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Extracellular excystation and development of Cryptosporidium: tracing the fate of oocysts within Pseudomonas aquatic biofilm systems.

Koh W, Thompson A, Edwards H, Monis P, Clode PL - BMC Microbiol. (2014)

Bottom Line: Here we used confocal laser scanning microscopy, scanning electron microscopy (SEM), and flow cytometry to identify and describe various Cryptosporidium developmental stages present within aquatic biofilm systems, and to directly compare these to stages produced in cell culture.We also show that Cryptosporidium has the ability to form a parasitophorous vacuole independently, in a host-free biofilm environment, potentially allowing them to complete an extracellular life cycle.Correlative data from confocal and SEM imaging of the same cells confirmed that the observed developmental stages (including trophozoites, meronts, and merozoites) were Cryptosporidium.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary and Life Sciences, Murdoch University, South Street, Murdoch 6150, WA, Australia. wan.koh@ryerson.ca.

ABSTRACT

Background: Aquatic biofilms often serve as environmental reservoirs for microorganisms and provide them with a nutrient-rich growth environment under harsh conditions. With regard to Cryptosporidium, biofilms can serve as environmental reservoirs for oocysts, but may also support the growth of additional Cryptosporidium stages.

Results: Here we used confocal laser scanning microscopy, scanning electron microscopy (SEM), and flow cytometry to identify and describe various Cryptosporidium developmental stages present within aquatic biofilm systems, and to directly compare these to stages produced in cell culture. We also show that Cryptosporidium has the ability to form a parasitophorous vacuole independently, in a host-free biofilm environment, potentially allowing them to complete an extracellular life cycle. Correlative data from confocal and SEM imaging of the same cells confirmed that the observed developmental stages (including trophozoites, meronts, and merozoites) were Cryptosporidium. These microscopy observations were further supported by flow cytometric analyses, where excysted oocyst populations were detected in 1, 3 and 6 day-old Cryptosporidium-exposed biofilms, but not in biofilm-free controls.

Conclusions: These observations not only highlight the risk that aquatic biofilms pose in regards to Cryptosporidium outbreaks from water distribution systems, but further indicate that even simple biofilms are able to stimulate oocyst excystation and support the extracellular multiplication and development of Cryptosporidium within aquatic environments.

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Cryptosporidiumgamont-like cell identified within 6 day-oldCryptosporidium-exposed biofilms. (A) Confocal image; (B) Superimposed confocal and brightfield images. Scale bars = 2.5 μm.
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Fig3: Cryptosporidiumgamont-like cell identified within 6 day-oldCryptosporidium-exposed biofilms. (A) Confocal image; (B) Superimposed confocal and brightfield images. Scale bars = 2.5 μm.

Mentions: From day 1, fluorescent oocysts containing internal sporozoites (Figure 1A and B) and free sporozoites (Figure 1C and D) with a rounded posterior and a pointed tapered anterior end were routinely observed. By day 3, well formed, individual trophozoites (3 × 3 μm: Figure 1E and F) were routinely observed, and occasionally, aggregations of trophozoites were also seen (Figure 1G and H). At day 6, microgamonts (Figure 2; 4.5 × 4.5 μm) presumably containing masses of microgametes were identified, confirming that the process of merogony had occurred within the aquatic biofilm environment. Interestingly, gamont-like cells were observed in the 6 day-old biofilms (Figure 3). This gamont-like stage in biofilms was also blunt-ended and rod-shaped (2 × 4 μm), which complements the description by Hijjawi et al. [14]. The internal structure of the cell was intensely labelled by Sporo-Glo™ antibody (Figure 3B).Figure 1


Extracellular excystation and development of Cryptosporidium: tracing the fate of oocysts within Pseudomonas aquatic biofilm systems.

Koh W, Thompson A, Edwards H, Monis P, Clode PL - BMC Microbiol. (2014)

Cryptosporidiumgamont-like cell identified within 6 day-oldCryptosporidium-exposed biofilms. (A) Confocal image; (B) Superimposed confocal and brightfield images. Scale bars = 2.5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4236811&req=5

Fig3: Cryptosporidiumgamont-like cell identified within 6 day-oldCryptosporidium-exposed biofilms. (A) Confocal image; (B) Superimposed confocal and brightfield images. Scale bars = 2.5 μm.
Mentions: From day 1, fluorescent oocysts containing internal sporozoites (Figure 1A and B) and free sporozoites (Figure 1C and D) with a rounded posterior and a pointed tapered anterior end were routinely observed. By day 3, well formed, individual trophozoites (3 × 3 μm: Figure 1E and F) were routinely observed, and occasionally, aggregations of trophozoites were also seen (Figure 1G and H). At day 6, microgamonts (Figure 2; 4.5 × 4.5 μm) presumably containing masses of microgametes were identified, confirming that the process of merogony had occurred within the aquatic biofilm environment. Interestingly, gamont-like cells were observed in the 6 day-old biofilms (Figure 3). This gamont-like stage in biofilms was also blunt-ended and rod-shaped (2 × 4 μm), which complements the description by Hijjawi et al. [14]. The internal structure of the cell was intensely labelled by Sporo-Glo™ antibody (Figure 3B).Figure 1

Bottom Line: Here we used confocal laser scanning microscopy, scanning electron microscopy (SEM), and flow cytometry to identify and describe various Cryptosporidium developmental stages present within aquatic biofilm systems, and to directly compare these to stages produced in cell culture.We also show that Cryptosporidium has the ability to form a parasitophorous vacuole independently, in a host-free biofilm environment, potentially allowing them to complete an extracellular life cycle.Correlative data from confocal and SEM imaging of the same cells confirmed that the observed developmental stages (including trophozoites, meronts, and merozoites) were Cryptosporidium.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary and Life Sciences, Murdoch University, South Street, Murdoch 6150, WA, Australia. wan.koh@ryerson.ca.

ABSTRACT

Background: Aquatic biofilms often serve as environmental reservoirs for microorganisms and provide them with a nutrient-rich growth environment under harsh conditions. With regard to Cryptosporidium, biofilms can serve as environmental reservoirs for oocysts, but may also support the growth of additional Cryptosporidium stages.

Results: Here we used confocal laser scanning microscopy, scanning electron microscopy (SEM), and flow cytometry to identify and describe various Cryptosporidium developmental stages present within aquatic biofilm systems, and to directly compare these to stages produced in cell culture. We also show that Cryptosporidium has the ability to form a parasitophorous vacuole independently, in a host-free biofilm environment, potentially allowing them to complete an extracellular life cycle. Correlative data from confocal and SEM imaging of the same cells confirmed that the observed developmental stages (including trophozoites, meronts, and merozoites) were Cryptosporidium. These microscopy observations were further supported by flow cytometric analyses, where excysted oocyst populations were detected in 1, 3 and 6 day-old Cryptosporidium-exposed biofilms, but not in biofilm-free controls.

Conclusions: These observations not only highlight the risk that aquatic biofilms pose in regards to Cryptosporidium outbreaks from water distribution systems, but further indicate that even simple biofilms are able to stimulate oocyst excystation and support the extracellular multiplication and development of Cryptosporidium within aquatic environments.

Show MeSH