A histology-based fish health assessment of farmed seabass (Dicentrarchus labrax L.)

Aquaculture 448 (2015) 375–381

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A histology-based fish health assessment of farmed seabass (Dicentrarchus labrax L.) Aurélia Saraiva a,b,⁎, Joana Costa b, Joana Serrão b, Cristina Cruz a,b, Jorge C. Eiras a,b a b

Faculdade de Ciências, Universidade do Porto, Departamento de Biologia, Rua do Campo Alegre, Edifício FC4, 4169-007 Porto, Portugal CIIMAR — Centro Interdisciplinar de Investigação Marinha e Ambiental, Rua dos Bragas, 4050-123 Porto, Portugal

a r t i c l e

i n f o

Article history: Received 14 May 2015 Received in revised form 8 June 2015 Accepted 16 June 2015 Available online 18 June 2015 Keywords: Aquaculture Histological semi-quantitative system Welfare Fish

a b s t r a c t The aim of the present study was to assess the health status of farmed seabass (Dicentrarchus labrax L.) using as a tool the histological semi-quantitative system proposed by Bernet et al. (1999). Gills, liver, kidney and intestine were fixed and processed for histological analysis using standard techniques. Organ index values were determined and used to classify the severity of histological response using classes based in the scoring scheme proposed by Zimmerli et al. (2007). The gills were the most histologically affected organ with 56.7% of the fish presenting severe histopathological alterations consisting mainly of hyperaemia, hypertrophy, hyperplasia and necrosis. The kidney was the organ which in most of the fish (58.6%) presented a normal tissue structure. Nevertheless hyperaemia, vacuolar degeneration of renal tubules, necrosis of the haematopoietic interstitial tissue and nephrocalcinosis were observed in some specimens. 71.4% of the fish showed normal or slightly modified liver but hyperaemia, high vacuolization and hypertrophy of hepatocytes were observed in some cases. In general the histology of the intestine was normal or presented slight enteritis. Statement of relevance: The aim of the present study was to assess the health status of farmed seabass (D. labrax L.) using as a tool a histological semi-quantitative system. This tool is being extensively used in wild fish to assess aquatic pollution but rarely applied in farmed fish. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Aquaculture remains the fastest-growing animal-food-producing sector, now accounting for almost half of the total food fish supply (FAO, 2010), and it is the best alternative to the world-wide collapse of commercial fishery stocks. The welfare of fish in captivity has been a matter of growing concern and, in contrast to many terrestrial species, there is a paucity of scientific information on the welfare of fish raised under aquaculture conditions (Ashley, 2007; Chandroo et al., 2004; Huntingford and Kadri, 2014; Tort et al., 2011). Despite the paucity of knowledge about their welfare, welfare of fish is considered in the legislation of most European countries (Galhardo and Oliveira, 2006). One important component of fish welfare is, without a doubt, maintenance of a good health status, and this is a universally accepted measure of welfare (Ashley, 2007; Segner et al., 2012). It is important to remember that poor health can be both a cause and a result of poor welfare. Fish farm health maintenance is a concept in which fish should be reared under conditions that optimise the growth rate, feed ⁎ Corresponding author at: Faculdade de Ciências, Universidade do Porto, Departamento de Biologia, Rua do Campo Alegre, Edifício FC4, 4169-007 Porto, Portugal. E-mail address: [email protected] (A. Saraiva).

http://dx.doi.org/10.1016/j.aquaculture.2015.06.028 0044-8486/© 2015 Elsevier B.V. All rights reserved.

conversion efficiency, and survival while minimising problems related to infectious, nutritional, and environmental diseases (Plumb and Hanson, 2011). The interaction of all these factors may be clearly different from the sum of their individual effects. The overall effect of all these factors is reflected by the histological status of an organ (Baeverfjord and Krogdahl, 1996; Salamat and Zarie, 2012; Van der Oost et al., 2003; Zimmerli et al., 2007). This tool is being extensively used in wild fish to assess aquatic pollution (Handy et al., 2002; Lukin et al., 2011; Marchand et al., 2009; McHugh et al., 2011, 2013; Torres et al., 2014; Van Dyk et al., 2009; Zimmerli et al., 2007) but rarely applied in farmed fish (Coz-Rakovac et al., 2005; Raskovic et al., 2013). The aim of the present study was to evaluate the suitability of the use of a histological tool, the semi-quantitative system proposed by Bernet et al. (1999), to unravel the health condition of farmed fish. 2. Material and methods Seabass of commercial size (n = 30) were randomly sampled in a semi-extensive fish farm. Fish were sacrificed via concussion (procedure approved by Directive 2010/63/EU of the European Parliament and of the Council). For each fish the total weight (g), the hepatic weight (g) and the total length (cm) were recorded. Fulton's condition

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factor (CF = 100 × total weight / length3) and hepatic somatic index (HSI = 100 × liver weight / total weight) were determined. External and internal macroscopic examinations were conducted on each fish. The heads of the specimens were removed and frozen in individual plastic bags. After thawing, the gills were removed and searched for parasites under a stereomicroscope. Portions of gills, liver, kidney and intestine were preserved in ethanol for molecular studies whenever there was a suspicion of microparasites or bacterial infection. Pieces of gills, liver, kidney and intestine were fixed in 10% neutral buffered formalin for 48 h and stored in 70% ethanol until processing. The samples were processed for histological analysis using standard techniques in an automatic tissue processor, sectioned at 5 μm thickness and stained with haematoxylin and eosin (H&E). Histological changes were assessed according to the semi-quantitative system proposed by Bernet et al. (1999). Briefly, according to this method, histological changes are classified into five reaction patterns (circulatory, regressive, progressive, inflammatory and neoplastic). Each reaction pattern contains several alterations per organ. These alterations are assessed individually using a score value ranging from 0 (unchanged) to 6 (severe/ diffuse occurrence). To each alteration an importance factor ranging from 1 (minimal pathological importance) to 3 (marked pathological importance) is attributed. The final value for each alteration results from the multiplication of the score value with the importance factor. Summing up these final values for one reaction pattern or organ gives the index for the respective reaction pattern i.e. index for circulatory disturbance (IC), index for regressive changes (IR), index for progressive changes (IP), index for inflammation (II) and index for tumour (IT) for each organ or organ index (OI). Organ index values were used to classify the severity of histological response using classes based in the scoring scheme proposed by Zimmerli et al. (2007): Class I (index ≤ 10) — normal tissue structure with slight histological alterations; Class II (index 11–20) — normal tissue structure with moderate histological alterations; Class III (index 21–30) — moderate modifications of normal tissue; Class IV (index 31–40) pronounced histological alterations of the organ; Class V (index N 40) — severe histological alterations of the organ. Histopathological assessment tools for intestine are given in Table 1 following the same methodology used by Bernet et al. (1999) for the other organs.

Data analysis was carried out using IBM SPSS statistic software. The histological reaction indices for each organ were compared by nonparametric Kruskal–Wallis test followed by multiple comparisons. Statistical significance was accepted when p b 0.05. 3. Results The macroscopic observation of fish performed during sampling for histological studies did not reveal any macroscopic abnormality. The host parameters (weight, length, condition factor, hepatosomatic index) are depicted in Table 2. All the histological indices determined in the present study are indicated in Fig. 1 and Table 3. The organ indices were significantly different (Table 3).

3.1. Gill The gills were infected with the monogenean Diplectanum aequans (Wagener, 1875) Diesing, 1858, a few specimens of the copepod Caligus minimus Otto, 1821 and some unidentified copepodite and calimus stages (see Saraiva et al., 2015). Gills were the most histologically affected organ with a mean histological organ index of 46.7 (Table 3), with 56.7% of fish presenting severe histopathological alterations (Fig. 1A). The most usual observed histological changes were hyperaemia (circulatory changes), hypertrophy and hyperplasia (progressive changes) and necrosis (regressive changes) of gill tissues and inflammatory cell infiltration (see Saraiva et al., 2015). 3.2. Kidney The kidney was the organ which in most of the fish presented a normal tissue structure (Table 3, Figs. 1B and 2A). Nevertheless some specimens (27.6%) presented hyperaemia (Fig. 2B), vacuolar degeneration of renal tubules epithelial cells (Fig. 2C) and necrosis of the haematopoietic interstitial tissue (Fig. 2D). In two fish nephrocalcinosis caused architectural and structural alterations of renal tubules (Fig. 2E).

Table 1 Histopathological assessment tools in fish intestine (I) following the same methodology used by Bernet et al. (1999) for other organs. An importance factor (WI rp alt) ranging from 1 to 3 is assigned to each alteration: it is composed of the reaction pattern (rp) and the alteration (alt). The score value has to be rated for every alteration with a score ranging from 0 to 6. Addition of supplementary alterations could be done according to the specific needs. However, these should not be considered for index calculation. Reaction pattern (rp)

Functional unit of the tissue

Circulatory disturbances (C) Regressive changes (R)

Epithelium

Lamina propriaa

Progressive changes (P)

Epithelium a

Lamina propria Inflammation (I)

Tumour (T)

Alteration (alt)

Importance factor (W)

Score value (a)

Index (I)

Haemorrhage/hyperaemia/aneurysm Intercellular oedema Architectural and structural alterations Plasma alterations Deposits Nuclear alterations Atrophy Necrosis Architectural and structural alterations Plasma alterations Deposits Nuclear alterations Atrophy Necrosis Hypertrophy Hyperplasia Hypertrophy Hyperplasia Exudate Activation of RES Infiltration Benign tumour Malignant tumour

WIC1 = 1 WIC2 = 1 WIR1 = 1 WIR2 = 1 WIR3 = 1 WIR4 = 2 WIR5 = 2 WIR6 = 3 WIR7 = 1 WIR8 = 1 WIR9 = 1 WIR10 = 2 WI11 = 2 WIR12 = 3 WIp1 = 1 WIp2 = 2 WIp1 = 1 WIp2 = 2 WII1 = 1 WII2 = 1 WII3 = 2 WII1 = 2 WII2 = 3

aIc1 aIc2 aIr1 aIr2 aIr3 aIr4 aIr5 aIr6 aIr7 aIr8 aIr9 aIr10 aIr11 aIr12 aIp1 aIp2 aIp3 aIp4 aII1 aII2 aII3 aIt1 aIt2

IIC IIR

IIP

III

IIT

a The wall of fish intestine does not include a well-defined mucosa and submucosa. The term lamina propria should be understood as including the stratum compactum and stratum granulosum referred to several authors.

A. Saraiva et al. / Aquaculture 448 (2015) 375–381 Table 2 Mean and range of seabass parameters (CF — condition factor; HSI — hepatosomatic index; Ht — haematocrit). CI — bootstrap confidence interval for the mean (for 95% confidence level).

Mean (CI) Range N

Weight (g)

Length (cm)

CF

HSI

301.3 (276.9–326.6) 140.0–76.0 30

31.7 (30.9–32.5) 26.4–36.3 30

0.93 (0.88–0.97) 0.52–1.13 30

1.14 (1.06–1.23) 0.87–1.86 30

Table 3 Histological reaction indices (IC — index for circulatory disturbances; IR — index for regressive changes; IP — index for progressive changes; II — index for inflammation; IT — index for tumour) and organ index (OI) for each organ and for all the organs examined (Total). Significant differences (p b 0.05) among histological reaction indices for each organ (small letters), and among organ indices (capital letters) analysed by Kruskal–Wallis non-parametric test followed by multiple comparisons. Similar letters indicate no significant differences.

3.3. Liver

3.4. Intestine

A

IC

IR

IP

II

IT

OI

Mean (s.d.)

Mean (s.d.)

Mean (s.d.)

Mean (s.d.)

Mean (s.d.)

Mean (s.d.)

Range

Range

Range

Range

Range Range

26.8 (8.2) 17–50 d 8.6 (10.8) 0–26 b 11.0 (7.6) 4–24 b 9.9 (1.5) 7–14 c 57.9 (15.6) 36–86

9.4 (3.5) 4–16 c 0.0 0 a 4.6 (1.7) 0–6 b 0.0 0 a 13.7 (3.8) 8–22

6.5 (2.6) 2–12 bc 0.0 0 a 0.0 0 a 2.1 (0.9) 1–4 b 9.0 (2.5) 4–14

0.0 0 a 0.0 0 a 0.0 0 a 0.0 0 a 0.0 0

GILLS (n = 29) 4.1 (2.0) 2–8 b KIDNEY 0.8 (1.8) (n = 29) 0–6 ab LIVER 1.4 (1.6) (n = 28) 0–6 ab INTESTINE 0.4 (0.8) (n = 30) 0–2 a Total (n = 28) 6.8 (3.4) 2–16

The liver mean histological organ index was low (Table 3) as most of the fish (71.4%) showed a normal or slightly modified liver for fish in farming conditions (Figs. 1C and 3A). However, some fish presented slight hyperaemia and high vacuolization (Fig. 3B) or very high vacuolization (Fig. 3C) that caused hypertrophy of hepatocytes probably related to lipid accumulation (steatosis). In more affected livers necrotic foci were also observed (Fig. 3D).

In general the histology of the intestine was considered normal or with slight alterations (Table 3; Fig. 1D). Mast cells/Eosinophilic Granule Cells (EGCs) were very frequent and were considered normal components of the intestinal lamina propria. However in some fish they were also present in the epithelial lining, together with lymphocytes and between enterocytes (Fig. 4A and B). These cases were considered an inflammatory response (enteritis). Coccidians (Apicomplexa) were present in very low amount in the intestine of some fish with no

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46.7 (13.6) 27–78 B 9.4 (11.7) 0–30 A 17.0 (9.4) 6–36 A 12.5 (2.7) 8–20 A 87.4 (21.0) 59–130

inflammatory response (Fig. 4C). So far we were not successful in its detection and identification by molecular techniques. The histological reaction indices were significantly different in all analysed organs. In all organs, the highest index was the one caused by regressive changes, although not significantly different in kidney for circulatory changes and in liver for progressive changes (Table 3).

B

D 100

C Relative frequency (%)

90

Intestine

80 70 60 50 40 30 20 10 0 Class I (<10) Class II (11 - 20) Class III (21-30) Class IV (31-40) Class V (>40)

Fig. 1. Severity degree of histological changes observed (Class I — normal to slight alterations; Class II — normal with moderate alterations; Class III — moderate alterations; Class IV — pronounced alterations; Class V — severe alterations) observed in gills (A), kidney (B), liver (C.) and intestine (D) of farmed seabass.

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Fig. 2. Representative photos of histological features of kidneys of farmed seabass. A. Normal kidney; B. kidney with hyperaemia (arrows); C. vacuolar degeneration of renal tubules epithelial cells; D. necrosis of the haematopoietic interstitial tissue; E. nephrocalcinosis caused architectural and structural alterations of renal tubules (H&E).

4. Discussion Seabass CF and HSI values were slightly lower than those generally reported in farmed seabass (see Saraiva et al., 2015). These values are probably associated with expense of energy to address adverse factors, namely the parasites observed in gills. The gills are among the most delicate structures of the teleost body and are very sensitive to environmental conditions and pathogens including parasites. There are several reports of histological effects caused by biotic and abiotic factors on seabass gills. Among gill parasites the monogenean D. aequans is probably the most common detected parasite. This parasite causes frequently hyperaemia, haemorrhages, oedema, hyperplasia, leucocyte infiltration and gill necrosis (Dezfuli et al., 2007; González-Lanza et al., 1991; Oliver, 1977; Yardimci and Pekmezci, 2012). Oedema, hyperplasia and necrosis were also reported in parasite infections caused by the copepod Lernanthropus kroyeri (Manera and Dezfuli, 2003; Yardimci and Pekmezci, 2012). Further these alterations have been related to the presence of toxic products

in water namely cadmium and the herbicide terbuthylazine (Dezfuli et al., 2006; Giari et al., 2007). In this study gills were the most histologically affected organ. Probably several factors contributed to it but we believe that the infection by D. aequans contributed decisively to the histological changes observed (Saraiva et al., 2015). Concerning kidney histology, Kurtovic et al. (2008) referred to a higher number of melanomacrophage centres and atrophy of glomerulus as common features of kidney of farmed seabass, compared to wild ones. In this study we never detected these features and the glomeruli were similar to the ones reported by these authors in wild seabass. According to the same authors, vacuolar degeneration of tubular epithelial cells is common in seabass both farmed and wild. Renal tubular degeneration and circulatory disturbances including haemorrhages in farmed fish have been associated with antibiotic therapy (Roberts, 2012; Smith et al., 1973) and necrosis of renal haematopoietic tissue, which is a very sensitive tissue, may occur in many biotic and toxic situations (Roberts, 2012) including treatments. Nephrocalcinosis is frequently associated with fish farming. Particularly in marine fish it is

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Fig. 3. Representative photos of histological features of liver of farmed seabass. A. Normal liver; B. liver with slight hyperaemia and high hepatocytes vacuolization; C. liver with pronounced hepatocyte vacuolization (in upper right side normal pancreatic tissue); D. necrotic hepatocytes near pancreatic tissue (H&E).

very often related to unsuitable levels of calcium and magnesium in the diet or exposure to high levels of carbon dioxide in the water (causing metabolic acidosis which results in calcium precipitation in urine), or interpreted as side effects of excessive antibiotic use (Roberts, 2012). Cytoplasm alterations in fish hepatocytes is a very early and unspecific signal of disturbance of hepatocellular homeostasis (Braunbeck, 1998). Hypertrophy, vacuolar degeneration and increase of lipid droplets in hepatocytes of fish exposed to toxicants have been reported by many authors (Biagianti-Risbourg and Bastide, 1995; Braunbeck, 1998; Dezfuli et al., 2006; Giari et al., 2007; Strmac and Braunbeck, 2002). In farmed fish it is known that commercial feed causes lipid droplet accumulation, hepatic cell membrane degeneration, and hepatocyte vacuolization and can cause circulatory disturbances (Bilen and Bilen, 2013; Coz-Rakovac et al., 2002, 2005). In this study all these histological changes were observed and were not considered pathological if they did not cause extensive hepatic necrosis. However it is very difficult to establish a threshold for what should be considered a fish farm healthy liver. Pathological changes in the intestine of fish have been only poorly studied (Roberts, 2012). It is known that the intestinal epithelium is an important site for the absorption of nutrients, immunity, osmotic balance, recycling of enzymes and macronutrients (Alvarez-Pellitero, 2011; Urán et al., 2008) and the distal intestine is the principal site for the endocytosis of proteins (Rombout et al., 1985). On the other hand the increased use of plant feedstuffs in farmed fish diets can affect the gut integrity and increase the deleterious effect of gut pathogens (Couto et al., 2014; Estensoro et al., 2011; Mourente et al., 2007; Oliva-Teles, 2012; Urán et al., 2008). Integrity of intestine is assumed to be a key factor for the growth and welfare of farmed fish. Although the composition of the food given to the examined fish was not known we can say that it did not cause relevant histological alterations although

the increase of mast cells/EGCs in intestinal mucosa could be related to it (Baeverfjord and Krogdahl, 1996; Roberts, 2012; Urán et al., 2008). In seabass several parasites, namely Shaerospora dicentrarchi and the economically important myxosporean Enteromyxum leei cause serious disorders in intestine (Fioravanti et al., 2004; Mladineo, 2003; Sitja-Bobadilla et al., 2007). Oocysts of the coccidian type, probably Eimeridae similar to the one reported by Daoudi and Marques (1987) and Alvarez-Pellitero et al. (1993) occurred in some specimens without causing pathogenic effects. It was not possible to confirm their presence and identity through molecular techniques probably due to the very low levels of infection. The advantage of the use of histopathology as biomarker lies in its intermediate location in the hierarchy of biological organisation once it is able to integrate the effects of both abiotic and biotic factors upon organ function and fish health (Handy et al., 2002; van Dyk et al., 2009; Zimmerli et al., 2007). Moreover, histopathological survey in fish populations usually refers to more serious and obvious changes even if they are present only in a few specimens. The use of Bernet et al. (1999) histopathological protocol to unravel the health condition of farmed fish proved to be an excellent tool. However it is important to bear in mind that healthy fish are not characterised by absence of histopathology, but may display moderate structural disorders or mild inflammatory reactions (Bernet et al., 2004). Acknowledgments This work was partially funded by the Project AQUAIMPROV (reference NORTE-07-0124-FEDER-000038), co-financed by the North Portugal Regional Operational Programme (ON.2 — O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF).

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Fig. 4. Representative photos of histological features of intestine of farmed seabass. A. Eosinophilic granule cells (EGCs) and lymphocytes in lamina propria and between enterocytes; B. high magnification of A with very visible EGCs (arrow) between enterocytes; C. oocyst with sporocysts (arrow) of unidentified coccidian in intestinal mucosa (H&E).

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