Cryptosporidium parvum oocysts preparation
Cryptosporidium parvum cattle isolate used in the study was originally obtained from the Institute of Parasitology, University of Zurich. The use of this isolate was approved by the Murdoch University Institutional Biosafety Committee (Australia). Fresh oocysts were purified and obtained by passage through infected ARC/Swiss mice as described by Meloni and Thompson . These purified oocysts were stored in 1 - phosphate buffered saline (PBS) with antibiotics (10,000 U penicillin G and 0.01 g streptomycin; Sigma) at 4°C before use. All oocysts used in this study were not more than 4 weeks old and similar batches of oocysts were used for parallel experimental controls. Oocysts used in this study were pre-treated with 2% household bleach in distilled water for 20 min at room temperature. They were then pelleted at 3500 rcf and resuspended with 200 μl of sterile 1 × PBS. This animal work was reviewed and approved by the Animal Ethics Committee of Murdoch University, Australia (Permit number: R2310/10).
Biofilm flow cell system
Wild type strain Pseudomonas aeruginosa (PA01) was used to produce biofilms and was maintained on Pseudomonas agar prior to incubation into flow cell systems. The flow cell biofilm system was operated as described previously . Three experimental conditions were used, with each replicated 3 times:
Cryptosporidium-exposed biofilm: oocysts were injected into the influent medium and the flow initiated and continued (60 ml h-1) for one, three or six days.
Biofilm-free control. No biofilm was developed. Decontaminated oocysts were injected into the flow system and maintained as above.
Biofilm-only control. Biofilms were grown in the flow system without the introduction of Cryptosporidium oocysts.
Cryptosporidium-exposed, biofilm-free and biofilm-only systems were dispersed with 500 nM of sodium nitriporusside (nitric oxide) overnight (Sigma, USA). This nitric oxide was pumped into the flow cell overnight and undisturbed for 24 h. After this, these flow cells were gently washed with 1 × PBS several times to completely detach the biofilms from the flow cell surface . The cell suspensions were further washed several times with sterile 1 × PBS to remove any residual nitric oxide and immediately post-fixed with 2.5% paraformaldehyde in sterile 1 × PBS for 20 min at 4°C. The cells were pelleted at 3500 g for 10 min and resuspended in sterile 1 × PBS to a final volume of 400 μl. Similarly, the cells (unattached biofilms/bacteria/Cryptosporidium) in the effluent/waste bottle were also collected by centrifuging at 4°C and 1000 rpm for 1 hour and post-fixed with 2.5% paraformaldehyde in sterile 1 × PBS for 20 min at 4°C. The fixed sample was resuspended to a final volume of 2 ml. Prior to microscopic and flow cytometry analyses, an aliquot of flow cell (100 μl) and effluent (500 μl) samples were subjected to qPCR analysis for determination of Cryptosporidium number as described previously .
Cell-free culture was performed to follow morphological changes in oocysts during the excystation process. Cryptosporidium oocysts (4 - 106) were excysted with excystation media (0.5% trypsin-EDTA, pH 2.5) for 30 min at 37°C. An aliquot (1 - 106) of solution was immediately post-fixed with 2.5% paraformaldehyde in sterile 1 × PBS for 20 min at 4°C. The cells were pelleted and resuspended to a final volume of 200 μl. This sample was stored at 4°C for further flow cytometry analysis and is hereafter referred to as excysted oocysts. The remaining excysted oocysts were kept in maintenance medium for a further 60 min or 24 h at 37°C. These samples are referred to as 60 min and 24 h culture samples respectively. The maintenance medium consisted of 10% RPMI media, 0.03 g l-1 glutamine, 0.3 g l-1 sodium bicarbonate, 0.02 g l-1 bovine bile, 0.1 g l-1 glucose, 25 μg g l-1 folic acid, 100 μg l-1 4-aminobenzoic acid, 50 μg g l-1 calcium pantothenate, 875 μg g l-1 ascorbic acid, 1% fetal calf serum (FCS), 0.36 g l-1 HEPES buffer, 10,000 U penicillin G and 0.01 g l-1 streptomycin, adjusted to pH 7.4.
HCTcell line infections
For direct comparison with the Cryptosporidium extracellular stages in biofilms, human colon carcinoma cell line (HCT-8) was routinely passaged and maintained in RPMI 1640 medium (pH 7.2) containing 2 g L-1 sodium bicarbonate, 0.3 g L-1 L-glutamine, 3.574 g L-1 HEPES buffer (15 mmol L-1) and 10% heat inactivated FCS, at 5% CO2 and 37°C. HCT8 cells infected with Cryptosporidium were prepared as previously described in Borowski et al. . The infected cultures were sampled and processed for SEM analysis at 6, 24, 55, and 120 h post inoculation.
Developmental stage-specific antibody
Confocal laser scanning microscopy was used to monitor the subsequent development of Cryptosporidium in aquatic biofilms. Currently, Sporo-Glo™ (Waterborne Inc.) is the only commercially available antibody which specifically targets Cryptosporidium developmental stages ,. The binding pattern of Sporo-Glo™ antibody was previously studied in detail and it was shown that the antigen is not expressed or cannot be detected until the oocysts have begun excystation ,. Additionally, intense fluorescence expression can be detected from several developmental stages ,,. An aliquot of dispersed biofilm suspension (20 μl) was fixed with 2.5% paraformaldehyde in PBS for 20 min at 4°C and immunolabeled with quantum dot (#Q-11621MP, Invitrogen) conjugated Sporo-Glo™ antibody as described previously .
As Sporo-Glo™ antibody is only expressed after excystation, it is not suitable for tracking the changes to oocyst morphology ,. As such, the changes of oocyst morphology in cell-free cultures, Cryptosporidium-exposed biofilms, and biofilm-free systems were determined by labelling with the Cryptosporidium oocyst-specific antibody, Cy5-Crypt-a-Glo™ (Waterborne, Inc), which is constantly expressed on the oocyst wall regardless of excystation status. All samples (200 μ1) including unexcysted Cryptosporidium oocysts (1 - 106), Cryptosporidium-exposed biofilms (flow cell biofilms and waste samples), biofilm-free controls and Cryptosporidium cell-free cultures were immunolabeled with Crypt-a-Glo™ antibody (5.0 μg ml-1) at room temperature in a dark room for 30 min. The cells were then washed twice with 1 × PBS for 5 min each and pelleted at 3500 rcf for 10 min. Except for cell-free culture samples, all other samples were resuspended to a final volume of 200 μl. Cell-free culture samples were resuspended in 400 μ1 of sterile 1 × PBS and an aliquot of each cell-free culture sample (200 μ1: 5 - 105 oocysts) was taken and processed for morphological analysis by confocal microscopy. The remaining samples were analysed by flow cytometry on the same day.
During flow cytometry analysis, an unknown population was detected in 3 and 6 day-old Cryptosporidium-exposed biofilms. Previous studies had shown that biofilm matrix tends to trap and prevent the penetration of antibodies into biofilms ,. Therefore, to determine whether this population could also be detected in the biofilm-only controls, immunolabelling with Cy5-Crypt-a-Glo™ antibody as described above was performed on the 6 day-old biofilm-only sample.
For examination of Cryptosporidium biofilm samples or cell-free cultures by confocal laser scanning microscopy, an aliquot of the immunolabeled suspension was immobilised and attached to coverslips using 0.01% poly-L-lysine (Sigma, USA). The samples were then analysed directly by confocal microscopy (Leica SP2 inverted) using excitation/emission wavelengths of 488/525 nm (quantum dots) or 649/688 nm (Cy5). Both confocal and bright field images were acquired simultaneously. For correlative analyses, serial sections on the xy plane were obtained at 0.44 μm along the z-axis for meront stage and the three dimensional Z-stack images were constructed using Volview™.
Scanning electron microscopy
An aliquot (20 μl) of dispersed flow cell biofilm sample was further fixed in an equal volume of 5.0 % glutaraldehyde in sterile 1 × PBS solution. The samples were immobilised and attached onto 0.01% poly-L-lysine (Sigma, USA) coated round coverslips (12 mm). To prevent sample dehydration at room temperature, the sample was left to stand in a humidified box for 30 min. For the infected cell line, RPMI medium was removed and the coverslips with adherent cells were fixed in 2.5% glutaraldehyde in sterile 1 x PBS solution. These samples were then dehydrated through a series of ethanol concentrations and critical point dried as described in Borowski et al. .
Dried coverslips were mounted on stubs with carbon tape. These were then coated with 4 nm carbon and 5 nm platinum for stable, high resolution imaging using the in-lens secondary electron detector at an accelerating voltage of 3 kV (Zeiss 55 VP field emission SEM).
An SEM finder grid was used to locate the same fluorescent cells from the confocal, in the SEM. The SEM grid finder pattern was made on a sample mould that could be seen using both confocal and SEM, allowing for the establishment of directly correlative studies. The SEM finder grid pattern was transferred from the mould onto a 12 mm circular coverslip as described by Powell et al. . Essentially, an SEM finder grid was placed flat on the 12 mm circular coverslip and sputter-coated with Pd/C for 3 min to produce a clear outline of the SEM finder grid visible under both confocal microscopy and SEM.
For this experiment, Cel-Tak™ coated round coverslips (12 mm) were used to immobilise and adhere dispersed flow cell biofilm samples (20 μl). Again, the sample was left to stand in a humidified box for 15 min. During the incubation period, a clean sterile square coverslip (22 - 22 mm) was placed onto the square coverslip holder in preparation for confocal analysis. The SEM coverslip was removed from the humidified box and placed onto the square coverslip. As we were using inverted confocal microscope, the SEM coverslip was gently turned over after 15 min of incubation so that the side with the experimental sample was facing down towards the square coverslip. After analysis by confocal microscopy and the location of the cells of interest had been recorded, the sample was fixed in 2.5% glutaraldehyde in sterile 1 × PBS and immediately processed for SEM by freeze drying.
Prior to freeze drying, the coverslip was briefly rinsed in 150 mM ammonium acetate and blotted on filter paper to remove the excess liquid. The sample was then rapid frozen in liquid nitrogen slush and freeze dried (Emitech K775X turbo pumped freeze drying system) in a step wise fashion (-120°C to -65°C over 14 h; -65°C to +25°C over 10 h; hold at 25°C for 24 h). The samples were then mounted on stubs with carbon tape and coated with 4 nm carbon and 5 nm platinum. It was observed that the Pt/C pattern could be visualised at 10 kV with the SE2 detector. Although the in-lens detector provides better image resolution at 3 kV, no grid pattern underneath the sample could be observed at this lower voltage. Therefore, 10 kV was used, with the SE2 and in-lens detector selected for grid and sample observation respectively.
Due to the immense numbers of bacteria relative to Cryptosporidium within the biofilm, several attempts at cutting thin (~100 nm) sections for transmission electron microscopy observation failed to locate life stages within the masses of biofilm at this scale. Therefore, Cryptosporidium developmental stages as described in this study were correlated with, and identified based on previous detailed morphological descriptions in in-vivo, cell-free, and in-vitro systems ,,,,,-.
All samples were analysed using flow cytometry (FACSCalibur, Becton Dickinson, Australia). Logarithmic signals were used for all parameters and the forward light scatter detector was set at E00 for all assays. The forward scatter detector was set to 396 V and the red fluorescent detector to 597 V.
To investigate the possibility of Cryptosporidium excystation in the biofilm system and cell-free culture, gating was used to discriminate and differentiate Cryptosporidium oocyst populations from mixed populations. The acquisition gate was determined based on the fluorescence (intensity) and forward scatter characteristics of Cy5- Crypt-a-Glo™-labelled pure bacteria and Cryptosporidium oocysts (unexcysted/excysted). In addition, cell free culture samples were also used to identify excysted oocyst populations. As such, two different populations were similarly used to differentiate gated areas for each of the physiological states of oocysts - intact and excysted. The collected data was analysed using FlowJo software (TreeStar).