Microbiology of Fish and Shellfish

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Microbiology of Fish and Shellfish

• FISH/MICROM 490
• Sage Chaiyapechara
• Spring 2005
• 5/9/05

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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

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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

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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

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Microbiology of bivalve mollusks

• Microorganisms as food
• Natural microflora
• Filter feeders and the ecosystem

Hansen and Olafsen, 1999 Maeda, 2002
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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
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Oyster anatomy

• Draw water in over its gills through the beating
  of cilia
• Suspended food (plankton) and particles are
  trapped in the mucus of the gills
• Sort by labial palps and transport 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
• gt 50F (10C)

Labial palps
Visceral mass
Lower intestines
Rectum and anus
Oyster anatomy lab- http//www.mdsg.umd.edu/oyster
s/anatlab/index.htm
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Oyster filtering mechanism lab-
http//www.mdsg.umd.edu/oysters/oysfilt.htm
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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
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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/
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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
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Microbiology of Fish

• Eggs, skin, gills microflora
• Intestinal microflora

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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

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Bacteria on mucosal surface (2)
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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
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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
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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
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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
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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
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Intestinal microflora (3)
Endocytosis of bacteria in the hindgut of spotted
wolffish fry
Bacteria
TEM of Atlantic salmon gut epithelium
Ringo et al., 2003
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Aquaculture of marine larval fish

• More difficult to raise compared to freshwater
• Smaller egg size
• Smaller size at hatching
• Longer larval duration
• Higher mortality rates
• Mass mortality often with unknown cause
• Nutrition?
• Disease?
• Little is known about the role of intestinal
  microorganisms

Yolk-sac
First feeding
Larvae
Juvenile
Adult
Fuiman, 2002
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Fish Anatomy
Larva
Adult
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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-Jeffreys, 2002
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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
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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
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Disease

• Disease triangle concept
• Pathogenesis
• Types of pathogens

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Diseases triangle concept

• For a disease to develop
• Susceptible host
• Pathogens
• Specific environment conditions

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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

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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
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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

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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 mm 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
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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
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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

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Thank you
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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. 1021-41.
• 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 381-26.
• 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 14527-532.
• 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 44175-185
• 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 491891-1898
• Kueh, C.S.W., and K. Chan. 1985. Bacteria in
  bivalve shellfish with special reference to the
  oyster. Journal of Applied Bacteriology 5941-47.

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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/oysclas
  s.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
  227395-415.
• Ringo, E., and T.H. Birkbeck. 1999. Intestinal
  microflora of fish larvae and fry. Aquaculture
  Research 3073-93.
• Skjermo, J., and O. Vadstein. 1999. Techniques
  for microbial control in the intensive rearing of
  marine larvae. Aquaculture 177333-343.

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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
  64655-671.