical microscopy image of spherical structures (white arrows) present in fresh feces samples after 4 days of T. foetus K strain oral administration. ( C ) A representative image analyzed by epifluorescence microscopy of pseudocyst-like structures stained with WGA in fresh feces samples after 4 days of T. foetus oral administration.

Article Snippet: The parasites were centrifuged, resuspended in a 40 ug/mL propidium iodide solution, incubated for 30 min at 37°C and counted using an epifluorescence microscope (Zeiss Axio Observer 7 inverted fluorescence microscope).

Techniques: Microscopy, Epifluorescence Microscopy, Staining

Journal: Microbiology Spectrum

Article Title: Prolonged survival of venereal Tritrichomonas foetus parasite in the gastrointestinal tract, bovine fecal extract, and water

doi: 10.1128/spectrum.00429-23

Figure Lengend Snippet: Parasite growth in an extract from bovine feces. ( A ) Analysis of the T. foetus kinetic growth curve. Replication curves of parasites' growth in extracts of bovine feces were compared. Parasite counts were collected at the indicated times on the X -axis. Each point represents the average parasite concentration in 10 different tubes containing different feces samples. ( B ) Growth rates of parasites grown in standard culture media (TYM) were compared to parasites grown in an extract of bovine feces (BFE). Error bars represent standard error, and asterisks denote statistically significant differences determined by t-Student ( P < 0.05). ( C ) An example of a pseudocyst stained with Calcofluor white (CFW) in a T. foetus grown in bovine feces extracts for 10 days. The samples were analyzed by epifluorescence microscopy. ( D ) Quantification of the number of nuclei per parasite. Fifty parasites from each population were counted in triplicate in three independent experiments. The percentages of parasites with one nucleus (1N), two nuclei (2N), or more than two nuclei (>2N) are shown. T. foetus grown under standard culture media (TYM) and parasites grown in extracts from bovine feces (BFE) were compared. The results represent the average of three independent experiments and the standard error (SE). ( E ) Comparative images of T. foetus parasites with one nucleus grown in standard culture media (TYM) (left panel) and parasites grown in extracts from bovine feces (BFE) (right panel). Nuclei were stained with DAPI (blue). PC, phase-contrast.

Article Snippet: The parasites were centrifuged, resuspended in a 40 ug/mL propidium iodide solution, incubated for 30 min at 37°C and counted using an epifluorescence microscope (Zeiss Axio Observer 7 inverted fluorescence microscope).

Techniques: Concentration Assay, Staining, Epifluorescence Microscopy

Journal: Microbiology Spectrum

Article Title: Prolonged survival of venereal Tritrichomonas foetus parasite in the gastrointestinal tract, bovine fecal extract, and water

doi: 10.1128/spectrum.00429-23

Figure Lengend Snippet: T. foetus water survival analysis. ( A ) A representative image of the viability of T. foetus maintained in water was determined by propidium iodide (PI) staining. Parasites were stained with PI and photographed under a fluorescent microscope. Alive parasites are not stained, whereas dead cells are stained red. ( B ) Quantification of the viability of T. foetus when they were incubated in water for 24, 48, 72, 96, and 120 h. One hundred parasites were counted in triplicate in three independent experiments. Bars represent the means of three independent assays. Samples were analyzed with ANOVA test. Mean differences were calculated using Tukey’s test with an alpha = 0. 05. ( C ) Estimation of the size of the nuclei of T. foetus grown for 24, 48, and 72 h in water. TYM: parasites T. foetus grown in TYM culture media (control). The images of 50 nuclei staining with DAPI were analyzed using Image J’s threshold tool and the “Analyze Particles” function. Experiments were performed in triplicate and repeated three times. Mean differences were calculated using the Kruskal-Wallis test with a P value of 0.05. ( D ) Indirect immunofluorescence using an anti-alpha tubulin antibody shows spherical parasites with internalized flagella (white arrow). DAPI (blue). PC, phase-contrast image ( E ) Representative images of a pseudocyst or cyst-like structure stained with Calcofluor white (CFW) in a T. foetus grown in vitro for 72 h, maintained in water. The samples were analyzed by epifluorescence microscopy. ( F ) A graph representing the DNA content profile of T. foetus grown in standard culture media conditions for 24 h (TYM) and T. foetus incubated in water for 24 h (Water). DNA content was measured by flow cytometry. ( G ) Image representative of an indirect immunofluorescence image showing PCNA-positive nuclei (green) of T. foetus parasites that were grown in water for 24 h. DAPI (blue). PC, phase-contrast image.

Article Snippet: The parasites were centrifuged, resuspended in a 40 ug/mL propidium iodide solution, incubated for 30 min at 37°C and counted using an epifluorescence microscope (Zeiss Axio Observer 7 inverted fluorescence microscope).

Techniques: Staining, Microscopy, Incubation, Immunofluorescence, In Vitro, Epifluorescence Microscopy, Flow Cytometry

Journal: Microbiology Spectrum

Article Title: Prolonged survival of venereal Tritrichomonas foetus parasite in the gastrointestinal tract, bovine fecal extract, and water

doi: 10.1128/spectrum.00429-23

Figure Lengend Snippet: T. foetus pH survival analysis. ( A ) Simplified schematic diagram of the ruminant digestive tract and pH conditions in various regions. The pH of the digestive tract is variable in the different regions: pH 5.5 and 7 (esophagus-reticulum-rumen), pH 2–2.5 (abomasum), and pH 6–7 (large intestine). The digestive transit times are as follows: 24–48 h for the esophagus, reticulum, and rumen; 1–3 h for the abomasum; and 10–20 h for the large intestine. ( B ) The growth rates of parasites grown in culture medium at pH 5 and 7 were compared. Error bars represent standard error, and asterisks denote statistically significant differences determined by t -Student ( P < 0.05). ( C ) A representative image of a parasite stained with Calcofluor white (CFW) in a T. foetus grown in culture media at pH 2 for 3 h. The samples were analyzed by epifluorescence microscopy. ( D ) Quantification of viable T. foetus incubated for 3 h in TYM culture media at pH 2. One hundred parasites were counted in triplicate in three independent experiments. Bars represent the means of three independent assays. Samples show significant differences when analyzed with the Kruskal-Wallis test. LP (live parasites). DP (dead parasites). ( E ) A representative image of the viability of T. foetus maintained in water was determined by propidium iodide (PI) staining. Parasites were stained with PI and photographed under a fluorescent microscope. Alive parasites are not stained (white asterisks), whereas dead cells are stained red. ( F ) T. foetus kinetic growth curve depicting the percentage of motile trophozoites. T. foetus parasites were incubated in water for 24 h, then incubated for 3 h in culture media at pH 2, and then recovered in Diamond’s medium for 24, 48, and 72 h. Parasite counts were collected at the indicated times on the X -axis. Each data point on the graph represents the average percentage of motile parasites from three independent experiments. The error bars indicate the standard deviation.

Article Snippet: The parasites were centrifuged, resuspended in a 40 ug/mL propidium iodide solution, incubated for 30 min at 37°C and counted using an epifluorescence microscope (Zeiss Axio Observer 7 inverted fluorescence microscope).

Techniques: Staining, Epifluorescence Microscopy, Incubation, Microscopy, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of Nucleotide Sugar Transport in Trypanosoma brucei Alters Surface Glycosylation *

doi: 10.1074/jbc.M113.453597

Figure Lengend Snippet: TbNST4 is localized at the Golgi apparatus. A, genomic PCR analysis of in situ C-terminal HA-tagged tbnst4 in T. brucei BSF. Genomic DNA was isolated from untagged (lane 1) and tagged (lane 2) TbNST4 cells. In situ tagging locus was PCR amplified with a forward primer complementary to TbNST4 coding sequence and a reverse primer complementary to the 3′ UTR (outside the recombination sequence). The expected genomic PCR products are indicated. B, Western blot analysis of the in situ tagged TbNST4HA protein expression. Cell lysates from untagged (lane 1) and tagged TbNST4HA (lane 2) cells were subjected to SDS-PAGE and Western blotting with anti-HA antibody detection. The position of the expressed TbNST4 is indicated. C, immunofluorescence analysis of TbNST4HA localization. BSF cells carrying the tagged TbNST4 were stained with anti-HA for TbNST4 (green), anti-Ty for TbGT15 (red, upper panel), or anti-p67 (red, lower panel) antibodies. Cell images were captured with Nikon wide-field epifluorescence microscope. Blue, DAPI staining for nuclei and kinetoplasts.

Article Snippet: Cell images were captured with a Nikon wide-field epifluorescence microscope.

Techniques: In Situ, Isolation, Amplification, Sequencing, Western Blot, Expressing, SDS Page, Immunofluorescence, Staining, Microscopy