Bull. Eur. Ass. Fish Pathol., 30(5) 2010, 160
Severe mortality in common sole (Solea solea) at post-weaning stage following environmental stress
A. Paolini1*, G. E. Magi2, L. Gennari3, C. Egidi1, M. Torresi1, M. Vallerani1, M. Berti1, A. Pavone1 and G. Giorgei1
1Istituto Zooprofila ico Sperimentale dell’Abruzzo e del Molise “G.Caporale”, Teramo, Italy; 2 Dipartimento di Scienze Veterinarie, Facoltà di Medicina Veterinaria, Università di Camerino, Matelica, Italy; 3 Consultant
Abstract Common sole (Solea solea) is one of the most interesting species for diversification of aquaculture production in the Mediterranean area, but production of sole on an industrial scale has not yet begun. In the present study, larval rearing was performed in 1 cubic meter tanks with a re-circulating system, using the “green water” technique. AVer an initial mortality occurred during the first 10 days of larval rearing, no particular mortality was registered during weaning period until 69 dph (day post hatching). On 66 dph, because of technical problems, fish were transferred in a new tank with open system where temperature control was not possible and water temperature increased from 19°C to 24 °C in three days. Four days later and for the following ten days we registered a mortality rate of 84.5%. Microbiological analysis revealed the absence of Betanodavirus and the presence of Vibrio anguillarum O2 and of Tenacibaculum maritimum; the former represents the first isolation on common sole in Italy. Histology revealed necrotic-haemorrhagic lesions in brain, kidney and gut, and severe skin erosions, the laer especially on fish collected at the end of the outbreak. Moreover the pathogens have been detected by immunohistochemistry (IHC). This severe mortality could be interpreted as a multi-factorial event where post-weaning weakness, manipulation, higher temperature and pathogens could have had a synergic effect. The extreme stress susceptibility of S. solea, pointed out in this and previous papers is discussed.
Introduction Common sole (Solea solea) is one of the most viral encephalopathy and retinopathy (VER), in interesting species for mariculture diversification fact VER outbreaks with mass mortalities were in the Mediterranean area, but this species is not reported, in the farm where broodstock parents currently cultured in Italy on an industrial scale. of larvae described in this paper spawned During the last ten years, the major limiting (Borghesan et al., 2003) and breeders have to factor for farming development resulted in be considered as potential carriers of the virus the difficult to wean sole larvae (Imsland et al., (Zanella, 2004). 2004). Moreover, S. solea is highly susceptible to
* Corresponding author’s e-mail: [email protected] Bull. Eur. Ass. Fish Pathol., 30(5) 2010, 161
The present work, performed in the experimental algae were added once a day. Enriched rotifers hatchery of the “Istituto Zooprofilattico (INVE Culture Selco and DHA Protein Selco) dell’Abruzzo e del Molise” (IZSA&M) in were added every two hours in order to maintain Pineto (Teramo, Italy), describes a severe a constant concentration of 10-20 rotifers/litre. mortality in post-weaning stage common sole From 20 dph artemia nauplii (INVE AF 430 μm during experimental feeding trials. During the artemia) were added 6 times a day in order to experiment the organogenesis and the possible maintain a constant concentration of 0.5-1 A1/ presence of abnormalities and/or pathological litre. In the second feeding period, from 26 dph conditions were monitored by means of to 58 dph, feeding was carried out with enriched virological, bacteriological, histological and metanauplii (INVE EG artemia and A1 DHA immunohistochemical analysis. Selco) and an artificial diet (Ewos Aglonorse). Artificial diet distribution and weaning started Material and methods respectively on 33 dph and on 51 dph. Artemia Larval rearing and weaning conditions supply stopped on 58 dph (Table 1). Water flow Two batches of eggs, each obtained from the in the tank was progressively increased from 2 to spawning of 3 successive days, were used in 4 litre/min. Water renewal in the system ranged this experiment. Egss were obtained from a between 10 and 20% per day. Central aeration group of 10 breeders with 1:1 sex ratio. During was provided and oxygen concentration was 7.2 the spawning period, the farm which supplied ± 0.3 mg/l. the eggs experienced water quality problems (turbidity and +/- 1° temperature variations) On 66 dph, because of technical problems, fish which had a negative impact on broodstock and were transferred in a new tank, from closed eggs. system to an open one without temperature control. Water temperature increased from 17.9°C Larval rearing and weaning were conducted in ± 0.83 to 22.2°C ± 1.9 °C in three days (Figure 1). two 1 cubic meter circular and flat boom tanks, with re-circulating system and temperature Health investigation control (17.9 °C ± 0.83 °C). Larval densities at Virus hatching were 50 and 75 larvae/litre respectively. Eggs samples, collected before introducing them On day 2-3 dph (day post hatching), at first to the experimental hatchery, then larvae and feeding, larval density in the two tanks was fish were checked for Viral Encephalopathy reduced respectively to 15 and 30 larvae/litre. and Retinopathy (VER), caused by a virus of During the first week of feeding, mortalities did Betanodavirus genus, on 0 and 10 dph and during not stop and in consideration of the low density mortality occurrence on 73 dph. Each sample in the tanks, the two batches were transferred in was made of 50-100 eggs or animals. Confluent the same tank at 12 dph. SSN-1 monolayers grown in EMEM at 25°C were inoculated with homogenated material The first feeding period, from 3 dph to 25 dph, from embrionated eggs or larvae according to was carried out with the “green water” method standard methods (Maltese and Bovo, 2007). The and a 16 hours daily lighting. Nannochloropsis same material was processed in parallel by nested Bull. Eur. Ass. Fish Pathol., 30(5) 2010, 162
Table 1. Larval rearing and weaning conditions.
Period Feeding Total (day post (Feed quality and Total length Number Mortality hatching) quantity) (mm) of fish (%) Observations 63.500 (1 1 and 2 dph Small and slow 1-2 2.09 (3 dph) dph) 63.8% swimming larvae Swim-bladder From 3 to 21 dph From 3 to 12 development Algae Nannochloropsis sp. 23.000 dph 300.000-750.000 cell/cc 5.25 (11 dph) (3 dph) From 20 dph Rotifers Brachionus 74.0 % boom swimming plicatilis 3-25 7.19 (18 dph) behaviour 7.5-25 rotifers/cc 7.66 (21 dph) From 13 to Metamorphosis From 20 to 25 dph 5.970 25 dph with eye migration Artemia nauplii 8.64 (25 dph) (12 dph) and pigmentation 0.5-1 A /cc 0 16.2 % process
From 26 to 58 dph 10.19 (28 dph) Digestive track Enriched Artemia From 26 to differentiation 0.5-1 A /cc 11.46 (33 dph) 1 5.005 59 dph 26-59 (26 dph) End of From 33 dph 14.15 (40 dph) 22% metamorphosis Artificial diet 16.25(57 dph)
3.900 on 60 dph From 60 to 65 dph 5.1% Artificial diet 17.79 (63 dph) 60-82 3.700 on regular increasing 66 dph 18.28 (69 dph) From 66 to On 66 dph transfer 82 dph in an open system 570 on 84,6% tank 82 dph
PCR to detect the presence of Betanodaviruses organs (kidney, liver, spleen) and diluted and (OIE, 2006). In addition formalin fixed larvae was homogenized in phosphate buffered saline processed for detection of betanodaviruses by (PBS). The suspensions were inoculated onto immunohistochemistry (Mutinelli et al., 1998). non-selective medium (Columbia blood agar containing 2% NaCL) and selective medium Bacteriology TCBS (Thiosulfate Citrate Bile Sucrose Agar, Twenty post-larvae collected during mortality Merck). Mucous obtained from skin scrapings (at 75-80 dph) were examined. Samples for was also plated on FMM (Flexibacter Marine culture were aseptically obtained from internal Medium) (Pazos et al. 1993). The isolated strains Bull. Eur. Ass. Fish Pathol., 30(5) 2010, 163
Figure 1. Larval growth and rearing conditions. Bull. Eur. Ass. Fish Pathol., 30(5) 2010, 164 were identified using standard morphological, described (Magi et al., 2007). Briefly, sections physiological, biochemical probes and API were incubated with 3% H2O2 (45 min) in order to 20-E miniaturized system, BioMérieux, France block the endogenous peroxidase activity. AVer (Table 2). Phenotypic tests were performed microwave antigen retrieval treatment (600 W), as previously described (Bauman et al., 1984; the slides were incubated overnight in a moist Austin and Austin, 1999; Buller 2004). All the test chamber at 4°C with the following antibodies: media were incubated at 22°C. The serological anti-V. anguillarum O2 (polyclonal antibody characterization of the Vibrio anguillarum strain Microtek, Canada) and anti-Tenacibaculum isolated was carried out by slide agglutination maritimum (polyclonal antibody). DAB solution test (Toranzo et al., 1987). Three polyclonal was used as chromogen and the sections were antisera, obtained as described by Larsen et al. counterstained with haematoxylin. (1994) against the representative V. anguillarum strains of serotypes O1-O3, were employed. Heat Results stable O-antigens were obtained by heating the Larval rearing and weaning bacterial suspensions (109 cells mL-1) at 100°C For the two eggs batches in this study, the hatching for 60 min. A drop of O-antigen suspension was rate was estimated at about 70%. Because of the mixed with a similar volume of the 1:10 diluted poor quality of the eggs, and due to the transport rabbit antiserum on a multi-well glass slide. conditions, post-hatching mortality was 63.8% Controls were made using PBS and rabbit serum (mean) at 3 dph. During the first week of feeding, normal (Vector). The observation of a strong and mortalities did not stop and reached 74% (mean) rapid agglutination was considered as a positive of total first feeding larvae. result. In both tanks, feeding was regular from the Histology beginning and 100% of larvae presented full Larvae samples were collected on 0, 1, 3, 8, 17, digestive tracts from 2 days aVer the start of 27, 31, 41, 45, 53, 58, 71, 73, 75, 80, 81 dph. Each exogenous feeding. From 11 dph we could sample was constituted of 25 larvae (0-17 dph observe a continuous and regular growth (Table samples), 15 larvae (27–45 dph samples) and 1 and Figure 1), normal developmental features, 10 (31-81 dph samples). Animals were stored in homogenous pigmentation of all fish and low Bouin fixative and then embedded in paraffin daily mortality (less than 0.5-1.5%). wax for histological study of larval development; 4 μm thickened sections were stained with On 66 dph, the fry were transferred to a new haematoxylin and eosin (Bucke, 1989). tank, from closed system to open ones with some changes in rearing conditions and a water Immunohistochemistry (IHC) temperature increase from 19°C to 24°C in three AVer the mortality episode, larval histological days. Furthermore the handling during transfer samples, kept in Buoin fixative, were used to detect was considered to be stressful for the fish because V. anguillarum O2 by means of IHC techniques of problems in harvesting for some of them. with the avidin-biotin-peroxidase complex Four days later and for the following 10 days, method (Vector Lab., U.S.A.) as previously a total 84.5% mortality was registered (Table 1 Bull. Eur. Ass. Fish Pathol., 30(5) 2010, 165 and Figure 1). Clinically moribund fish showed reddening areas of the head.
Health investigation Virus All samples processed by cell culture isolation, nested PCR and immunohistochemistry for detection of Betanodaviruses were negative.
Bacteriology From the internal organs of 15 larvae only one type Figure 2. Solea solea, 80 dph, necrotic epidermis with cluster of filamentous bacteria, HE, Bar 12,5 of colony could be observed on TCBS agar aVer μm. 48h incubation, whereas there was polymicrobial growth from samples inoculated onto Columbia blood agar. The bacteria which grew on TCBS 71 dph: increase of melanophores and mucous was identified as V. anguillarum serotype O2 cells in the skin; edema and congestion of by phenotypic characterization (Table 2). The interstitial tissue of kidney together with biochemical and physiological characteristics of degenerative changes in renal tubular cells. the bacteria isolated from the internal organs are given in Table 2. Serological characterization of 73 dph: increase of melanophores and mucous this isolate by slide agglutination assay showed cells in skin; worsening of edema and congestion only a strong reaction between the antiserum of interstitial tissue in kidney associated with raised against the V.anguillarum serotype O2 areas of tubular necrosis. and the O-antigen extracted from the isolated demonstrating that the V. anguillarum isolates 75 dph: increase of melanophores and mucous belonged to this serotype. From two larvae with cells in skin together with erosion condition of skin erosions pure culture of bacterial isolated epidermis; necrosis of gill epithelium associated were obtained on FMM. The morphological with severe congestion and heterophil and biochemical charatcteristics of this isolated infiltration; brain characterized by extreme allowed their presumptive identification as T. dilatation of vessels and hemorrhagic areas maritimum close to meninges; severe tubular necrosis and depletion of haematopoietic tissue was observed Histopathology in kidney; severe diffuse necrotic changes were Samples collected on 0, 1, 3, 8, 17, 27, 31, 41, 45, seen in the gut together with sloughting of the 53, 58 dph, showed normal tissues. In the tissue associated mucosa and presence of masses of of fish sampled on 73, 75 dph, severe septicaemic bacteria spread all over the tissue. lesions were observed, while in sample of 80, 81 dph skin erosion affected the survivals (Figure 80 dph: severe increase of melanophores and 2). The main histopathological findings observed erosion of epidermis associated with heterophils were the following. and macrophages infiltration were observed in Bull. Eur. Ass. Fish Pathol., 30(5) 2010, 166
Table 2. Biochemical characteristics of the bacteria isolated. nt = not tested
Vibrio anguillarum Tenacibaculum sp. Gram stain - - Cell shape rod Long slender rods Motilità + - Cytocrome-Oxidase + + Catalane + + O/129 (150 ug) sensitive nt O/F F O Arginine dihydrolase + - Lysine decarboxylase - - Ornitihine decarboxylase - - ONPG + nt Gas production from glucose - - Indole + - Voges Proskauer + - Nitrate reduction + + Simmon’s citrate - nt Urea hydrolysis - nt Gelatinose + + Aesculin hydrolysis - - Amilase + -
H2S production - - Flexirubin production nt - Congo Red nt + Fermentation of: Inositol, , - nt Sucrose + nt Sorbitol - nt Arabinose - nt Lactose - nt Mannitol + nt Maltose + nt Mannose + nt Growth on: nt T.C.B.S. + nt Growth at 4°C - - 28°C + + 37°C + - 0% NaCl - nt 2% NaCl + nt 6% NaCl + nt >8% NaCl - nt ++------+++++---+-+++ API 20 E results nt (3047127) Bull. Eur. Ass. Fish Pathol., 30(5) 2010, 167 the skin; in the subcutaneous muscle layer areas myolisis together with clusters of filamentous bacteria were ascertained.
Immunohistochemistry (IHC) Fish younger than 73 dph were negative, V. anguillarum serotype O2 was firstly ascertained in the gut, both in the lumen and aached to the epithelium (Figure 3), of the fry sampled at 73 dph. Brain and liver were also positive for V. anguillarum O2 in the sample collected at 75 dph Figure 3. Solea solea 73 dph, gut. Presence of numeorous bactria immunoposite for Vibrio (Figure 4). anguillarum. O2 in the intestinal lumen and aached to the necrotic mucosa IHC. Bar, 8 μm. In fish outliving the Vibrio outbreak, clusters of filamentous bacteria, penetrating derma and involving the subcutaneous muscle layer, demonstrated in other flatfish (Scophthalmus resulted immunopositive for the presence of T. maximus).Recent study suggested that maritimum. glycosphingolipids on the intestinal mucosa of rainbow trout act as a receptor for V. anguillarum Discussion (Irie et al., 2004). The vibriosis described in the present study occurs aVer a 65 days of flawless larval rearing At the end of the outbreak few soles showed signs and weaning. It occurred after a stressful of marine tenacibaculosis, an important disease transfer and the consequent changes in rearing of marine fish caused by T. maritimum (formerly conditions. V. anguillarum is well recognized as a Flexibacter maritimus). This pathogen primarily highly pathogenic bacterium and has become a aacks skin, mouth, fins and tail of fish, causing severe problem for fish farming industry during severe necrotic and ulcerative lesions on the body the last decades. surface (Toranzo et al., 2005). Losses associated with this disease have been previously reported V. anguillarum serotype O2 detected during this in common sole (McVicar and White, 1979). Also mortality episode represent the first isolation of in our case a great mortality was associated with this pathogen on common sole in Italy. Previously, the V. anguillarum infection, only a low number a report of Vibriosis in reared common sole in of fish survived though and then was affected by Italy was documented but in that episode the T. maritimum. V. anguillarum isolated belonged to serotype O1 (Manfrin et al., 2003). The described mortality could be interpreted as a multi-factorial event in which post-weaning The immunohistochemical findings suggest that weakness, handling, raising of temperature and the gut may have acted as an entrance for the pathogens have had a synergic effect. V. anguillarum infection, as Olsson et al. (1998) Bull. Eur. Ass. Fish Pathol., 30(5) 2010, 168
Acknowledgements The present work has been carried out in the framework of European Financial Instrument for Fisheries Guidance 2000-06.
We would like to express our gratitude to the experimental hatchery of Veneto Agricoltura (Venice - Italy) for S. solea eggs supply and to “Istituto Zooprofilaico delle Venezie” (Padua- Italy) for Betanodavirus analysis and for preparation of polyclonal antisera against Vibrio Figure 4. Solea solea, 75 dph, brain, extreme anguillarum. dilatation of blood vessels immunopositive for V. anguillarum O2 (*), some bacteria were detected outside the blood vessels,(arrows). IHC. Bar 8 μm References Austin B and Austin DA (1999). In “Bacterial fish pathogens. Disease in farmed and wild fish” Springer Praxis Publishing. As transfers are usual in aquaculture practices and similar temperature changes are well Baynes SM and Howell BR (1993). Observation tolerated by other farmed species (Eaton et al., on the growth, survival, and disease resistance of juvenile common sole Solea solea fed Mytilus 1995), this work points out the extreme stress edulis. Aquaculture and Fisheries Management 24(1), susceptibility of S. solea. 95-100.
Further efforts should be done to enhance the Bauman P, Furniss AL and Lee JV (1984). In: Krieg NR and Holt JG (eds) “Bergey’s Manual immune system (vaccination, immunostimolants) of Systematic Bacteriology, Vol. 1”. Williams & and to verify the effects of any nutritional Wilkins, Baltimore. unbalance on the weakness of the fry (Rueda Jasso et al., 2005). Baynes and Howell, (1993) Borghesan F, Palazzi R, Zanella L, Vascellari M, Mutinelli F, Montesi F, Manfrin A, Selli L and Bovo referred that mortalities of S. solea fed with G (2003), Viral encephalopathy and retinopathy heat-treated mussel during an outbreak of T. infection in reared common sole (Solea solea). 11th maritimum skin necrosis was 97%, while in International Conference “Diseases of fish and groups reared in the same conditions, but fed shellfish” of the European Association of Fish Pathologists (EAFP), 21st–26th September 2003, with fresh mussel, mortalities was less than 10% St Julians (Malta). and fish did not develop the symptoms, whereas Logue et al. (2000) demonstrated a higher stress Bucke D (1989). Histology In: “Methods for susceptibility in soles fed with a diet with low the microbiological examinaiton of fish and shellfish” (B. Austin and D.A. Austin, Ed.), 69-97 level in n-3 polyunsaturated fay acid (PUFA). pp. Ellis Horwood Ltd., Chichester. Therefore future studies are needed to determine dietary requirements of S. solea in order to lead to Buller N.B. (2004). “Bacteria from Fish and other the commercial availability of a specific artificial aquatic animals”. Cabi publishing. diet for this new species. Bull. Eur. Ass. Fish Pathol., 30(5) 2010, 169
Eaton JG, McCormick JH, Goodno BE, O’Brien Montero L, Quartieri K, Ramazzo E, Vascellarei DG, Stefany HG, Hondzo M and Scheller RM M, Mutinelli F, Melchioi E, Bozza MA, Selli L (1995). A Field Information-based System for and Bovo G. (2003). Primo isolamento di Vibrio Estimating Fish Temperature Tolerances. Fisheries anguillarum sierotipo O1 dalla sogliola comune 20, 10-18. (Solea solea, L.) in Italia. Bolle ino Società Italiana di Patologia I ica 37, 13-15. Irie T, Watarai S, Iwasaki T and Kodama H (2004). Binding of Vibrio anguillarum to Neutral McVicar AH and White PG (1979). Fin and skin Glycosphingolipids from Intestinal Mucosa of necrosis in cultivated dover sole, Solea solea (L.). Rainbow Trout (Oncorhynchus mykiss). Journal of Journal of Fish Diseases 2, 557-562. Veterinary Medical Science 66, 205-208. Mutinelli F, Bozza MA, Basilicata L and Bovo G Imsland AK, Foss A, Conceicao LEC, Dinis MT, (1998): Diagnosi istologica ed immunoistochimica Delbare D, Schram E, Kamstra A, Rema P and della Encefaloretinopatia virale della spigola White P (2004). A review of the culture potential di allevamento (Dicentrarchus labrax). Bolle ino of Solea solea and S. senegalensis. Reviews in Fish Società Italiana Patologia I ica 23, 1-10. Biology and Fisheries 13, 379-407. OIE (2006) Manual of Diagnostic Tests for Johansen R, Sommerset I, Torud B, Korsnes Aquatic animals Chapter 2.1.7. K, Hjortaans MJ, Nilsen F, Nerland AH and Dannevig BH (2004). Characterization of Olsson J C, Jöborn A, Westerdahl A, Blomberg Nodavirus and encephalopathy and retinopathy L, Kjelleberg and Conway PL (1998) Survival, in farmed turbot, Scophtalmus maximus. Journal of persistence and proliferation of Vibrio anguillarum Fish Diseases 27, 591-601. in juvenile turbot, Scophalmus maximus (L.), intestine and faeces. Journal of Fish Diseases 21, Larsen JL, Pedersen K and. Dalsgaard A.(1994) 1-9. Vibrio anguillarum serovars associated with vibriosis in fish. Journal of Fish Diseases 17, 259- Palazzi R, Richard J., Bozzato G, Zanella L (2006) 267. Larval and juvenile rearing of common sole (Solea solea L.) in the Northern Adriatic (Italy). Logue JA, Howell BR, Bell JG and Cossins AR Aquaculture 255, 495-506. (2000). Dietary n-3 long-chain polyun-saturated fay acid deprivation, tissue lipid composition, Pazos F, Santos Y, Macías AR, Núñez S and ex vivo prostaglandin production, and stress Toranzo AE (1996). Evaluation of media for the tolerance in juvenile Dover sole (Solea solea L.). successful culture of Flexibacter maritimus. Journal Lipids 35, 745-755. of Fish Diseases 19, 193-197.
Magi GE, Avendaño-Herrera R, Magariños B, Rueda-Jasso RA, Conceicao LEC, Coen W, Rees Toranzo AE and Romale JL (2007). First reports JF and Sorgeloos P (2005). Diet and weaning age of flexibacteriosis in farmed tub gurnard affect the growth and condition of Dover sole (Chelidonichthys lucernus L.) and wild turbot (Solea solea L.). Sciencias Marinas 31(3), 477-489. (Scophthalmus maximus) in Italy. Bulletin of the European Association of Fish Pathologists 27, 177- Toranzo AE, Baya A, Robertson BS, Barja JL, 184. Grimes DJ and Hetric FM (1987). Specificity of slide agglutination test for detection bacterial fish Maltese C and Bovo G (2007). Viral pathogens. Aquaculture 61, 81-97. encephalopathy and retinopathy/ Encefalopatia e retinopatia virale. I iopatologia 4, 93-146 Toranzo AE, Magariños B and Romalde JL (2005). A review of the main bacterial fish diseases in Manfrin A, Doimi M, Antonei P, Delgado mariculture system. Aquaculture 246, 37-61.