Development of a Microfluidic Diagnostic Kit for Field Detection of Cryptosporidium Oocysts in Calves

PROJECT TITLE:

Development of a Microfluidic Diagnostic Kit for Field Detection of Cryptosporidium Oocysts in Calves

PROJECT COORDİNATOR:

Prof. Dr. Ahmet Çiçek

PROJECT TOPIC:

In rural areas of Turkey, particularly where healthcare services are limited, diagnosing various significant microbial, infectious, and non-infectious diseases is quite challenging. The lack of timely access to accurate diagnostics and trained personnel in these regions has led to a substantial increase in bacterial and infectious diseases, resulting in irreversible material losses, including significant emotional distress. Many bacterial and infectious diseases that can be diagnosed early can also be treated rapidly and cost-effectively.

Bacterial and infectious diseases are commonly observed both globally and in Turkey. Diarrhea is a prevalent clinical symptom that negatively impacts animal health, and various factors, including bacteria, viruses, parasites, and others, can cause it. Cryptosporidium is a significant protozoan parasite that leads to parasitic diarrhea in animals. This parasite has a wide distribution in both developing and developed countries and can cause disease in various hosts, including humans, domestic animals, and wildlife.

In particular, Cryptosporidium infection in cattle manifests with clinical symptoms such as diarrhea, abdominal pain, nausea, and weight loss, and if not diagnosed and treated promptly, it can lead to severe mortality. While C. parvum typically infects humans and cattle, C. andersoni and C. bovis have been reported in humans in rare instances. Therefore, infected cattle are considered an important reservoir for humans. Studies have shown that zoonotic transmission may occur due to close contact between cattle and humans.

C. parvum can infect many species of mammals and is transmitted through the fecal-oral route in its oocyst stage. The main symptoms of cryptosporidiosis include acute, watery, and bloody diarrhea. Particularly in immunocompromised patients, diarrhea can reach 10-15 liters per day, and C. parvum is regarded as a significant waterborne pathogen in developed countries.

C. parvum oocysts are highly resilient, making it difficult to control the parasite. They can withstand low temperatures (sometimes as low as -22 °C), indicating that fecal contamination can persist for an extended period. Additionally, oocysts can tolerate pH changes during some water treatment processes.

Detecting C. parvum oocysts can be challenging due to their small size. ELISA tests can be used to identify the presence of oocysts in fecal samples, but this method is not practical for field use. Rapid multiplex diagnostic kits are available, but they tend to be expensive and may require fluorescent microscopy.

The project focuses on developing more suitable, low-cost, and field-applicable early diagnostic systems for detecting Cryptosporidium oocysts. Three different diagnostic approaches with varying complexity and costs are being developed. All these kits are designed to be used by veterinarians in the field, enabling rapid and effective diagnosis of cryptosporidiosis without the need for laboratory facilities or microscopes. The products developed in the project are planned to be manufactured entirely with local capital. The expected common impacts of the project results are as follows:

i) The developed kits will be patented and converted into commercial products. A product has already been patented, and field tests are currently being conducted. Commercialization of the product is planned in the short term.

ii) On-site diagnostic systems can be used by veterinarians to diagnose cases practically without needing laboratory facilities or microscopes.

iii) Veterinarians will have the option to purchase and use the developed systems according to their budget.

iv) Early diagnosis will help prevent mortality cases in calves, significantly contributing to minimizing losses for the regional and national economy.

v) The production of on-site diagnostic systems with local capital will reduce dependence on foreign sources.

vi) Animal owners will no longer need to transport their calves to veterinary hospitals or laboratories, eliminating transportation costs.

vii) On-site diagnostic systems can be easily applied to other factors as well.

PROJECT OBJECTIVE:

The main goal of the project is to enable the rapid detection of Cryptosporidium protozoan oocysts in fecal samples and water using field-deployable systems. To achieve this objective, a series of specific targets have been established:

Development of a portable light microscope that integrates with a smartphone camera for the detection of Cryptosporidium oocysts stained with Carbol fuchsin. This microscope allows for the rapid and accurate identification of oocysts. This phase of the project has been successfully completed, yielding positive results in the field.

Development of image processing software to automatically count detected oocysts, minimizing human error.

Design of a portable fluorescent microscope that can be integrated with a smartphone, enabling the rapid detection of Cryptosporidium oocysts using fluorescent labeling and autofluorescence methods.

Design and optimization of microfluidic systems for the purification of Cryptosporidium oocysts, along with their production using numerical analysis methods. These chips will facilitate sample preparation and oocyst purification.

Testing of the produced microfluidic chips and statistical analysis of the purified samples.

Development of a suitable system for field use by integrating microfluidic chips with portable microscopes.

Provision of prototype kits for pre-use testing by veterinarians, forming a sample for field applications.

Utilizing the statistical data obtained to access crucial information about the geographical and temporal distribution of specific animal diseases, primarily cryptosporidiosis.

Ultimately, significantly reducing animal mortality rates through early diagnosis and intervention.

The success of this project relies on the rapid and effective detection of Cryptosporidium protozoan oocysts and the development of a diagnostic system that can be used directly in field conditions. Our main objective is to convert body samples such as blood, feces, urine, and skin into integrated systems that actively utilize smartphones through a national and local Microscopic Diagnostic System, enabling the rapid diagnosis of various bacterial and infectious diseases.

When used correctly, this enhanced system will provide an invaluable and cost-effective solution, particularly in rural areas, military units with limited healthcare services, regions with restricted diagnostic capabilities, and rural areas where early disease detection is crucial. Many diseases can be easily diagnosed on-site without the need for advanced investigations through the microscopic examination of blood, feces, urine, and other body samples.

In addition to the many advantages of traditional microscopes, this national and local technology system stands out due to its compact size, smartphone integration, and advanced software support. Its portable design makes it suitable for use in any environment. Key features include portability, smartphone integration, specialized data transfer software, magnification options of 200X, 400X, and 1000X, low cost, and high performance. The diagnostic systems developed within the project are designed as powerful and portable tools that can be utilized in various important application areas.

REGIONAL CONTRIBUTION OF THE PROJECT:

Protozoan parasites, particularly Cryptosporidium spp., are among the primary agents causing calf mortality, which poses a significant problem in the livestock sector in our country. Known colloquially as "crypto disease," cryptosporidiosis results in considerable calf losses annually, leading to economic and social repercussions. Statistically, it is estimated that around 750,000 calves are lost each year in our country, with approximately 50% of these deaths attributed to diseases caused by protozoan parasites. These losses translate to an annual Gross Domestic Product (GDP) loss of about 5 billion TL.

The project, structured in three phases, has already begun to yield positive results and has made tangible contributions to regional livestock. In the first phase, a mobile light microscope and diagnostic software were developed, allowing for field testing by veterinarians affiliated with the Burdur Chamber of Veterinarians. As part of a cooperation protocol between Burdur Mehmet Akif Ersoy University (MAKÜ) and the Burdur Chamber of Veterinarians, 20 prototype mobile microscopes were delivered to veterinarians during the Regional Development-Oriented Livestock Projects Sector Council meeting held on December 30, 2022, attended by the Governor of Burdur, Mr. Ali Arslantaş.

With these mobile microscopes, veterinarians can capture images of samples taken from the field and send them to MAKÜ Veterinary Faculty faculty members for analysis via the mobile diagnostic system. Hardware and software improvements have been made based on feedback from veterinarians during this pilot application. Additionally, on January 6, 2023, practical training on the use of the mobile microscope and application was provided to 20 veterinarians from the Burdur Chamber of Veterinarians. The training was conducted by the project team at the Burdur Chamber of Veterinarians, where Prof. Dr. Ahmet Çiçek informed participants about the features of the mobile microscope, Prof. Dr. Nurettin Körözü demonstrated its use, and Assoc. Prof. Dr. Ahmet Biçer explained how to use the mobile application practically. Furthermore, Prof. Dr. Şima Şahinduran provided information on sample collection, staining, imaging, and image analysis necessary for identifying intestinal parasites.

The data obtained from the project is collected in the MAKÜ Farm Monitoring and Management System (ÇİYS) database. This data offers significant insights into the fight against animal diseases for both scientific research and policymakers. Statistical analysis of the data can lead to the production of theses, projects, and publications regarding the regional and temporal distribution of specific animal diseases.

The mobile diagnostic system has been included in the investment support program of the Ministry of Agriculture and Forestry. As stated in the announcement made by MAKÜ on April 14, 2023, “Burdur Mehmet Akif Ersoy University’s ‘Mobile Microscope and Diagnostic System Integrated with Smartphones,’ which stands out among the Regional Development-Oriented Livestock Projects, has qualified for support under the Ministry of Agriculture and Forestry's Program for Supporting Rural Economic Infrastructure Investments in the 'Smart Agriculture' category. Through this support, partial grants will be provided to those wishing to purchase the mobile microscope developed by MAKÜ, aiming to accelerate its widespread use, particularly among veterinarians and farms.”

The concept of the mobile diagnostic system developed for animal diseases has opened up opportunities for collaboration with the private sector firm TeknoTıp Analitik Sistemler A.Ş. Initial contacts with this company began in 2019. As a result of this collaboration, a joint venture was established between MAKÜ Technology Transfer Office (TTO) A.Ş. and the company, named "T1 Diagnosis," which conducts the production, marketing, sales, communication, and technical support activities for the mobile diagnostic system. In the first phase, 20 products were purchased under the Social Development Support Program (SOGEP) by the Western Mediterranean Development Agency (BAKA) and delivered to veterinarians affiliated with the Burdur Chamber of Veterinarians for pilot applications. The joint venture also continues its international marketing efforts, aiming for at least 10% of approximately thirty thousand veterinarians to use the product within the first two years. Additionally, the mobile diagnostic system is planned to be offered at competitive prices in international markets, with the goal of generating export revenue. It is also anticipated that the system will be used in developing African countries through TİKA to combat diseases like malaria. The applicability of the system for human diseases is also among the targets.

SOCIAL CONTRIBUTION OF THE PROJECT:

The inability to locally detect parasitic diseases that emerge immediately after birth and can lead to death within 1-2 days in calves is a significant shortcoming. The mobile diagnostic system addresses this gap by enabling on-site, rapid, and accurate diagnosis. Additionally, it contributes to addressing the equipment deficiency for the professional practices of veterinarians.

In our country, it is known that the calf mortality rate is alarmingly high at around 25%, which is a primary source of problems for breeding heifers and fattening bulls. Enterprises suffer losses due to increased replacement heifer rearing costs, decreased total meat and milk yields, and higher veterinary and medication expenses resulting from calf losses. This situation also constitutes a significant loss for the national economy. The main cause of calf deaths is diarrhea due to parasitic infections. In this context, the diagnosis of diseases caused by intestinal parasites such as Cryptosporidium spp. and Giardia spp., which can be easily detected through proper staining under a microscope, is very low due to reasons like veterinarians lacking microscopes, the difficulty and cost of transporting animals to health institutions. The fundamental hypothesis of the project is that significant reductions in calf losses can be achieved by making a diagnosis rapidly and accurately through simple microscopic examinations and applying the correct treatment immediately, thereby minimizing losses for both enterprises and the country. This hypothesis has been validated by results obtained in laboratory, field, and pilot applications. Moreover, this hypothesis is also applicable to human diseases that can be easily diagnosed with blood samples, such as malaria.

The project has contributed to raising the level of social welfare by significantly reducing the labor and financial losses caused by calf deaths. Treatments administered without identifying the disease agent do not lead to the recovery of the animal and cause waste of medications. The outputs of the project play a crucial role in preventing this situation and addressing the harmful effects of incorrect treatments on both animal and human health. Furthermore, reducing losses is expected to positively impact the prices of animal products, thus contributing to the economic welfare of society. In the near future, it is anticipated that significant contributions to human health will be made by enabling on-site diagnoses of diseases, including malaria, particularly in areas where access to healthcare services is limited both domestically and globally.

Images taken through the mobile diagnostic system by users are initially reviewed and analyzed by faculty members at MAKÜ. In subsequent stages, the aim is to facilitate disease diagnosis using artificial intelligence applications. This approach represents a unique innovation that is unmatched worldwide and offers significant scientific and economic advantages for our country. Additionally, the success of the project has fostered advancements in diagnostic studies and imaging techniques. Work is ongoing to develop three different fluorescent mobile microscopes.

Efforts are continuing to develop unique advanced on-site diagnostic systems that can be obtained through the integration of project outputs and these systems. For example, the project leader is providing consultancy for a TÜBİTAK 3501 project being carried out at Istanbul University, where imaging is performed at the single-cell level using a mobile microscope. The mobile microscope developed in the project serves as a significant component with a broad application spectrum across various scientific fields, including microbiology, mineralogy, metallurgy, food engineering, agricultural engineering, and criminology.

With the widespread adoption of project outputs, it is targeted to reduce calf deaths from parasitic diseases by 50%, thereby preventing approximately 187,500 calf deaths annually. Assuming the value of a three-month-old calf is 20,000 TL, this could prevent an annual GDP loss of approximately 3.75 billion TL. When considering the long-term loss of products such as meat and milk, even greater losses can be averted. Including other ruminants (goats, sheep, etc.) in this calculation, it is expected that the GDP loss could exceed 5 billion TL annually. The national and international sales of the mobile microscope, which possesses superior features compared to its counterparts, are also expected to contribute to the GDP.