Evaluation of a combined immunomagnetic separation/flow cytometry technique for epidemiological investigations of Cryptosporidium in domestic and Australian native animals.

A combined immunomagnetic separation (IMS) and flow cytometry (FC) technique was developed for the sensitive detection of Cryptosporidium in faecal samples. The IMS/FC technique was found to be approximately 50-fold more sensitive than formol-ether concentration, which is commonly used for Cryptosporidium epidemiological investigations. Of 31 faecal samples from captive animals 16 were found to contain Cryptosporidium oocysts when analysed using the IMS/FC compared to four when using formol-ether concentration (FEC). In a wild population of eastern grey kangaroos Macropus giganteus 66.3% of infected animals were shedding <500oocysts/gfaeces when analysed using IMS/FC. This is below the detection limit for the FEC method. The dispersal of Cryptosporidium in host populations is aggregated, with many individuals shedding low numbers of oocysts and few individuals shedding numbers of oocysts sufficiently high to be detected by FEC. This research demonstrates that the prevalence and oocyst shedding intensity of Cryptosporidium in animal populations will be significantly underestimated using standard detection methods.

Interaction between Cryptosporidium oocysts and water treatment coagulants.

The electrokinetic properties of gamma-irradiated Cryptosporidium oocysts in the presence of coagulants (ferric chloride and alum) and coagulant aids (DADMAC based cationic polyelectrolytes) have been studied. The zeta potential of the oocysts was unaffected by the addition of ferric chloride at all pH values (3-10) studied. Addition of alum resulted in reversal of the oocysts charge, which suggests that the initial stage in the coagulation process leading to floc formation proceeds via the adsorption of hydrolysed aluminium species. The cationic polyelectrolyte Magnafloc LT35 was adsorbed onto iron flocs at doses of 0.1 mg/L even against an electrostatic barrier. The cationic polyelectrolyte only adsorbed and caused charge reversal at the oocyst surface at around 0.4 mg/L, suggesting a lower affinity for this surface. These results indicate that the oocysts, unlike inorganic colloidal materials such as metal oxides, appear to possess a lower surface density of active or charged sites. The lower density of sites, combined with the rapid precipitation of iron salts, may be responsible for the lack of specific adsorption of either hydroxylated ferric species or primary iron hydroxide particles on the oocysts. Further, this suggests that a process of sweep flocculation, where oocysts are engulfed in flocs during coagulation and floc formation, is the more likely mechanism involved. By comparison, it is likely that the specific interaction of hydrolysed aluminium species with the oocysts surface would result in a stronger link at the oocyst-floc interface and that the flocculation process may initially proceed via charge neutralisation.

Flow cytometry and RT-PCR for rotavirus detection in artificially seeded oyster meat.

A flow cytometry (FC)-based method was developed for the detection of rotavirus in oyster meat using simian rotavirus SA11 as a model. To study virus recovery, oyster meat was injected with rotavirus and the oyster extract used to infect MA104 cell monolayers. Following varying periods of infection, the cells were recovered and reacted with the monoclonal antibody M60 which is specific for the rotavirus group A serotypes 1-4 outer capsid protein, VP7, followed by a second antibody (anti mouse IgG-FITC). A FACScan FC was used to estimate the number of infected cells as well as the level of infection. To evaluate the sensitivity of the method, non-inoculated oysters were processed following the same extraction protocol and, at the end, they were seeded with the same amount of virus used for oyster inoculation. This seeded oyster extract was then used to infect MA104 cells and the number of infected cells determined using the same FC procedure. A semi-nested two-step PCR for detection of rotavirus nucleic acid was undertaken to compare the sensitivity of FC with RT-PCR. Using FC, as little as 0.02 flow cytometry units (fcu) (number of infected cells counted by FC) could be detected after 72 h of cell infection. This is a very similar limit of sensitivity to that obtained with RT-PCR. Both methods are approximately 100 times more sensitive than the plaque-forming units (pfu) assay.