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

Abstract

BackgroundAquatic 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.ResultsHere 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.ConclusionsThese 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0281-8) contains supplementary material, which is available to authorized users.

Life without a Host Cell: What is Cryptosporidium ?

“…[6,14,17,20], and a greater degree of pleomorphism than reported previously, particularly of gamonts and gamont-like stages [11,20,21,26], all of which are gregarine characteristics. Of particular significance are observations from both in vitro and in vivo studies that, in addition to epicellular multiplication, Cryptosporidium can also complete its life cycle without apparent epicellular attachment with host cells (Figure 1, Key Figure) [6][7][8]10,12,18,20]. Thus, two types of multiplication and development processes -epicellular and extracellular -could occur simultaneously in the host for mass production of new oocysts.…”

“…In the absence of host cells, two unique Cryptosporidium extracellular morphotypes have been observed; these are commonly referred to as gamont-like or gigantic gamont-like stages [6,7,9]. These stages probably develop from the trophozoite stage (also a key stage in gregarines) with the purpose of producing more merozoites and trophozoites to maximize reproductive potential without the need for sexual reproduction (Figure 1), but their true nature and function remain unknown (see Outstanding Questions).…”

“…Type II meronts containing many type II merozoites develop into either microgamonts (male) or macrogamonts (female). Microgamonts in the biofilm model also undergo binary fission and syzygy processes [7]. Microgamonts were observed to contain many microgametes, but the production of new oocysts in aquatic biofilms has not been recorded and requires further investigation (see Outstanding Questions).…”

“…The studies of Carreno et al [5] using 18S sequencing demonstrated a closer phylogenetic relationship to gregarine protozoa than to coccidians and these have since been complemented by observations of Cryptosporidium's developmental biology and metabolism (Box 1) [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. In particular, the initial controversial images and theories of Hijjawi et al [6] and Karanis et al [10], which showed that Cryptosporidium oocysts, like gregarines, could excyst and produce pleomorphic (see Glossary) stages without host cells or triggers, were widely disputed within the Cryptosporidium research community.…”

Waterborne Giardia and Cryptosporidium: contamination of human drinking water by sewage and cattle feces

“…However, recent phylogenetic studies have shown closer proximity of Cryptosporidium with gregarine protozoan than with coccidia, because they have a feeding organelle and absence of apicoplast, which allowed their reclassification in the Gregarinomorphea class, Cryptogregaria subclass (KOH et al, 2013(KOH et al, , 2014CAVALIER-SMITH, 2014;HUANG et al, 2014;CLODE et al, 2015;ALDEYARBI;RYAN et al, 2016).…”