It seems this disease is Enteric redmouth. Diagnostic techniques as staining bacteria by Gram's method and chemical methods are suggested.

What about differential diagnosis and which diagnosis method could be done as confirmation method?

The signs that you named, are according to signs of ERM. Such as lethargy, darkness of skin, hemorrhages around the mouth and isthmus, exophthamos, hemorrhages on internal organs and ... .

Agent of this disease is Yersinia ruckery that is a negative gram bacteria. This bacteria is differentiated from other negative gram bacteria by negative oxidase reaction.

Also other usoual laboratory tests, PCR, IFA and ... can help to diagnosis exactly.

All of the mentioned sings are characteristic for ERM caused by Yersinia ruckeri. As a representative of Enterobacteriaceae, I suggest isolation on BA, TSA or XLD agar, followed by Gram staining and API kit for identification. 

What was the water temperature during the outbreak? Above 15-160C or less?

Dear Aleksandar,

Thanks for your valued comment. Temperature in outbreak duration was 18-20oC.

Environmental parameters measuring revealed low water quality i.e.; pH = 5, high temperature (18-20 oC), excess ammonia (NH3 = 0.05 mg/L), dissolved oxygen (O2 = 4-5 mg/L), marginal carbon dioxide (CO2 = 8 mg/L) and H2S gas bubbling. The fish were also subjected to low quality feed, overcrowding, handling stress, and high aquatic plant growth.

This additional data are supportive for a yersiniosis outbreak. When you test the isolates for antibiotic sensitivity, I suggest to include fluoroquinolones and sulfa/trimetoprim combinations.

Dear colleagues,

What about commercial available vaccine? what do you think about application of some immunostimulators in prevention of similar outbreak in the future?

There are commercially available vaccines for Yersinia ruckeri, depending on the strain or variant. Some of them are AQUAVAC® ERM, AquaVac® Relera, Ermogen®, etc.

You can try producing an autogenous vaccine and test its effect.

Hello to all of you. In general terms, vaccine against Yersinia ruckeri shows very good result but I think that in terms of the time the use of commercial vaccine could be the first measure to prevent new outbreak in healthy population. However, other external factor should be resolved such as ERM is a exotic or enzootic disease in the country?, what about the rules in this terms?, if is exotic disease perhaps need to inform to the government to receive the authorization to import and use of commercial vaccine??? etc. Perhaps all of these things are not necessary and everything is simple to do it (?), but is important to know going on with all of these aspects. Anyway,  maybe in parallel could work in auto-vaccine depending efficacy of commercial vaccine, availability to do it, etc. Of course all of this in case Y. ruckeri be confirmed as causative agent of this disease, in such case the isolation, bacterial characterization and identification by biochemical, serological and mostly molecular confirmatory diagnosis is need. Considering the poor water quality condition described is need to exclude other opportunistic bacteria which in "special conditions" like this become pathogenic, mainly the high temperatures, as is the case of Aeromonas spp (ie Aeromonas hydrophila) or for instance Rainbow Trout Fry Syndrome (Flavobacterium psychrophilum) which looks be less feasible because not sign was mentioned in kidney (swollen).  Florfenicol is an excellent alternative for these cases, but it depends on the drug sensibility test.

Don't rule out Viral Hemorrhagic Septicemia or VHS. Which I hope isn't the case.  Signs described "hemorrhagic gills and fin rays areas, hemorrhaging into the eye orbits " can also be seen with VHS.

As you say: Intestine is inflamed and filled with a thick yellowish or whitish fluid is one of the importantant clinical signs of ERM caused by Yersinia ruckeri

Dear colleagues,

Let me appreciate all of you for your valued responses.

I agree with Dr.Patricio.

As he mentioned before, using of commercial vaccine against ERM is very critical and it depended on exotic or endemic feature of disease in the country. Unfortunately, ERM  could be considered as second important bacterial diseases in Coldwater fish farms in Iran. In fact, Yersinia ruckeri was isolated and identified in many provinces in the country. So, more study for comprehensive program is needed in order to identification and confirmation of main causative agent in order to prepare an effective and native vaccine against ERM disease in the country.