Fasciola gigantica and Paramphistomum cervi have been discovered to be composed of three important types of proteins these structural proteins that make them more adapted to their habitats and increase their immunity against their destruction by antibodies. This project is aimed at identifying these proteins contained in these two fluke genera using an analysis of soluble protein that elicit an immune response.
Biological research has shown that proteins like phosphagens contain high amounts of phosphates that release high amounts of energy that allow muscle movements like contraction and expansion. Other proteins like phosphocreatine and phosphoargine are also found in many invertebrates. Trematodes for example are known to contain phosphoarginine and phosphogens. One way of differentiating between phosphosphoarginine and phosphogens is using the N-Methyl group. The Proteomic analysis used in this research helps distinguish the N-Methyl groups in all these proteins so as to identify all the kind of proteins that compose these fluke genera.
Paramphistomum cervi and Fasciola gigantica are collected from the bile ducts of infected livestock that re butchered in slaughterhouses for meat (Boukli, 2011, p.852).The next important step is to collect these flukes from the gut of these animals by washing them with cold water that is highly concentrated with solutes. This can be done in a bottle or another container. These flukes should then be weighed so as to record the initial weight of these flukes.
These fluke genera are then homogenated in a solution that has a pH of about 8.5 so as to buffer the pH of their bodies. This solution could preferably be mercapt ethanol. However, it is important to use a small manageable amount of this solution which could be about I millimeter. The next important step is to take a centrifuge the solution for about 20 -25 minutes making sure that the temperature of the solution is favorable enough for proteins(Anuracpreeda, 2011, p.341). Bearing in mind that proteins denature at a temperature of about 40 degrees Celsius, it is important to keep the solution contains the flukes at a low temperature of about 3-4 degrees Celsius so as to avoid denaturing them using the high temperatures. After centrifuging the solution, the solution collected is diluted by about 50 times using the same ethanol solution. This is used to measure any activity of proteins especially phosphagen, phosphoarginine, phosphocreatine. The SI units used to measure the activity of these proteins are Watts.
The use of electro methods to detect protein activity has come to be known as the 2DE analysis. Boukli et.al explains that the 2DE Analysis uses pH gradient strips that turn blue in spots that F.gigantica flukes are discovers. This method indicates that at most times flukes like F.gigantica are discovered at a pH of about 3-10( Boukli, 2011, p.853). This suggests that this range of pH is favorable for their existence.
Another important method that this project is going to use is the use of western blots. In this method protein activity is going to be dected by introducing pH changes to the enzymes catalyzing proteins such that one can identify when the reactions are rising and when the reactions are slowly decreasing. This project intends to diaminobenzidine as the substrate to be catalyzed in that its shape forms favorable active sites for protease.
Mass spectrometry is also important method that this project intends to use. In this method, protein activity is going to viewed using microbial equipments that can detect catalytic activity that is not visible to the human eye. This increases the accuracy of the project in that the results are not only based on hypothesis.
Bioinformatics will also play a big role in this project in that this project intends to analyze the protein structures detected on these two fluke genera using a wide research of body structural proteins. This will include the arrangement of the peptides within the protein structure and also that of the phospholipids bi-layer.
Enzyme activity is also a key are to look for while measuring the activity of these proteins. A dilute extract of this solution is expected to produce 0.05 M of phosphoargenine for every 0.10M of phosphagen (Anuracpreeda, 2008, p.205).The important substrate from this reaction is ATP which should be about 0.005 Ml for every 0.1 Ml solution that is used for the process.
The results should be based on a series of experiments so as to enhance the accuracy of the data collected. One thing that is important to note is that if all the procedures are followed carefully three kinds of proteins should be identified in these two fluke genera. These include phosphoarginine, phosphoglycocyamine and phosphocreatine. However, it is important to make sure that the substrate –enzyme concentration is kept in a way that the enzyme concentration does not exceed the substrate concentration so as to maintain the protein activity at a constant paste. A scenario whereby enzymes exceed the substrate would cause a phenomenon whereby all the active sites of substrates are occupied by enzymes thereby caused the shape of the substrate to be altered. The alteration of the shape of the substrate might slow down the reaction or may at times stop protein activity completely.
The presence of availability of phosphocreatine in these two fluke genera indicate that the muscles of these flukes are under high activity. The amount of this kind of protein is the same in both kinds of flukes which means that they have a chemical relation. However, it is important to note that these flukes can easily convert phosphocreatine to phosphoarginine and phosphagens in that the chemical compositions of these two proteins are almost the same. Therefore, the key immune system that occurs within these flukes is that they can convert their body proteins to a form that is favored by the condition in which they are living.
F.gigantica has been known to be able to use this adaptive measure to grow to a length of about 340 amino acids. The N-Methyl group of F.gigantica. This fluke species releases a recombinant name proFhCatB which helps them to attach themselves properly on enzymes of poly-proteins. This is a defense mechanism that prevents them from being digested once they reach the gut of animals. This is the reason as to why it is sometimes difficult to discover this fluke genera in adult humans in that the more proteins a human body possesses the more these fluke genera attach themselves to protein enzymes therefore making them seem as if they are part of the human body. The juvenile stage of a fluke like F.gigantica is the most active stage in that in they are able to dominate the substrate catalyzed by enzyme CatB proteases. This increases the penetration power of these juveniles down the guts and lumens of the
Another adaptation of these two fluke genera that makes them more suited for the environment in which they live in based on their protein structure is the fact that during the first month of the juvenile stage acquire immunity against substances like MOAb and PoAb. This means that these fluke genera are not digested by these enzymes in such an area stage. This increases their rate of survival in the guts and rumens of most of ruminants because there is a lower risk of the juveniles dying due to digestive enzymes. Scientists have discovered that the mRNA transcript of these species is slowly changing making them to become more adaptable in their environments. First of all, it is important to understand that the chemical components of mRNA are proteins. The mRNA of these fluke species has over time genetically specialized to form CatB3 proteins that have the same combination as the proteins that form the lining of the walls of the caecal epithelium of most animal hosts. (Panyarachun, 2010, p.99).This means that digestive substances like MoAb and PoAb detect the proteins that compose the bodies of these fluke as being part of the epithelium cells thereby not digesting them. This allows the translocation of these flukes down the guts of animals possible. Therefore, the activity of proteins detected in terms of volts in this experiment indicates that these two fluke genera contain poly-proteins that serve an immunity and adaptation purposes to ensure their survival in the gut and rumens of the hosts in which they live in.
IV. Importance of this Project
The economic importance of the project is the fact that F.gigantica is known to be the causative agent of the Fascilosis disease (Anuracpreeda, 2011, p.340). This fluke species has been estimated to lead to the death of many livestock species around the world for a long time. In fact, it is estimated that $ 3 billion is set aside annually to curb this disease all over the world (Chemale, 2010, p.4940).The most affected continents in the world are Africa and the Asian continents. In continents like Europe about 30% of the livestock die annually due to infections resulting from these two fluke genera (Morphew, 2007, p.963). Biologist argues that these fluke species are difficult to control in that their adaptation strategies make it difficult to detect them. They attach themselves in the substrate molecules of key digestive enzymes like CatB protease. Due to the increased secretion of enzyme protease for digestive purposes, the likelihood of the survival of these flukes is higher in that their habitat environments are constantly produced make it easier for them to easily translocate to other molecules in case the conditions within the substrate like pH or temperatures are not favorable for their survival. This adaptation strategy makes the control measures of these fluke genera to require sophisticated equipment which means that a lot of resources have to be invested into the process. However, it is important to understand that continents like Africa and Asia that are most affected lack enough resources to curb this disease .Therefore, the identification of the specific kind of proteins that constitute these two fluke genera opens up a research of drugs and other substances that are specialized to destroy specific kind of proteins in an infected animals body so as to either completely destroy these fluke genera in the guts and lumens of affected animals. In addition, the identification of the proteins that constitute the bodies of these fluke genera also opens up preventive measures to reduce the spread of diseases like Fasciolosis. This is because water sources and other external habitats where these flukes are known to inhabit can be disinfected therefore reducing the chances of animals to contract diseases related to these two fluke genera.
Understanding the genetic transformations that are taking place in the bodies of these fluke genera in terms of the change of the m-RNA transcript makes it easier for disease control centers to device substances that can ensure that m-RNA transcript do not successively adapt themselves with the conditions in the gut and the lumen of the hosts that these fluke genera infest. This can be achieved by conducting immunizations on livestock in the infested areas so that the immune systems of these animals detect the proteins that constitute these fluke genera is as antigens within their bodies thereby producing antibodies to counter their multiplication.
In conclusion, this project opens up more research on better disease control of disease caused by F.gigantica and P.cervi by analyzing their protein body compositions thereby creating drugs and substances that can destroy the kind of proteins that constitute their body structures.
Anuracpreeda, P. (2008). Paramphistomum cervi: Antigenic profile of adults as recognized by infected cattle sera. Experimental Parasitology. 118 (1), p203-207.
Anuracpreeda, P. (2011). Fasciola gigantica: Production and characterization of a monoclonal antibody against recombinant cathepsin B3 .Experimental Parasitology. 127 (1), p340-345.
Boukli, N. (2011). Fasciola hepatica and Schistosoma mansoni: Identification of Common Proteins by Comparative Proteomic Analysis .Journal of Parasitology. 95 (5), p852-861.
Chemale, G. (2010). Comparative Proteomic Analysis of Triclabendazole Response in the Liver Fluke Fasciola hepatica. Journal of Proteome Research. 9 (10), p4940-4951.
Morphew, R. (2007). Comparative Proteomics of Excretory- Secretory Proteins Released by the Liver Fluke Fasciola hepatica in Sheep Host Bile and during in Vitro Culture ex Host . Molecular & Cellular Proteomics . 1 (6.6), p963-972.
Panyarachun, B. (2010). Paramphistomum cervi: Surface topography of the tegument of adult fluke. Experimental Parasitology. 125 (1), p95-99.