WISER Objective 1: Water treatment against cercariae
There is surprisingly little information on the best ways to treat water to remove or inactivate schistosome cercariae. Cercariae only persist in water for a few days, dying out if they do not find a human host to infect, though this is highly water temperature-dependent and not all species of Schistosoma have been studied. A basic water supply strategy would be to pump or otherwise divert the contaminated water into a holding tank and maintain a storage time long enough to achieve cercaria die-off.
However, a multi-barrier treatment approach would be preferable. Chlorine kills Schistosoma mansoni cercariae, though previous studies have only been conducted in relatively pure waters (buffered or distilled water), under a limited range of temperatures and pH conditions, and have only considered S. mansoni. WISER will build on these studies to develop a comprehensive data set to inform the design of chlorination processes for community water supplies. We plan to collect water from freshwater bodies in at least eight endemic regions of Ethiopia and Tanzania (four in each country), in the aim of capturing some samples with S. mansoni and some with S. haematobium, the two most common species in Africa.
Cercarial presence in the water samples will be confirmed using the methods described in Objective 2, i.e. using the biosensor and eDNA. We will assess the survival of cercariae after exposure to various concentrations of chlorine, for various durations.
We will also conduct trials to determine the ideal sand filter grain sizes, bed depths, and filtration rates to achieve consistent cercaria removal. Slow sand filtration with sand of an effective size of 0.2 and 0.3 mm has been shown to catch most cercariae, but some passed through a filter of effective size 0.4 mm. Locally available sand with a range of effective sizes and uniformity coefficients will be sourced for the trials. The trials will be conducted using water samples exhibiting a range of turbidities. Cercaria presence/absence and viability will be assessed in the filtrate and the filter backwash water when the filters are cleaned.
Ultraviolet (UV) radiation is detrimental to cercariae. We will build on past studies by investigating the influence of water quality characteristics (e.g. UV absorbance and turbidity), in order to to understand the UV sensitivity of cercariae alongside other waterborne pathogens, as tested by modern day UV disinfection experimental protocols. In bench-scale collimated beam experiments we will expose water samples to uniform, quantifiable UV fluences, measured by a calibrated radiometer. With each UV source, varying fluences will be applied (in triplicate), to cover the typical range applied in drinking water disinfection to control other pathogens; higher fluences will be applied if the cercariae appear to be very UV-resistant. We will also investigate the effectiveness of solar disinfection (SODIS), for cases without reliable power supplies; samples will be placed in shallow UV-translucent containers and exposed to sunlight for varying durations, with the temperature of the water and cercaria survival assessed at equal intervals.
Cercariae counts and viability will be assessed by motility observation under microscope and using the new biosensor described in Objective 2, and next-generation sequencing results will also be used to confirm results. The effectiveness of the processes will be considered individually and in combinations, e.g. filtration followed by chlorination or UV disinfection.
WISER Objective 2: A rapid and cheap cercaria biosensor
Schistosome cercariae are phototaxic and thus move towards the surface of shallow waters where they can maximise the chance of contact with humans. The cercariae also follow a thermal gradient to find their potential hosts, and when they make contact with human skin, the presence of chemical signals, including medium-chain fatty acids such as linoleic acid, act as a stimulator for skin invasion. The first step in the invasion process is the release of gland contents from the acetabular gland complex of the posterior of the cercarial head. One of the factors secreted from the glands is a specific enzyme activity, cercarial elastase, which facilitates and is required for invasion by degrading the dermal elastin (skin).
Although there is a wealth of information on the life and transmission cycles, there is still a lack of a rapid and cheap technique for detection of these parasites at the site of infection, i.e. freshwater bodies. Detection in situ is essential to help break the transmission cycle, and to provide a rapid way to test for re-emergence of the parasite in watercourses previously cleared of these parasites. Classically, detection methods have relied on diagnostic tests for people, not the environment, to see if they are infected with schistosomes and then subsequent treatment to eliminate reservoirs of infection. This is a key challenge since many infections occur in resource-poor rural areas that lack access to diagnostic tests, expensive equipment or trained staff. We will address this in the current application, and will use a bioengineering approach to build Schistosoma species-specific biosensors that can be used to map populations of schistosomes at the site of infection. This will confer the ability to track and monitor re-infection of water courses previously cleared of the parasites as well as providing data on the spread of Schistosoma species.
A synthetic recognition motif for Schistotoma mansoni cercarial elastase activity has been identified. We recently used a bioengineering approach, to build upon the identification of this synthetic recognition motif for the elastase activity and incorporated it into our proof-of-principle whole-cell-based biosensors that have been designed to target this S. mansoni elastase activity. Our cercarial elastase biosensors were able to detect for the presence of cercarial elastase in several independent S. mansoni-derived biological samples that contained soluble cercarial antigens, termed cercarial transformation fluid (SmCTF).
We now plan to build on our successful proof-of-principle biosensor designs. In both Ethiopia and Tanzania, our proof-of-concept and novel biosensor designs will be further validated with water samples infested with different schistosome species in situ at water courses. We will design whole-cell-based biosensors that give an observable 'gain of colour' output upon positive detection of cercarial elastase. Our published biosensor designs produced 'a loss of colour' output as a positive indication for schistosome detection. In contrast, for our new designs we will fuse enzymes similar to and including LacZ to our surface display anchor, which will be housed on Bacillus subtilis. We will continue using B. subtilis as the bacterial host for our biosensors as we have proven that this organism can survive, and maintain the biosensor-encoding plasmids, during the process of lyophilisation. This will enable cheap and easy storage of the biosensors.
We will generate methods for the purification of S. mansoni cercarial elastase to enable rapid biosensor design validation and to test the sensitivity of the biosensors. We plan to over-express the S. mansoni cercarial elastase in insect cell-lines using baculovirus transfection. Once the elastase has been purified we will test its activity using a rapid colourimetric assay we have used previously. We will determine substrate specificities of cercarial elastase from S. mansoni, as well as from other species of schistosomes.
With a panel of purified recombinant cercarial elastases obtained and verified, we will carry out substrate profiling in terms of the specific peptide recognition motifs, using synthetic combinatorial libraries of diverse tetrapeptides coupled to fluorogenic leaving groups, allowing spectrophotometrical quantification of cercariae. These recognition motifs will be engineered into our whole-cell-based biosensors designs.
We will also design an alternate biosensor system that uses the recognition motifs identified for the different cercarial elastases but which is based on using cell-free transcription-translation reactions. Here, a similar design will be used where a fusion protein will be expressed from a plasmid in an Escherichia coli or Bacillus subtilis derived cell-free system. We have recently reported a cell-free transcription-translation system for B. subtilis, and since lacZ is not present in this organism, a similar design using lacZ could be used. We will also design and test biosensor traps (i.e. point-of-test device) so that we can attract and concentrate cercariae in the water test site. The traps could be engineered to include the biosensor to ease in aid of detection of the parasite.
Finally, we will also use state-of-the-art molecular techniques to validate the biosensor outputs, using environmental DNA (eDNA) and next-generation sequencing (NGS). Water samples will be tested for eDNA to yield information on the full range of larval cercariae present in the water samples, and to allow comparison with the biosensor outputs.
WISER Objective 3: Water infrastructure design recommendations
We will also examine the non-technical factors that are important when implementing water infrastructure in schistosomiasis-endemic communities, which are often populated by the poorest people who lack the skills and education to operate or maintain a water supply system, have limited monetary resources, and do not have access to treatment chemicals or spare electricity. We will assess the supporting education programmes, financial instruments, and supply chains that are necessary through site investigations and questionnaires in four case study communities, in both Ethiopia and Tanzania. We will consider the appropriate ownership and responsibility for the infrastructure and how to ensure equitable access to the water supply (by gender, age and socio-economic status).