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Practical 3

The document outlines proper techniques for collecting samples from live and moribund fish and shrimp, detailing both lethal and non-lethal methods. It specifies the types of tissues and organs to be sampled for various viral detections, as well as preservation methods for histopathology and molecular diagnosis. Emphasis is placed on the importance of quick handling and appropriate fixatives to ensure sample integrity.

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chinna rajesh
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4 views2 pages

Practical 3

The document outlines proper techniques for collecting samples from live and moribund fish and shrimp, detailing both lethal and non-lethal methods. It specifies the types of tissues and organs to be sampled for various viral detections, as well as preservation methods for histopathology and molecular diagnosis. Emphasis is placed on the importance of quick handling and appropriate fixatives to ensure sample integrity.

Uploaded by

chinna rajesh
Copyright
© All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd

3.

Sample collection and handling: Proper techniques for collecting samples from live and
moribund fish, including external swabs and tissue biopsies.

1 Sampling of shrimp Sampling can be either lethal or non- lethal. Non-lethal sampling is usually
carried out with broodstocks or cultured adult shrimp where a piece of pleopod is cut and used
for PCR without sacrificing the animal. For lethal sampling, entire animal (in case of larvae) or any
tissue material can be collected based on the type of viral pathogen needed for detection.
Different parts those are usually collected for nucleic acid extraction include hemolymph, gill,
muscle, pleopod, lymphoid organ, hepatopancreas, eye stalk and faecal matter. While moribund
shrimp are usually preferred to detect the actual disease status, samples can also be collected
from healthy shrimp to find out whether a particular virus is present or not. The collected samples
may be transported in 95 % ethyl alcohol. If the samples are intended for detection of RNA
viruses, then it can be preserved in any RNA preservation media (e.g. RNA later). For PCR analysis,
the sample to be transported in frozen or chilled condition preferably in dry ice.

For example: for WSSV and IHHNV, gill can be taken as a sample. If IMNV has to be detected,
telson is preferred as a sample. For HPV, MBV, AHPND and EHP, hepatopancreas, stomach, midgut
etc. are taken as samples. Lymphoid organ is a preferred sample for many of the shrimp viruses.

The sample for histopathology: For histopathological analysis, the moribund shrimp should be
selected. The shrimp should have preferable the clinical symptom and is about to die. Davidson’s
fixative is the most suitable media for preservation of shrimp for histopathology. The shrimp
should be injected with Davidson’s fixative at the rate of 10% of the bodyweight. If the weight of
the shrimp is 20 g, t hen 2 ml of Davidson’s fixative is required.

The sample for molecular diagnosis: For molecular diagnosis, dissected tissues should be fixed
in 95% ethyl alcohol for detection of DNA viruses and in RNA later for detection of RNA viruses.
Shrimp larval samples can be fixed in these fixatives whereas bigger animals have to be fixed in a
proper manner.

2. Sampling for finfish

In general, the skin and gills should be sampled first, before being dried or damaged by handling.
Then, the visceral organs are sampled. Subsequently, the kidney and heart are sampled. Lastly,
the brain is sampled, since the brain is covered with a cranium and it often takes time to sample
the organ. Agitate the fixative briefly, at each time you put a piece of tissue into the fixative. The
overall sampling should be carried out as quickly as possible.

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