Isolation and Identification of Fish Pathogens

Importance of Fish:

• Marine sources provide about 20% of the animal protein eaten by humans.

• 60% of fish consumption is by the developing world.

• The advantage of fish as food is as a result of its easy digestibility & high nutritional
value.

• The fishing enterprise employ some 200 million people worldwide.

Fish pathogens & diseases:

1. Viral infections

2. Bacterial infections - which cause diseases like fin rot and tail rot ,such as
Pseudomonas fluorescens leading to fin rot and fish dropsy

3. Fungal infections -woolly or cottony patches on the surface of fish, and gill rot
causing asphyxia

4. Water mould infections, such as Saprolegnia sp .

5. Parasitic

 Ectoparasites – Those that occur outside the fish body for example those
that cause Black spot, white spot, fish louse and Nematode.

 Endoparasites – Those that get into the body of the fish

like Contraceacum, and Ligula intestinalis

6. Metazoan parasites, such as copepods

7. Unicellular parasites, such as Ichthyophthirius multifiliis leading to ich

8. Certain parasites like Helminths.

Procedure for Examination of Fish:

Necropsy Procedure:

A necropsy is an examination of an animal body after death to determine cause of death and
changes produced by disease.
Reasons for Necropsy:

• Determine cause of death

• Determine accuracy of clinical diagnosis

• Evaluate effects of therapy

• In herds, 1 or more animals may be sacrificed to determine the cause of disease or

toxicity

• Routine in pharmaceutical studies

Necropsy procedure:

1. External Examination

2. Internal Examination

3. Bacterial infections

External Examination:

Sampling from eyes, Opercula, Gills, Fins, Skin surface

Examine immediately by microscope

Internal Examination:

Sampling internal organs and musculature

Examine immediately by microscope

Bacterial infections:

Collection of samples taken immediately after the fish is opened

Streaking in labeled plate

Incubation
 Colonies shape and pigmentation are examined

The bacteria from fish only become pathogens when fish are:

 Physiologically unbalanced,
 Nutritionally deficient, or
 Other stressors, i.e., poor water quality, overstocking etc.

Isolation of Bacteria:

OIE (Oficina Internacional de Epizootias) suggested method for the isolation of the bacterial
pathogens is:

Collection and dissection of specimens

Transfer of affected tissue (skin lesions/muscle) in a test tube

Homogenization

Spreading over the nutrient agar

Incubation at 30±10º C for 24 hours

Observation of bacterial growth

Isolation of pure colonies

Storage of pure cultures at -200C

Nonselective Isolation:

The Purpose of nonselective medium is the primary isolation of variety of organisms, including
some fastidious ones. The nonselective isolation of bacteria is commonly conducted by enriched
media such as Blood agar.
Selective Isolation of Disease Causing and Fish Spoilage Microflora:

In microbiology, selective isolation refers to the separation of a particular group of microbes.

Media that are involved in these types of isolation are known as selective media.

Selective Media:

These media are used to select (isolate) specific groups of bacteria. They incorporate chemical
substances that inhibit the growth of one type of bacteria while permitting growth of another,
thus facilitating bacterial isolation.

In case of isolating fish pathogen, several types of selective media are available.The most
common disease causing and fish spoilage microflora are:

• Mycobacterium spp.

• Streptococcus spp.

• Vibrio spp.

• Aeromonas spp.

• Salmonella spp. Etc.

Group of microorganism Selective media

Mycobacterium spp. Lowenstein-jensen media

Streptococcus spp. Streptococcal selective agar

Vibrio spp. Thiosulphate citrate bile sucrose (TCBS)

Aeromonus spp. Aeromonus selective agar (BSIBG)

Salmonella spp. Xylose lysine deoxycholate agar(XLD)

Specific Spoilage Organism (SSO)

Properties:

• Exogenous microbiota.
• Grows faster than the rest of microorganisms.
• Dominant spoilage microbiota.
• Spoilage potential (qualitative ability to produce off-odors).
• Spoilage activity (quantitative ability to produce metabolites).
Factors Affecting the Selection of SSOs:

• Initial microbiota composition in fish.

• Fish origination & storage condition.
• Preservation hurdles applied.
• Temperature.
• Atmospheric condition during storage & distribution.

Identification of Specific Spoilage Organism (SSO):

1. Culture based methodologies

2. Culture – independent methodologies

Culture Based Methodologies:

Various phenotypic tests can be done. Such as,

• Gram reaction

• Oxidase test

• Hugh & Leifson reaction

• Production of acid and/or gas from glucose

• Motility

• Sensitivity to various compounds

For example, Gram et al. (1987) isolated “black” colonies on Iron Agar (IA) with the ability to
reduce TMAO (Trimethylamine oxide) to TMA and produce H2S, from spoiled fish at 0°C and
identified as Alteromonas putrefaciens.

SSOs identified by culture-based methodologies

• Pseudomonas sp.
• Vibrio sp.
• Aeromonas sp.
• Achromobacter sp.
• Flavobacterium sp.
• Coryneforms sp.

Culture- Independent Methodologies: It involves use of phylogenetic 16s RNA gene.

 Extraction of DNA from the samples

PCR amplification of the nucleotide sequence of 16s RNA gene

Analysis of 16sRNA gene fragments from the mixed amplification products