Many thanks, Amedeo for sharing your experience with us. Do you have any information coming from farmers, or people in charge of the health management on marine fish farms what are the environmental conditions for disease outbreaks due to "flexibacteriosis" or "myxo" for Croatian farmers? Do they apply any preventive measures or treatment?

Hi dear Amedeo, it is nice hearing from you! Your comments are interesting because they show that isolation of several Tenacibaculum species from internal organs, though uncommon, is possible. I think more sampling of internal organs should be done systematically in parallel with the sampling of external lesions.

My apologies to Zeljko, I just realized I forgot to answer his question! For 16S rRNA identification of Tenacibaculum strains, we indeed used standard universal primers. We are currently developing specific primers based on the whole-genome sequences we obtained for the type strains of most Tenacibaculum species.

Jean-François

Dear JF andAmadeo

I must say that tenacibaculum discolor lesions are usually slightly different from tenacibaculum maritimum or other tenacibaculum, affecting deeper skin layer and giving more ulcerative lesions. When looking at them, we can see that this bacterium is going deeper in the muscular septa but we did not found it on the internal organs. It happens time to time with tenacibaculum maritimum in the head kidney and the liver. 

We will pay more attention from now on and sample internal organs at least in case of recurrent outbreaks. Another limiting factor for the detection of such bacterium in internal organs is that most ield diagnisis is done on fresh smears with or without staining and eventually organ imprints. But we often see that in case of presence of tenacibaculum in internal organs, their shape is slightly different being shorter. And it might be that in some cases they are not properly identified on smears as it could be difficult to distinguish them. Moreover, when plating organs on classic media (Marine agar, TSA) it is not always easy to growth them and specific media or swabs with transport media are required to be sure to grow them

So 'technical problematic' might do as well that we miss part of the potential septicemic forms or at least intrnal organs infection. The main consequence of that being unappropriate prophylactic method or curative method applied. 

Many thanks Alain, but could you advise us precisely, how to take sample from lesion, how to seed material on the plate and how to obtain pure isolate? What transport media do you use for isolation and what solid media is good for growing Tenacibaculum?

Alain knows much better than me how to isolate Tenacibaculum strains, but there are a few things I can say. External lesions are usually heavily contaminated by many opportunistic bacteria; this is why it is frequently difficult to obtain pure cultures of Tenacibaculum. What I can recommend is to sample the margins of the lesion, not the center, and to try to obtain material from under the skin. The sample may then be resuspended in about 1 ml of marine broth such as Marine Broth 2216 (Difco). A few serial dilutions of the suspension can then be made and inoculated on Marine Agar 2216. This may improve the chance to get rid of the contaminants and to obtain at least a few colonies of Tenacibaculum that can then be subcultivated for purification. One must keep in mind that Tenacibaculum cultures (either broth or agar) degenerate within a few days and that they must be subcultivated at least every 3 days and preserved as soon as possible.

Many thanks, Jean-Francois, for your instruction. Surely, Alain will have something to add. I am wondering if you or anyone else have experience with FMM agar. How selective is it and does it prevent the overgrowing of saprophites from the skin or gills?

Another questions is regarding the primers designed to diagnose T. maritimum, MAR 1, MAR 2 (Toyama et al. 1996). Unfortunately, we had false positives results using mentioned set of primers.

Dear All,

Tenacibaculosis is considered as one of the main bacterial diseases of sea bass and sea bream in aquaculture.

Bacteriological analyses are performed from the kidneys, liver, spleen and external lesions of the skin of infected fish.

Mortality is high in sea bass weighing less than 60g

As for sea bream, Tenacibaculum is generally associated with Vibrio alginolyticus Vibrio.sp and Photobacterium.

Tenacibaculosis disease is also a mixed infection in association with a parasitosis such as sparicotylosis and enteromyxosis.

  Isolations are performed on seawater agar and soy agar (TSA) supplemented with 1.5% Na Cl and incubated at 22-24 ̊C for 48 to 72 hours. The identification of presumptive T. maritimum was performed using morphological, physiological and biochemical methods (API 20 E and API ZYM).

 In molecular diagnosis we have successfully used the primers MAR1 MAR 2 (Toyama et al 1996) however when we used  a nested PCR to confirm the species we didn't find any positive results.

thank you

Many thanks Kaouthar for sharing your experience. It seems that we have similar results with MAR1 and MAR2. Using this primers we used to confirm isolated bacteria as Tenacibaculum. However, when we sequenced isolates, they did not show similarity to Tenacibaculum, but to some Vibrio species.

Dear all,

Unfortunately, Dolors Furones had technical problems with registering. On behalf of Dolors I am asking several questions:

In the context of Sea bream and Sea bass  culture:

Is there evidences of Tenicibaculum  being part of biofilms in RAS’s biofilter  or in the  pipes in general ? If so,  how to treated the system before restocking. ( chlorine?) .

Is it probable that  TM survives long term in the RAS or open flow system , producing recurrent  outbreaks  when reloaded with  fish ?.

Is  there  information on the  level of prevalence  in asymptomatic carrier fish? .

Is  oxygen peroxide a good choice to  do preventive bath in fish stoked  both RAS or in open flow system? Which will be the dose range ( min to max) depending on the suspicions of having infected fish  or clean, fingerlings/ juveniles ; etc.

Is Tm  colony morphology very variable comparing  Marine agar with FMM ?, and the  cells  types observed?, can we see a wide range of  morphologies , from  colonies form apparently pure cultures?

I am hoping that Dolors will ovecome the technical issues soon and include in the discussion.

Dear all,

I would like to add my opinion and personal experience on some of the comments and questions previously issued in this forum. As you probably know, I am not a microbiologist and my knowledge on that is limited, so I only can say that when 'Flexi' is suspected in seabream or seabass, my choice media is FMM (using aged marine water) and when I detect any apparent growth I immediately send it to the specialized microbiology lab. Transportation is also very critical from my experience: bacteria growth potential (in broth and plates) can be easily affected by transport (I suspect temperature susceptibility is very important). Posterior isolation and identification are done by the lab. Results about species are similar to the previously indicated here by different colleagues: maritimum & dicentrarchi & discolor, but I am not able to establish clear differences between the pathogenic pattern of each one. 

Also from my experience, it is very difficult to find "flexi" from smears and also from histopathology is very difficult as Alain says, at least in seabream and seabass compared to other species (mainly flatfish). I have no explanation for that: different pathogenic trends and/or different species strains? 

Most 'pure flexi' cases are found in seabass postlarvae and juveniles: in these cases, the progression of the disease tend to be fast, in terms of mortality and also a progression from the skin to the dermis and muscular tissue. In the most severe cases sometimes I saw seabass still alive, swimming, without the caudal fin but with the caudal vertebrae visible and without muscle (quite creepy). 

In larvae and postlarvae the isolation is very difficult, as usually, the lesions present mixed infection with other bacteria (mainly Vibrionaceae) and frequently with accompanying long, thick, segmented-aspect filaments (bacteria? ).

Outbreaks can be found in open systems, even in tanks recently cleaned and disinfected so the previous presence of biofilms is not necessary. 

My experience about the use of H2O2 in treatments is a conundrum: I never know if the treatment will succeed or not: maybe too many different factors associated and many of them difficult to assess: water temperature, early stage or advanced stage of the disease, organic matter present...

I also would like, if possible, to set up another relevant topic: efficacy of vaccination. Some experimental vaccines / autogenous vaccines are available in different places, but the information about results is sometimes a 'black hole'. Surprisingly, most of the available information is from salmonids....

Best regards

Jean François is right. We usually sample from the border of the lesion under the skin or with tenacibaculum from under the scales. We avoid sampling from the necrotic parts of the lesions from where we will isolate several opportunistic bacteria which will cover the growth of the primary pathogen. 

Regarding the media used for transport, different soft media can be used (FMN, cytophaga agar,..). The main constraint is to avoid the growth of opportunistic bacteria and that is done by adding antibiotics in the transport media where the swab is put.

Then soft media can be used for isolation as well as salted TSA (or TSA prepared with sterile seawater)

Dear Kaouthar,

thanks for your valuable remarks.

Effectively using TSA prepared with sterile salt water allow growing Tenacibaculum, but soft media remains more selective. 

You can use morphological characteristic to identify the presence of Tenacibaculum sp. But to differentiate Tenacibaculum species, the best way remain the identification by mass spectrometry in routine work or by sequencing (longer and more expensive). Physiological and biochemical methods are not from what I experienced very performant to identify the strains at species level. 

Many thanks Sito for your comprehensive consideration. I just want to raise a question regarding the H2O2. I am not sure that it is a good solution to treat sea bass by bath. we had experience that H2O2 bath on the farm improve the course of the disease shortly, but at the same time remove the mucus from the skin and enable even more severe clinical outbreak.

Alain I'd like to ask you which antibiotics are the most appropriate for addition to transport media? Could you please, consider Dolor's questions. Maybe you have something to comment

Dear Fransec

Nice to hear you and you said almost every thing

Those segmented aspect filaments, could it be filamentous segmented bacteria such as Arthromyces?

Regarding vaccination, different autologus vaccines have been applied in marine species (solea, turbot, seabass) with various results as far as I experienced. But RPS are not very high even in lab trials (50 to 60% in the best cases). It helps reducing the prevalence of the disease especially if combined with C&D procedures.