Download (A.) salmonicida

Bacteria in aquaculture - fish
Infectious diseases
Henning Sørum
Overview of bacterial pathogens
•
•
•
•
•
Aeromonads
Vibrios
Enterobacteria
Pseudomonads
Flavobacterium, Flexibacter,
miscellaneous bacteria
• Gram-positive bacteria
Section 5
Facultative anaerobic gramnegative rods
Divided in 3 families:
• Fam. Enterobacteriaceae
• Fam. Vibrionaceae
• Fam. Pasteurellaceae
Family Vibrionaceae
• Divided in 4 genera:
Genus Vibrio
Genus Listonella
Genus Aliivibrio
Genus Moritella
Genus Aeromonas
Genus Plesiomonas
Genus Aeromonas
Genus Aeromonas
Divided into 18 species, ex.:
A. salmonicida
A. hydrophila
A. caviae
A. media
A. sobria
A. trota
A. veronii
Relevant diseases caused by
bacteria in Section 5
Genus Aeromonas
Bacterium
A. hydrophila
A. salmonicida ss salmonicida
A. salmonicida ss achromogenes
Disease
Sepsis in fish, food poisoning
Furunculosis in salmonids
Atypical furunculosis in fish
Aeromonads
• Vibrionaceae Véron 1965, familia.
Type genus: ¤ Vibrio Pacini 1854 (Approved Lists 1980).
• Proposal still not accepted:
• Aeromonadaceae Colwell et al. 1986, fam. nov.
• Type genus: ¤ Aeromonas Stanier 1943 (Approved Lists 1980).
• Etymology: N.L. fem. n. Aeromonas, type genus of the family; suff. aceae, ending to denote a family; N.L. fem. pl. n. Aeromonadaceae, the
Aeromonas family.
•
Reference: COLWELL (R.R.), MACDONELL (M.T.) and DE LEY (J.): Proposal to
recognize the family Aeromonadaceae fam. nov. Int. J. Syst. Bacteriol., 1986, 36, 473-477.
Genus Aeromonas
• Type species: ¤ Aeromonas hydrophila (Chester 1901) Stanier 1943
(Approved Lists 1980).
References: SKERMAN (V.B.D.), McGOWAN (V.) and SNEATH
(P.H.A.) (editors): Approved Lists of Bacterial Names. Int. J. Syst.
Bacteriol., 1980, 30, 225-420. [STANIER (R.Y.): A note on the
taxonomy of Proteus hydrophilus. Journal of Bacteriology, 1943, 46,
213-214.]
• Species: 20
• Subspecies: 12
• Species within genus primarily connected to fresh water environments.
• Cause diseases in warm-blooded animals, diarrhea and in reptiles and fish
• Motile Aeromonas; A. hydrophila group
• Non-motile Aeromonas; A. salmonicda group
Aeromonas hydrophila
• Synonyms: "Bacillus hydrophilus fuscus" Sanarelli 1871,
"Bacillus hydrophilus " Chester 1901, "Proteus hydrophilus"
(Chester 1901) Bergey et al. 1923, "Bacterium hydrophilum"
(Chester 1901) Weldin and Levine 1923, "Pseudomonas
hydrophila" (Chester 1901) Breed et al. 1948.
• Gram-negative, straight, motile rod, 0.8 – 1.0 x 1.0 – 3.5 mm in
size, fermentative, motile with single polar flagellum, growing
well at 370C
• Isolated from kidney of diseased fish on standard media, with
incubation of 30oC for 1 day or 20oC for 2 days, round,
convex, shiny colonies of 2-3 mm
Aeromonas hydrophila
• Disease:
Haemorrhagic septicaemia; small surface lesions,
bleedings in the gills, ulcers, abscesses, exophtalmia,
abdominal distension
• Tail and fin rot; often in mixed culture with other bacteria
when cultured from wounds
• Occurrence: Global in fish farming in fresh water
systems including ornamental fish
• Laboratory diagnosis:
hemolysis, microscopy, susceptibility to O/129, motility,
oxydase, growth temperature, protease, sugar
fermentation, serology often cause cross-reactivity if
polyclonal
Aeromonas hydrophila
Aeromonas hydrophila
infection in rainbow trout with
skin wound
(photo courtesy of D. Bruno)
Aeromonas hydrophila
• Virulence factors:
Exotoxins as hemolysin, protease, elastase
Endotoxins contribute to pathogenesis
• Control of disease:
• Antibacterial drugs have been commonly used resulting
in widespread drug resistance in isolates from farmed
fish including in particular ornamental fish
• Vaccines have not been developed for a useful
commercial utilization but the potential for vaccine
protection is promising
Aeromonas hydrophila
•
Control and treatment: Environmental
improvement, including reduction of
pollutant, regularly drying and
disinfection, avoidance of
overcrowding, removal of dead fish.
Antibiotic and chemotherapeutic
measures are often used.
Aeromonas salmonicida
• Aeromonas salmonicida (Lehmann and Neumann 1896) Griffin et al.
1953, species.
Synonyms: "Bacterium salmonicida" Lehmann and Neumann 1896,
"Bacillus salmonicida" (Lehmann and Neumann 1896) Kruse 1896,
"Proteus salmonicida" (Lehmann and Neumann 1896) Pribram
1933.
[1 LEHMANN (K.B.) and NEUMANN (R.): Atlas und Grundriss der
Bakteriologie und Lehrbuch der speziellen bakteriologischen
Diagnostik, 1st ed., J.F. Lehmann, München, 1896. 2 GRIFFIN
(P.J.), SNIESZKO (S.F.) and FRIDDLE (S.B.): A more
comprehensive description of Bacterium salmonicida. Transactions
of the American Fisheries Society, 1953, 82, 129-138.]
• The most intensively studied fish pathogen
• Divided in subspecies
Aeromonas salmonicida ss salmonicida
Aeromonas salmonicida ss achromogenes
Aeromonas salmonicida ss masoucida
Aeromonas salmonicida ss smithia
Aeromonas salmonicida ss pectinolytica (polluted water)
Subspecies salmonicida
• Causes the disease furunculosis in salmonid fish
• Before and after 1900 it became a problem in
wild stocks of Great Britain and Central Europe
rivers
• In North America similar disease occurred but a
few years later.
• Fish has been transferred between the two
continents.
• Today the disease occurs globally in cold or
temperate waters also in marine waters with
farming of salmonids.
Subspecies salmonicida
• Diagnosis
• Gram-negative, straight, non-motile rod, 0.8 – 1.3 x 1.3 –
2.0 mm in size, fermentative, growing well at 200C
• Isolated from kidney of diseased fish on standard media,
with incubation of 20oC for 2 days or 15oC for 3-7 days,
hemolytic, round, convex, shiny colonies of 1-2 mm,
production of brown water soluble pigment after 2 or
more days
• Cells auto-aggregate in salt solution because of
production of a proteinaceous layer (A-layer) on the
surface of the cells.
• After sub-cultivation the A-layer may be lost irreversibly
because of a mutation in the gene, particularly after
cultivation above 22oC
Subspecies salmonicida
• Diagnosis, cont’d
• Cultures are fermentative, oxydase positive,
vibriostatic agent (O/129) resistant
• Commercially available monoclonal antibodies
may be useful in diagnosis, but do not separate
between typical and atypical isolates
• Strong genetic homology within the subspecies,
making a clonal appearance; while moderate but
clear genetic relation to atypical isolates
Oxydase - test
• This is a biochemical test to detect production of
the enzyme cytochrome oxydase. Cytochrome
oxydase is an enzyme (heme protein) that
participate in oxydative phosphorylation in the
respiratory chain.
• The enzyme exist in bacteria that can use
oxygen as the final electron acceptor in the
energy metabolism
Oxydase - test
• We use strips that are impregnated with a
substrate (tetramethyl-p-phenylenediamine
dihydrochloride) that is oxydated by cytochrome
oxydase to a blue violet/purple coloured
compound.
Oxydase negative
Escherichia coli
Oxydase positive
Pseudomonas aeruginosa
TYA (Trypticase Yeast Extract
Agar)
• Pepton (from caseine)
• Yeast extract
• Agar
Aeromonas salmonicida produce a brown
black, water soluble pigment on this
medium with the amino acid tryptophan
as source
Atypical A. salmonicida
• Similar to typical isolates except from
• less and slower production of water soluble
pigments
• Slower growth
• Growth at more differentiated temperatures
• Large heterogeneicity among strains in
biochemical reactions and virulence
• Clear genetic homology with other typical
isolates and other atypical isolates both on
chromosomal and on plasmid level.
Virulence factors A. salmonicida
• A-layer, a proteinaceous array of the
surface, protecting the cell against the
immune system
• Hemolysin, a cytolysin
• Proteinase, digesting proteins from the
host as part of the pathogenesis
Vaccination against furunculosis
• Vaccines containing typical isolates producing A-layer
and extracellular proteins results in 60 – 70 % protection
in salmonids with regular adjuvants
• Vaccination already in the 1940’s
• Adding mineral oil to the vaccine increases the
protection to 95% and prolongs the effect for the whole
production cycle
• Injecting mineral oil alone triggers the immune response
to protect the salmonids against furunculosis
• Before effective vaccines, drug resistance developed
dramatically in Northern Europe, Japan and North
America
Aeromonas salmonicida
• Host specificity
• Subspecies salmonicida produce disease in
salmonids
• Atlantic salmon is particularly susceptible
compared to rainbow trout
• Atypical isolates may produce disease in various
species also in marine waters
• Variation in pathogenicity between atypical
isolates is considerable different compared to
the stable virulence in typical isolates
Aeromonas salmonicida
– disease, contagiosity
• Furunculosis,
• typically abscesses an wounds in the muscles
both on the surface and in the depth
• Healthy carriers are the main reservoar of
bacteria in both wild and farmed populations,
contributing to an endemic situation
• Atypical isolates of A. salmonicida seem to be
ubiquituous and remain a potential reservoar of
disease to fish farming
• Atypical isolates seem to be more often drug
resistant than typical isolates
Microcolony of atypical Aeromonas salmonicida in heart muscle of salmon
Photo Trygve Poppe
Old Genus Vibrio
Genus Vibrio
V. cholerae
V. parahaemolyticus
V. anguillarum (Listonella anguillaria)
V. salmonicida (Aliivibrio salmonicida)
V. viscosus (Moritella viscosa)
V. wodanis (Aliivibrio wodanis)
V. vulnificus
Genus Vibrio
• Vibrio cholera, Vibrio parahaemolyticus, Vibrio
anguillarum and Vibrio salmonicida have importance in
human- and veterinary medicine.
Features
• Gram-negative, straight or bent rods. Motile with polar
flagella. Facultative anaerobic. Grow easily on plain
media, but some species need extra salt in the growth
medium. Optimal temperature for growth is for several
species lower than 30°C. Susceptible to pteridin
(O/129, vibriostatisk agent).
Epidemiology
• Common in seawater
Relevant diseases caused by
bacteria in Section 5
Genus Vibrio
Bacterium
V. cholerae
V. parahaemolyticus
V. anguillarum
V. (A.) salmonicida
M. viscosa (V. viscosus)
Disease
Cholerae (human)
Enteritis (human)
Vibriosis (fish)
Cold water vibriosis
Winter ulcer (fish)
Differensiation between
species within genus Vibrio
• To separate between species within genus
Vibrio the ALO-test is used. By this test
produktion of enzymes that degrade the amino
acids arginine, lysine and ornithine is detected.
These enzymes are decarboxylases or
dihydrolases. If the amino acids are degraded,
alkaline products are made resulting in colour
change by a pH-indicator.
Differensiation between
species within genus Vibrio
Ornithine
Alkaline amines (putrescine, cadaverine)
Odecarboxylase
Lysine
Ldecarboxylase
Adihydrolase
Arginine
Gelatin - medium
• The gelatin medium is used to detect if bacteria
produce enzymes (proteinase/gelatinase) that
degrade gelatin. A regular pepton-medium supplied
with gelatin (in tube) is used. The medium is solid by
temperatures below 22°C. If gelatin is degraded, the
medium will turn liquid at this temperature. When
incubation is performed at 37°C, the medium has to
be cooled (tap water) before reading. Often
incubated at 20°C for several days before reading.
Gelatin
Hydrolysis
Proteolytic enzymes
Peptides and
amino acids
Vibrio anguillarum
• Features
• V. anguillarum is a Gram-negative facultatively
anaerobic bent/straight rod that grow well on regular
media and with hemolytic colonies on blood agar.
Optimal temperature are ca. 25°C. The bacterium is
heavily motile. V. anguillarum is subdivided into 10
serovariants based on O-antigens.
• Epidemiology
• V. anguillarum occurs freely in salt water, brackish
water and on fish.
Vibrio anguillarum
Importance
• V. anguillarum cause vibriosis in fish both in
brackish and salt water and is detected in
salmonids, flat fish and cod in Norway. The
disease occurs endemic all over the world in
temperate marine waters. Effective vaksines
are used profylactic against vibriosis. Isolates
from salmonids belongs normally to serovar 1
or 2. Serovar 2 and other serovariants are
normally detected from the environment or
from various fish species.
Vibrio ordalii
• Occurs in the Pacific Ocean and cause
disease in the farmed Pacific salmon
species
• Closely related to V. anguillarum
• Detected in Norway in 2005
Aliivibrio salmonicida
Features
• Al. salmonicida is a Gram-negative,
facultatively anaerobic, motile, bent/straight
rod. The bacterium are cultivated on blood
agar added 2% extra NaCl (Na-ions).
Al.salmonicida does not grow at temperatures
above 22°C. Common incubation
temperature is 15°C. In the cell wall there is a
layer consisting of lipopolysaccharides and a
protein (Al. salmonicida protein 1 shortened
VSP1). Detection of VSP1 is used
diagnostically.
Aliivibrio salmonicida
Epidemiology
• The bacterium occurs freely in salt water and on fish.
In salt water the bacterium survives long as so-called
micro cells that is a live but not culturable state of the
bacterium.
Importance
• V. (Al.) salmonicida causes cold water vibriosis in
Atlantic salmon. The disease occurs endemically in
Finnmark and Troms counties and along the entire
coast of Norway. The disease occurs in Scotland,
Iceland and on the western coast of North America.
The disease also occurs in farmed cod.
• A complete bacterial cell vaccine is developed which
is very effective in the prophylaxis against cold water
vibriosis.
Lipopolysaccharides and
protein in the cell wall of Al.
salmonicida
• A 40 KDa outer surface molecule (VSP1)
• 90 % of the antibodies produced in
salmon were specific for one single
epitope on the molecule
• Espelid, S., Hjelmeland, K., and Jørgensen, T. 1987
Dev. Comp. Immunol. 11: 529-537.
Lipopolysaccharide structure
• It is concluded that the oligosaccharide has the
following structure, in which:
• L-alpha-D-Hep p is L-glycero-alpha-D-mannoheptopyranose,
• D-alpha-D-Hep p is D-glycero-alpha-D-mannoheptopyranose,
• alpha-D-Fuc p4N is 4-amino-4,6-dideoxy-alphaD-galactopyranose,
• alpha-NonA is 5-acetamidino-7-acetamido-3,5,7,
9-tetradeoxy-L-glycero-alpha-D-galactononulosonic acid,
• BA is (R)-3-hydroxybutanoyl, and
• PEA is phosphoethanolamine.
Edebrink P, Jansson PE, Bøgwald J, Hoffman J. 1996. Carbohydr
Res. 1996 287: 225-45
Vaccination
• Effective vaccines with formalin-killed V. (Al.)
salmonicida controlled the disease from
1988.
• Today V. (Al.) salmonicida is one component
of multivalent vaccines with up to six
components.
• Common bacterial vaccine components:
Aliivibrio salmonicida, Vibrio anguillarum,
Aeromonas salmonicida ss salmonicida,
Moritella viscosa
•
Press, CM; Lillehaug, A, Br Vet J. 1995 Jan-Feb;151(1):45-69.
Vaccination in European salmonid aquaculture: a review of
practices and prospects.
Iron metabolism and
temperature
• Cold water vibriosis is observed only at 10oC
or lower.
• In iron-limited media growth of Aliivibrio
salmonicida at 12oC and below.
• A single dihydroxamate siderophore,
bisucaberin, is produced only at 10oC and
below.
• High molecular weight iron-regulated outer
membrane proteins are expressed at 6 and
10oC but not at 15oC.
Colquhoun, D.J. and Sørum, H. 2001. Microb. Pathog. 31: 213-19.
Expression of OMP’s during in
vivo cultivation of Al.
salmonicida
• Containers of dialysis tubing with Al.
salmonicida cultures implanted in the
peritoneal cavity of rainbow trout resulted in
1010 cfu after 4 days at 10 – 11.5oC
• Expression of three proteins of approx. 99, 90
and 78 kDa was up-regulated in vivo
• A capsule was not produced in vivo
Colquhoun, D.J. & Sørum, H. 1998. Fish and Shellfish Immunol.
8: 367-77
Incubation temperature for Al.
salmonicida in vaccine production
• Cold water vibriosis occurs below 10oC
• The highest cell division rate occurs at
15oC on solid media and at 10oC on
liquid media
• Growth temperature for diagnostic
purposes should be 15oC
• Growth temperature for vaccine
production should be at 10oC or below
Colquhoun et al. 2002. J. Appl. Microbiol. 92: 108796
Coasts of the North Atlantic
Ocean
H. Sørum, E. Myhr, B. M. Zwicker, and A. Lillehaug
Canadian Journal of Fisheries and Aquatic Sciences, 1993, 50: 247250
• Plasmid profiling of Al. salmonicida
isolates shows homogenic plasmid
profiles in all areas with no clear relation
to the origin of the isolate.
• Plasmid profiling is the most convenient
method to separate between isolates of
this homogenic cold water pathogen
Plasmids in Vibrio salmonicida isolated from salmonids with
hemorrhagic syndrome (Hitra disease).
H Sørum, T T Poppe, and O Olsvik
84 Vibrio spp.
from 1982-84:
A 41.7 %
B 27.4 %
C 11.9 %
D 2.4 %
E 16.7 %
J Clin Microbiol. 1988 September; 26(9): 1679–1683.
Geographic origin
Year
Strain
West coast of Norway
1983
NCMB 2262T
21, 3.4
Shetland Islands
1985
NCMB 2245
21, 3.4
Shetland Islands
1985
MT 160
21, 3.4
Faroe Islands
1988
881129-1/1
21, 3.4
Faroe Islands
1988
881129-1/2
21, 3.4, 2.8
Faroe Islands
1988
891129-1/13
21, 3.4
Faroe Islands
1989
880206-1/4
21, 3.4, 2.8
Faroe Islands
1989
890811-1/7
21, 3.4, 2.8
New Brunswick
1989
Can 89457
21, 3.4
Nova Scotia
1990
Can 90321
21, 3.4, 2.8
Northern Norway
1982
A 2181/82
Northern Norway
1987
1509/87
21, 5.5
West coast of Norway
1987
1512/87
21, 3.4
West coast of Norway
1987
1513/87
21, 3.4, 2.8
Northern Norway
1988
2933/88
61, 21, 3.4, 2.8
Plasmid profile
61, 21
Plasmid profiling of Vibrio salmonicida for epidemiological studies of cold-water
vibriosis in Atlantic salmon (Salmo salar) and cod (Gadus morhua).
H Sørum, A B Hvaal, M Heum, F L Daae, and R Wiik
A Salmo salar 1983 West (NCMB2262T)
B Salmo salar 1983 West
C Gadus morhua 1988 North
D Salmo salar 1988 North
E Salmo salar 1982 North
F Gadus morhua 1988 North
Appl Environ Microbiol. 1990 April; 56(4): 1033–1037.
Environmental detection of V.
salmonicida in marine
sediments.
• In marine fish farm sediments V.
salmonicida may be detected in
relatively high numbers (104 – 107
cells/g) during and after a disease
outbreak.
• In farms with no disease or in control
sediments the same number of V.
salmonicida cells are detected.
• Ø. Enger, B. Husevåg, J. Goksøyr, 1989, Appl. Environ.
Microbiol. 55: 2815-2818.
Environmental detection of Al.
salmonicida in fish-farm water.
• In marine water samples from 12 fish farms
collected twice monthly in 9 months Al.
salmonicida was detected in all farms in a
number of 12 to 43 cells/ ml water.
• The number of farms with detection of Al.
salmonicida was in general highest during
winter
• The total bacterial counts varied between
4x104 and 9x105 cells/ml water.
• Ø. Enger, B. Husevåg, J. Goksøyr, 1991, Can J
Microbiol. 8:618-623.
Use of antimicrobial drugs in
Norwegian fish farming
60
450
400
50
40
300
250
30
200
20
150
100
10
50
0
0
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
År
Antimicrobial agents
Salmon produced
95
96
97
98
99
00
Production (1000 ton)
Antimicrobial agents (1000 ton)
350
Identification and
cloning of a
tetracycline resistance
gene from the fish
pathogen Vibrio
salmonicida.
H Sørum, M C Roberts, and J H
Crosa
170 MDa plasmid,
pRVS1, R plasmid
Antimicrob Agents Chemother. 1992
March; 36(3): 611–615.
Transposons in various strains
TetE
Vs 75
sulII
Vs 250
dfr1
Vs 476
dfr1
sulII
Vs 420
dfr1
TetE
Vs 88
TetE
Vs 201
Vs 311
TetE
TetE
sulII
Winter ulcer
• ”Winter ulcer" is a disease that occures in farmed salmon and
trout at temperatures below ca. 7-8°C. The disease occurs
along the coast of Norway from the Oslo fjord to Finnmark. In
addition the disease occurs in Scotland, Iceland and the Atlantic
coast of North America. The mortality during an outbreak is
normally low, however the prevalence may be quite high.
Affected fish is widely classified as reduced quality, and as a
consequence the economic losses connected to the disease are
large.
Moritella viscosa (Vibrio viscosus)
and Aliivibrio wodanis
• Two «Vibrio» species is connected with winter
ulcer, Moritella viscosa and Aliivibrio wodanis, of
which M. viscosa probably have a major
importance. Both species may be characterized
as psychrophilic, salt demanding and bhemolytic vibrios:
– Grow normally at temperatures up to 15oC (M.
viscosa) and 25°C (Al. wodanis), do not grow at 30°C
– Need extra salt in the medium to grow (2% NaCl).
Biochemical features for M. viscosa
and Al. wodanis
M. viscosa
V. wodanis
ALO -testen trehalose mannose casein
( V. viscosus)
-/-/+
-/+/+
+
-
Moritella viscosa phylogenetic
relations
• Winter ulcer
Vibrio parahaemolyticus
Features
• Cellular morphology as typical of the genus. Many
serotypes (based on O- and K-antigens). Extra salt
(NaCl) is needed in the medium for growth. Saltconcentrations up to 8-9% is tolerated. Optimal
temperature for growth is 30-37°C. Anhemolytic on
regular blood agar. Human pathogenic strains are
normally hemolytic on blood agar with human blood.
Importance
• Cause acute gastroenteritis in humans. Cause
disease in marine organisms, but is so far no major
pathogen to fish farms in warm waters.
Vibrio parahaemolyticus
Epidemiology and pathogenesis
• The bacterium occurs in seawater, in sediments, on
plankton, fish and shellfish. In largest concentration
along the coast of countries in tropic and sub-tropic
areas where the sea temperature may reach 2530°C. V. parahaemolyticus is detected in Norway.
The concentration of the bacterium in the water
varies considerably through the year and is largest in
the summer. The source of human disease is almost
invariably infected seafood.
Family Enterobacteriaceae
(H-antigener)
(K-antigen)
(Outer membrane)
(Fimbrier)
F-antigener, noen K-antigener
Enterobacteriaceae
have peritrich
flagellae (figur to the
left shows one polar
flagella)
Family Enterobacteriaceae
Occurring globally:
Soil and water
Plants, trees, fruit and vegetables
Insects - Humans (feces)
Medical important:
Gastroenteritis among animals and humans
Extraintestinal diseases
Opportunistic pathogens (urinary tract inf. m.m.)
Economical important:
Diseases in fruit, grain and vegetables
Diseases in fish (also medical important)
Important research tool:
Genetical manipulation
Family Enterobacteriaceae
Escherichia
Edwardsiella
Citrobacter
Salmonella
Shigella
Klebsiella
Enterobacter
Hafnia
Serratia
Proteus
Yersinia
Providencia
Morganella
Erwinia
Family Enterobacteriaceae
Divided into genera based on biochemical features
• Ex. lactose fermentation
yellow = coliforme bacteria, lactose positive
blue = lactose negative
• Ex. IMVIC, TSI, Urea
Divided into
• Species based on biochemical features
• Serotypes based on antigenic features
• Patogroups based on patogenicity factors
• Genetical groups
• Etc.
Identification of bacteria
Bacterium
Gram +
Gram -
Coccus
Rod
Spores
Bacillus
Clostridium
No spores
Catalase +
Listeria,
Corynebacterium,
Actinomyces
Catalase -
Lactobacillus,
Erysipelothrix,
Arcanobacterium
pyogenes
Catalase +
Micrococcus
Staphylococcus
(Rhodococcus)
Rod
Catalase -
Streptococcus
Enterococcus
Oxydase +
Coccus
Oxydase -
Oxydase +
Pseudomonas,Vibrio, Enterobacteriaceae
Aeromonas,
Brucella, Bordetella
bronchiseptica
Pasteurella
(Actinobacillus)
(Haemophilus)
Lactose +
IMViC
Escherichia,
Klebsiella,
Citrobacter,
Enterobacter
Neisseria
Moraxella
Lactose -
IMViC
Proteus,
Salmonella,
Shigella,
Yersinia
Oxydase -
Identification enterobacteria
Cultivation blood plate (an/aer) and BTB
Gram-negative rods, Oxydase-negative
Lactose positive (BTB)
Escherichia, Klebsiella
Citrobacter, Enterobacter
Lactose negative (BTB)
Salmonella, Proteus, Shigella,
Edwardsiella, Yersinia, Serratia
Motility, IMViC, TSI, Urea, Enterotube, Serology,
Phage typing, DNA typing
Bromothymol blue lactose
agar (BTB)
Indicator system: Lactose og bromothymol blue.
Selective factors: Crystal violet and sodium thiosulphate
Stimulate growth of Enterobacteriaceae
and repress gram positive bacteria.
Use:
Cultivation of enterobacteria
Differentiate between lactose positive
and lactose negative bacteria
Enrichment medium and selective indicator medium often
used together
Lactose negative, left
Lactose positive (coliforme), right
LPS, endotoxin
Lipid A is the
toxic part
Part of cell wall in Gramnegative bacteria
Enteric red mouth disease
Yersinia ruckeri
• Enteric Red Mouth Disease
(ERM), Yersiniosis
• Often the disease is induced by
stress. The source of the
bacterium may be latent
infected fish. Transfers between
fish via feces.
• Clinical symptoms varies. May
look like other septicemic
diseases as furunculosis and
vibriosis (Vibrionaceae).
Yersinia ruckeri
• Occurs globally, and attacks both wild and farmed
fish
• Occurs in fresh water, salt water and brackish
water.
• Vaccines are available
Edwardsiella piscicida
•
•
•
Edwardsiella septicaemia:
Gram-negative, motile and peritrichously
flagellated straight rod. Facultatively anaerobic
and a pathogen of warm water fishes (eels).
Catalase-positive and cytochrome oxidase
negative. Usually found in organically polluted
water.
Isolation and diagnosis: Isolation from kidney of
diseased eels with 24h on brain-heart infusion agar
(BHI) or trypton soya agar (TSA), and indolepositive. Diagnosis can be done by ELISA, PCR, or
serological methods.
Edwardsiella septicaemia
Occurrence: Most commonly during summer
months in USA and Japan, and in Taiwan during
spring.
Clinical signs: Severe cutaneous lesions deep into
the musculature, sometimes progress to necrotic
abscesses and bullae, loss of skin pigmentation.
Diseased eels often develop internal hyperaemia
and bloody congestion of fins with petechial
haemorrhage.
Control and treatment: Necessary to make
improvement in health and hygiene management,
and control water quality and stocking density.
Sulphonamide and tetracycline can be used to
reduce losses. Vaccine is still on experiment.
Seksjon 4
Gram-negative aerobic rods
and cocci
Family
Genus
Pseudomonadaceae
Pseudomonas
Burkholderia
Xanthomonas
Frateuria
Zoogloea
Azotobacteriaceae
Rhizobiaceae
Methylococcaceae
Halobacteriaceae
Acetobacteriaceae
Legionellaceae
Legionella
Neisseriaceae
Neisseria
Moraxella
Acinetobacter
Kingella
Other genera
Flavobacterium
Alcaligenes
Brucella
Bordetella
Francisella
Beijerinckia
Derxia
Xanthobacter
Thermus
Thermobacterium
Halomonas
Alteromonas
Serpens
Janthinobacterium
Paracoccus
Lampropedia
Genus Pseudomonas
• Many species. Pseudomonas aeruginosa og
Pseudomonas fluorescens are important in veterinary
medicine and human medicine.
Features
• Gram-negative, straight or bent, clear rods. Motile by
polar flagella. Most species are strict aerobic. Several
species produce pigments (water soluble, not water
soluble). Several species grow well at low temperatures
(psychrophilic). Grow well on regular media.
Genus Pseudomonas
Epidemiology
• Several species occur in soil, water (also
seawater) and food. Many species are
pathogenic to plants and a few are pathogenic to
animals and humans. Some species cause food
spoilage.
Pseudomonas fluorescens
Features
• Gram-negative, motile rods. Aerobic. Grow on
agar media with round or uneven, often opaque
colonies of varying size. Grow on blood plates.
Produce water soluble, fluoresceing pigments.
Occurrence and importance
• Common in soil and water. Cause spoilage of
food. Is causing disease in farmed salmonids in
Norway.
Pseudomonas anguilliseptica
• Pseudomonas anguilliseptica Wakabayashi and
Egusa 1972, species.
Type strain: strain ATCC 33660 = CCUG 35503 = CIP
106711 = DSM 12111 = NCIMB 1949.
References: SKERMAN (V.B.D.), McGOWAN (V.) and
SNEATH (P.H.A.) (editors): Approved Lists of Bacterial
Names. Int. J. Syst. Bacteriol., 1980, 30, 225-420.
[WAKABAYASHI (H.) and EGUSA (S.): Characteristics
of a Pseudomonas sp. from an epizootic of pondcultured eels (Anguilla japonica). Bulletin of the
Japanese Society of Scientific Fisheries, 1972, 38, 577587.]
Red Spot Disease in eels
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•
•
Pseudomonas anguilliseptica and Red Spot
Disease:
Gram-negative, motile and straight or
slightly curved rods with long polar
flagellae. It is strictly aerobic and never
fermentative. It is oxidase-positive.
Isolation and diagnosis: Growth on
common lab media, such as BHI TSA, but
not on Pseudomonas isolation agar.
Catalase and oxidase positive but does not
produce acid. Specific rabbit serum can
help identification.
Red Spot Disease in eels
Occurrence: A major disease of Japanese
eels and less infectious to European eels.
It happens often in low temperature during
spring.
Clinical signs: Diseased eels have dark skin,
inappetite. Some red spots can be seen on
the skin, which are deep haemorrhagic
ulcers. Features of acute septicaemia, like
petecchial haemorrhage over visceral
organs and musculature. The kidney is
sometimes severely necrotic.
Red Spot Disease in eels
Control and treatment: Since infection of
Pseudomonas anguilliseptica is closely
associated with poor environment, it is
also necessary to make improvement in
health and hygiene management, control
water quality and stocking density. Raising
water temperature to 27oC can effectively
treat eels only. Furthermore, kanamycin
injection and oral oxytetracycline have
been recorded to be efficacious.
Other genera
Genus Flavobacterium
• Occurs in water. Pathogenic to fish. F.
meningosepticum pathogenic to humans
Genus Flexibacter
• Flexibacter/Flavobacterium psychrophilum
• Important fish pathogen of different species of fish in temperate to
cold water. The bacterium often infects the skin of the fish and may
cause considerable losses in fish farming.
• In Japan the bacterium is the causative agant of bacterial coldwater disease and rainbow trout syndrome
Piscirickettsia salmonis
• A rickettsia causing a major disease of
salmonids in Chilean aquaculture.
• More sporadic isolation in Norwegian
Atlantic salmon aquaculture.
• Confirmation of Piscirickettsia salmonis as
a pathogen in European sea bass
Dicentrarchus labrax and phylogenetic
comparison with salmonid strains.
Photobacterium damsela ss
(Pasteurella) piscicida
• Pasteurellosis or pseudotuberculosis, as are the
clinical designations of the disease is a serious
disease causing severe losses in the farming of
marine yellowtail (Seriola quinqueradiata) in
Japan since 1969.
• In the Mediterranean marine farming of mainly
gilthead sea bram (Sparus aurata) and sea bass
(Dicentrarchus labrax), pasteurellosis became a
serious problem with the advent of epizootics in
the farms at the beginning of the 1990s.
Francisella spp.
• Farmed cod in Norway with outbreaks of
disease caused by a Gram-negative
bacterium with close similarity to
Francisella philomiragia isolated in
Japanese aquaculture.
• Proposed name is Francisella philomiragia
subspecies noatunensis
• Zoonotic considerations must be taken.
Francisella philomiragia subspecies
noatunensis
• A novel systemic granulomatous inflammatory disease in
farmed Atlantic cod, Gadus morhua L., associated with a
bacterium belonging to the genus Francisella. J Fish Dis.
2006 May;29(5):307-11. Olsen et al.
• Francisella philomiragia subsp. noatunensis subsp. nov.,
isolated from farmed Atlantic cod (Gadus morhua L.). Int
J Syst Evol Microbiol. 2007 Sep;57(Pt 9):1960-5.
Mikalsen et al.
• New species in the genus Francisella
(Gammaproteobacteria; Francisellaceae); Francisella
piscicida sp. nov. isolated from cod (Gadus morhua).
Arch Microbiol. 2007 Nov;188(5):547-50. Epub 2007 Jul
10. Ottem et al.
Genus Renibacterium
• Only one species, Renibacterium
salmoninarum
Features
• Gram-positive short rods.
• Aerobic. Does not grow on blood agar.
• Cultivated on KDM medium with growth
after days/weeks.
• Needs cystein for growth.
• Optimal temperature for growth 15–18° C.
• Catalase positive
• Does not produce acid from sugar
Genus Renibacterium
Importance
• Bacterial kidney disease (BKD), a chronical
disease in salmonids often with ascites.
Epidemiology and pathogenesis
• Have been considered as an obligate
pathogen to salmonids.
• Occurs in infected environment.
• Occurs intracellularily
• Granulomes in kidney and other organs
Renibacterium salmoninarum
Diagnostic
• Direct microscopy (gram-staining)
• Cultivation from granulomes on KDM2 medium. Incubation
up to 12 weeks at 15°C. The bacterium grows with greywhite to whipped cream yellow, shiny colonies of varying
size. Suspicious colonies are Gram-stained and tested on
API-zym. NB!! The bacterium does not grow on blood
agar with or without extra salt. Serological methods,
ELISA, FAT og IFAT
Streptococci
• Relatively uncommon cause of disease in fish
• Abdominal infections in artificially spawning
salmonids in Norway
• Disease outbreaks in farmed yellowtail in Japan
• Drug resistance against macrolides, lincomycin,
tetracyclines and chloramphenicol in
streptococci from Japanese yellowtail
• Transferable drug resistance demonstrated
Mycobacteria
• Granulomas caused by Mycobacterium
spp. are not uncommon in marine fish
species
• Zoonotic importance is sporadic and
relatively unknown
• New fish species in aquaculture may
increase the impact of mycobacteria in fish
farming
Mycobacteria
• 1. Mycobacterium salmoniphilum (earlier
M.chelonae, separate species in 2007) occurs in
salmonids, but also in fresh water fish in general.
Isolated from farmed Atlantic salmon and lake
herring. Atlantic cod is susceptible by
challenge.
• 2. Mycobacterium marinum occurs in marine
fish species (mackerel, turbot, eel, etc.).
• 3. Mycobacterium chelonae and
Mycobacterium gordonae important in
ornamental fish.