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Tenacibaculosis in marine fish - 11 - 22 February 2019  

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(@snjezana)
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Joined: 6 years ago
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Dear MedAID Health Forum members,

After a pause, we are starting today with a new topic, tenacibaculosis, the disease of sea bass and sea bream indicated as one of the most serious problems in Mediterranean aquaculture. Jean-François Bernardet and Alain Le Breton will moderate this topic, but even more, they will share their knowledge and practical experience with us. I am sure that there are a lot of questions regarding the choice of proper diagnostics methods, prevention, epidemiology and treatments to be discussed. Therefore, go ahead, start with your questions but also, you are all kindly requested to share your own experience with us.


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(@zeljko)
Joined: 5 years ago
Posts: 2
 

Hi everyone!

To start the discussion, I'd like to know which diagnostic procedures do you employ in your lab to diagnose tenacibaculosis?

 

 


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(@jfbernardet)
Joined: 5 years ago
Posts: 7
 

I usually receive in my lab, not diseased fish, but pure bacterial strains that have been isolated by French or foreign aquaculture veterinarians or bacteriologists. Among the several Tenacibaculum species that are pathogenic to marine fish, T. maritimum is the one that has been known for several decades (mid-seveties in Japan), the most common and the easiest to identify, essentially thanks to its colony morphology, on which I very much rely. Colonies on marine agar (e.g., Marine Agar 2216 from Difco) at 25-28°C are cream to very pale yellow, irregular, with rhizoid margins and a mucoid consistency. The most particular trait, however, is the fact that colonies display varying degrees of adherence to the agar; they can sticky and in some cases they are nearly impossible to remove from the agar without taking some agar together with bacterial growth, or they may be removed as kind of a membrane. In any case, what is removed from the agar is impossible to resuspend in a liquid. In Marine Broth 2216, bacterial growth is seldom homogenous; rather, it forms many clumps as well as a ring of growth at the meniscus. Cells are rods 2 to 10 micrometers long but filamentous cells may also occur. Agar and broth cultures degenerate within a few days; in broth cultures, rods are progressively replaced by round degenerative forms ("speroplasts"). The odour of bacterial growth is also characteristic. Other Tenacibaculum species are rather similar but colonies do not adhere to the agar and their yellow colour is usually darker than that of T. maritimum. We use 16S rRNA sequencing to identify the isolates at the species level. We also have developped Multi Locus Sequence Typing and Maldi Tof, but these are used to type the strains for epidemiological applications, not for identification. I forgot to mention that rods of most Tenacibaculum species usually display gliding motility in broth cultures. Though not so easy to observe and not consistent, this particular type of motility can help with identification.

This post was modified 5 years ago by jfbernardet

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(@snjezana)
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Joined: 6 years ago
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Topic starter  

Many thanks for the very interesting information. I am sure that someone from our registered members will share with us what are challenges of proper sampling and isolation of the Tenacibaculum species.

 

This post was modified 5 years ago by Snježana Zrnčić

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(@zeljko)
Joined: 5 years ago
Posts: 2
 

Thank you dr. Bernardet for the information! I'm also interested in which primers do you use for Tenacibaculum species identification? Do you use "standard" universal primers (e.g. 16S_27F and 16S_1492R), do you use them with some modifications or something else entirely?

I would also very much like to hear the answers to dr. Zrnčić last post 🙂

This post was modified 5 years ago by Zeljko

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(@snjezana)
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Joined: 6 years ago
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Topic starter  

Dear Forum members,

With the aim of stimulating discussion, the topic moderators would very much appreciate your answering the following questions:

1. In France, the survey done within the PathoTrackfish on Tenacibaculum infections has demonstrated that almost 50% of the species isolated during clinical outbreaks were not Tenacibaculum maritimum. Have you ever noticed any differences in the clinical symptoms of Tenacibaculum infections which could suggest that different species are present in your area or your country?

2. Co-infections (with different species of Tenacibaculum or with Vibrio, such as Vibrio harveyi) are being reported, which can affect the therapy efficacy. Have you noticed any increasing pitfalls of treatments which could be eventually explained by co-infections?

3. When performing a cost-benefit analysis for Tenacibaculum infection in your epidemiological context, would you consider developing a vaccination a worse strategy?

4. Would you propose any other issue related to this topic (Tenacibaculum) that has not been addressed and that you would like the moderators to address?

All members who want to discuss about above-quoted topics in the forum are very welcome. However, those who prefer to answer the questions using the completely anonymous form are kindly requested to visit the following site and answer the questions:

https://docs.google.com/forms/d/1Nz-N7gang08fyoLodHxS8gnyZwYRFyfxxLPH5wqF7Qw/edit?usp=sharing

Your inputs will be transferred into the discussion. Once again, I'd like to point out that all registered members are welcome to answer the question in mother tongue and MedAID partners will translate it to English to become available to others.

 

This post was modified 5 years ago by Snježana Zrnčić

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(@le-breton)
Joined: 6 years ago
Posts: 39
 

Thank you Jean François for your detail answer; 

To answer one previous question, the first challenge is to get to the lab a proper sample. it is quite easy to run a field diagnosis and observe the presence of Tenacibaculum from lesions , looking at fresh smears from the lesions and eventually staining the smears with Gram stain or MGG (quick coloration RAL555 for example). It does not allow to identify which tenacibaculum species are present. The challenge is to send them viable to the lab as we often loose them during transport or they are overgrown on culture by other colonies and not easy to isolate. 

It is why swabs with specific transport media have been developped which make life much easier.

When received in the laboratory and isolated , identification of the various isolates of Tenacibaculum  can be done with a good accuracy  by mass spectrometry (Maldi toff) allowing to differenciate nowaday most pathogenic species (maritimum, discolor, aesturiani, gallaicum, soleae, dicentrarchi)

Most of the time it is very difficult to differentiate them from clinical symptoms and rather difficult if not experienced from the phenotypic caracteristic of the culture. Moreover, we often are in presence of co-infection with two or three species of tenaci. 

This post was modified 5 years ago 3 times by Snježana Zrnčić

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(@jfbernardet)
Joined: 5 years ago
Posts: 7
 

I think we must be very careful about the way we use the terms "strain", "isolates" and "species". One bacterial species comprises many strains that can be distinguished (i.e. typed) according to a variety of phenotypic (serotypes, morphotypes, etc) or molecular characteristics (for instance Multilocus Sequence Typing = MLST). It has been shown that different pathogenic Tenacibaculum species (e.g., T. maritimum and T. mesophilum) can occur on the same individual fish, but so far it has not been formally demonstrated whether different serotypes or MLST types of the same Tenacibaculum species may be retrieved from the same fish. Hence, when several isolates belonging to the same bacterial species are retrieved from a single or several fish during an outbreak, it is usually assumed that they merely represent different isolates of the same strain.

This post was modified 5 years ago by Snježana Zrnčić

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(@snjezana)
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Joined: 6 years ago
Posts: 41
Topic starter  

Many thanks, Jean François, for your comment. I've corrected all our mistakes to avoid confusion among the readers.

 


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(@le-breton)
Joined: 6 years ago
Posts: 39
 

Yes jean françois,I should be more precise with those terms when I write

One remaining interrogation I have  is the occurence of recurrent outbreaks after biocid immersion treatment. Does that mean that some strains are developping resistance or that we are in presence of septicemic forms or at least internal organs infection requiring a systemic treatment? 

I remember jean françois that once looking at the spleen we found  some long rot bacteria in the spleen of such infected fish with Tenacibaculum suffering from a recurrent infection. Most of the time in diagnosis procedures we are looking at skin or lesion samples on fresh smears but not at internal organs (organ print or culture). Will that means that systemic infection are more frequent than we suppose and that in case of recurrent infection, we should lsample internal organ to retrieve the bacterium. In that case, it might be of interest to compare the strains isolated from external lesions with the strains eventually isolated from internal organs.

Is anyone having detected some septicemic form of Tenacibaculum infection? 


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(@jfbernardet)
Joined: 5 years ago
Posts: 7
 

When reading the literature on T. maritimum, there are very few instances when the bacterium was isolated from internal organs. However, it is not clear whether this is due to the fact that people preferentially sample external lesions rather than internal organs (as Alain said) or because the bacterium indeed seldom causes septicemia. I think the only way to know is to sample internal organs more frequently in addition to external lesions. If the bacterium is found in both types of samples, then it would indeed be most interesting to compare them using some kind of typing method to know whether different phenotypes or genotypes of T. maritimum may occur outside and inside the same individual fish. Recurrent outbreaks could be due to bacterial cells that either persist in internal organs or remain hidden and protected from biocids in biofilms at the surface of the farming environment (tanks, sea cages...).


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(@le-breton)
Joined: 6 years ago
Posts: 39
 

Is there anyone having sampled internal organs during outbreaks and experienced septicemic forms. That will be very interresting to share that experience and to progress on the prophylactic and currative methods to control Tenacibaculum infections; 


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(@snjezana)
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Joined: 6 years ago
Posts: 41
Topic starter  

Unfortunately, we have no answer to your question Alain. However, I am sure that many colleagues are reading comments and I am very sorry that they do not want to ask questions. It is widely known that bacteria from the genus Tenacibaculum are causing problems worldwide and it will be useful for all us to share experiences. I am curious very much how does infection with Tenacibaculum look like in salmon farming?

Inputs from Northern European colleagues are highly appreciated.


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(@le-breton)
Joined: 6 years ago
Posts: 39
 

I agree with you Snjezana. 

It is quite interesting to notice that tenacibaculum is spread worldwide but not much information from the field in cold water species. 

And we still have a lot of question regarding, for example, the reservoir of the bacteria. Some studies have shown that jellyfish were the carrier of Tenacibaculum in their buccal part: could that be a reservoir of the pathogen in some cases; 

We have been looking at the presence of the pathogen in the biofilm which is considered as the main reservoir of the bacterium in a land-based farm. As shown in fresh water with Flavobacterium, strong and accurate cleaning and disinfection procedures help to reduce the prevalence of the disease. And we are currently testing the use of bacterial barrier flora to re-inoculate the biofilm prior introduction of the fish, which is showing some promising results. Has anyone done similar tests or have some experience to share in that field of prophylactic approach?

This post was modified 5 years ago by Snježana Zrnčić

toffana liked
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(@manfrina)
Joined: 5 years ago
Posts: 1
 

Dear all, nice to hear some comments from international experts... we have isolated a few tenacibaculum in the last 10 years as usually our colleagues, belonging to the feed companies, with a simple smear and according to clincial symptoms and fish farmers information can give them the diagnosis: flaxibacteriosis (easier to pronounce than tenacibaculosis  😊 )

We usually found Tenacibaculum from skyn lesion in Marine Agar, but also T. discolor and T. mesophilum from head kidney, brain and spleen (sea bass coming from Turkey) and T. maritimum/discolor from spleen in sea bass juvenile from our wet facilities....but I'm speaking about a few specimens in ten years so without any epidemiological value.

all the best

 

amedeo


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