LECTURE NOTES ON DISEASES AND PARASITES IN AQUACULTURE

DISEASES AND PARASITES IN AQUACULTURE

Disease is a serious limiting factor in aquaculture, Caused by pathogens, genetic disorders, physical injuries, nutritional imbalances, pollution etc or combinations of these reasons.
Outbreak of diseases in aquaculture occur due to overcrowding, unbalanced nutrition, reduced oxygen content, increase in organic matter, & changes in pH and temperature.
Diseases of fishes can be classified as
        - Communicable  diseases Infectious(caused by microbes) & contageous (caused by           Parasites)
Non- communicable diseases   caused by  Environmental, Nutritional & Genetic factors or unknown factors
Neoplastic  - Tomours  & cancers.
Among these genetic diseases and neo plastic diseases are very rare in Asian countries.
  Viral diseases:-
Viral diseases are limited to young stock & depend on environmental factors and conditions of fish. Cause tissue damage and rapid death of fish.
Once fish is infected, it develops antibodies and protects the fish from second attack. Recovered fish act as carriers of the virus.
Viral diseases are rare in tropical region and seen mostly in temperate regions.
Viral Haemorrhagic Septicaemia is an example.
Infectious pancreatic necrosis, spring viremia of carp, catfish viral disease are other examples.
Iodine treatment, Vitamin administration, exposure to UV and vaccination are some of the treatment measures against viral diseases.
Bacterial Diseases
1. DROPSY(Bacterial Haemorrhagic Septicaemia).
Common bacterial disease, caused by Pseudomonas fluorescens. Symptoms include extreme enlargement of belly due to fluid accumulation, Scale protrusion and inflammation of intestine.
In India it is reported on Clarias batrachus, C.macrocephalus & Osphronemous gourami.
                        
2) TUBERCULOSIS  ( MYCOBACTERIOSIS).
Caused by Mycobacterium marinum( Marineform cause skin lesions in man), M. fortuitum, & M. salmoniphilum.(Fresh Water).
Sympyoms are retarded growth, Extreme emaciation, (weight loss and thinness by loss of sub cutaneous fat), incomplete gonad development & lesions in the internal organs.
3) COTTON WOOL (COLUMNARIS) DISEASE:-
Caused by Flexibacter columnaris. Greyish white spots appear on the body, especially around mouth.
This disease is Common in young fishes.
4) FIN ROT:-
Caused by Aeronomas hydrophila, A. punctuata,  A.pseudomonas fluorescens.  Affecting both young and adult. It appears as a white line on the margin of the fin. Erosion of tissues start at the extremities of fin and progress towards the base  only the fin ray remains in extreme cases.
Fungal Diseases:-
Fungi attack all stages of fish life.
In India fungal diseases are common among carps.
Saprolegnia cause External mycosis in fishes. Here minute, white hair-like out growths appear on the affected parts followed by ulceration.
Branchiomyces cause Gill rot- common in summer, while ponds rich in organic decaying matter.
Protozoan Diseases:-
1)TRYPANOSOMIASIS:-
Caused by Trypanosoma, a flagellate mostly seen in  Carps & Eels, like Clarias batrachus, Heteropneustes fossilis, Wallago attu.
Affected fish lies in a sleeping posture, floating laterally on water surface, breathing slowly and show very little movement.
Disease is transmitted by aquatic leeches.
2)WHITE SPOT DISEASE (ICHTHYOPHTHIRIASIS):-
Caused by Ichthyphthirius multifilis.
Appearance of white small, white specks all over the body and gills. Each spot represent the encysted parasite

DISEASE BY WORMS:-
       Trematodes, Cestodes, Nematodes, leeches.
Larval stages of worms are more injurious. Cause tissue disruption, Metabolic disturbances, poor gonad development., Growth retardation  and lead to death.
Prarasitic infeststion  reduce the market value.
DISEASE BY CRUSTACEANS:-
Argulus or fish louse  have leaf like body. It attaches to the fish and suck blood- wound caused invite secondary bacterial and fungal infection.
Lernaea or Anchor worm- anterior part is modified like an anchor which is buried deep in to the skin of the fish. It feeds on the tissues and erythrocytes, leads to death or secondary infections.
EPIZOOTIC ULERATIVE SYNDROME(EUS)
Most dangerous disease of fresh water fishes of Asian countries.
Apperaed in Thailand in 1980 followed by Philippines and Indonesia. 1990 reached Srilanka.
Appeared in Kerala & Tamil Nadu in 1991. First appeared in Kerala in July- Aug 1991.
The Channa fish in many water bodies of Kerala disappeared due to this disease.
Appear as grey patches on the body surface- becomes red and ulcerated- spreads to internal organs. At later stages fish stop feeding, swims erratically on surface water- wound on body become deeper - succumb to infection.
Gram negative bacteria, Aeromonas and Pseudomonas were believed to cause EUS. Recent sudies prove sapropythic fungus, Aphanomyces as the causative agent.
Deterioration of environmental factors may enhance the disease outbreak. No effective treatment devised.

LECTURE NOTES ON GENE SEQUENCING

GENE SEQUENCING

 DNA sequencing is a method of  sequencing for determining the order of the nucleotide bases - adenine, guanine, cytosine, and thymine - in a molecule of DNA of an organism. Knowledge  of DNA sequences has become important for basic biological research and in numerous applied fields like  diagnostic service,biotechnology,forensic biology and Taxonomy.

The  basic unit of the genome is a single deoxyribo nucleotide.  It is a complex process.DNA sequencing involves the determination of the order of DNA bases.

Sanger sequencing("Next- Generation“ sequencing)

It is method to find out the nucleotides Sequence of unknown DNA strand. Sanger sequencing has been known as as "Next- Generation“ sequencing methods, especially for large scale genome analyses and for obtaining especially long DNA sequence reads (>500 nucleotides).

This method generally is an In-Vitro synthesis of DNA strand and by using terminators (di-deoxynucleotide) the growing strand terminates at specific site.Upon termination the strands are overlap to got original sequence of unknown DNA Strand.

REQUIREMENTS FOR SANGER SEQUENCING

Single Stranded DNA  template

Primer

DNA polymerase enzyme

Di-Deoxynucleotides:The 3′-OH group necessary for formation of the phosphodiester bond is missing in ddNTPs).Every nucleotide have its specific ddNTP form i.e., ddATP, ddGTP etc

Procedure

1. Denaturation of DNA

2. Primer attachment and extension of bases

3. Termination of Sequence

4. Gel electrophoresis for separation of DNA fragments

The double stranded DNA template is treated with heat so that it becomes single stranded.A short, single-stranded radioactively labeled primer  is added to the end of the DNA templateThe  template DNA and primer are added in four seperate tubes.Then  ddNTPs were added in tubes in the way that single tube contain one type of ddNTP.Extension is start and band of various sizes are formed.The DNA fragments  are separated by electrophoresis.Overlap these sequences to find out sequence of Target DNA.

 

Maxam–Gilbert sequencing

Maxam–Gilbert DNA sequencing is a method of sequencing developed by Allan Maxam and Walter Gilbert in 1976–197.Maxam–Gilbert sequencing was the first widely adopted method for DNA sequencing, and, along with the Sanger dideoxy method.method based on chemical modification of DNA and subsequent cleavage at specific nitrogenous bases.

PRINCIPLE

It involves the purification of the DNA fragment that to be sequenced and labeled with radioactive material.Chemical treatment generates breaks at a specific nitrogenous bases and thus a series of radio labelled fragments is generated. The fragments in the four reactions are arranged side by side in gel electrophoresis for size separation.These fragments are  visualize in X-ray for autoradiography. Procedure

This sequencing utilises  radioactive labeling at one 5′ end of the DNA fragment to be sequenced (gamma-32P).Chemical treatment generates breaks at a small proportion of one or two of the four bases of nucleotide in each of four reactions (G, A+G, C, C+T). For example,

1. the purines (A+G) generated by using formic acid,

2. the guanines and to some extent the adenines are generated  by dimethyl sulfate,

3. the pyrimidines (C+T) generated  by using hydrazine.

4. NaCl add to hydrazine for generating Cytosine.

Each chemicals are then added in separate tube to generate series of labeled fragments .Electrophorosis of the fragments in the four reactions are done  side by side for size separation.To see the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each showing the location of identical radiolabeled DNA molecules.

APPLICATIONS:

By DNA sequencing  one  can understand the function of a specific sequence and the sequence responsible for any disease.

By  comparative DNA sequence study we can detect any mutation.

It is used in DNA fingerprinting.By DNA sequencing the human  genome sequence, Human genome project get completed.

In forensic study DNA sequencing is used to identify particular individual because every individual has unique sequence of his/her DNA. It is particularly useded to identify the criminals by finding some proof from the crime scene in the form of hair, nail, skin or blood samples.

In agriculture DNA sequencing used  in mapping and sequencing of the whole genome of microorganisms has allowed the agriculturists to make them useful for the crops and food plants.

In medical research, DNA sequencing can be used to detect the defective  genes which are associated with some heredity or acquired diseases. Researchers  use different techniques like gene therapy to identify the defected genes and replace them with the healthy ones.