For the first research area, we developed a quantitative Polymerase Chain Reaction (PCR) technology that detects the presence of Cryptosporidium parasites in DNA extracted from water samples. This method can distinguish between the main Cryptosporidium species that can infect people from other Cryptosporidium species that are not usually found in people. During the last year we have shown that the PCR can detect a single oocyst and can distinguish the 3 main Cryptosporidium species’ infecting people from 20 other Cryptosporidium species not usually seen in humans. We also have shown that none of the numerous common parasite and animal species tested gave false positive results.

Following amendments to the second research area last year, we focused on the identification of parasite egg proteins that could be used to generate new antibodies that specifically bind to the parasite eggs. This year we evaluated six parasite proteins for their potential to produce suitable antibodies. Four of these proteins were found to be suitable and the DNA, of the genes encoding these proteins, was amplified and cloned. The DNA products of the four genes were sequenced to confirm that they were correct and that they did not contain errors. The DNA products of the four genes were then inserted into circular pieces of bacterial DNA constructs (called expression vectors), which will allow us to make proteins from these genes in bacteria. These proteins can then be purified, which is scheduled for next year. These purified proteins can then be used to make antibodies for isolating and detecting T. gondii oocysts from water.

The third research area established and optimised methods that allowed the specific detection and enumeration of Cryptosporidium and Giardia parasite eggs in water, confirming that this technology has potential. The limit of detection was determined for both parasite species, but currently the sensitivity may not be sufficient to allow replacement of current methodologies used by the drinking water industries.

The project involved the following Knowledge Exchange activities:
A meeting with stakeholders from Scottish Water, DWQR and RESAS was held to discuss the progress of the project in June, and the project was presented to Scottish Government policy advisors, with an interest in water, at an in-person event in July. An article featuring the project was included in the SEFARI Spark Newsletter in January (2026). 
 

Impact

Policy: 
During the last year we met with members of the DWQR to discuss our project and they reiterated the importance of looking at technological developments that can be used by the water industries to make their drinking water safer for consumers. The DWQR also stressed the importance to develop a specific concentration method for Toxoplasma parasite eggs from water as currently there is no method available for the water industries to monitor Toxoplasma contamination in drinking water. The project was also presented to policy advisors, who have a water remit, at an in-person meeting within the Scottish Government where the policy advisors commented that the wider impact and costs associated with Cryptosporidium drinking water outbreaks, like the one in Devon in 2024, clearly demonstrate the need for better monitoring tools and the importance of this project.

Industry: 
The technologies that are being developed in this project have been discussed with Scottish Water, who expressed their interest in these technologies particularly the quantitative PCR for the detection and enumeration of Cryptosporidium, as this methodology is closer to full evaluation. However, the PCR would require further validations against industry standards before it could be adopted by the water utility companies. Another water utility company, Welsh Water, also expressed an interest in the quantitative PCR. Three water utility companies have provided a letter of support for a BBSRC grant application (value £1.2 million), in which the quantitative PCR will be used to monitor two catchments. The companies also offered in-kind contributions of their time to be part of the steering committee and Scottish Water has offered to provide access to catchments that are known to have Cryptosporidium present in their untreated water.

Academia: 
The results from the quantitative PCR have been discussed with colleagues from the Scottish Microbiology Reference Laboratories (Glasgow) and the Cryptosporidium Reference Unit (Swansea), who expressed an interest in the technology and the possible application in Public Health settings.
The Toxoplasma detection technology and the specific need for a tool to detect T. gondii in water, was discussed at a symposium (“One Health Approach to Tackling Toxoplasmosis”) held at the Moredun Research Institute in November. The symposium involved researchers and key stakeholders involved in public and veterinary health, food and water safety, and policy development to discuss the current situation surrounding toxoplasmosis in Scotland and to identify knowledge gaps. The lack of a Toxoplasma concentration method from food, water and environmental samples was identified as such a limitation.
The Toxoplasma work conducted in this project has resulted in a NERC grant application (value £828K), which was submitted in January 2026.
 

Objective1: Quantification and characterisation methods for Cryptosporidium and Giardia.

DNA segments, which contain both variable and conserved sequence regions within the Cryptosporidium and Giardia genomes, were used as targets for the development of specific DNA detection assays. In order to achieve this, small DNA sequences (primers) were identified (O1-4-D9) that could bind to specific DNA sequences in the genomes of target parasite species. The specificity of these primers was evaluated in molecular detection assays (O1-5-D10, M5).

Objective2: A technique to extract Toxoplasma eggs from drinking water.

This year has focused on evaluating the sensitivity of the proposed assay (O2-4-D11) but it became apparent that this technique was not sensitive enough and the available antibodies were not binding to the parasite eggs well enough for the assay to work. In discussions with RESAS and the Drinking Water Quality Regulators (DWQR) it was decided to change the approach to enable the generation of new antibodies for this test. Since this meeting, we identified six potential parasite egg proteins (new O2-4-D11) that can be taken forward for expression in bacteria. Subsequently, these proteins could be used to generate new antibodies.

Objective3: Digital analysis methods for detection and quantitation of protozoan eggs.

We assessed digital microscopy and computer-based screening methods to identify parasite eggs within water samples; however, the equipment available to us was not sensitive enough to allow reliable/sensitive identification of parasite eggs (O3-3-D12). Following engagement with RESAS, DWQR and Scottish Water it was decided to use a different digital technology called Flow Cytometry, which uses a laser to determine the size/density of cells or particles. Preliminary findings indicate that Flow Cytometry can be used to detect and count Cryptosporidium eggs in water (new O3-3).

Knowledge Exchange:

A meeting with stakeholders from Scottish Water and DWQR (KE3) was held to discuss the progress of the project in July. In addition, we had a follow up meeting with RESAS and DWQR to discuss changes to the project also in July. Finally, we had a further stakeholder meeting in December with Scottish Water, DWQR, RESAS for us to provide an update of progress with the changed objectives and to update the new RESAS lead of the project (KE7). All deliverables and KE activities were completed on time and the overall objectives are on track for completion.

Impact

Policy: During this financial year, we had six meetings, which involved representatives from Drinking Water Quality Regulator (DWQR); two were scheduled meetings (KE2 & KE7), one meeting on Teams, visits to the labs at Moredun, a water treatment plant and the microbiology labs at Scottish Water. During all meetings, the progress of MRI-D2-1 was discussed. Colleagues from the DWQR were very complimentary about the work conducted at Moredun, including the agreed changes to the project objectives. They stated that the “presentations [by Moredun scientist] were fascinating as ever”, that they were very happy with our progress and that there was a real need to investigate new/better technologies to keep Scottish drinking water safe.

Industry: During this financial year, we had four meetings, which involved representatives from Scottish Water; two were scheduled meetings (KE2 & KE7), the third was the visit to a water treatment plant and fourth the visit to the microbiology labs at Scottish Water. During all meetings, the progress of MRI-D2-1 was discussed as well as how the project aligns to the needs of the Water Industries.

Public and Professional: Research outputs, related to MRI-D2-1, were discussed in six presentations at meetings that included academics, health professionals and representatives from water utility companies. These presentations focused mostly on alerting the audience to the risk of Cryptosporidium in water for livestock and humans and included detection methodologies. The presentations were well received and resulted in invitations to future events.

Year 2 progress:

• DNA detection methods: Variable regions of the Cryptosporidium and Giardia genomes have been identified as excellent targets for developing sensitive DNA-based assays. Sequence data has been generated and will be used to design new detection tests in Year 3.

• Extracting Toxoplasma eggs: Work is underway to create a magnetic separation technique that can isolate Toxoplasma eggs from water samples. Optimisation trials revealed challenges with antibody performance, but a new antibody has been sourced from collaborators and will be tested next.

• Digital image analysis: Digital microscopy tools are being developed to automatically screen water samples for parasite eggs. Positive and negative slides have been successfully scanned using two different slide scanners, with a third test planned. This will allow the team to select the best system for producing high-quality images quickly and efficiently.

Engagement:
Stakeholder meetings with Scottish Water and the Drinking Water Quality Regulator (DWQR) have ensured the research stays closely aligned with industry needs.

All deliverables and milestones were achieved on time, and the project remains on track.

The project is developing new tools to help water providers, including Scottish Water, detect harmful parasites more quickly and effectively in drinking water. Current testing methods are slow, costly, and limited – focusing mainly on Cryptosporidium and occasionally Giardia. However, research has already shown that Toxoplasma can also be present in Scottish water supplies, and there is currently no method available for water companies to test for it. This project is addressing these gaps.

Year 1 progress:

• Faster parasite detection: Work is underway to develop a sensitive, specific, and fast DNA-based test (qPCR) for Cryptosporidium and Giardia. Promising genetic targets have been identified, and a reference bank of parasite DNA has been created to support test development.

• Detecting Toxoplasma in water: Efforts have started to create a new immunomagnetic separation (IMS) technique – similar to the one already used for Cryptosporidium. Early trials have highlighted challenges, but the method is being refined with international collaborators.

• Digital screening: A new image analysis system is being designed to automatically detect parasite cysts and oocysts in water samples, reducing the need for manual microscopy and speeding up results. Algorithms have been identified, and image capture methods are being optimised.

Engagement:
The team has met regularly with Scottish Water, the Drinking Water Quality Regulator (DWQR), and scientific collaborators to share progress and gather feedback.

All project milestones and knowledge exchange activities were delivered on time, keeping the research firmly on track.