Development of Neuraminidase Diagnostic Kits for Rapid Diagnosis of Enterotoxemia Disease in Animals

PROJECT NAME: Development of Neuraminidase Diagnostic Kits for Rapid Diagnosis of Enterotoxemia Disease in Animals
PROJECT MANAGER: Prof. Dr. Özlem ÖZMEN
PROJECT SUBJECT:
Enterotoxemias are infections caused by various toxins of Clostridium perfringens, resulting in sudden deaths in many animals, especially lambs, goats, and cattle, both young and adult. Enterotoxemias are a significant group of diseases characterized by general toxemia, formed by the excessive proliferation of microorganisms in the intestines of animals. C. perfringens is an anaerobic bacterium commonly found in nature. It is always present in soil, fresh and polluted waters, and the digestive systems of humans and animals. The agents cause disease together with various predisposing factors. There are 5 types of enterotoxemias: A, B, C, D, and E. Each type is caused by multiple toxins. In our country, the most common types are B, C, and D. Enterotoxemias are typed according to major toxins, and clinically, detecting the presence of the disease is more important than typing the disease. This is because the most effective treatment of the disease is done through vaccination. Therefore, rapid diagnosis in the disease is a very important issue in controlling the disease. There are 16 different virulence factors effective in the formation of enterotoxemic disease patterns caused by C. perfringens, which are usually fatal. Twelve of these are toxins produced by the agent. Other important virulence factors include enterotoxin, hemolysin, neuraminidase (sialidase), and the dynamic metabolic activity of the agent.

Enterotoxemia vaccines implemented for disease control are generally polyvalent vaccines against many types. Therefore, type detection is not very necessary for rapid diagnosis; animals are vaccinated against many types in cases of enterotoxemia diagnosis or suspicion. Neuraminidase is an enzyme found in all types, and kits created with antiserum specific to neuraminidase for C. perfringens will significantly reduce economic losses with rapid diagnosis and vaccination programs. The main goal in this disease is to immunize healthy animals against the disease through vaccination. This test will also allow the planning of vaccination programs by detecting the absence of this enzyme in healthy animals. No kit has been found for the detection of neuraminidase in the studies conducted.

Neuraminidase is an enzyme produced by all types of C. perfringens and is also called sialidase. Neuraminidase acts by breaking down receptor molecules on the cell surface or connective tissue. Neuraminidase secreted by bacteria, called exo-α-sialidase, hydrolyzes the α-glycosidic bonds of terminal sialic acid residues found on oligosaccharides, glycoproteins, and glycolipids in host cells. Sialic acid plays an important role in various cell-cell interactions and cell-molecule recognition processes and is a nutrient source for bacteria. Bacteria can obtain sialic acid in two ways: by synthesizing it anew or by collecting it from the host. Neuraminidase secreted by bacteria catalyzes the hydrolysis of terminal sialic acids of sialoglycoconjugates in different organisms, and free sialic acid is directly transported to the bacterial membrane. Sialic acid is then used as an energy and carbon source for bacterial proliferation. Additionally, neuraminidase plays an important role in the early stages of bacterial infection and contributes to biofilm formation on glycoprotein-coated surfaces. Recent scientific studies emphasize that neuraminidase inhibitors can be used as a new target for developing therapeutic agents to treat bacterial infections. Neuraminidases play important functions in the early stages of infections and have significant roles in bacterial proliferation and colonization. In this study, rapid neuraminidase diagnostic kits will be developed for the rapid diagnosis of enterotoxemia disease, which is important for farm animals and causes high mortality, allowing for quick intervention in this important disease. Because the clinical symptoms of this disease can be confused with many other diseases. However, in this disease, antibiotic treatment is not required, but vaccination is necessary. Therefore, rapid diagnosis is very important to reduce the mortality rate in farm animals.

PROJECT PURPOSE:
Rapid tests or rapid diagnostic kits are tests that provide quick results in less than 20 minutes and are easy to use. Unlike most standard tests, rapid tests are applied immediately at the animal's bedside without being sent to the laboratory and show results in a short time. Thus, not only is time lost for diagnosis prevented, but it also provides information for quick treatment. The material to be used for rapid tests is practical as it consists of samples collected without harming the animal such as blood, urine, feces, saliva, or nasal discharge. Many substances such as bacteria, viruses, proteins, and chemicals can be detected with rapid test kits. These kits, which work on the basis of antigen-antibody reaction for protein-based substances, are rapidly entering the use of veterinarian.

Similarly, diagnostic kits produced for animal diseases are very useful for veterinarians for rapid diagnosis in hospital and field conditions. The use of these tests, which are easy and practical in terms of making a correct diagnosis in a short time in field conditions, is rapidly increasing in veterinary medicine. However, the absence of rapid tests for all diseases and dependence on foreign countries for those that exist are the most important problems. Dependence on foreign countries also increases the price of these kits and significantly reduces their cost-effectiveness. The average price of an imported kit used for diagnosing a disease in farm animals is around 10 dollars. Additionally, differences between local strains and foreign strains, especially in viral agent diagnoses, can lead to incorrect results. For all these reasons, the production of domestic and national kits is essential.

In this project, BALB/c mice will be immunized 3-4 times with neuraminidase protein in the presence of an adjuvant for the production of a diagnostic kit for enterotoxemia disease, and the animals with the highest antibody activity against this protein will be subjected to fusion studies. Lymphocytes obtained from the spleen and lymph nodes of the mice will be fused with myeloma cells under in vitro conditions, creating hybrid cells that can continuously proliferate and simultaneously synthesize antibodies in the cell culture environment. Hybrid cells producing antibodies specific to neuraminidase protein will be determined using the ELISA test. After obtaining the anti-neuraminidase antibody, monoclonal antibodies will be produced on a large scale under in vitro conditions and purified using various protein purification columns. Purified monoclonal antibodies will be labeled with various enzymes for use in diagnostic kits, and the prototype of the diagnostic kit will be created, and studies will be conducted to determine the working sensitivity of the diagnostic kit and the shelf life of the product in blood, feces, and body fluid samples taken from sick animals.

Neuraminidases function directly as virulence factors during bacterial pathogenesis. C. perfringens produces three different neuraminidases (sialidases), named NanH, NanI, and NanJ. NanH (43 kDa) does not contain a secretion signal peptide and therefore has a cytoplasmic location in log-phase cultures. In contrast, NanI (77 kDa) and NanJ (129 kDa) are secreted exosialidases. The catalytic modules of the three sialidases show conserved amino acid sequence identity and have family 33 carbohydrate-binding modules. Compared to NanH, which contains only one catalytic domain, NanI and NanJ also contain additional carbohydrate-binding modules. These carbohydrate-binding domains are thought to increase the binding affinity between NanI and NanJ and multivalent substrates. NanJ has a complex multi-modular structure consisting of a central catalytic module and five accessory modules. The two N-terminal modules show amino acid sequence identity with family 32 and family 40 CBMs. NanI, on the other hand, has a simpler structure consisting of a catalytic module and an N-terminal family 40 CBM. It also includes the chemical structure of NanJ.

In the stages of the project carried out so far, all the above-mentioned stages have been completed by purchasing commercial neuraminidase, but the desired level of accuracy could not be achieved in the field due to the low purity of the purchased substance. For this reason, the section of neuraminidase known as NanI, which plays the most important role in disease development, will be produced recombinantly, and the study will be repeated on this protein to produce kits with high accuracy.

PROJECT'S REGIONAL CONTRIBUTION:
Enterotoxemia in ruminants is a bacterial disease that can cause serious problems in the gastrointestinal systems of animals. This disease is more commonly seen in sheep, goats, and cattle. Deaths due to enterotoxemia can vary depending on many factors. Mortality rates can vary depending on factors such as the degree of disease transmission, the immune system status of the animals, geographical location, feeding practices, and access to treatment. Especially vaccination and early diagnosis of the disease are important for the success of the treatment. These factors in the disease and whether rapid diagnosis can be made can significantly change mortality rates. This situation shows that economic loss can reach very large proportions.

The fact that animals can die in a very short time in this disease shows that time is very important in the correct diagnosis of the disease. Although the toxins of C. perfringens cause the disease, it is thought that the detection of neuraminidase, which is considered a pathogenicity factor for the disease, is more important for diagnosis, and this project has been designed.

If the production of monoclonal antibodies that will specifically recognize the neuraminidase protein with high specificity and the development of a diagnostic kit within the scope of the proposed project are successfully completed, the produced kits will be a very practical and effective product in the diagnosis of this disease. The fact that the production will be done with national resources will significantly reduce the cost and increase its use. The few imported and expensive commercial enterotoxemia kits currently used are complex kits made according to types. With the rapid diagnostic kits planned to be developed in the project proposal, simple and accurate diagnosis will be made quickly, and ELISA, strip, biosensor, and immunohistochemistry kits that can be used both in field conditions and in the laboratory environment will be produced. With this study, it is aimed to produce kits that can be used in different ways in live and dead animals to detect enterotoxemias, which cause serious economic losses, especially in ruminants.

With the rapid tests to be developed within the scope of this project, not only will rapid diagnostic kits be produced for veterinarians to use in field conditions and quickly diagnose the disease, but also practical and cheap kits will be produced. In this way, an infrastructure will be created for the production of these kits, which we are almost entirely dependent on abroad. Thus, an infrastructure will be created for a center where kits that can quickly detect the diseases of large and small farm animals, which are intensively raised in our region, will be made. This situation will increase the interest of scientists and companies in the region. Additionally, the breeding of farm animals, which constitutes a large part of the regional economy, will be done under more modern and healthy conditions, which will increase production quality. Situations such as the high economic value of quality and healthy products and the increase in yield in healthy animals will be an added value for regional livestock.

With this project, since enterotoxemia disease, for which antibiotic treatment is not effective, will be diagnosed quickly and accurately, unnecessary antibiotic use will also be prevented. This situation will significantly reduce the economic loss caused by unnecessary antibiotic use and antibiotic residues. The likelihood of developing antibiotic resistance will also be significantly reduced. Additionally, drug residues in meat and milk will not form, which will minimize economic losses due to the destruction of meat or milk. The rapid and accurate diagnosis of this disease, which can cause significant losses in both young and adult animals, will also provide significant contributions to reducing the disease agent both within the herd and in the environment. For all the reasons mentioned above, the importance of producing and using domestic, national, cheap, and practical kits that can be used for the rapid and accurate diagnosis of enterotoxemia disease in field conditions is clear. This project has the capacity to meet all these criteria and will provide significant contributions to the necessary infrastructure and training of experienced personnel for the production of diagnostic kits for other diseases, making our region a center of attraction.

PROJECT'S SOCIAL CONTRIBUTION:
Animal diseases are generally important problems from a social perspective. Because it is known that animal diseases can cause a series of negative effects. Many diseases, such as enterotoxemia, can be important in terms of food safety, in addition to causing death and economic loss in animals. Animal diseases can generally negatively affect the livestock economy and cause product losses. Especially diseases like enterotoxemia can reduce animal populations, which can negatively affect food production. The production purpose of farm animals is to meet the food needs of humans. Therefore, all fatal diseases like enterotoxemia negatively affect food production.

In general, diseases also negatively affect animal welfare. Animal welfare is important in terms of both productivity and food safety. Therefore, the prevention of diseases, or if this is not possible, their rapid diagnosis, is also important in terms of animal welfare. For these reasons, the control and prevention of animal diseases are of great importance for the general health of society, food safety, and economic stability.

Additionally, the fact that experienced personnel will be trained in many areas such as monoclonal antibody production, laboratory techniques, and kit production within the scope of this project is also an important plus of the project. The data obtained in this study will be turned into publications and will also contribute to science.

Rapid diagnostic kits allow diseases to be detected in their early stages. Early diagnosis ensures the correct treatment of the disease and a better response to treatment. Rapid diagnosis also helps determine the correct treatment methods. Correct treatment increases the patient's chance of recovery and prevents complications from incorrect treatment. The rapid diagnostic kits to be produced with this project are aimed at veterinarians and will allow rapid and accurate diagnosis of enterotoxemia in field or hospital conditions. Thus, the prevalence of the disease will be determined more clearly. Because the most important clinical symptoms of this disease, diarrhea and sudden deaths, can be seen in many diseases. Additionally, since many cases cause animal deaths without being fully diagnosed, the prevalence of the disease cannot be fully determined.

The rapid diagnosis of enterotoxemia disease is of great importance for protecting the health of both animals and society. Veterinarians can use rapid diagnostic kits for the rapid and accurate diagnosis of the disease and take treatment and prevention measures.

With this study, an important infrastructure will also be created for the production of diagnostic kits, which we are dependent on abroad. This situation will provide significant support to our university, which is a pilot university in the field of livestock, in fulfilling this mission.

With this study, different products such as biosensors, strip kits, and ELISA kits will be developed. Four separate patent applications have also been made within the scope of this project. All these rapid diagnostic kits will provide significant contributions to both regional and national livestock and animal welfare.

This project will also lead to the team gaining experience, training new researchers, and perhaps new thesis projects for many future projects and kit production. Because kit production is becoming a very important industry branch in our country. Therefore, there is a great need for trained human resources and infrastructure in this field. Additionally, this production branch will lead to the formation of a wide industrial infrastructure and many subsidiary productions. Thus, this project has the potential to provide significant contributions to veterinarians, the livestock sector, and the academic field.

Therefore, the social benefit of this project is very important in terms of providing significant contributions to both the country and the country's livestock and scientific community.