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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 • • • 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.