Microbiology of Fish and Shellfish

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

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

Deposit feeders
Feed on microorganisms that coats the surface of sediments and soil particles

Clams, oysters, barnacles, sponge

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

Visceral mass

Lower intestines

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
Rectum and anus
Greatest when water temperature > 50°F (~10°C)

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



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.)


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


TEM of Atlantic salmon gut epithelium

Ringo et al., 2003

Aquaculture of marine larval fish
Yolk-sac First feeding Larvae Juvenile Adult 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

Fuiman, 2002

Fish Anatomy


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
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 triangle concept Pathogenesis Types of pathogens

Diseases triangle concept
For a disease to develop: 1. Susceptible host 2. Pathogens 3. Specific environment conditions




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
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 nonopportunistic 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

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

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

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.

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. 6178. 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.

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.

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