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Microbiology of Fish and Shellfish

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Title: Microbiology of Fish and Shellfish


1
Microbiology of Fish and Shellfish
  • FISH/MICROM 490
  • Sage Chaiyapechara
  • Spring 2005
  • 5/9/05

2
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

3
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

4
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

5
Microbiology of bivalve mollusks
  • Microorganisms as food
  • Natural microflora
  • Filter feeders and the ecosystem

Hansen and Olafsen, 1999 Maeda, 2002
6
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
7
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
8
Oyster filtering mechanism lab-
http//www.mdsg.umd.edu/oysters/oysfilt.htm
9
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
10
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/
11
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
12
Microbiology of Fish
  • Eggs, skin, gills microflora
  • Intestinal microflora

13
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

14
Bacteria on mucosal surface (2)
15
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
16
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
17
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
18
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
19
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
20
Intestinal microflora (3)
Endocytosis of bacteria in the hindgut of spotted
wolffish fry
Bacteria
TEM of Atlantic salmon gut epithelium
Ringo et al., 2003
21
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
22
Fish Anatomy
Larva
Adult
23
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
24
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
25
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
26
Disease
  • Disease triangle concept
  • Pathogenesis
  • Types of pathogens

27
Diseases triangle concept
  • For a disease to develop
  • Susceptible host
  • Pathogens
  • Specific environment conditions

28
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

29
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
30
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

31
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
32
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
33
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

34
Thank you
35
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.

36
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.

37
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.
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