A STEP BY STEP GUIDE ON DIAGNOSIS OF FRESHWATER FISHES IN NIGERIA

USING MOLECULAR TECHNIQUES AND ITS PCR ANALYSIS: A REVIEW

Jibrin, H., Omotoyo, I.A. Adam, A.A., Muhammad, U. and Ibrahim, K.

ABSTRACT
Pathogens can be detected from asymptomatic fish using molecular diagnostic methods so disease
outbreak could be prevented. Several years ago, molecular techniques have been employed to
diagnose fish diseases. These methods include the polymerase chain reaction (PCR), restriction
enzyme digestion, probe hybridization, in situ hybridization, and microarray. This paper reviews a
step by step guide for carrying out molecular diagnosis of fish disease. However, the application of
molecular methods as a routine tool in a diagnostic laboratory in areas where relevant literatures is
scarce is important for the adoption of these methods.
Keyword: Molecular methods, fish disease, molecular diagnosis, aquatic environment, climate
change.

1. INTRODUCTION
Molecular methods can be used to increase sensitivity and specificity of pathogen detection. These
techniques include polymerase chain reaction (PCR), in-situ hybridization, micro-array among
others. Since molecular methods are more sensitive than the conventional diagnostic techniques,
pathogens can be detected from asymptomatic fish, so outbreak of disease could be prevented,
thus; antibiotic treatment can be reduced [1].
Several years ago, great advances have been made in understanding the molecular biology of fish
pathogens and their hosts, and molecular biology has become a routine tool in search for improved
methods of diagnosis and control of fish diseases. The detection of nucleic acid molecules has
demonstrated its usefulness in detection and diagnosis of fish diseases [1].

1.1 Impact of fish disease to aquaculture

Freshwater fish have been model hosts in the study of the community and evolutionary ecology of
parasites for the last few decades. However, species identification is one of the main challenges in
freshwater fish parasites. These parasites are usually small, soft bodied with few morphological
characters. For instance, tapeworms are obligate internal parasites of vertebrates that display a
wide range of body forms, life history and host associations [1].
In an ideal environmental condition, healthy fish without lesion can carry pathogens capable of
creating an avenue for the spread of diseases in the fish populations. However, disease may
become eminent only when stressful conditions occur. In intensive aquaculture systems, the risk of
stress may increase with a significant proportion of the stock infected. Therefore, it is imperative to
detect pathogen from carrier fish for an effective fish disease control, since most often, the
prevalence of diseases may change depending on factors such as seasons and temperature [2].
Centuries ago, fish has been one of the main foods for humans and is still an important part of the
diet in many countries [3]. One of the advantages of fish as food source to man is because it is
easily digested and has a high nutritional and health benefits [3]. Fishes are important natural
resources to humans world over, most especially in the rural areas [4], where fishes are hunt for
both commercial and subsistence purposes.

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2.0 IMPACT OF CLIMATE CHANGE ON AQUATIC ECOSYSTEMS
Many years ago, there has been a global report of diseases affecting aquatic organisms of different
taxa [5]. Climate change has affected the aquatic ecosystem, thus; making it susceptible to many
anthropogenic disturbances such as pollution, habitat destruction and overfishing. Global warming
has led to increased pathogen development and survival rates, disease transmission and host
susceptibility [6].
However, environmental conditions play a pivotal role not only in the transmission of pathogens,
but also as risk factors for the occurrences of clinical diseases. Unlike mammals that regulate their
internal environments (homeostasis), most fishes are cold blooded (poikilotherms), with little or no
ability to regulate their body temperature [7].
In situations such as this, both the microbes and the host are physiologically tied to the
environment they live in with an optimum temperature range for their survival, and extended
periods beyond the optimal range could result in death [6].
Aquaculture production has dramatically increased; fish consumption is largely dependent on
fisheries [8]. Wild fish plays an important ecological and economic role in the ecosystem as a
major protein source for humans [9].
There are varieties of aquatic pathogens from aquaculture is well documented, there is still lack of
baseline information with regards to pathogenic agents and their prevalence in wild fish population
[10]. Although aquaculture is a fast growing industry for the production of high protein-sources
foods, his growth is accompanied by concerns from both public and private sectors [11], because
fish production is commonly associated with serious environmental impacts such as water
pollution, transmission of pathogens and changes in temperature [12].
2.1 Molecular tools for diagnosing fish disease
The utilization of molecular methods has brought new insights to different biomedical areas.
However, molecular parasitology laboratory specializes in the use of molecular methods for
investigations on the field of host-parasite interactions [13].
Other aspects of the use of molecular methods include the characterization and determination of
species, sub-species and strain of generated mutations during the treatment of anthelminthic
animals [14].
The efficiency of DNA extraction methods for any biological sample is determined through DNA
quality and recovery rate. Consequently, the extraction of DNA with high quality and quantity is a
key step in the genetic analysis [13].
Several DNA extraction methods have been employed in the preparation of DNA from various
organisms, ranging from the isolation of genomic DNA from small amounts of biological materials
such as single worms or blood smears is not always applicable using the traditional DNA isolation
methods that is based on phenol/chloroform/isoamyl alcohol, which is one of the most utilized
methods for DNA extraction in developing countries of the world [13].
2.2 Fish disease diagnosis
Fish disease management and assessment is a major concern to commercial aquaculturists.
However, the ability to identify the presence or absence of a pathogen in fish would be of
significant economic benefits. If the concentration of the infectious organism is determined in the
fish or the water environment, the changes in abundance of these organisms could be monitored as
well. However, developing a system that could access the carrier state of fish accurately within an
area that harbours a disease causing organism would help in developing management programs
[1].

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Several advances have been made in improving the sensitivity and specificity of diagnosis of fish
disease. In molecular methods, a typical DNA is extracted from the sample and probed by DNA
hybridization and analyzed using a restriction fragment length polymorphism (RFLP). Moreover,
DNA is amplified using the polymerase chain reaction (PCR), which employs specific primers for
diagnostic sequences. The polymerase chain reaction (PCR) is hybridized with specific
oligoprobes or non-specific primers used in producing random amplified polymorphic DNA
(RAPD) [15] and [16].
3.0 DETECTION AND LABELING OF NUCLEIC ACIDS
Radioisotopes were once the trend several years ago, but in the safety interest of the researcher,
other methods were adopted. A variety of detection and labeling methods presently can provide a
dynamic system suitable for any application, beginning from dot blots to in situ hybridization.
[17]. these include labeling with biotin or digoyygenin and detection by antibody binding coupled
with fluorescent, chemiluminescent or colorimetric detection methods [18].
4.0 MOLECULAR ANALYSIS
4.1 DNA Extraction
The extraction of DNA will be made possible using the procedures below:
 Pipette 50μl Protease (or proteinase k) into the bottom of a 1.5ml micro-centrifuge tube.
 Add 200μl samples to the micro-centrifuge tube. About 200μl of whole blood, plasma,
serum, buffy coat, or body fluids, or about 5 x 106 lymphocytes in 200μl PBS.
 Add 200μl Lysis Buffer to the sample. Mix by vortexing for about 15 seconds to yield a
homogenous mixture.
 Incubate the mixture at 750C for 10 minutes to allow DNA yield reach a maximum after
lysis for about 10 minutes at 750C.
 Centrifuge the 1.5 ml micro-centrifuge tube briefly to remove drops from the inside of the
lid.
 Add 250μl ethanol (96 – 100%) to the sample, and mix again by pulse-vortexing for about
15 seconds. After mixing, the 1.5 ml micro-centrifuge tube, briefly centrifuged to remove
drops from the inside of the lid.
 Carefully apply the mixture obtained to the Mini spin column (in a 2 ml collection tube)
without wetting the rim.
 Close the cap and centrifuged at 12,000 x g (8000 rpm) for 1 minute.
 Place the Mini spin column in a clean 2 ml collection tube and discard the tube containing
the filtrate.
 Open the Mini spin column carefully and add 500μl de-ionized solution without wetting
the rim.
 Close the cap and centrifuged at full speed 12,000 x g for 1 miunte.
 Place the spin column in a new 2 ml collection tube and discard the old collection tube
containing the filtrate.
 Centrifuged at 14, 000 g for 3 minutes at room temperature to remove the residual ethanol.
 Place the spin column in a clean 1.5 ml micro-centrifuge tube, and discard the collection
tube containing the filtrate.
 Carefully open the Mini spin column and add 200μl Ethanol Buffer.
 Incubate at room temperature (720C) for 1minute and then centrifuged at 14, 000 x g for 1
minute.
4.2 PCR Experiment

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  Place the template DNA in a mixture containing the four DNA nucleotides, pair of primers
flanking the target sequence, Taq DNA polymerase, buffer and water.
 Heat the mixture at 940C to separate (denaturation) the hydrogen bonds holding the two
stands of double-stranded DNA molecules together.
 Cool the mixture down to 50 – 60 0C, and allow the primers to anneal to specific target on
the single stranded DNA molecules.
 Raise the temperature again to 740C to enable the Taq DNA polymerase attach to one end
of each primer and synthesizes new strand of DNA complementary to the template DNA
molecules.
 Raise the temperature back to 940C to denature the double-stranded DNA into one strand of
the original molecule and one new strand of DNA into single strands.
 This will lead to a second cycle of denaturation-annealing-synthesis at the end of which
there will be eight DNA strands.
 Repeat the cycle 25 times to enable the double stranded molecule convert to 50 million new
double-stranded molecules, with each one of them being a copy of the starting molecule
delineated by the annealing sites of the two primers.
 Perform the PCR reaction in a thermo-cycler machine, which is programmable heating
block that cycle between melting, annealing and polymerization temperatures.
4.3 PCR Procedure
 Label the PCR tubes for samples and controls.
 Thaw the PCR reagents and prepared PCR reaction mix.
 Aliquote the reaction mix in an individual PCR reaction tube.
 Add the template i.e. sample/ control in the appropriate labeled tube.
 Add 1μl template to each tube to achieve 25μl total reaction volume.
 Open the PCR machine’s software and edit parameters e.g. run ID, user ID, sample ID’s,
sample volume and cycling conditions according to desired protocol.
 Place the sample tubes in the thermal cycler
 Close the lid and the program will run simultaneously.
 On completing the PCR, remove the tubes from the thermal cycler and proceed for agarose
gel electrophoresis or other downstream application. Otherwise, store the PCR products at -
200C.
4.4 Gel Electrophoresis Procedure
 Weigh 1.5g of agarose and dissolve in 100ml of 0.5X TBE buffer by heating using
microwave oven and swirl flask to mix until clear solution is obtained.
 Dissolve the agarose completely and cool to ~500C.
 Add 5ml of 20,000X RedSafe into the agarose solution and mix thoroughly or use an
alternative method of staining agents such as GelRed.
 Prepare the gel tray using a comb with enough wells for number of PCR reactions and two
wells for the markers.
 Pour the gel solution into the gel tray carefully to avoid bubbles trapping in the gel and
allow cooling for ~30 minutes.
 Carefully remove the comb and rinse immediately with water.
 Place the tray in a tank containing 0.5X TBE buffer just enough to submerge gel.
 Pipette 5μl of 5X orange G loading dye onto a parafilm paper for each PCR reaction.
 Mix 25μl of PCR reaction with 5μl of 5X orange G loading dye (5:1 proportion) on the
parafilm paper and load separately into each well.

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  Once all samples have been loaded, load the first and last empty well on the gel with 10 -12
μl of ladder and cover the lid of the gel tank.
 Connect the leads to their respective sockets (black to black, and red to red) on gel tank to
the power supply.
 Electrophores sampless at 4 – 10V per cm distance between electrodes for the desired
length of time.
 Turn off the power pack before removing the gel tray from the tank and DNA bands will be
visualized on the UV transilluminator in a dark room (chamber).
 Capture the photograph of the gel picture using a gel documentation system.

5.0 CONCLUSION AND RECOMMENDATION

In conclusion, molecular tools are increasingly relevant to fish diseases. However, sequencing the
complete genome of pathogens allows great advances in studying the biology, and improving
diagnosis and as well as control of pathogens. Conversely, using new methods of analyzing
polymorphism in nucleic acids improves specificity, sensitivity and speed of diagnosis and offer
ways of examining the genotype and phenotype of various pathogens.
Molecular methods aids epidemiological studies, as well as identify causes of disease outbreak or
the presence of pathogens. It is therefore recommended that molecular tools can be a routine tool
in the search for improved methods of diagnosis and control of fish pathogens and the
epidemiology of infected fishes. However, in Nigeria, the application of these methods on a
routine basis in diagnostic laboratories is few. It is time to apply these methods in the diagnosis of
diseases in aquaculture.

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