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Identification and treatment of diseases in the common sole (Solea solea L.)
Aquaculture 16 (1979) 271-274 o Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
271
Short Communication IDENTIFICATION AND TREATMENT OF DISEASES IN THE COMMON SOLE (SOLEA SOLEA L.) JtiRGEN
FLtiCHTER
Bayer&he
Landesanstalt
fiir Fischerei,
Starnberg
(Federal Republic
of Germany)
(Accepted 16 October 1978)
ABSTRACT Fliichter, J., 1979. Identification and treatment of diseases in the common sole (Solea solea L.). Aquaculture, 16: 271-274. Three bacterial Pseudomonas was furunculosis could could not because probably facilitate
diseases (tailrot, furunculosis and red-spot disease) are described. A associated with the first two and a Vibrio with the third. Tailrot and be cured by treating the fish with antibiotics, but red-spot disease of its very rapid progression and late on-set of signs. Ectoparasites the entry of disease organisms.
Long-term attempts to rear sole in captivity were defeated repeatedly by the extermination of the stock of sole, acclimatized to captivity, by disease epidemics and plagues of parasites. The development of methods to combat these calamities and to find prophylactic measures was therefore a prerequisite for continuation of the sole rearing experiments. Three main diseases occurred repeatedly: (1) An irritation starting at the edge of the caudal fin and progressing forward to the integument and muscle tissue. In advanced stages, the caudal fin was lost and spines of the body skeleton became visible. Generally, at this stage, about 2 or 3 weeks after the first irritation occurred, the fish died. Similar signs have been described for several other species by other authors and named “tailrot”. (2) Sole developed several swellings of about 10 mm in diameter, on both sides of the body, which burst to form open furuncles. This disease may thus be termed “furunculosis”. The sole survived with the abscesses for more than a month and sometimes the furuncles healed up spontaneously if the fish was transferred to clean water early enough. (3) While tailrot and furunculosis progress slowly and are identifiable early enough for treatment, there is a disease with few signs, manifest only in a haemorrhagic reddish discoloration of the blind side and in paleness of the gills. These signs appear only 2 days before death. This disease may thus be termed “red-spot disease”. As it was very contagious and progressed rapidly, it was the main danger to stocks of acclimatized sole.
272
The mortality
of acclimatized
sole caused by disease became
catastrophic
when many dabs had to be kept in the same small, closed water circulation. The organic pollution of the water by wastes from the dabs, which feed on fish, obviously encouraged the outbreak of diseases. As a first step in bringing these diseases under control, a bacteriological investigation was started. Firstly, the skin was sterilized by ethanol and burning, then the body cavity was opened under sterile conditions and a smear was taken with a platinum wire. Next, the pericardium was opened and blood taken under sterile conditions from the sinus venosus. Later on, kidney was included in this procedure. In the same way, smears were taken from the musculature and from the connective tissue between the musculature and the skin. A simple seawater nutrient agar, developed for the culture of non-pathogenic marine bacteria, was used as culture medium (Table I). Firstly, acclimatized fish with signs of disease were investigated. In all eases many colonies developed when the smears were taken from the body cavity, from blood and from the kidney. No colonies developed when the smears were taken from the musculature or from connective tissue. TABLE I Composition
of culture medium (Zobell 2216 E)
5 g Bacto peptone 5 g Bacto yeast extract 15 g Bacto agar 0.01 g FePO * 4Hz 0 750 ml Stale sea water 250 ml Distilled water (PH 7.6)
The type of bacterial colony was different depending on the disease. The moderate nutrient requirements of these pathogenic bacteria should be stressed. On the given culture agar, tailrot and furunculosis resulted in cream-coloured colonies about 2 mm in diameter, while red-spot disease was characterized by small (diameter ca. 0.5 mm), hyaline colonies with an acrid smell of purulence. Tailrot and furunculosis bacteria were determined as belonging to the Pseudomonas group. The organism causing red-spot disease was identified as a near relative of Vibrio anguillarum. Much larger colonies were obtained when an extract of tryptic digested fish tissue was added to the agar. To assess bacterial infection in the natural environment, sole freshly caught at sea and appearing healthy were included in the investigation. It was surprising that about 80% of these sole had a latent infection. Evidence that isolated bacteria were indeed the cause of death could be produced with certainty within 4 days of infection but the typical signs became visible only 1 or 2 days before death. Vibrios stored for 1 year on nutrient agar
273
were fully infective. This indicates the special danger presented by this organism. In-vitro methods had to be used to bring these contagious diseases under control by antibiotics, for adult acclimatized sole were too precious to use live in extensive contagion experiments. To measure the sensitivity of the pathogens to antibiotics, so-called sensitivity discs and stars were used: sterile paper discs containing known amounts of antibiotics. A single colony was evenly distributed upon an agar plate and the sensitivity disc or star was applied and incubated at 20°C (later at 25°C to obtain the results more rapidly). The antibiotics diffused into the nutrient agar and zones became visible where susceptible bacteria did not grow. In this way, the susceptibility of the pathogens to the different antibiotics could be determined. This test furthermore allowed a very rough (but very rapid and for this purpose sufficient) classification to be made, based upon specific sensitivity of the different pathogenic bacteria to antibiotics (Table II). The incitants of the diseases described here are all sufficiently susceptible to chloromycetin. However, chloromycetin could not be injected, because it is inadequately water soluble. Oral administration was complicated by the very rapid sedimentation of the chloromycetin-water suspension, which made exact dosage difficult. When chloromycetin was suspended in 1% carboxy-methylcellulose-water solution, the suspension was stable enough for exact dosage. While medical treatment of furunculosis and tailrot by the administration of 100 mg chloromycetin per fish was successful, the red-spot vibrio disease could not be treated in practice, even by those antibiotics successful in vitro. The rapid progress of this disease and the late appearance of its signs hindered control by antibiotics. Administration of chloromycetin to sole following vibrio infection even accelerated the death of the fish. The virulence of these vibrios enabled them to infect not only feeble sole but also those in excellent condition. TABLE II Sensitivity of the pathogenic bacteria to some antibiotics Antibiotic
Tailrot
Furunculosis
Red-spot disease
Penicillin Dihydrostreptomycin Ampicillin Tetracyclin Chloramphenical* Spirarnycin Kanamycin Colistin
t-)
++ +
(-) (+) (-) (-)
(-)
I?) :-) (+) ++
;=, -k-k + + ++
& (--) (-)
Resistant; +, sensitive; (+), sensitive but not all strains; +f, highly sensitive.
*Chloramphenical
= chloromycetin.
274
The ability of red-spot disease vibrios to survive for long periods and to multiply rapidly on organic wastes made them a steady menace to the experiments , to such an extent that prophylactic action was absolutely indispensable. Artificial immunization was attempted as a means of solving this problem. The injection of inactivated pathogens to cause specific defensive reactions against virulent pathogenic bacteria has been a proven method for many years. One method of gently inactivating pathogenic bacteria is to heat them up to 56°C for 1 h. The bacterial mass which overgrew a nutrient agar plate (about 60 cm2) - sufficient to dose one fish -was scraped off, suspended in 1 ml physiological saline solution, heated as described, and this vaccine finally injected intramuscularly. After vaccination, the sole did not feed and were very lethargic for 1 week. Two weeks after vaccination, the sole were infected by injection of a high dose of living vibrios. The dosage for each fish was the growth from half a petri dish (about 30 cm*) suspended in 1 ml sea water. Although such a massive infection never occurred in the natural environment, none of the 10 vaccinated soles died after artificial infection. In each of two experiments, five un-vaccinated sole were infected as a control. They all died within 4 days of injection. Observations suggest that zooparasites could be instrumental.in these diseases. By perforating the skin of the sole, they allow the pathogenic bacteria to invade the fish. The extermination of parasitic crustaceans and leeches was possible by bathing fish in 2,2,2-trichlor-l-hydroxy-ethylphosphoneaciddimethylester (Masoten Bayer, Leverkiisen), 25 g/l sea water, for 5 min. However, an ectoparasitic trematode, Entobdella solea, which was resistant to all usual chemical treatments, remained. Mechanical control was not practicable in the frequently occurring massive attacks by this parasite. Finally, a special warm water treatment was successful. When the temperature was raised gradually (3OC/day) to a maximum of 30°C, the trematodes disappeared, and the sole survived. The description of signs of diseases and methods of controlling bacterial and parasitic outbreaks may be a step in the direction of long-term screening of such diseases.
271
Short Communication IDENTIFICATION AND TREATMENT OF DISEASES IN THE COMMON SOLE (SOLEA SOLEA L.) JtiRGEN
FLtiCHTER
Bayer&he
Landesanstalt
fiir Fischerei,
Starnberg
(Federal Republic
of Germany)
(Accepted 16 October 1978)
ABSTRACT Fliichter, J., 1979. Identification and treatment of diseases in the common sole (Solea solea L.). Aquaculture, 16: 271-274. Three bacterial Pseudomonas was furunculosis could could not because probably facilitate
diseases (tailrot, furunculosis and red-spot disease) are described. A associated with the first two and a Vibrio with the third. Tailrot and be cured by treating the fish with antibiotics, but red-spot disease of its very rapid progression and late on-set of signs. Ectoparasites the entry of disease organisms.
Long-term attempts to rear sole in captivity were defeated repeatedly by the extermination of the stock of sole, acclimatized to captivity, by disease epidemics and plagues of parasites. The development of methods to combat these calamities and to find prophylactic measures was therefore a prerequisite for continuation of the sole rearing experiments. Three main diseases occurred repeatedly: (1) An irritation starting at the edge of the caudal fin and progressing forward to the integument and muscle tissue. In advanced stages, the caudal fin was lost and spines of the body skeleton became visible. Generally, at this stage, about 2 or 3 weeks after the first irritation occurred, the fish died. Similar signs have been described for several other species by other authors and named “tailrot”. (2) Sole developed several swellings of about 10 mm in diameter, on both sides of the body, which burst to form open furuncles. This disease may thus be termed “furunculosis”. The sole survived with the abscesses for more than a month and sometimes the furuncles healed up spontaneously if the fish was transferred to clean water early enough. (3) While tailrot and furunculosis progress slowly and are identifiable early enough for treatment, there is a disease with few signs, manifest only in a haemorrhagic reddish discoloration of the blind side and in paleness of the gills. These signs appear only 2 days before death. This disease may thus be termed “red-spot disease”. As it was very contagious and progressed rapidly, it was the main danger to stocks of acclimatized sole.
272
The mortality
of acclimatized
sole caused by disease became
catastrophic
when many dabs had to be kept in the same small, closed water circulation. The organic pollution of the water by wastes from the dabs, which feed on fish, obviously encouraged the outbreak of diseases. As a first step in bringing these diseases under control, a bacteriological investigation was started. Firstly, the skin was sterilized by ethanol and burning, then the body cavity was opened under sterile conditions and a smear was taken with a platinum wire. Next, the pericardium was opened and blood taken under sterile conditions from the sinus venosus. Later on, kidney was included in this procedure. In the same way, smears were taken from the musculature and from the connective tissue between the musculature and the skin. A simple seawater nutrient agar, developed for the culture of non-pathogenic marine bacteria, was used as culture medium (Table I). Firstly, acclimatized fish with signs of disease were investigated. In all eases many colonies developed when the smears were taken from the body cavity, from blood and from the kidney. No colonies developed when the smears were taken from the musculature or from connective tissue. TABLE I Composition
of culture medium (Zobell 2216 E)
5 g Bacto peptone 5 g Bacto yeast extract 15 g Bacto agar 0.01 g FePO * 4Hz 0 750 ml Stale sea water 250 ml Distilled water (PH 7.6)
The type of bacterial colony was different depending on the disease. The moderate nutrient requirements of these pathogenic bacteria should be stressed. On the given culture agar, tailrot and furunculosis resulted in cream-coloured colonies about 2 mm in diameter, while red-spot disease was characterized by small (diameter ca. 0.5 mm), hyaline colonies with an acrid smell of purulence. Tailrot and furunculosis bacteria were determined as belonging to the Pseudomonas group. The organism causing red-spot disease was identified as a near relative of Vibrio anguillarum. Much larger colonies were obtained when an extract of tryptic digested fish tissue was added to the agar. To assess bacterial infection in the natural environment, sole freshly caught at sea and appearing healthy were included in the investigation. It was surprising that about 80% of these sole had a latent infection. Evidence that isolated bacteria were indeed the cause of death could be produced with certainty within 4 days of infection but the typical signs became visible only 1 or 2 days before death. Vibrios stored for 1 year on nutrient agar
273
were fully infective. This indicates the special danger presented by this organism. In-vitro methods had to be used to bring these contagious diseases under control by antibiotics, for adult acclimatized sole were too precious to use live in extensive contagion experiments. To measure the sensitivity of the pathogens to antibiotics, so-called sensitivity discs and stars were used: sterile paper discs containing known amounts of antibiotics. A single colony was evenly distributed upon an agar plate and the sensitivity disc or star was applied and incubated at 20°C (later at 25°C to obtain the results more rapidly). The antibiotics diffused into the nutrient agar and zones became visible where susceptible bacteria did not grow. In this way, the susceptibility of the pathogens to the different antibiotics could be determined. This test furthermore allowed a very rough (but very rapid and for this purpose sufficient) classification to be made, based upon specific sensitivity of the different pathogenic bacteria to antibiotics (Table II). The incitants of the diseases described here are all sufficiently susceptible to chloromycetin. However, chloromycetin could not be injected, because it is inadequately water soluble. Oral administration was complicated by the very rapid sedimentation of the chloromycetin-water suspension, which made exact dosage difficult. When chloromycetin was suspended in 1% carboxy-methylcellulose-water solution, the suspension was stable enough for exact dosage. While medical treatment of furunculosis and tailrot by the administration of 100 mg chloromycetin per fish was successful, the red-spot vibrio disease could not be treated in practice, even by those antibiotics successful in vitro. The rapid progress of this disease and the late appearance of its signs hindered control by antibiotics. Administration of chloromycetin to sole following vibrio infection even accelerated the death of the fish. The virulence of these vibrios enabled them to infect not only feeble sole but also those in excellent condition. TABLE II Sensitivity of the pathogenic bacteria to some antibiotics Antibiotic
Tailrot
Furunculosis
Red-spot disease
Penicillin Dihydrostreptomycin Ampicillin Tetracyclin Chloramphenical* Spirarnycin Kanamycin Colistin
t-)
++ +
(-) (+) (-) (-)
(-)
I?) :-) (+) ++
;=, -k-k + + ++
& (--) (-)
Resistant; +, sensitive; (+), sensitive but not all strains; +f, highly sensitive.
*Chloramphenical
= chloromycetin.
274
The ability of red-spot disease vibrios to survive for long periods and to multiply rapidly on organic wastes made them a steady menace to the experiments , to such an extent that prophylactic action was absolutely indispensable. Artificial immunization was attempted as a means of solving this problem. The injection of inactivated pathogens to cause specific defensive reactions against virulent pathogenic bacteria has been a proven method for many years. One method of gently inactivating pathogenic bacteria is to heat them up to 56°C for 1 h. The bacterial mass which overgrew a nutrient agar plate (about 60 cm2) - sufficient to dose one fish -was scraped off, suspended in 1 ml physiological saline solution, heated as described, and this vaccine finally injected intramuscularly. After vaccination, the sole did not feed and were very lethargic for 1 week. Two weeks after vaccination, the sole were infected by injection of a high dose of living vibrios. The dosage for each fish was the growth from half a petri dish (about 30 cm*) suspended in 1 ml sea water. Although such a massive infection never occurred in the natural environment, none of the 10 vaccinated soles died after artificial infection. In each of two experiments, five un-vaccinated sole were infected as a control. They all died within 4 days of injection. Observations suggest that zooparasites could be instrumental.in these diseases. By perforating the skin of the sole, they allow the pathogenic bacteria to invade the fish. The extermination of parasitic crustaceans and leeches was possible by bathing fish in 2,2,2-trichlor-l-hydroxy-ethylphosphoneaciddimethylester (Masoten Bayer, Leverkiisen), 25 g/l sea water, for 5 min. However, an ectoparasitic trematode, Entobdella solea, which was resistant to all usual chemical treatments, remained. Mechanical control was not practicable in the frequently occurring massive attacks by this parasite. Finally, a special warm water treatment was successful. When the temperature was raised gradually (3OC/day) to a maximum of 30°C, the trematodes disappeared, and the sole survived. The description of signs of diseases and methods of controlling bacterial and parasitic outbreaks may be a step in the direction of long-term screening of such diseases.