Post on 17-Nov-2014
Microbiology of Fish and Shellfish
FISH/MICROM 490Sage Chaiyapechara
Spring 20055/9/05
Outline
BackgroundMicrobiology of bivalve mollusks
Microorganisms as foodFilter feeders and the ecosystem
Microbiology of fishEggs, skin, gills microfloraIntestinal microflora
DiseasesApplication of bacteria in aquacultureSummary
Microbial Interactions with Macroorganisms
Aquatic environment is relatively rich in microorganismsUp to 105 to 106 cells / mLCilliates, other protists, and
viruses
Macroorganisms in aquatic environmentConstantly 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 bacteriaHumanFish & Shellfish
Increasingly, more focus on normal microflora and their interactions with the host organisms
Microbiology of bivalve mollusks
Microorganisms as foodNatural 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 lab- http://www.mdsg.umd.edu/oysters/anatlab/index.htm
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 expelledPseudofeces = particles which are
not sorted as food and are rejected through the mouth
Affect by temperatureGreatest when water temperature
> 50°F (~10°C)
Labial palps
Visceral mass
Lower intestines
Rectum and anus
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 environmentSuggests 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 phytoplanktonReduce 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/
Newell, 2004
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
Microbiology of Fish
Eggs, skin, gills microfloraIntestinal microflora
Bacteria on mucosal surface (1)
Host-parasite relationshipHost = 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
animalTransient = 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, PseudomonasDead eggs: fluorescent Pseudomonas
Not the cause of dead, but rather attracting to nutrient leaching
Overgrown of bacteria can hamper eggs development
Cahill, 1990; Hansen and Olafsen, 1999
Leucothrix mucor on cod eggs Flavobacterium ovolyticus on halibut eggs
Skin Microflora
Reflect that of surrounding waterMay have from 102 to 104 bacteria/ cm2
Unit of measurement per areaSurface sampled by using a sterile swabMuscle 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/ gThe number is quite low considering its high surface area and being
continual flushed by waterExtensive 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, MicrococcusInfluenced by stages of life, diets, feeding, water
temperature, habitatLarge number when feeding, very few when not feedingOrganic content of the environmentVibrio dominates in seawater, Aeromonas dominates in freshwater
Cahill, 1990; Hansen and Olafsen, 1999
Intestinal microflora (2)
Ringo et al., 2003
Microvilli of the epithelial cells of common wolffish (A. lupus L.)
SEM of the enterocytes in the midgut of Artic charr
Bacteria
Intestinal microflora (3)
Ringo et al., 2003
Endocytosis of bacteria in the hindgut of spotted wolffish fry
TEM of Atlantic salmon gut epithelium
Bacteria
Aquaculture of marine larval fish
More difficult to raise compared to freshwaterSmaller egg size Smaller size at hatchingLonger larval durationHigher mortality rates
Mass mortality often with unknown causeNutrition?Disease?
Little is known about the role of intestinal microorganisms
Fuiman, 2002
Yolk-sac
First feeding
Larvae
Juvenile
Adult
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 environmentMarine larvae needs to “drink” to osmoregulateInfluence 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 complexpH change (lower)O2 tension (more anaerobic)
Receptors for bacteria
Ringo and Birkbeck, 1999; Birkbeck and Verner-Jeffreys, 2002
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
NutritionPolyunsaturated fatty acids, amino
acids and vitaminsExtracellular enzymes: chitinase
Preventing infection from fish pathogensCompetitive attachmentNeutralization of toxinsBacteriocidal activity
Survival and growthBacterial load impact on survival &
digestive organ developmentPresence of certain species influence
survival
Stimulation of the immune systemProvide antigens to trigger
development of immune responses in the gut
Ringo and Birkbeck, 1999; Photo by Mark Tagal
Pre-release China rockfish
Disease
Disease triangle conceptPathogenesis
Types of pathogens
Diseases triangle concept
For a disease to develop:1. Susceptible host2. Pathogens3. 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 hostExposure to pathogensAdherence to skin or mucosal
surfaceInvasion through epithelium
Colonization and growthLocalization (boil, ulcer, etc)Systematic infection
Production of virulence factorsTissue damage via toxins or
invasiveness
Types of pathogens
Obligate pathogensCause disease in healthy
organismsContagious disease
Aeromonas salmonicidaSalmonids and other fishesFurunculosis, skin lesions
Opportunistic pathogensFound in the environmentDo not cause disease unless the
host immune response is suppressed (stress, environmental factor, etc)
Listonella anguillarumFish, mollusks, shrimp, crabsVibriosis
Buller, 2004
Application of bacteria in aquaculture
BiofiltersThe 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 antibioticChange in the composition of microbial population Create more resistant strains of bacteriaTypes of bacteria more important than numbers
Water that has been treated to select for non-opportunistic bacteriaNon-opportunists (K-strategists) is competitive at low substrate
availabilityFiltration with 0.2 m membrane to remove most bacteria and
particulate organic nutrientsSelective 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
ProbioticsProbiotic = 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.
Verschuere et al., 2000
Hmm, yogurt!
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
ReferencesBirkbeck, 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.
Referencesvan 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.
ReferencesThompson, 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.