Microbiology of Fish and

Shellfish

FISH/MICROM 490
Sage Chaiyapechara
Spring 2005
5/9/05
 Outline
Background
Microbiology of bivalve mollusks
Microorganisms as food
Filter feeders and the ecosystem
Microbiology of fish
Eggs, skin, gills microflora
Intestinal microflora
Diseases
Application of bacteria in aquaculture
Summary
Microbial Interactions with
Macroorganisms
Aquatic environment is
relatively rich in
microorganisms
Up to 105 to 106 cells / mL
Cilliates, other protists, and
viruses

Macroorganisms in aquatic
environment
Constantly exposed to
microorganisms
 Historical perspectives
Changes during storage
Effects on spoilage
Relationship between
environmental and fish
microflora
Basis for monitoring changes in fish
farms
Disease causing bacteria
Human
Fish & Shellfish

Increasingly, more focus on normal

microflora and their interactions
with the host organisms
Microbiology of bivalve mollusks

Microorganisms as food
Natural microflora
Filter feeders and the ecosystem

Hansen and Olafsen, 1999; Maeda, 2002

 Microorganisms as food
Filter feeders
(Suspension feeders)
Feed on microorganisms that
they filter out of the
environment
Clams, oysters, barnacles,
sponge
Deposit feeders
Feed on microorganisms that
coats the surface of sediments
and soil particles
Worms, fiddler crab

Larval forms of animals may require smaller

microorganisms such as bacteria, while an adult
may prefer larger microorganisms such as
flagellated protists and algae
 Oyster anatomy
Labial palps Draw water in over its gills
through the beating of cilia
Suspended food (plankton) and
Visceral mass particles are trapped in the
mucus of the gills
Sort by labial palps and transport
Lower intestines to the mouth, eaten, digested,
and feces expelled
Pseudofeces = particles which are
not sorted as food and are
rejected through the mouth
Affect by temperature
Greatest when water temperature
> 50°F (~10°C)
Rectum and anus

Oyster anatomy lab- http://www.mdsg.umd.edu/oysters/anatlab/index.htm

Oyster filtering mechanism lab- http://www.mdsg.umd.edu/oysters/oysfilt.htm
Natural microflora of mussels and
oysters
A majority of isolates are gram-negative (68%) and
aerobic (76%) bacteria
Predominant flora: Vibrio, Pseudomonas, Shewanella,
Aeromonas, Acinetobacter, and Flavobacterium
Gram-positive bacteria: Staphylococcus, Bacillus,
Streptococcus
Predominant Vibrio species includes:
V. alginolyticus, V. splendidus, and V. (Listonella) anguillarum*
Not always reflect external environment
Suggests selective process to sequester and maintain certain
species

Kueh and Chan, 1985 ;Hariharan et al., 1995

Filter feeders and the ecosystems
An adult oyster can filter as much as 60 gallon per day

Oysters can filter out sediments and nutrients

(nitrogen) and deposit them on the bottom

“Top-down" grazer control on phytoplankton

Reduce turbidity, increasing the amount of light reaching the
sediment surface
Extending the depth to which ecologically important benthic
plants (seagrasses and benthic microalgae) can grow

Newell, 2004 ;Chesapeake Bay Foundation- http://www.cbf.org/

 Filter feeders bivalves removing inorganic and organic particles from water
column and transferring undigested particulate material to the sediment in the
form of their biodeposits
Newell, 2004
Microbiology of Fish

Eggs, skin, gills microflora

Intestinal microflora
Bacteria on mucosal surface (1)
Host-parasite relationship
Host = an organism which harbors parasite (microorganisms)
Parasite = an organism that lives on or in a second organism
Surfaces such as eggs, skin, gills, and intestinal tract
Mucus layer as an adhesion site and protective layer
Indigenous vs. transient (autochthonous vs.
allochthonous)
Indigenous = able to grow and multiply on the surface of the host
animal
Transient = not able to grow or multiply on the surface of the host
animal; does not persist for a long period of time
Bacteria on mucosal surface (2)

Loose association Adhesion Invasion

 Eggs microflora
Fish embryos secret inorganic and low molecular weight
organic compound, which can diffuse out through the
shells
Attract bacteria utilizing these compounds and colonize
egg surface
Normal healthy eggs flora: Cytophaga, Pseudomonas
Dead eggs: fluorescent Pseudomonas
Not the cause of dead, but rather attracting to nutrient leaching
Overgrown of bacteria can hamper eggs development

Leucothrix mucor on cod eggs Flavobacterium ovolyticus on halibut

eggs
Cahill, 1990; Hansen and Olafsen, 1999
 Skin Microflora
Reflect that of surrounding water
May have from 102 to 104 bacteria/ cm2
Unit of measurement per area
Surface sampled by using a sterile swab
Muscle tissue should be sterile
Gram negative: Pseudomonas, Moraxella, Vibrio,
Flavobacterium, Acinetobacter, Aeromonas
Gram positive: Micrococcus, Bacillus

Cahill, 1990
 Gill Microflora
May contain 102 to 106 bacteria/ g
The number is quite low considering its high surface area and being
continual flushed by water
Extensive colonization of certain types of bacteria (Flavobacterium)
Gram negative: Pseudomonas, Flavobacterium, Vibrio,
Moraxella, Cytophaga
Gram positive: Micrococcus, Bacillus (in warmer water)

Cahill, 1990
 Intestinal microflora (1)
Established at the larval stage
Developed into a persistent flora at the juvenile stage
Population of microorganisms tends to increase along the
length of the GI tract
Largest number of bacteria in the intestines (up to 108
CFU/g)
Gram negative: Pseudomonas, Vibrio, Achromobacter,
Flavobacterium, Corynebacterium, Aeromonas
Gram positive: Bacillus, Micrococcus
Influenced by stages of life, diets, feeding, water
temperature, habitat
Large number when feeding, very few when not feeding
Organic content of the environment
Vibrio dominates in seawater, Aeromonas dominates in freshwater

Cahill, 1990; Hansen and Olafsen, 1999

Intestinal microflora (2)

Microvilli of the epithelial cells

of common wolffish (A.
lupus L.)

Bacteria

SEM of the enterocytes in the

midgut of Artic charr

Ringo et al., 2003

 Intestinal microflora (3)

Endocytosis of bacteria in the

hindgut of spotted wolffish
fry

Bacteria

TEM of Atlantic salmon gut

epithelium

Ringo et al., 2003

Aquaculture of marine larval fish
More difficult to raise compared
Yolk-sac to freshwater
Smaller egg size
Smaller size at hatching
First Longer larval duration
feeding Higher mortality rates
Mass mortality often with
Larvae unknown cause
Nutrition?
Disease?
Juvenile
Little is known about the role of
intestinal microorganisms
Adult

Fuiman, 2002
 Fish Anatomy

Larva

Adult
 Development of the intestinal
microbiology
At the time of hatching, the digestive tract of most fish
species is an undifferentiated straight tube
Prior to first feeding, microbiology reflects that of the
rearing environment
Marine larvae needs to “drink” to osmoregulate
Influence by eggs, live feed, and rearing water

Once feeding begins, microbiology is derived from live feed

ingested rather than water
As the digestive tract becomes more developed, the
intestinal microbiology becomes more stable and more
complex
pH change (lower)
O2 tension (more anaerobic)
Receptors for bacteria

Ringo and Birkbeck, 1999; Birkbeck and Verner-

 Development of the intestinal
microflora (2)
Criteria for testing whether or not microorganism is
indigenous to the intestinal tract of fish:
• Found in healthy individuals
• Colonize early stages and persist throughout life
• Are found in both free-living and hatchery-cultured fish
• Can grow anaerobically
• Are found associated with the epithelial mucosal in the stomach,
small intestine or large intestine

Ringo and Birkbeck, 1999

 Roles of intestinal microflora
Nutrition
Polyunsaturated fatty acids, amino acids
and vitamins
Extracellular enzymes: chitinase

Preventing infection from fish

pathogens
Competitive attachment
Neutralization of toxins
Bacteriocidal activity

Survival and growth

Bacterial load impact on survival &
digestive organ development
Presence of certain species influence
survival
Stimulation of the immune system
Provide antigens to trigger development
of immune responses in the gut

Pre-release China rockfish

Ringo and Birkbeck, 1999; Photo by Mark Tagal

 Disease
Disease triangle concept
Pathogenesis
Types of pathogens
 Diseases triangle concept
For a disease to develop:
1. Susceptible host
2. Pathogens
3. Specific environment conditions

Host Pathogen

Environment
Pathogenesis
Pathogenesis = the origin and
development of a disease
Pathogenicity = the ability of a
parasite to inflict damage on
the host
Entry of the pathogen into the
host
Exposure to pathogens
Adherence to skin or mucosal surface
Invasion through epithelium
Colonization and growth
Localization (boil, ulcer, etc)
Systematic infection
Production of virulence factors
Tissue damage via toxins or
invasiveness
Types of pathogens
Obligate pathogens
Cause disease in healthy organisms
Contagious disease
Aeromonas salmonicida
Salmonids and other fishes
Furunculosis, skin lesions

Opportunistic pathogens
Found in the environment
Do not cause disease unless the
host immune response is
suppressed (stress,
environmental factor, etc)
Listonella anguillarum
Fish, mollusks, shrimp, crabs
Vibriosis

Buller, 2004
 Application of bacteria in
aquaculture
Biofilters
The use of bacteria to remove
ammonia and nitrite- toxic
at high concentration to fish
Nitrosomonas and Nitrobacter sp.
Aerobic process
Microbial matured water
Probiotics
 Microbial matured water
Problems with treatment to completely eliminate
bacteria such as antibiotic
Change in the composition of microbial population
Create more resistant strains of bacteria
Types of bacteria more important than numbers
Water that has been treated to select for non-
opportunistic bacteria
Non-opportunists (K-strategists) is competitive at low substrate
availability
Filtration with 0.2 µ m membrane to remove most bacteria and
particulate organic nutrients
Selective recolonization of these non-opportunists in biofilters
help controlled microbial community in water
Increase survival, faster growth rate, higher intestinal
bacteria at first feeding

Skjermo and Vadstein, 1999

 Probiotics
Probiotic = a live microbial feed supplement which
beneficially affects the host by improving its intestinal
balance
A broader definition might also include:
Other forms of addition (submerged bath, add to the rearing water)
Beneficial effects such as preventing pathogens from proliferating,
improving nutritional values of feed, enhancing the host responses
towards disease, improving rearing environment
Interactions other than in the intestinal tract (skin, gills)
Can be used for fish (all life stages), crustaceans, bivalve
mollusks, live food (rotifers, Artemia, and algae)
Vibrio sp., Streptococcus lactis, Lactobacillus,
Carnobacterium, Pseudomonas fluorescens, Bacillus sp.
Hmm,
yogurt!

Verschuere et al., 2000

 Summary

Diverse population of microorganisms associated with

fish + shellfish
“Association of marine archaea with the digestive tracts of two
marine fish species”- Maarel et al., 1998
“Carnobacterium inhibes sp. nov., isolated from the intestine of
Atlantic salmon (Salmo salar)”- Joborn et al., 1999
“Phylogenetic analysis of intestinal microflora indicates a novel
Mycoplasma phylotype in farmed and wild salman”- Holben et
al., 2002
“Vibrio tastmaniensis sp. nov., isolated from Atlantic salmon
(Salmo salar L.)”- Thompson et al., 2003

Several types of interactions between microorganisms

and fish + shellfish
Thank you
 References
Birkbeck, T.H., and D.W. Verner-Jeffreys. 2002. Development of the intestinal
microflora in early life stages of flatfish, p. In C. S. Lee and P. O'Bryen (ed.),
Microbial Approaches to Aquatic Nutrition within Environmentally Sound
Aquaculture Production Systems. The World Aquaculture Society, Baton
Roughe, Louisiana.
Cahill, M.M. 1990. Bacterial flora of fishes: A review. Microb. Ecol. 10:21-41.
Fuiman, L.A., and R.G. Werner. 2002. Fishery science: the unique contributions of
early life stages. Blackwell Science, Oxford UK; Malden MA.
Hansen, G.H., and J.A. Olafsen. 1999. Bacterial interactions in early life stages of
marine cold water fish. Microbial Ecology 38:1-26.
Hariharan, H., J.S. Giles, H.S. B., G. Arsenault, N. McNair, and D.J. Rainnie. 1995.
Bacteriological studies on mussels and oysters from six river systems in
Prince Edward island, Canada. Journal of Shellfish Research 14:527-532.
Holben, W.E., P.Williams, L.K. Sarkilahti, and J.H.A. Apajalahti. 2002. Phylogenetic
analysis of intestinal microflora indicates a novel Mycoplasma phylotype in
farmed and wild salmon. Microbial Ecology 44:175-185
Joborn A., M. Dorsch, J.C. Olsson, A. Westerdahl, and S. Kjelleberg. 1999.
Carnobacteria inhibens sp. nov. isolated from the intestine of Atlantic salmon
(Salmo salar) International Journal of Systematic Bacteriology 49:1891-1898
Kueh, C.S.W., and K. Chan. 1985. Bacteria in bivalve shellfish with special
reference to the oyster. Journal of Applied Bacteriology 59:41-47.
 References
van der Maarel, M.J.E.C, R.R.E. Artz, R. Haanstra, and L. J. Forney. 1998.
Association of marine archaea with the digestive tracts of two marine fish
species. Applied and Environmental Microbiology 64: 2894-2898
Maeda, M. 2002. Microbial Communities and Their Use in Aquaculture, p. 61-
78. In C. S. Lee and P. O'Bryen (ed.), Microbial Approaches to Aquatic
Nutrition within Environmentally Sound Aquaculture Production Systems.
The World Aquaculture Society, Baton Rough, Louisiana.
Maryland Sea Grant. 2004. Oyster in the classroom.
http://www.mdsg.umd.edu/oysters/oysclass.htm
Newell, R. I. 2004. Ecosystem influences of natural and cultivated populations
of suspension feeding bivalve molluscs: a review. Journal of Shellfish
Research 23: 51-61
Ringo, E., G.J. Olsen, T.M. Mayhew, and R. Myklebust. 2003. Electron
microscopy of the intestinal microflora of fish. Aquaculture 227:395-415.
Ringo, E., and T.H. Birkbeck. 1999. Intestinal microflora of fish larvae and fry.
Aquaculture Research 30:73-93.
Skjermo, J., and O. Vadstein. 1999. Techniques for microbial control in the
intensive rearing of marine larvae. Aquaculture 177:333-343.
 References
Thompson, F.L., C.C. Thompson, and J. Swings. 2003. Vibrio tasmaniensis sp.
nov. isolated from Atlantic salmon (Salmo salar L.). Systematic and Applied
Microbiology 26: 65-69
Verschuere, L., G. Rombaut, P. Sorgeloos, and W. Verstraete. 2000. Probiotic
bacteria as biological control agents in aquaculture. Microbiology and
Molecular Biology Reviews 64:655-671.