Cyprinus carpio belongs to the family of Cyprinidae and order Cypriniformes. Although C. carpio belongs to the family of freshwater fish, they also inhabit in brackish-water environment. C. carpio are commonly found in lakes, rivers and ponds. They are widely distributed in the world, mainly in the region of Europe and Asia. Rivers that are flowing into Black, Caspian and Aral Sea are natural habitat of the wild stocks. C. carpio is one of the oldest domesticated aquatic species for food.
In 2002, farmed C. carpio production was nearly 14 percent of the total global freshwater aquaculture production. The production of C. carpio has been gradually increasing throughout the years from 343 739 tonnes in 1978 to 4 556 621 tonnes in 2016. (Fig. 1) The number of diseases and problems caused by pathogens such as bacteria, viruses, fungi and parasites are proportional to the increase of fish production.
Aeromonas infection is one of the common diseases in aquaculture industry. It is a zoonotic disease that is life threatening for both human and aquatic species.
Aeromonas spp. are widely distributed in the environment, mainly in freshwater and terrestrial environment.
In this study we will be focusing on Aeromonas hydrophila. A. hydrophila is one of the most common zoonotic bacteria found in the carp farms. It commonly exist as a secondary pathogen and as opportunistic pathogen. Host can be infected by A. hydrophila when exposed to stress or through ingestion of contaminated food. A. hydrophila can also enter the host body through open wounds or sores caused by diseases or other pathogens.
Disease management such as application of antibiotic and chemotherapy had resulted in development of drug resistant in pathogen. Also, due to limited drugs that can be used for aquaculture industry and long withdrawal period of drug, prevention from such disease is needed. Prevention methods such as using of vaccinations and keeping water parameter in optimum were performed in many fish farms. In this study, we will be focusing on using immunostimulants, chitin and chitosan, to improve the immunity and disease resistance of C. carpio against A. hydrophila.
Immunostimulants are substances that activate the immune system of humans and animals for prevention of diseases and improvement of the bodys natural resistance to various viral and bacterial infections. Chitin and chitosan are non-specific immunostimulants that do not have antigenic properties and enhances immune response to other antigens. Research shows that feeding of supplemented diet with chitin and chitosan shows a significant decrease in mortality when host is exposed to A. hydrophila.
The aim of this experiment is to identify the optimum concentrations of chitin and chitosan in the diet of C. carpio which leads to higher immunity against A. hydrophila without decreasing its growth rate.
In this experiment, 5 set ups with 3 repetitive for each set up will be conducted. The experiment will include one control, two set up with different concentration for both chitin and chitosan respectively. C. carpio will be fed with respective diet for a period of two months. Body weight and length of C. carpio will be measured and recorded biweekly. By the end of two months, challenging test will be conducted with A. hydrophila. Mortality rate will be recorded for each set up and immune assay of C. carpio will be conducted. Polymerase Chain Reaction will also be conducted for expression of MX gene. Results recorded for each set up will be compared and the optimum concentration of chitin and chitosan will be determined.
H0: High concentration of Chitin result in no significant difference in immunity of C. carpio
H1: High concentration of Chitin result in significant difference in immunity of C. carpio
H0: Low concentration of Chitin result in no significant difference in immunity of C. carpio
H1: Low concentration of Chitin result in significant difference in immunity of C. carpio
H0: High concentration of Chitosan result in no significant difference in immunity of C. carpio
H1: High concentration of Chitosan result in significant difference in immunity of C. carpio
H0: Low concentration of Chitosan result in no significant difference in immunity of C. carpio
H1: Low concentration of Chitosan result in significant difference in immunity of C. carpio
3. Literature Review
3.1 Aeromonas hydrophila
Aeromonas hydrophila is a heterotrophic, facultative anaerobic Gram-negative rod-shaped bacterium which causes opportunistic infections in fishes, amphibians, mammals and human. It is one of the three species that are commonly referred as motile aeromonads. The genus, Aeromonas were discovered more than a century ago. However, related diseases were only studied a few decades back. Although A. hydrophila is a common pathogen among the cold-blooded animals and is normally transmitted to human via water related injuries, there is a case report that infection is developed in human after a large feline bite. Aeromonas infection may not be deadly in human due to immediate treatment with antibiotic and other drugs. However, it will cause high mortality rate in aquaculture since in acute infection, fishes died before any gross clinical sign are formed. Clinical signs are often present with symptoms caused by primary pathogen, resulting in inaccurate diagnosis. Also, limited drug can be use for treatment due to the laws and regulations for aquaculture.
3.1.1 Clinical Signs
In aquaculture, A. hydrophila can result in various type of infection, acute, chronic and latent. One of the infections commonly caused by A. hydrophila is Motile Aeromonas Septicaemia (MAS).
For acute infection, the most common symptoms are exophthalmia, reddening of the skin, and ascites. The abdomen may become distended and scales may bristle out from the skin. Liver may turn green or pale and kidneys may become swollen and friable. Hepatomegaly may also be observed.
For chronic infection, significant clinical signs are haemorrhage on fins and tail, dermal ulceration lesions, anaemia and swimming abnormalities. Severe necrosis in liver and various haemorrhages in the interstitial tissues of visceral organs may be observed.
As mentioned earlier, A. hydrophila is an opportunistic pathogen, host are commonly infected under high level of stress or weakened immunity. The pathogenesis of A. hydrophila is known to involve multiple determinants, including adhesins, enterotoxins, hemolysin, aerolysin, and proteases. MAS is one of the most common and fatal disease in aquaculture industry. A. hydrophila enters the host body through wounds or sores caused by primary pathogens or diseases. Phagocytes such as neutrophils and monocytes will migrate to the infected area to eliminate A. hydrophila through phagocytosis. At the same time, extracellular trap (ETs) were released by the leucocytes. This mechanism was found to have a antimicrobial effect. However, nucleases produced by bacteria can break down the DNA backbone of ETs, allowing the bacteria to rapidly escape from the infection site and into other parts of the body such as the blood vessels. Due to weakened immunity, phagocytosis activity will decrease along with the release of ETs. Shorter time will be required for increase of bacteraemia level, as bacteraemia level reaches and maintain at a certain concentration, it will result in MAS.
Immunostimulants are classified into two major group, specific and non-specific immunostimulants. Specific immunostimulants acts as antigens for stimulation of immune responses. Non-specific immunostimulants that do not contain antigenic properties enhances the immune response of other antigens. These immunostimulants are then further classified according to their origin and function. Immunostimulants are often used in prevention of disease due to drug resistance in bacteria, allergic reaction to antibiotics, poor effect on viral infection and immunosuppressant effect of antibiotic.