FRY DISEASES: INTRODUCTION TO THEIR OBSERVATION, ANALYSIS AND FIRST TREATMENT (E-book)

When the cause of fish mortality is a bacterial disease (Annex 30), any successful therapy requires the identification of the pathogen. Administering drugs without knowing the etiological agent may result in a total failure in the treatment and in the selection of drug resistant strains.
Most fish farmers are not trained to diagnose bacterial diseases. However, a fish pathologist is not often at hand, nor it is always possible to send samples to a certified diagnostic laboratory in a reasonably short time. In these cases, the advantage of being able to carry out a rapid initial screening of disease possibilities should convince the farmer of the necessity to acquire some basic knowledge and tools to perform an initial analysis by himself.
This section provides a minimal basic knowledge to observe the main bacterial diseases in farmed seabass and gilthead seabream. It should be pointed out that this section does not intend to replace the experience of a professional fish pathologist, who bears the final responsibility as the only source of reliable advice and expertise in this field. However, if properly performed, such initial investigations can provide an amount of information that often becomes essential in the decisional process of a professional that has to decide rapidly upon a suitable therapy in case of acute outbreaks associated with mass mortality.
In examining fish from an affected population, standard operating procedures must be adopted to avoid mistakes and to facilitate the exchange of information among farmers and specialists. These standard operating procedures can be summarised as follows:

• collection of environmental data (main parameters);
• observation of fish behaviour;
• in vivo bacteriological observation (smears of possible lesions, blood, skin, gills, spleen and kidney, either stained or fresh),
• in vitro bacteriological examination (cultures on Petri dishes of samples from spleen, anterior kidney, dorsal aorta and swim-bladder),
• identification of the pathogenic bacterium and culture of a pure strain,
• screening essays to identify which drugs are effective on the pathogen,
• selection of a suitable therapy.

In vivo tests to trace bacterial pathogens on the skin surface and
in some of the internal organs, can be a quick and often decisive step to provide a first answer for many diseases. Then, the in vitro culture from a sample of the most infected organs may be carried out. Most bacterial colonies develop after 24 to 36 h and their examination under a microscope (x1000 with oil objective) can be helpful to prepare an antibiotic essay. In this way, it should be possible to determine the most appropriate treatment 48 hours after having observed the presence of the disease for the first time.
Minimum time required for therapeutic treatment determination

• In vivo examination of skin and main organs 1 h
• In vitro culture of sample in Petri dish +24 h
• Antibiotic assay results +48 h

To diagnose a disease, identify the responsible bacterial pathogen and determine the most efficient treatment for the fish proceed as follows:

1. sample at least 10 to 30 moribund fish;
2. examine their external surfaces and wounds;
3. obtain some skin and gill scrapings; examine (after staining) and culture samples in vitro;
4. examine main internal organs looking for: appearance and presence of lesions. In particular observe and culture in vitro samples of spleen, kidney, and stomach/intestine contents. Take samples for analysis of bacteria and viruses, to be performed later by a specialist;
5. examine results of in vitro cultures: type of colonies; in vivo microscopical study; staining;
6. perform an antibiotic test.

Studying lesions at the skin surface

Several opportunistic bacteria can be found in the skin surface together with pathogenic bacteria and it is very difficult to distinguish them. It is best to reduce your study to ascertain the presence of Myxobacteria (immobile bacillus, Gram negative). For this, proceed as follows:

1. scrub the lesion with a cover slide; place the latter together with the scraping over a slide; observe the presence of bacteria under a microscope (x400 and x1000 with oil immersion);
2. take off the cover slide and dry the material over a Bunsen burner. Make a Gram stain;
3. take a Pasteur pipette, break the tip and sterilize on the burner flame; let it cool;
4. obtain a first sample for in vitro culture:

• using sterile tweezers, lift one margin of the skin lesion, detaching it from the muscle; introduce the pipette between lesion margin and muscle and suck some liquid;
• place one drop on a TSA/salt (Tryptophan Soya Agar with 1.5 percent NaCl) culture medium in a Petri dish;
• incubate at ambient temperature (20 to 25°C) for 24 to 48 h.

5. obtain a second sample for in vitro culture following the instructions given in the previous point;
6. in the two sample cultures, observe the growth of germ colonies for colour, size and shape.

Observing internal organs of diseased fish

Open the fish abdomen:

1. using sterile scissors, make a first cut just in front of the pectoral fin, at ventral line level;
2. cut along the ventral line, stopping just before the anus;
3. return to the first cut and continue to open in the direction of the lateral line, cutting around the pectoral fin;
4. continue to cut along the lateral line until you reach the previous ventral cut at the anus;
5. remove the piece of skin and flesh;
6. carefully observe the appearance of the swim bladder;
7. using sterile tweezers pull swim bladder out and move it forward, to give access to the kidney.

ATTENTION

• swim bladder and kidneys are sterile inside; avoid opening them and always use sterile equipment;
• do not cut open the digestive tract;
• do not open any nodule.

Then observe internal organs for abnormalities:

1.	Loss of colour(LOC)	colour intensity is far below standard for a given organ
2.	Atrophy (ATR)	significant decrease of the volume of a given organ
3.	Hypertrophy (HYP)	significant increase of the volume of a given organ
4.	Red Spots (RSP)	presence of small red spots, easily identified
5.	Redness (RED)	presence of an abnormal red stain over part of or the whole surface of an organ
6.	Haemorrhage (HAE)	presence of blood at the surface of an organ
7.	Nodule (NOD)	an identifiable mass in an organ having such a consistency and a colour that it can be clearly distinguished from that organ tissue
8.	Ascitis (ASC)	presence of liquid, usually clear, in the abdominal cavity

Look for the presence of these abnormalities according to the following example:

Abnormality (see above)
Location	LOC (1)	ATR (2)	HYP (3)	RSP (4)	RED (5)	HAE (6)	NOD (7)	ASC (8)
Abdominal cavity	-	-	-	-	-	-	-	*
Swim bladder	-	-	-	*	*
Digestive tract	-	*	-	*	*	-	*
Kidney	*	-	*	*	*	*	*
Liver	*	*	*	*	*	*	*
Muscle	-	-	-	*	*	-	*
Pylonic caeca	-	-	-	*	*
Spleen	*	*	*	-	*	*	*

Record all the information described following as much as possible the above mentioned procedures (see Annex 31) and contact a fish pathologist to proceed further.