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.