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The interaction of cryptosporidium with aquatic biofilm systems

Koh, Wan Hon (2013) The interaction of cryptosporidium with aquatic biofilm systems. PhD thesis, Murdoch University.

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Cryptosporidium parvum is a common, opportunistic, diarrhoeal-causing, apicomplexan pathogen in humans, of which water is an important transmission vehicle. Recently, aquatic biofilms have been recognised as environmental reservoirs for the infective stage (oocysts) of Cryptosporidium, yet their fate after being trapped within biofilms is unknown. Previous cell-associated and cell-free studies have demonstrated, controversially, that Cryptosporidium may be able to multiply extracellularly, indicating that Cryptosporidium is not an obligate intracellular microorganism, and that the environment may play an important role in shaping its life cycle. Previously published data raise the question as to whether Cryptosporidium can survive and multiply within biofilms, resulting in an increase in numbers before release into water systems, leading to possible disease outbreaks.

This study, therefore, aimed to investigate the ability of biofilms to support Cryptosporidium multiplication. This was achieved using a combination of quantitative polymerase chain reaction (qPCR), flow cytometry (immunolabeled with Cryptosporidium oocysts-specific antibody), confocal microscopy (immunolabeled with Cryptosporidium developmental stage-specific antibody) and scanning electron microscopy (SEM) techniques. To mimic a water distribution system, Pseudomonas aeruginosa biofilm flow cell systems were established and unexcysted C. parvum oocysts were constantly supplied over a 6-day period. Prior to analysis, the four analytical methods were designed and empirically optimised according to the nature of the experimental sample studied.

Quantitative PCR results showed a significant increase (P<0.001) in Cryptosporidium as the biofilm matured, with the total number of C. parvum multiplying 2-3 fold during this period. Flow cytometry analysis also revealed that the captured oocysts had undergone excystation in biofilms, confirming that the increase in Cryptosporidium number was due to Cryptosporidium multiplication. From this, various Cryptosporidium developmental stages (sporozoites, trophozoites, meronts, and merozoites) were also identified from the biofilm using confocal microscopy and SEM. A correlative study using both SEM and confocal imaging determined that the observed developmental stages were Cryptosporidium, rather than degenerate/accumulated oocysts or yeast contamination. Furthermore, SEM analysis also revealed that Cryptosporidium may form a parasitophorous vacuole independently, potentially allowing it to complete its life cycle extracellularly. In addition, certain stages of the Cryptosporidium life cycle (trophozoites, meronts, and some previously undescribed gamonts) in biofilms were identified, and shown to closely resemble stages reported in the gregarine life cycle, emphasising the possibility that Cryptosporidium has inherited the capability to multiply extracellularly from their gregarine ancestor.

In conclusion, this study has successfully shown that biofilms can support Cryptosporidium multiplication in aquatic environments and thus, also demonstrated a role for biofilms in outbreaks and spreading of this disease. The generated results are novel, offering new insights into the role of biofilms in the C. parvum life cycle, providing additional information for water authorities, aiding in the control of Cryptosporidium and biofilm-contaminated drinking water.

Publication Type: Thesis (PhD)
Murdoch Affiliation: School of Veterinary and Life Sciences
Supervisor: Thompson, Andrew
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