Departamento de Microbiología y Parasitología Fac. de Biología/CIBUS e Instituto de Acuicultura Universidad de Santiago de Compostela Tenacibaculosis of farmed fish in Southern Europe Alicia E. Toranzo Group of Ichthyopathology (USC) Tenacibaculum maritimum Workshop Maritime Heritage Centre, British Columbia, Canada. February 2015
Aquaculture Institute (IA) Biological Research Center (CIBUS)
ACTIVITY OF THE USC GROUP (From more than 25 years) More than 500 peer-review papers published Participation in National and European Research Projects and Contracts (AQUAGENOMICS, SANCO, SEAFOOD PLUS, REPROSEED, MAXIMUS ) Service contracts & Know-how transfer for Fish and Shellfish farms Patents on Fish vaccines, probiotics and PCR diagnostic protocols Technical support for Official Regulatory Agencies Training for Private Companies
Coastline and River system of Galicia (NW, Spain)
Tenacibaculosis Wide distribution in fish species cultured in seawater in Southern European countries: - Turbot - Sole - Seabass - Gilthead Seabream - Salmon (only NW Spain)
Main marine finfish pathogens Turbot * Vibrio anguillarum *** Tenacibaculum maritimum/spp * Aeromonas salmonicida * Edwardsiella tarda IPNV VHSV Sole * Photobacterium damselae subsp. piscicida *** Tenacibaculum maritimum/spp * Vibrio anguillarum Nodavirus IPNV Gilthead seabream * Photobacterium damselae subsp. piscicida * Vibrio anguillarum * Tenacibaculum maritimum/spp Nodavirus Seabass * Photobacterium damselae subsp. piscicida * Vibrio anguillarum *** Tenacibaculum maritimum/ spp Nodavirus
Main Pathogens in the Salmon cultured in Norwest of Spain (1975-2015 ) Vibrio anguillarum Aeromonas salmonicida ***Tenacibaculum maritimun Amoeba spp (AGD) Renibacterium salmoninarum SPDV (Salmon Pancreas Disease)
Main Tenacibaculum species recovered for marine fish cultures in southern Europe T. maritimum T. soleae T. discolor T. dicentrarchi T. gallaicum From our experience, only T. maritimum and T. soleae have the capacity to produce a systemic infection and can be recovered from internal organs
External signs of Marine Tenacibaculosis (T. maritimum) Skin Ulcers Eroded mouth Frayed fins Tail rot Gill necrosis Some variation depending on the fish species and age of fish
T. maritimun in Turbot (Scophthalmus maximus)
T. maritimum in Sea bass (Dicenthrarchus labrax)
T. maritimum in sole (Solea senegalensis)
Tenacibaculosis in Atlantic Salmon in Northwest, Spain (first isolations in the 1990 decade) In some cases a mixed of T. maritimum and Aeromonas salmonicida was diagnosed
Evolution of skin lession diseased Atlantic salmon by T. maritimum in NW, Spain (last mortalities in 2014-2015)
In some cases a mixed infections of T. maritimum and Vibrio anguillarum O1 was present The systemic infection of V. anguillarum causes an inhibition in the growth of T. maritimum which difficult its recovery from internal organs
al., Fac. Veterinary, USC) (Quiroga et Skin of healthy fish showing homogeneous surface of epidermis without lesions (bar = 200 mm ). Severe ulceration in the central area of the scale (bar = 100 mm ) Large number of groups of filamentous bacteria located on the surface of scales and between the radii of scales (bar = 10 mm )
Tenacibaculum maritimum - Fastidious filamentous (5-40 mm ) gliding bacterium. Ocasionally cells up to 100 mm in length can be observed. In older liquid and solid media cells tend to become spherical. - Presumptive diagnosis: Microscopic examination of abundant long, thin, rod-shaped bacteria in wet mounts or Gram preparations obtained from skin lesions and gills. Giemsa stain (ulcer smear) Gram stain
Tenacibaculum maritimum The pathogen is an obligate marine microorganism which does not growth on media prepared only with NaCl. It must be cultured in oligothrophic media elaborated with seawater (30-100% strength seawater) Marine Agar and FMM (Flexibacter maritimum medium) are the most appropriate for the succesful isolation of this pathogen However, FMM allows a better recognization of the typical colonies of this species: flat, pale-yellow with irregular uneven edges and adherent to the medium
Pros and Cons of AM and FMM Marine Agar (or broth) - Rapid growth - More density - Difficult to recognize the T. maritimum colonies - Dfficult to recognize the typical long filamentous FMM (Agar and broth) - Slow growth - Low density - Useful to recognize the typical T maritimum colonies - The cells have the typical filamentous morfology
The importance to recognize the T. maritimum colonies and differenciate from unrelated and related species (i.e., T. soleae, T. discolor, T. gallaicum) TM TM TS TG TS TG TD TD Marine Agar FMM medium
Phenotypic characterization On solid media colonies absorb Congo red and the cells do not contain cell wall-associated flexirubin-type pygment All strains produce cytochrome oxidase and catalase. Gelatin is hydrolyzed but starch, esculin an chitin are not All strains gives a similar enzymatic profile in the API ZYM System in which all enzymes related to the metabolism of carbohydrtaes are absent. The phenotypic characterization does not allow the differenciation of T. maritimum from the other Tenacibaculum species associated to fish diseases
PCR detection of T. maritimum - Specific PCR is necessary for an accurate diagnosis of the pathogen por PCR. It allows the differentiation of T. maritimum from other taxonomic related Tenacibaculum species (primers MAR1/MAR2, Toyama et al.) * MUCUS: ideal non destructive sample for a sensitive detection by PCR in carrier fish (detection limit,100 cels/ml)
Rapid Identification by Proteomic (MALDI-TOF) from colonies T. maritimum Other Tenacibaculum
Serological studies in T. maritimum * Antigenically heterogeneous species: Three O serotypes described in marine fish: There are not a strict host specificity, but. The strains from seabream and seabass the serotype O1 is the predominant. In strains from turbot, serotype O2 is the predominant In strains from sole, O1 y O3 are the predominant serotypes, although the prevalence of O2 strains is increasing In salmon, whereas the first isolations in NW Spain belonged to serotype O2, recent isolates belonged to serotype O3 A continuos monitoring of the serotypes of the strains isolated in each fish species in a zone along the time is recommended
Immunoblot (LPS analysis) O1 strains O2 strains Antiserum anti O1 Antiserum anti O2
Dot Blot analysis : More simple and accurate method for serotyping Antiserum anti-o2 Antiserum anti-o3 O1 O2 O3 As cross-reactions among serotypes exist, the use of absorbed antisera are needed for an accurate serotyping scheme of T. maritimum
Regardless of the O serotype all strains have the same surface Protein pattern
The Marine Agar is not useful to distinguish T. maritimum serotypes Serotype O1 Serotypes O2 & O3
The FMM is the appropriatte medium to distinguish T. maritimum serotypes Serotype O1 Serotypes O2 and O3
Genotyping studies RAPD-PCR analysis: The strains can be separated into distinct genotypes that are strongly associated with the serogroups described. Some subgroups within each clonal lineage can be detected associated to the host and/or geographical origin Strains O1 Strains O2 Strains O1 Strains O2 RAPD (primer 2) RAPD (primer 6)
Genotyping studies (cont.) MLSA : the concatenated analysis of 11 loci from 73 strains revealed that An statistically significant asociation between genotypes and information related to the host fish, geographical origin and year of isolation. The same bacterial lineages can be found in distinct hosts in the same geographical area, which points to the possibility of crossspecies contamination. An endemic colonization of fish farms by local strains with little contribution of long-distance dissemination linked to fish movements.
Virulence and Challenge Model in turbot Intraperitoneal injection and 1-2 h immersion : No successful infection Prolonged immersion of fish for 18 h is an effective model to induce the disease (LD50 = 103-104/ml for 5-10 g fish): Inoculated fish reproduced some of the external signs of the natural infection (eroded mouth and skin ulcers) and bacteria can be recovered from external lesions and kidney. In asymptomatic fish surviving exposure, the bacteria can be only recovered from mucus which support the idea that T. maritimum can persist within aquatic environments utilizing mucus as reservoir in fish hosts. - These results indicates that it is necessary a prolonged exposure for the development of a biofilm on the skin and/o gills which must be the first step in a succesful infection
Survival Experiments In sterile sea water T. maritimum remained viable and culturable for more than 160 days. Under natural conditions the pathogen remained viable for only 5 days which can be due in part to the inhibitory effect of the natural aquatic microbiota. Therefore, these results seem to sugest that seawater would not be an important route of transmission of T. maritimum However the bacteria can remain in the aquatic environment for a long time, utilizing fish mucus as a reservoir because T. maritimum is resistant to the inhibitory activity of the skin mucus.
Risk factors of disease Several factors increase the prevalence and severity of the disease : - high temperature (above 15ºC) and salinity (30-35 ppt) - Low water quality - High density - Management stress These factors contributes to the formation of a T. maritimum biofilm within the skin mucus necessary to express the virulence associated factors which facilitates the entrance and systemic infection involving internal organs. In addition, these biofilms can be also formed attached to the tanks and net pens constituying an important reservoir of the pathogen.
Virulence associated factors Adherence : T. maritimum produces capsular material that can facilitate the cell adherence to surfaces including the fish skin (first step of the infection) Quorum-sensing system: This cell-cell comunication system mediated by AHLs molecules as autoinductors is present in T. maritimum which regulates the expression of genes involved in virulence factors such as the exoenzyme production
Virulence associated factors (cont.) Secretion of enzymes : T. maritimum produces extracellular enzymes such us proteases and chondoitinase which could facilitate the alteration of the host tissue, contributing to the colonization and invasion High-affinity iron-uptake mechanisms: T. maritimum possesses two different systems of iron acquisition which can compete with the host ironbinding proteins: one involving the synthesis of siderophores another that allows the utilization of heme groups as iron sources by direct binding
Vaccination studies (T. maritimum) * Only commercial vaccine for turbot (serotype O2) * Autovaccines designed for sole (serotypes O1 & O3 of T. maritimun or T. maritimum + T. soleae) Vaccination Strategy recommended: Bath immunization at 1-2 g followed by a IP booster at 20-30 g. * No vaccines for seabream and specially for seabass were developed.
Humoral response of vaccinated sole against T. maritimum (O1 and O3) 6 months 2 months Two baths with a month interval Revaccination by ip or bath, six month after the two baths A cross protection with the serotypes not included in the vaccine was demonstrated
Vaccination studies (cont): * Polivalent vaccine (tenacibaculosis/vibriosis) is also available for turbot and it is included in the vaccination calendar for this species. * However, antigenic interference occurs in the tenacibaculosis/photobacteriosis formulation developed to prevent these diseases : - The presence of antigens of T. maritimum causes a decrease in the protection of sole against a weak antigen as Photobacterim damselae subsp piscicida
Cumulative mortality Antigenic interference in the divalente formulation Tenacibaculosis/Photobacteriosis Monovalent vaccine: Photobacterium damselae subsp. piscicida Divalent vaccine: Tenacibaculum maritimum Photobacterium damselae subsp. piscicida Days Days Non Vaccinated sole Vaccinated sole
Control (Chemotherapy) Enrofloxacin This drug was employed during many years as the election compound for controlling outbreaks by T. maritimum However a rapid appearance of resistant strains was evidenced Florfenicol In the last years this drug proved to be the best compound to arrest mortalities due to T. maritimum with no resistances being detected.
Prophylatic treatments Hydrogen peroxide (H2O2): Experiments in turbot demonstrated that 240 ppm/30 min of H2O2 is necessary to kill T. maritimum when it is colonizing the skin mucus. However this high concentration increase dangerously the level of stress of fish Therefore, the use of 240 ppm of H2O2 can be regarded as a general preventive method for treating surface of tanks before the introduction of the fish. Fresh water baths: Preliminary experiments indicated that a decrease of salinity until 5ppt during at least 24 h can be an useful strategy to reduce the prevalence of T. maritimum in the farm.
FINAL REMARKS Despite the considerable effort made during the past years to understand the pathogenesis of T. maritimum, there are yet several gaps concerning: The route of transmission, survival strategies and reservoirs The genetic basis of the virulence mechanism The establishement of adequate vaccination programmes for economically important marine fish as seabass and salmon. The knowledge of the predominant serotypes affecting Atlantic salmon in the different countries