Page 1 F Fish Pathology, 46 (3), 87–90, 2011. 9 © 2011 the Japanese

魚病研究 Fish Pathology, 2011.(3), 46987–90, © 2011 The Japanese Society of Fish Pathology

BloodFluke Infection of Cage 2 to 10 months (average of 6 months) fed with highly fat content fish, mainly chub mackerel Scomber japonicus, R Atlantic eared Bluefin Tuna A tla n tic mackerel S c o mber scombru s , European pil- Th u n n u s thynnusThunnus in chard C lupea pilchartus and round sardinella S a rd in e lla aurita. After this period, tunas are sacrificed in the Wes t Mediterranean floating cages a n d immediately commercialized fresh or frozen. R ocio Ruiz de Ybañez1, José Peñalver2, Among pathological problems reported in reared C arlos Martínez-Carrasco1, Laura del Río1, tuna, a blood fluke Cardicola forsteri (Digenea: 2 1 Aporocotylidae), has been pointed out as a significant Emilio María Dolores , Eduardo Berriatua risk of tuna health1). Initially identified in the Australian 1 and Pilar Muñoz * population of farmed southern BFT Thunnus maccoyii 2), this blood fluke was later reported in Atlantic BFT3–5), 1 Animal Health Department, University of Murcia, being the only one aporocotylid repo rte d s o fa r in th is Murcia 30100, Spain 2 species. Aporocotylids are parasites of marine and Livestock and Fishery Departmen t, Murcia 6) fre s h wate r fis h . Most species are located in the R egional Gov e rn men t, Murcia 30201, heart, bulbus arteriosus, ventral aorta or branchial ves- S p a in sels, although the cephalic or dorsal vessels are not uncommon habitats7). Once established, adult flukes (Recieved November 24, 2010) lay eggs whic h tra v e l to th e g ills where they lodge. The eggs hatch there and break out of the gill as free liv- in g mira c id ia 6). These miracidia infect an intermediate ABSTRACT —Infection of a blood fluke, possibly C ardicola host into which they penetrate to undergo asexual s p . (Digenea: Aporocotylidae), in reared Atlantic b lu e fin reproduction. Bivalves and polychaetes have been tuna Thunnus thynnus was investigated. Parasitological reported to be intermediate hosts for some marin e analyses included visual examination of the heart for the 6,8) aporocotylids . C ercariae emerg e fro m the intermed i- presence of adult fluke and stereomicroscopic and histopa- ate host and actively search for the definitive host, a thological analyses of the gill to assess the presence of fish, penetrate the skin of the host and juvenile flukes lesions caused by parasite eggs. No adult flukes were a tte mpt to reach the circulatory system in whic h they found in the hearts. Some of the gills exhibited small w h ite 6) undergo a migration to the final site where they matu re . to y e llo w fo c i in v o lv in g s in g le fila men ts . B lo o d flu ke eggs Blood flukes are suspected cause of mor ta litie s in a w ere found in gill tissue sections of 29.6% o f s a mpled tuna. number of farmed fish s p e c ie s , fo r e x a mple a mberjack A s lig h t in fla mmatory response was observed around mos t 9) S e rio la d u merili in S p a in and tiger puffer Takifugu of these eggs, while occasionally individual eggs were 10) rubripes in Japan . These mortalities are generally encapsulated by a granulomatous reaction. D espite the considered to be due to the eggs of the blood flukes, absence of remarkable pathological effects in the infected w h ic h b lo c k c a p illa rie s in g ill la mellae and other organs. tuna, blood flukes combined with other agents may cause 11 ) Although Munday and H allegraeff briefly mentioned ‘a major problem. moderately severe, multifo c a l, g ra n u lo matous myocardi- tis’ associated with the presence of eggs (presumably of Key words: Thunnus thynnus, Atlantic bluefin tuna, C . fo rs te ri) during a mortality of southern BFT in 1996, Ca rd ic o la , blood fluke, Aporocotylidae more intensive histopathological investigations of the heart, gills and other organs of ranched southern BFT have not yet established a clear and consistent link The Atlantic bluefin tuna (BFT) (Thunnus thynnus) between the burden of eggs of C . fo rs te ri a nd mor ta lity is a fish e x p lo ite d b y c o mmercial fishing during centu- (Valdenegro and Now ak, unpublished data). R ecently, ries in the entire Mediterranean area. Due to the Hayward et al.12) associated high number of blood increasing deman d fo r th is fish b y th e s a s h imi-sushi flukes with increased lysozyme and decreased haemo- market, a “capture-based” aquaculture industry has globin levels in southern BFT. According to these been developed in the Mediterranean Sea over the last authors blood flukes might also be associated with the decade. This activity involves the capture of adult indi- onset of elevated mor ta litie s . viduals, during the months of May-June, w hich enter the Cardicola forsteri infection is present in wild Mediterranean Sea for reproduction. After capture, A tla n tic 13) and southern14) B F T a t low leve ls, a n d it is fish are introduced in floating cages where they stay for exacerbated during the rearing cycle in confined conditions10,15). In fa c t, A ik e n et al.16) suggest that the * Corresponding author infection in southern BFT occurs in the farming zone. E-mail: p ila r [email protected] C . fo rs te ri intensities and prevalences are very low in 88R. Ruiz de Ybañez, J. Peñalver, C. Martín e z -C a rra s c o , L . d e l R ío , E . María Dolores, E. Berriatua and P. Muñoz th e wild 14) and at transfer17) while an antibody Table 2. Es timate s fro m the logistic regression models of response against C . fo rs te ri is only initiated after trans- blood fluke infection in captive bluefin tuna in Spain 14) fe r in to th e fa rming zone from th e wild . A s th e 95% C onfidence Variables Odds ratio P value impact of aporocotylids in cage reared tuna must not be intervals neglected, the objective of the present work was to Body weight (Kg) study the prevalence of blood fluke infectio n in B F T 27–40 1.0 0 reared in west Mediterranean, in the Murcian coasts. 150–300 2.61 6.91, 0.99 0.0543 F a rm C artagena Materials and Methods 1 1.0 0 Between November 2006 and December 2008, a 3 2.23 8.18, 0.61 0.2270 to ta l o f 1 0 8 A tla n tic B F T fro m s ix B F T fa rms located in 4 0.59 5.34, 0.06 0.6369 Murcia region (South East Spain) were sampled. Tw o San Pedro fa rms are located in San Pedro del Pinatar (37°49¢ 1 0 .1 6 1.42, 0.02 0 .1 0 1 6 55.46² N , 0 °3 9 ¢42.33² W) w h ile th e re maining farms a re 2 2.37 6.42, 0.87 0.0919 located in “El Gorguel” (Cartagena, 37°34¢31, 692²N, 00°52¢30, 702²W). Tuna weights in the range of 27–40 kg, 150–210 kg and 240–300 kg for 77.4%, a te ly fixe d in 10 % neutral buffered formalin . The rest 1 8 .3 % and 4.3% of the animals, respectively (Table 1). of the gill arch and the heart of each animal w e re c o l- Fish were shot underwater and then hoisted by hook. lected in individual plastic bags, transported to the labo- Gills and hearts were removed on board immediately ratory and stored at –20°C, until examined with a stereo- after tuna were slaughtered. T h e first gill arch w a s microscope (Nikon, SMZ 800) for parasite eggs and obtained from each tuna. For histology, two to four 1 pathological changes. To do this, approximate ly , cm long pieces of the gill arch were cut out and immed i- 120–150 gill filaments per arch were in d iv id u a lly observed under the stereomicroscope, placed in a large P e tri dish with saline solution. H earts were dissected Table 1. Percentage of captive bluefin tuna individuals positive and flushed with water to dislodge any adult flukes and for blood fluke eggs in Spain in 2006–07 flushes were then poured into Petri dishes and simila rly Variables N o . o f fis h % p o s itiv e examined. To carry out the histopathological study, Location the fixed pieces of gill arch (2–4 pieces per tuna) were C artagena 71 31 d e c a lc ifie d in a mix tu re o f 5 % E D T A a n d 3 % H C l fo r 2 4 San Pedro 37 27 h , fo llow e d b y 6 0 min washing in fresh water, prior to F a rm performing routine histological processing as follow s; San Pedro sample s were e mbedded in paraffin blocks, cut and 1 20 5* stained with haemato x y lin a n d e o s in (H & E ). The sec- 2 17 53 tions were e x a mined under a microscope (NIKON C artagena Eclipse 50i) and lesions were photographed. 1 30 43 EpiInfo 2002 (CD C ) and SAS (SAS Institute) were 2 6 50 used to do statistical analysis. Yate’s corrected chi- 3 15 33 squared test was used to compare the frequency of 4 20 5 blood fluke egg positive tuna across independent cate- Time in c a p tiv ity (months) gorical variables (Table 1) and the non-parametric 3 6 50 Kruskal-Wallis te s ts w a s e mploye d to c o mpa re mean 4 37 27 blood fluke egg densities. Logistic regression was 5 50 28 used to investigate the relationship between tuna infec- 6 15 33 tion (Table 2) and body weight, adjusted for farm. Samplin g mon th Other variables including farm location, month and time October 6 50 in captivity were not included in the model because of N ovember 37 27 their strong correlation with each other and with Farm, D ecember 50 28 leading to model convergence failure18). The maximum January 15 33 likelihood estimatio n model was used, Ps were from Body weight (kg) likelihood-ratio chi-squared test and alpha was 5% (p < 27–40 81 20* 0.05) level for a double-sided test. 150–210 16 69 240–300 5 40 * Significant differences between variable levels at p < 0.05. Blood fluke in Atlantic bluefin tuna 89

ogy revealed the presence of eggs consistent with R esults those described for aporocotylids6), in g ills o f 2 9 .6 % o f H earts were macroscopically normal and no adult tuna sampled (Table 1). The number of eggs in parasites were observed. Some of the gills exhibited in fe c te d fila men ts w a s lo w (≤ 1 0 e g g s ), med iu m (11–21 small w h ite to y e llo w fo c i in v o lv in g s in g le fila ments pos- eggs) and high (≥ 22 eggs) in 46.3%, 43.9% and 9.8% sibly associated to C ardicola sp. infection. H istopathol- of infected gill samples, respectively. The Kruskall Wallis test indicated that egg density was highest in gill samples with the greatest number of infected filamen ts (p < 0.05). The percentage of tuna positive for eggs w ere significantly greater in 150–300 kg compared to 27–40 kg tuna and differed between farms (p < 0.05) (T a b le 1 ). There were no statistically significant differ- ences in the percentage of infected tuna according to time in c a p tivity o r s a mplin g mon th (p > 0.05). In th e logistic regression analysis, infection was marg in a lly s ig - nificantly greater in 27–40 kg tuna compared to heavier tuna (odds ratio = 2.61, p = 0.0543) (Table 2) and there w as similar differences between farms (T a b le 2 ). Eggs found in the gill displayed different degrees of miracidial developmen t (F ig . 1 A ). A s lig h t in fla mma- tory response was observed around most of the eggs (F ig . 1 A , B ) while very occasionally, individual eggs w ere encapsulated by a granulomatous reaction (Fig. 1C), which consisted of epithelioid cells and lympho- cytes surrounded by fibroblasts and fibrocytes in older lesions. Some of the eggs were observed in afferent fila mentary arteries but mos t o f th e m had traversed the c a p illa rie s o f th e g ill la mellae and therefore observed w ithin the lamella e .

D iscussion Infection of a blood fluke, possibly C ardicola s p p ., in reared Atlantic bluefin tuna was investigated. Although C. fo rs te ri is the only blood fluke identified fro m A tla n tic B F T , Card ic o la s p . w ith a d iffe re n t IT S 2 sequence has been reported from Atla n tic B F T in Spain14,19) which strongly suggests co-existence of at least two species of blood flukes in Atlantic BFT. The prevalence of eggs detected in the present study was similar to the prevalence reported by Deveney et al.20) in southern BFT who observed a 31% of infection. Mladineo4) detected C . fo rs te ri eggs, but no adult flukes, in 63.34% o f s a mpled Atlantic BFT harvested in the Adriatic Sea. Differences in the prevalence between these studies could be due to the fact that some o f th e tuna sampled in the latter study were specimens that w ere found dead in an Adriatic tuna farm associated to other pathogens whic h may have predisposed to C. fo rs te ri infection. Even though tuna sampled in the present study F ig . 1 . Blood fluke eggs in the gills of Atlantic bluefin tuna show ed a high number of eggs and disseminated granu- (H&E ). A. Different degrees of miracidial lo mas without isolation of any adult parasite, this finding developmen t. B. Secondary gill lamella e w ith a n individual egg. C. T y p ic a l in fla mmatory response to re mains only an assumption of possible Cardicola s p p . an egg (asterisk), and a focal granuloma (a rro w) infection. Other authors have also failed to find adult fo rmed around an individual egg. fo rms o f C . fo rs te ri in Atlantic BFT even though eggs 90R. Ruiz de Ybañez, J. Peñalver, C. Martín e z -C a rra s c o , L . d e l R ío , E . María Dolores, E. Berriatua and P. Muñoz w ere found in gills4,6,17). They observed that as water cantly higher final cumula tiv e mortality than the fish unin- te mperature decreased the intensity and prevalence of fested with blood fluke. As blood fluke infection may C. fo rs te ri infection of southern bluefin tuna declined combine with other agents to cause such effects further after an initial peak to an average of 3.1 flukes per host investigations into the epidemiology of this parasite are and a prevalence of 35% respectively. Simila r w arranted. declines in intensity and/or prevalence follow ing a peak has been observed in other cultured fish species A cknowledgement infected by aporocotylids. Ogaw a et al.21) investigated infection of amberjack S e rio la d u merili blood flukes This work was supported by the National Plan of Paradeontacylix spp. and suggested that the blood Aquaculture (JAC U MAR ) of Spain. fluke infection has an annual cycle; cercarial invasion starts in September, eggs accumula te in th e g ills a n d R eferences heart from November, and mortality, occurring in the w inter mon th s fro m D ecembe r to March, decreases with 1) Now ak, B. (2004): B ull. Eur. Assoc. Fish Pathol., 24, 45–51. increasing water temperature. E xcept six fish sample d 2 ) C rib b , T . H ., M. Daintith and B. Munday (2000): R ec. Aust. Mus., 124, 117–120. 3 ) Mladineo, I. and M. Tudor (2004): in October, all tuna used in the present study were sam- Bull. Eur. Assoc. Fish Pathol., 24, 144–152. 4 ) Mladineo, I. pled in winter months. The lack of adult blood fluke (2006): Acta Adriat., 47, 23–28. 5 ) N o w a k , B ., I. Mladineo, H. c o u ld b e d u e firs tly, to th e low te mperatures and sec- A ik e n , N. B o tt a n d C . Hayward (2006): Bull. E u r. A s s . F is h ondly, to their hypothetical establishment in other loca- Pathol., 26, 38–42. 6 ) S mith , J . W. (1997): H elminthol. Abst. tions not sampled in this study. Indeed, Aiken et al.16) Ser., 66, 254–294. 7 ) K irk , R. S . a n d J. W. L e wis (1996): Parasitology, 113, 279–285. 8) C rib b , T . H ., R . D . A d la rd , C . reported the presence of C . fo rs te ri in branchial arteries J . Hayward , N. J. B o tt, D . E llis, D . E va n s a n d B . F. Nowak of southern BFT while Sanguinicola inermis adults were (2011): Internat. J. Parasitol., 41, 861–870. 9 ) Mon te ro , F . E ., observed in the afferent branchial arteries, ventral aorta A. Kostadinova and J. A. Raga (2009): Aquaculture, 288, and bulbous arterious7) of common carp Cyprinus 132–139. 1 0 ) Ogaw a, K., T. Nagano, N. Akai, A. Sugita and K. c a rp io . A. Hall (2007): Fish Pathol., 42, 91–99. 11 ) Munday, B. L. and G. M. Hallegraeff (1998): Fish Pathol., 33, 343–350. 1 2 ) A s mentioned above, Aiken et al. suggested that Hayward , C. J ., D . E llis , D . F o o te , R. J . Wilk in s o n , P . B . fish commonly become infected with C. fo rs te ri o n c e in C ro s b ie , N . J . B o tt a n d B . F . N o w a k (2 0 1 0 ): Vet. Parasitol., 173, 16) c a p tiv ity . In the present study, infection was not 107–115. 1 3 ) B u lla rd , S . A ., R. J . Goldstein, R. H. Goodw in related to duration in captivity but to the size of the hos t, and R . M. Overstreet (2004): Comp. Parasitol., 71, 245–246. greater in 150–300 kg tuna than in 27–40 kg. The 14) Aiken, H., N. J . B o tt, I. Mladineo, F. E. Mon te ro , B . F . N ow ak and C. J. Hayw ard (2007): F is h F is h ., 8, 167–180. 1 5 ) apparent negative correlation between infection and C o lq u itt, S . E ., B . L . Munday and M. Daintith (2001): J. Fish w eight or age requires further investigation. Infection Dis., 24, 225–229. 1 6 ) A ik e n , H., C . Hay w a rd , A . C a mero n accumulating with age would suggest some degree of and B. Now ak (2009): Aquaculture, 293, 204–210. 17) Aiken, parasite tolerance. On the other hand, although no cor- H. M., C . J . Hay w a rd a n d B . F. Now ak. (2006): Aquaculture, relation between fluke intensity and grow th measured 254, 40–45. 18) Kleinbaum, D . G., L . L . K u p p e r, K . E . Mulle r 17) and A. Nizam. (1998): Applied regression analysis and other as condition index has been reported a negative multiv a ria b le methods. D uxbury Press at Brooks/Cole Publ. effect should be considered as white lesions that com- C o ., B o s to n , 7 1 8 p . 19) Oga w a , K ., K . Is h imaru, S. Shirakashi, monly occurred in the gills are presumed to be caused I. T a k a mi a n d D . Grabner (2011): C ardicola opisthorchis n . s p . by the lodgement and hatching of blood fluke eggs22). (T re matoda: Aporocotylidae) from the Pacific bluefin tuna, Despite the pathology observed was not sufficient to Thunnus orientalis (T e mminck & Schlegel, 1844), cultured in Japan. Parasitol. Internat., (in p re s s ). 20) Deveney, M. R ., T . lead to mor ta litie s , it is p o s s ib le th a t c o mbin a tio n w ith J . B a y ly, C . J . J o h n s to n a n d B . F . N o w a k (2 0 0 5 ): J . F is h D is ., other factors will be able to cause severe illness or 28, 279–284. 21) Oga w a , K ., H. Andoh and M. Y a maguchi 15) death . It is generally believed that a disease assists (1993): Fish Pathol., 28, 177–180. 22) Colq u itt, S . (1 9 9 9 ): in secondary infections. P rimary infection might com- Histopathologaical changes in, and immune responses of, p ro mise host biodefence mechanisms. Kumon et al.23), southern bluefin tuna (Thunnus maccovii) infected with Card ic o la s p p . (Digenea: Sanguinicolidae). Hons. Thesis. fo r e x a mple, found that blood fluke-infested yellow tail Univ . o f T a s mania, Launceston, Tasmania, Australia. 23) Seriola quinqueradiata, when challenged with the bacte- Kumon , M., T. Iida, Y. Fukuda, M. A rimoto a n d K . S h imizu rial fish pathogen Lactococcus garvieae, had a signifi- (2002): Fish Pathol., 37, 201–203.