Pr�prPRINCIPLES OF DIAGNOSIS OF DISEASE IN AQUACULTURE

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Dr. C.V.NARASIMHA MURTHY

ASSOCIATE PROFESSOR (CONTRACT)

V.S.U.P.G.CENTRE, KAVALI

Diagnosis of Aquaculture Diseases

Disease means…………deviation from normal condition either behavioral, structure or physiological state

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You must know normal condition to detect abnormal one……

………….easily to be done by continuous observation

The aim of diagnosis process to determine the accurate cause of the disease the select the proper treatment

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The owner should be advised to bring both the fish (live and freshly dead ) and a water sample in separate clean containers.

Fish's Well,.being

Disease Pote tial

Environmental

Stress

Presence of Pathogens

Steps of diagnosis

• Case history

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• Clinical signs

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• PM exam

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• Lab exam (Parasitological, Bacterial, Mycological, Virological and water quality)

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• Histopathological exam

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• Molecular methods to detect and characterize the agent’s nucleic acid

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• Biological test

I – Case History

1)Fish

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2)Environment

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1. Rearing unit

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• Water

1)Fish

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• Mortality

1. Recent rates

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• Time

….early morning or sudden

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3. Species involved

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• Size and age of dead fish

Chronic or parasitic especially when affect some species

Water problem O2 or toxic

Has considerable morbidity

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All run facilities

b)Sudden change in routine management

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b) Usual routine for feeding as well Change in diet quantity, quality or forms. (fry, fingerlings or broad stock). As well source and protein percentages.

c) prior treatments, including medications, have been given.

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• Abnormal behaviour or function of fish

A) Rearing unit

2) Environment

• Type, size and location of the

facilit

ies

2. System of fish management

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• Stocking rate /stocking time

4. Depth of water……….

5. Presence of aquatic plants or vegetations…O₂

6. Presence of natural enemies

7. Habitat utilization …weeks ???

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• The surrounding of rearing unite

…human habituations, neighbor infected farm

B) Water

1) Water source: sewages, underground water or surface water.

2. Water exchange rates

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• Physical examination of water

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• color and transparency… algae…

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• turbidity

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• smell…..amonia…..organic matter

4) Chemical examination of water

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-Dis O2

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• Ammonia, nitrite,

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• pH………..acidic……Branchyomycosis

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• Hardness……….soft water……Dee disease

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• heavy metals

II-Clinical sign

1- Behavioural Examination General Exploration

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• If a client submits live fish to the clinic, aeration should be immediately placed into the container that holds the fish.

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• (Examination of fish in water)

1- swimming and movement

2- escape reflex 3- sick fish easily caught 4- position of fish….TB

• flashing or itching (rubbing against objects suggesting skin problems)

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• piping (staying near the air - water interface to obtain more oxygen, suggesting gill problems)

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• Sick fish may exhibit other behavioral signs, including staying near the surface of the water

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= respiratory distress

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• Extremely sick fish may be in dorsal or lateral recumbency.

^•scraping the body or holding the fins close to the body ( “ clamped ” ) because of parasite irritation

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^•showing various behavioral abnormalities

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^•Sick fish often congregate together, separating themselves from healthier

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• Different fish species inhabit different parts of the water column (surface, bottom, shoreline, etc.), and this position often changes with sickness.

* Precautions during examinations

1. Fish should be carefully handled
2. Not allow fish to come in contact with hard objects that affect slim
3. Examine fish on moist substances
4. Examine in shade and avoid wind to prevent dryness
5. You examine by naked eye, stereo microscope or magnifing lence.

Clinical Examination

2- External examination:

- small fish can be examined at moist palm, small plat

- large fish put on their right side on moist glass or plastic diaper. Put fish on their right sides and held them by left hands. Save eyes with palm of hands and with other hands support tail.

* Body inspection

1- color of the body

• Sick fish are often abnormally colored, compared with the healthier
• Fish in breeding condition often have more brilliant colors than non breeding fish
• Darkness over whole body…..chronic disease, bad water quality

- blacking of the tail and peduncle region…

Reddening of the body is usually caused by hemorhage, which can result from systemic bacterial or viral infections

Hemorrhagic lesions on body surface or fins…..

- white spots

- Grayish patches……

Fin rot

Parasites or other irritating conditions may also elicit a thickening of the skin, leading to a whitish or bluish skin color. This change might be highly localized (pinpoint to larger foci) or cover nearly the entire body

local tissue irritation/damage, such as parasite feeding,

chronic wounds, or healing wounds, which cause a change in the pigment cell distribution at that site

5- abnormalities in the vertebral column, mandible, nose…

Streptococcus

swollen vent, hemorrhage

8- abnormalities in anus, uorogenital opening or operculum

2- Erosions or ulcers

3- Exophthalmia of the eye……

4- parasite can be seen by naked eye……..

5- Distended abdomen or lost scales

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Fluid filled body cavity- ascite

6. Necrosis- dead tissue within a living animal

9- fecal examination

• Anesthesia often causes involuntary defecation, allowing the collection of a fecal sample
• Fecal material can be obtained by siphoning debris from the bottom of the tank

III.PM exam Next sections in

details

Symptoms and signs of disease

Symptoms and signs of disease

Symptoms and signs of disease

Symptoms and signs of disease

Symptoms and signs of disease

Symptoms and signs of disease

7. Swollen kidney, liver, inflamed internal organs

SIGNS OF DISEASE

Brown gills

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Pale gills

Eroded gills/excess mucu

Dasyrhynchus sp.

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Cestode larvae in jack

IV. Histopathology

Special histological / Cytological techniques

1- Immunohistochemistry (IHC) / Immunofluorescent chemistry (IFC)

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Enables identification of agent or altered cell receptor expression in relation to localization within the body and within the tissue and in relation to alterations in histomorphology. Therefore, it facilitates the understanding of potential pathogenicity.

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3 - Electron microscopy

VI. Laboratory exam

(Parasitological, Bacterial, Mycological, Virological)

Start by killing of the fish by one of the following methods

1. cut spinal cord
2. over dose of anesthesia 3- electric current

4- Catch the fish with dry towel and hit to hard object

Not use chemicals in killing of fish especially in bacteriological examination

VII. Molecular methods

1. Polymerase chain reaction (PCR)

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The PCR reaction is ideal for detecting the presence of bacterial, viral, parasitic (protozoal or metazoal), or fungal pathogens. The principle of detection is to attempt PCR with primers that are complementary to a part of the genome of the pathogen, but not of the host.

• Conventional PCR protocols are for the detection and identification of DNA sequences
• Real-time Taqman® PCR is a variation of conventional PCR. This type of PCR can be used to detect and quantify the load of an infectious agent in clinical samples. It can detect RNA or DNA sequences.
• reverse transcriptase PCR (rtPCR) protocols are for the detection and identification of RNA sequences (RNA viruses, bacteria).

1- DNA or RNA will be extracted from the samples 2- PCR reaction mix contains specific primers

3. replication in thermal cycler
4. The resulting PCR mix will be analyzed using electrophoresis on a gel, to see if a band of the expected size

General lines

2. Restriction fragment length

polymorphism (RFLP)/DNA fingerprinting

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• Used to identify a change in the genetic sequence. Can be used to trace inheritance patterns, identify specific mutations, and identify bacterial strains

3. Microarray analysis

Used to detect gene expression. It assesses the expression or activity of a specific gene of an organism by quantifying the amount of mRNA the gene produces. not commonly for the detection of infectious agents.

4. In-situ hybridization (ISH)

• Detects presence and location of nucleic acids within lesions in order to establish a link between infection and disease.
• Based on identified labled nucleic acid prob on histological sections prepared by modified method.

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Stress may be a result of:

1. Poor water quality

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• Poor nutrition

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4) Handling

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What causes fish to get sick?

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Fish are generally more vulnerable to disease outbreaks during summer months because:

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• Dissolved oxygen

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• Disease organisms are more active at warm temperatures

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What causes fish to get sick?

Behavioral:

1) Slowing or Cessation of Feeding 2)

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3) Abnormal Distribution

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5) Lethargic Swimming

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Symptoms and signs of disease

Physical Symptoms and Signs

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I. Gross External Signs:

1. Production of Excess Mucus
3. Eroded Fins, Gills, and/or Sores on the Body
5. Swelling or Cysts on the Body, Head, or Fins
7. Hemorrhages on Body Surface

Symptoms and signs of disease

Physical Symptoms and Signs

II. Gross Internal Signs:

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1. Discoloration

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• Lesions

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• Changes in texture of Organs, Tissues, and Musculature

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Symptoms and signs of disease

1) Mortality due to

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3) Mortality due to

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5) Mortality due to

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Mortality Patterns

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Three major patterns:

1 12 24

Time (hrs)

Mortality(# fish)

A.

1 12 24

Time (days)

Mortality(# fish)

B.

1 12 24

Time (weeks)

Mortality(# fish) C.