1416

J. Parasitol., 93(6), 2007, pp. 1416–1423� American Society of Parasitologists 2007

HISTOPATHOLOGY AND ULTRASTRUCTURE OF PLATICHTHYS FLESUS NATURALLYINFECTED WITH ANISAKIS SIMPLEX S.L. LARVAE (NEMATODA: ANISAKIDAE)

Bahram S. Dezfuli, Flavio Pironi, Andrew P. Shinn*, Maurizio Manera†, and Luisa GiariDepartment of Biology and Evolution, University of Ferrara, Via Borsari, 46-44100 Ferrara, Italy. e-mail: dzb@unife.it

ABSTRACT: The histopathology, ultrastructure, and immunohistochemistry of the alimentary canal of flounder Platichthys flesus(L.), naturally infected with the nematode Anisakis simplex s.l. (Rudolphi 1809) from the River Forth (Scotland), were investigatedand described. Eight of the 16 flounders were infected with A. simplex s.l. larvae (L3); parasites were encapsulated by serosa onthe external surface of the host’s digestive tract (intensity of infection 1–8 parasites per host), although nematode larvae werefound encysted under the peritoneal visceral serosa of the host spleen and liver and, occasionally, in the liver parenchyma (intensityof infection 3–10 parasites per host). In all sites, different structural elements were recognized within the capsule surroundinglarvae. Among the epithelial cells of the intestine of 5 flounders with larvae encysted on external surface of the gut, the presenceof several rodlet cells (RCs) was observed. Furthermore, often the occurrence of macrophage aggregates (MAs) was noticed ininfected liver and spleen, mainly around the parasite larvae. Eight neuropeptide antisera were tested with immunohistochemistrymethods on gut sections of 4 P. flesus infected with extraintestinal nematodes. Sections from the gut of 5 uninfected flounderwere used for comparative purposes. In the tunica mucosa of parasitized P. flesus, several endocrine epithelial cells were im-munoreactive to anti-CCK-39 (cholecystokinin 39) and -NPY (neuropeptide Y) sera; furthermore, in the myenteric plexus, a highnumber of neurons were immunoreactive to antibombesin, -galanin, and several to -NPY and -PHI (peptide histidine isoleucine)sera.

Species of Anisakidae are found mainly in fish-eating ver-tebrates. Various fish species and squid serve as paratenic hostsfor Anisakis spp., with sea mammals as definitive hosts (Mo-ravec, 1994). There are many reports dealing the taxonomy,ecology, and zoogeography of nematode parasites of fish (Køie,1999; Guillen-Hernandez and Whitfield, 2004), but only a fewhave dealt with histopathology caused by tissue-penetratingnematodes (Margolis, 1970; Miyazaki et al., 1988). Species oflarval Anisakis invade various tissue and organs of fish hosts.Known sites of infection include the digestive tract from theesophagus to the posterior intestine, gonads, swim bladder, liv-er, somatic musculature, mesenteries, peritoneum, body cavity,blood vessels, subcutaneous and other connective tissues, fins,and orbit of the eyes (Margolis, 1970; Moravec, 1994).

The pathology caused by larval Anisakidae, particularly inthe liver, has been thoroughly investigated (Margolis, 1970).Accordingly, the occurrence of anisakid larvae in this organwas reported in Otolithus argenteus (Bablanik and Bilequees,1998), Pagrus pagrus (Eiras and Rego, 1987), and Merlangiusmerlangus (Elarifi, 1982). There are reports for several fish spe-cies that heavy infections reduce the size of liver (Kahl, 1938;Brian, 1958).

Macrophage aggregates (MAs), also known as melanomacro-phage centers, can be found in different tissues of heterothermicvertebrates. These pigment-containing cells in fish have beenencountered in hemopoietic tissue of the kidney, spleen, liver(Agius and Roberts, 2003), and occasionally brain, gonads, andgills (Macchi et al., 1992). Several studies suggest that the gen-eral function of the centers include focalization of destruction,detoxification, or recycling of exogenous and endogenous ma-terials (Ferguson, 1976; Ellis, 1980; Herraez and Zapata, 1986).Furthermore, MAs are involved with late stages of the chronicinflammatory response to severe tissue damage (Agius andRoberts, 2003). During the present investigation, the presence

Received 31 January 2007; revised 13 April 2007; accepted 16 April2007.

* Institute of Aquaculture, University of Stirling, Stirling FK9 4LA,Scotland, United Kingdom.

† Department of Food Sciences, University of Teramo, Viale Crispi,212-64100 Teramo, Italy.

of MAs was noted in a few infected livers and spleens of P.flesus near the nematode larvae; accordingly, this is the firstreport on occurrence of MAs in this organ parasitized with ahelminth, and the present article will attempt to establish therelationship between A. simplex s.l. larvae and macrophage ag-gregates.

Rodlet cells (RCs) inhabit the tissue, particularly the epithe-lium, of virtually every fish species (Morrison and Odense,1978) and several functions have been described. Recently, itwas suggested that the RCs represent an inflammatory cell type(Dezfuli et al., 2000). Moreover, an increase in their numberdue to the presence of metazoan parasites was reported by Reite(1997) and Dezfuli et al. (1998, 2000, 2007); herein, severalRCs were noted in gut epithelium of flounder with encystedlarvae on outer surface of the intestine.

The capsules around the A. simplex s.l. larvae in organs ofP. flesus presented the same structural features described byMargolis (1970) in Melanogrammus aeglefinus parasitized withAnisakis sp., namely the presence of 3 layers in which the thick-ness of the capsule depends on the abundance of connectivetissue and on the age of infection.

Previous studies have dealt with the presence of larval ani-sakids in fish and mechanical damage, although data are lackingabout the possible involvement of the neuroendocrine systemin host reaction. One of main goals of present investigation wasto use the immunohistochemical methods to ascertain the re-sponse of flounder neuroendocrine system to nematode larvae.Thus, 8 neuropeptide antisera were tested on gut sections ofinfected and uninfected flounders. In addition, data on host–parasite interface as well as histopathological information arepresented.

MATERIALS AND METHODS

During July 2003, 16 specimens of Platichthys flesus (ranging from5.2 to 22.3 cm in total length) were collected from the River Forth(Stirling, Scotland). Fish were brought alive to the laboratory. Theywere anesthetized with the use of MS222 (Sandoz, Basel, Switzerland),and the spinal cord was severed. At necropsy, the whole digestive tractwas removed and searched for externally visible parasites on surface ofliver, intestine, and other visceral organs; the body cavity was examinedfor the presence of helminths. The number and location of parasites

DEZFULI ET AL.—HISTOPATHOLOGY OF PARASITIZED P. FLESUS 1417

TABLE I. Primary antisera used in this study.

Antiseraraised in

rabbit Code Source Dilution Incubation

Bombesin 1400-0004 Biogenesis 1:200 Overnight at 4 CCCKa-39 2050-0004 Biogenesis 1:100 Overnight at RT*Galanin AB 1985 Chemicon 1:250 48 h at RTGlucagon 4660-0904 Biogenesis 1:50 24 h at 4 CNPYa 6730-0004 Biogenesis 1:50 24 h at 4 CPHIa 7260-0004 Biogenesis 1:100 24 h at 4 CSecretin 8240-0004 Biogenesis 1:50 24 h at 4 CVIPa CA-08-340 Genosys 1:500 24 h at 4 C

* CCK, cholecystokinin; NPY, neuropeptide tyrosine; PHI, peptide histidine iso-leucine; VIP, vasoactive intestinal peptide; RT, room temperature.

TABLE II. Peptides used for absorption controls.

Peptide Code Source

Bombesin B 4272 Sigma Chemicals, St. Louis, MissouriGastrin G 3131 Sigma Chemicals, St. Louis, MissouriGalanin G-112 Sigma Chemicals, St. Louis, MissouriGlucagon H 6790 Bachem AG, Bubendorf, SwitzerlandNPY* H 6375 Bachem AG, Bubendorf, SwitzerlandPHI* (PHM-27) H 6355 Bachem AG, Bubendorf, SwitzerlandSecretin S 7147 Sigma Chemicals, St. Louis, MissouriVIP* V 3628 Sigma Chemicals, St. Louis, Missouri

* NPY, neuropeptide tyrosine; PHI, peptide histidine isoleucine; VIP, vasoactiveintestinal peptide.

were recorded. Some live larvae were isolated from surface of liver andintestine and fixed for species identification. The alimentary canal wascut longitudinally and examined. Pieces of fish tissues with attachedparasites, measuring up to 15 � 15 mm, were excised and fixed inchilled (4 C) Bouin’s for 8 hr. The samples were then transferred to70% alcohol, dehydrated through an alcohol series, and prepared forparaffin embedding. Cut sections (5 �m thick) were stained with eitherhematoxylin–eosin (H&E), Azan-Mallory, or PAS stains, or used forimmunohistochemical analysis. Platichthys flesus–A. simplex s.l. tissuesections were processed with the use of the indirect immunohistochem-ical method (peroxidase–antiperoxidase immunocomplex), as outlinedin Dezfuli et al. (2002). The antisera used are reported in Table I. Thecontrols for the specificity of the immunohistochemical reactions wereperformed by the preabsorption of each antiserum with the correspond-ing antigen (Table II). Mammalian (swine, rat) tissue sections were usedas positive controls.

For light and electron microscopy, host tissues measuring up to 7 �7 mm were fixed for 2 hr in chilled (4 C) 2% glutaraldehyde solution,pH 7.2, with 0.1 M sodium cacodylate. After 2 hr, the pieces were rinsedfor 12 hr in 0.1 M sodium cacodylate buffer containing 5% sucrose.The specimens were then postfixed in 1% osmium tetroxide in the samebuffer for 2 hr, dehydrated in graded ethanol, transferred to propyleneoxide, and embedded in an Epoxy-Araldite� mixture. Semithin sections(1.5 �m) were cut on a Reichert Om U 2 ultramicrotome and stainedwith toluidine blue. Ultrathin sections (90 nm) were stained with asolution of 4% uranyl acetate in 50% alcohol and Reynold’s lead citrateand examined with the use of a Hitachi H-800 transmission microscope.For comparative purposes, the tissues of 5 uninfected flounders wereprocessed along with the parasitized material.

RESULTS

Eight of 16 specimens of P. flesus were infected. For iden-tification purposes, some live larvae were removed from hostorgans, fixed, and later compared with morphological featuresand sizes reported for this genus in Moravec (1994). Accord-ingly, a third of the larvae were L3 stages of A. simplex s.l.

Several larvae were encapsulated in the peritoneal serosa onthe outer surface of the gut wall (Fig. 1a); the intensity of in-fection ranged from 1 to 8 parasites per host. In 2 flounders, afew A. simplex s.l. larvae were encountered within the intestinalwall (Fig. 1b). Moreover, in 1 fish, a single larva was encystedin muscle of the inner side of the fish’s body wall very closeto the liver.

Among the visceral organs, mainly the liver (Fig. 1c) and, ina few instances, the spleen, were infected. In the liver, the in-tensity of infection ranged from 3 to 10 larvae, with the ma-jority of the nematodes encysted under the visceral serosa; afew penetrated the parenchyma and destroyed the tissue of the

organ (Fig. 1c). In all infected livers, there was a translucentspace between the parasite cuticle and host tissue (Fig. 1c); insome instances, tissue debris was observed in the space. Thenematode larvae were often on the surface spleen.

The occurrence of macrophage aggregates (MAs) was ob-served around the A. simplex s.l. larvae encysted on liver (Fig.1d); in some specimens, MAs were encountered within thespleen parenchyma as well as on surface of the organ near theparasite larvae.

All parasites were encapsulated on external surface of hostdigestive tract and on the surface of the liver and spleen bygranulomatous reactive tissue involving the visceral serosa (Fig.1a, c). Some pancreatic acini in the mesenteries were involvedin the reaction and displayed nuclear picnosis and cellularshrinkage. Within the capsule around the parasite, reactive cel-lular elements were recognized. Specifically, from the outer parttoward the inner zone, a corona of fibroconnective tissue withscarce collagen component was noted (Fig. 2a). Herein, the oc-currence of eosinophilic granule cells and some lymphocyteswere observed (Fig. 2b). Near the parasite’s cuticle, palisade-arranged macrophages were evident; moreover, they exhibitedvacuolar degeneration (Fig. 2c). Such reactive tissue partitionwas not evident in all capsules. In some instances, diffuse gran-ulomatous tissues lacking fibroconnective outer corona and pal-isade-arranged macrophages were encountered. Sometimes,mesenteric edema and serous fibrinous exudates were apparent.

Several rodlet cells (RCs) were found in the epithelia (Fig.2d, e) of the guts of flounders that harbored encysted larvae onouter sides of their intestines. Discharge activity of the RCswas observed in grids of the intestine of several infected floun-ders. In some instances, the RCs presented high cytoplasmicvacuolization (not shown).

Eight neuropeptide antisera were tested on gut sections of 4P. flesus infected with extraintestinal nematodes (Table I). Sec-tions from 5 uninfected flounder were used for comparativepurposes. In the tunica mucosa of both uninfected and parasit-ized P. flesus, several structures (endocrine cells and neurons)were immunoreactive to anti-CCK-39 (Fig. 3a), -NPY (Fig. 3b),and -PHI sera (not shown) (Table III). In the tunica propriasubmucosa of infected fishes (Fig. 3c) and near the parasitecuticle (Fig. 3d) as well as in the myenteric plexus, numerousneurons were immunoreactive to antibombesin serum (Fig. 3c,d), whereas for the same neuropeptide, a smaller number ofpositive elements was noticed in gut sections of healthy fish(Table III). Figure 3e shows some neurons positive to antiga-

1418 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 6, DECEMBER 2007

FIGURE 1. (a) Several larvae (arrows) of Anisakis simplex s.l. on outer surface of the gut wall of Platicthys flesus stained with haematoxylin–eosin (H&E), scale bar � 280 �m. (b) Larvae of A. simplex s.l. (arrowheads) within the thickness of the flounder intestine stained with H&E;arrows show fibroconnective tissue around the larvae, scale bar � 120 �m. (c) Anisakis simplex s.l. larva encysted under the peritoneal visceralserosa of liver (arrowhead), 3 larvae (arrows) penetrated deeply in the organ; note white spaces around each larva, H&E, scale bar � 250 �m.(d) Macrophage aggregates (MAs, arrows) near the nematode larva (asterisk), H&E, scale bar � 50 �m.

DEZFULI ET AL.—HISTOPATHOLOGY OF PARASITIZED P. FLESUS 1419

FIGURE 2. (a) Transmission electron micrograph of liver showing the interface between Anisakis simplex s.l. larva (white asterisk) and hosttissue: fibroconnective elements (white arrowheads) are scattered among flounder cells (white arrows), scale bar � 4.0 �m. (b) Near parasitetegument (white asterisk) eosinophilic granule cell (white arrow) and lymphocytes (white arrowheads) are evident, scale bar � 3.6 �m. (c) Acorona of macrophages (arrows) with hydropic vacuolar degeneration can be seen, scale bar � 2.0 �m. (d) Three rodlet cells (white arrows) ingut epithelium of an infected flounder, scale bar � 5.8 �m. (e) High magnification of a single rodlet cell in which basal nucleus (white asterisk),some rodlets (black asterisks), and distinctive cell cortex (arrow) are evident, scale bar � 1.9 �m.

lanin serum in myenteric plexus of the intestine of uninfectedflounders, and Figure 3f shows larger number of neurons in P.flesus with a nematode encapsulated on the external surface ofthe host’s digestive wall (see Table III). Anisakis sp. larvae didnot generate a response to glucagon, secretin, or VIP (see TableIII).

DISCUSSION

Anisakis simplex s.l. is a common nematode and its larvaeinfect many species of fish. The parasite is transmitted to fishby microcrustaceans or other invertebrates and in fish the larvaereach the L3 stage (Oshima, 1972). The life cycle of A. simplex

1420 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 6, DECEMBER 2007

FIGURE 3. (a) Tunica mucosa of infected Platicthys flesus, some cholecystokinin 39 (CCK-39) immunoreactive endocrine cells (arrows) areevident, scale bar � 30 �m. (b) A positive immunoreaction to the anti-NPY serum within endocrine cells (arrows) of parasitized flounder, scalebar � 30 �m. (c) In the myenteric plexus of infected fish, a high number of nervous fibers (arrows) immunoreactive to antibombesin can be seen,scale bar �100 �m. (d) High magnification of nervous fibers (arrows) near parasite (asterisk) that yield positive reaction to bombesin, scale bar� 10 �m. (e) Some neurons (arrows) positive to antigalanin serum in myenteric plexus of uninfected flounder, scale bar � 50 �m. (f) Highernumber of positive neurons (arrows) near and around encysted larva (asterisk), scale bar � 100 �m.

s.l. and its congeners is still inadequately known (Moravec,1994). The nematode has a complicated life cycle with manyintermediate hosts and may be transmitted several times fromfish to fish (paratenic hosts) before the final host is reached.The final hosts are fish-eating marine mammals.

Almost all endoparasitic helminths that use the vertebrates asparatenic hosts inhabit the body cavity, or visceral organs, orboth. The body cavity contains a nutrient fluid, and providessecurity from dislodgment. These 2 factors may be involved indetermining its suitability as a site of infection; nevertheless, it

DEZFULI ET AL.—HISTOPATHOLOGY OF PARASITIZED P. FLESUS 1421

TABLE III. Frequencies of elements immunoreactive (IR) to primaryantisera in the intestine of Platichthys flesus uninfected and infectedwith Anisakis simplex. ��: medium presence, �: low presence, and �:absence of components immunoreactive to the specified antiserum.

Polyclonal antirabbit peptide

Platichthys flesus

UninfectedInfected with

A. simplex

Endocrine cells IRCholecystokinin 39 � �

Neurons and endocrine cells IRNeuropeptide Y � �

Neurons IRBombesin � ��Galanin � ��Peptide histidine isoleucine � �Glucagon � �Secretin � �Vasoactive intestinal peptide � �

still presents some problems for parasites (Howell, 1976). Ex-perimental studies on migration of Anisakis sp. larvae in thebody cavity and flesh of fishes have been described by Smithand Wootten (1975) and Smith (1984), but these authors didnot discuss larval nematode migration at host necropsy.

It is well known that one of the reactions of the host to thepresence of extraintestinal parasite is the formation of a con-nective tissue capsule that sequesters the parasite. The occur-rence of such a capsule around larval nematode, Hysterothy-lacium dollfusi Schmidt, Leiby, and Kritsky 1974 in Polyodonspathula Walbaum, was reported by Miyazaki et al. (1988). Thefeatures of capsule formation and structure around larvae out-side the intestine and in visceral organs of different fishes weredescribed by Margolis (1970), Hauck and May (1977), Torresand Gonzalez (1978), Elarifi (1982), and Eiras and Rego(1987).

Several roles for macrophage aggregates (MAs) in fish havebeen described; their proliferation has been associated with bothphysiological and pathological factors such as aging, starvation,infectious diseases, and chemical exposure (Vogelbein et al.,1987; Couillard and Hodson, 1996; Couillard et al., 1999). Re-cently, the nature of macrophage aggregates and their role infish pathology was reviewed by Agius and Roberts (2003). Ac-cording to these authors, these centers develop focally in as-sociation with late stages of the chronic inflammation responseto severe tissue damage due to viruses (Roberts, 2001), bacteria(Hjeltnes and Roberts, 1993; Chinabut, 1999), fungal infections(Carmichael, 1996), or to protozoans (Vogelbein et al., 1987).In the parasitized liver and spleen of P. flesus, macrophageaggregates were observed very close to A. simplex s.l. larvae,which represents the first report of MAs in association with ahelminth larva. A previous study described the presence ofMAs in Gasterosteus aculeatus L., infected with protozoanGlugea anomala Moniez 1887 (Dezfuli et al., 2004). The pres-ent observations lend support to the view of Vogelbein et al.(1987) that macrophage aggregates may be linked to parasiteinfections and, in all likelihood, represent an inflammatory re-sponse different from the typical granulomatous reaction.

Enigmatic rodlet cells (RCs), the function and nature of

which are still a matter of debate, can be found in the visceraand epithelia of virtually every fish species (Morrison andOdense, 1978; Leino, 1996). A large number of these cells wereencountered in the gut of Anguilla anguilla L., at the site ofdigenean attachment (Dezfuli et al., 1998), and in the liver andpancreas of Phoxinus phoxinus L. infected with a nematode(Dezfuli et al., 2000). Moreover, the number of RCs increasedin the intestinal epithelium of G. aculeatus near a protozoanxenoma (Dezfuli et al., 2004) and in the brain of P. phoxinusinfected with metacercariae of a trematode (Dezfuli et al.,2007). In the present investigation, several rodlet cells wereobserved, but only in the gut epithelium of flounders that har-bored A. simplex s.l. larvae on outer surface of intestine. Resultsof our study and data obtained from experiments with a syn-thetic corticosteroid (dexamethasone) (Manera et al., 2001) andwith a herbicide (propanil) (Dezfuli et al., 2003), suggest thatthe rodlet cells represent an inflammatory cell type closelylinked to other piscine inflammatory cells, such as eosinophilicgranulocytes (EGCs) and epithelioid cells (see Dezfuli et al.,2000). Furthermore, new information, especially in the last de-cade, supports the notion that the RCs are of an endogenousorigin (Leino, 1996; Iger and Abraham, 1997; Dezfuli et al.,1998, 2003, 2004, 2007; Mazon et al., 2007).

The neuroendocrine system of vertebrates includes the en-teric nervous and the diffuse endocrine systems (DES), both ofwhich play important roles in coordinating several intestinalprocesses (Hansen and Skadhauge, 1995; Larsson, 2000; Palm-er and Greenwood-Van Meerveld, 2001). A component of theDES includes the endocrine cells of the gut, which represent ahighly specialized mucosal sub-population (Rindi et al., 2004).Gut endocrine cells are associated with the expression of sev-eral regulatory molecules. These regulatory peptides are in-volved in the modulation of digestive functions such as enzymesecretion, nutrient uptake, and peristalsis (Hansen and Skad-hauge, 1995).

In the P. flesus digestive tract, among 8 tested neuropeptides,2 were found, i.e., bombesin and galanin, in higher concentra-tion in parasitized flounder. A bombesin-like substance was ob-served throughout a network of subtle nerve fibers within thefibroconnective capsule enclosing A. simplex s.l. larvae. In otherfish-parasite studies, this neuropeptide was observed in the con-nective tissue capsule produced by the gut of Salmo trutta inresponse to the acanthocephalan Pomphorhynchus laevis (Dez-fuli et al., 2002). A bombesin-like substance was also encoun-tered within the thin nerve fibers in the inflammatory connec-tive tissue of Gasterosteus aculeatus infected with the proto-zoan Glugea anomala (Dezfuli et al., 2004). Our previous ob-servations and those of the present survey support thehypothesis of Dezfuli et al. (2004), who suggested that the pres-ence of bombesin at the site of tissue inflammation in fishesacts as a putative neurotransmitter in the newly formed networkof nerve fibers. Most studies on galanin have focused on thecentral nervous system of vertebrates, but there are conflictingopinions regarding the function of this neuromodulator. It isalso postulated that it may not exert the same consequencewithin the different vertebrate classes. For example, in mam-mals galanin is said to have an inhibitory effect on electrolytesecretion (Kiliaan et al., 1993). In fish, galanin has been impli-cated in olfactory and gustatory functions, central visual pro-cessing, somatosensory transmission, osmoregulation, sex-spe-

1422 THE JOURNAL OF PARASITOLOGY, VOL. 93, NO. 6, DECEMBER 2007

cific behavior, and in affecting the cardiovascular system (Corn-brooks and Parsons, 1991; Holmqvist and Carlberg, 1992; LeMevel et al., 1998). The presence of galanin has been recentlyreported in the neuroendocrine system of the gut from severaluninfected (Bosi et al., 2004), as well as in parasitized, fishes(Dezfuli et al., 2004; Bosi et al., 2005). Further informationmust be obtained on the real function of this peptide in infectedfish. However, there were no discernible differences betweenCCK-39, NPY, and PHI in uninfected and infected flounders.

ACKNOWLEDGMENTS

We thank Dr. F. Moravec from the Academy of Sciences of the CzechRepublic for the identification of the nematodes. We are indebted to Dr.G. Bosi from the University of Milan for his expert advice in immu-nohistochemical techniques and also to Dr. E. Simoni from the Univer-sity of Ferrara for her technical assistance throughout the duration ofthis project. This investigation was supported through an award toB.S.D. from the European Union Access to Research Infrastructures(ARI) Action of the Improving Human Potential (IHP) Programme(contract HPRI-CT-1999-00106).

LITERATURE CITED

AGIUS, C., AND R. J. ROBERTS. 2003. Melano-macrophage centres andtheir role in fish pathology. Journal of Fish Diseases 26: 499–509.

BABLANIK, K., AND F. M. BILEQUEES. 1998. Histopathology of the liverof Otolithus argenteus associated with nematode larva. PakistanJournal of Zoology 30: 156–158.

BOSI, G., A. DI GIANCAMILLO, S. ARRIGHI, AND C. DOMENEGHINI. 2004.An immunohistochemical study on the neuroendocrine system inthe alimentary canal of the brown trout, Salmo trutta, L., 1758.General and Comparative Endocrinology 138: 166–181.

———, A. P. SHINN, L. GIARI, E. SIMONI, F. PIRONI, AND B. S. DEZFULI.2005. Histopathology and alteration to neuromodulators of the dif-fuse endocrine system of the alimentary canal of farmed Oncor-hynchus mykiss (Walbaum) naturally infected with Eubothriumcrassum (Cestoda). Journal of Fish Diseases 28: 703–711.

BRIAN, L. 1958. Ricerche sul ciclo biologico e l’ecologia di Ascariscapsularia Rud. Atti dell’Accademia Ligure di Scienze e Lettere14: 249–263.

CARMICHAEL, J. W. 1966. Cerebral mycetoma in trout. Sabouraudia 6:120–123.

CHINABUT, S. 1999. Mycobacteriosis and nocardiosis. In Fish diseasesand disorders, Vol. 3, P.T.K. Woo and D. W. Bruno (eds.). CABInternational, Wallingford, U.K., p. 319–340.

CORNBROOKS, E. B., AND R. L. PARSONS. 1991. Sexually dimorphic dis-tribution of a galanin-like peptide in the central nervous system ofthe teleost fish Poecilia latipinna. Journal of Comparative Neurol-ogy 304: 639–657.

COUILLARD, C. M., AND P. V. HODSON. 1996. Pigmented macrophageaggregates: A toxic response in fish exposed to bleached-Kraft milleffluent? Environmental Toxicology and Chemistry 15: 1844–1854.

———, P. J. WILLIAMS, S. C. COURTENAY, AND G. P. RAWN. 1999. His-topathological evaluation of Atlantic tomcod (Microgadus tomcod)collected at estuarine sites receiving pulp and paper mill effluent.Aquatic Toxicology 44: 263–278.

DEZFULI, B. S., S. CAPUANO, AND M. MANERA. 1998. A description ofrodlet cells of alimentary canal of Anguilla anguilla (L.) and theirrelationship with parasitic helminths. Journal of Fish Biology 53:1084–1095.

———, L. GIARI, AND A. P. SHINN. 2007. The role of rodlet cells in theinflammatory response in Diplostomum infected Phoxinus phoxinusbrains. Fish and Shellfish Immunology 23: 300–304.

———, ———, E. SIMONI, D. PALAZZI, AND M. MANERA. 2003. Alter-ation of rodlet cells in chub caused by the herbicide Stam� M-4(Propanil). Journal of Fish Biology 63: 232–239.

———, ———, ———, A. SHINN, AND G. BOSI. 2004. Immunohis-tochemistry, histopathology and ultrastructure of Gasterosteus acu-

leatus (L.) tissues infected with Glugea anomala (Moniez 1887).Diseases of Aquatic Organisms 58: 193–202.

———, F. PIRONI, L. GIARI, C. DOMENEGHINI, AND G. BOSI. 2002. Effectof Pomphorhynchus laevis (Acanthocephala) on putative neuro-modulators in the intestine of naturally infected Salmo trutta. Dis-eases of Aquatic Organisms 51: 27–35.

———, E. SIMONI, R. ROSSI, AND M. MANERA. 2000. Rodlet cells andother inflammatory cells of Phoxinus phoxinus infected with Ra-phidascaris acus (Nematoda). Diseases of Aquatic Organisms 43:61–69.

EIRAS, J. C., AND A. A. REGO. 1987. The histopathology of Scomberjaponicus infection by Nematobothrium scombri (Trematoda: Di-dymozoidae) and of larval anisakid nematode infections in the liverof Pagrus pagrus. Memorias do Instituto Oswaldo Cruz 82: 155–159.

ELARIFI, A. E. 1982. The histopathology of larval anisakid nematodeinfections in the liver of whiting, Merlangius merlangus (L.), withsome observations on blood leucocytes of the fish. Journal of FishDiseases 5: 411–419.

ELLIS, A. E. 1980. Antigen-trapping in the spleen and kidney of theplaice Pleuronectes platessa L. Journal of Fish Diseases 3: 413–426.

FERGUSON, H. W. 1976. The relationship between ellipsoids and melano-macrophage centres in the spleen of turbot (Scophthalmus maxi-mus). Journal of Comparative Pathology 86: 377–380.

GUILLEN-HERNANDEZ, S., AND P. J. WHITFIELD. 2004. Intestinal helminthparasites in flounder Platichthys flesus from the River Thames: Aninfracommunity analysis. Journal of Helminthology 78: 297–303.

HANSEN, M. B., AND E. SKADHAUGE. 1995. New aspects of the patho-physiology and treatment of secretory diarrhoea. Journal of Phys-iological Research 44: 61–78.

HAUCK, A. K., AND E. B. MAY. 1977. Histopathologic alterations as-sociated with Anisakis larvae in pacific herring from Oregon. Jour-nal of Wildlife Diseases 13: 290–293.

HERRAEZ, M. P., AND A. G. ZAPATA. 1986. Structure and function of themelano-macrophage centres in the goldfish (Carassius auratus).Veterinary Immunology and Immunopathology 12: 117–126.

HJELTNES, B., AND R. J. ROBERTS. 1993. Vibrionaceae. In Bacterial dis-eases of fish, V. M. Inglis, R. J. Roberts, and N. R. Bromage (eds.).Blackwell Ltd., Oxford, U.K., p. 109–121.

HOLMQVIST, B. I., AND M. CARLBERG. 1992. Galanin receptors in thebrain of a teleost: Autoradiographic distribution of binding sites inthe Atlantic salmon. Journal of Comparative Neurology 326: 44–60.

HOWELL, M. J. 1976. The peritoneal cavity of vertebrates. In Ecologicalaspects of parasitology, C. R. Kennedy (ed.). North-Holland, Am-sterdam, The Netherlands, p. 243–268.

IGER, Y., AND M. ABRAHAM. 1997. Rodlet cells in the epidermis of fishexposed to stressors. Tissue and Cell 29: 431–438.

KAHL, W. 1938. Nematoden in Seefishen. II. Erhegungen ueber denBefall von Seefishen mit Larven von Anacanthocoheilus rotunda-tus (Rudolphi) und die durch diese Larven hervorgerufenen Reak-tionen des Wirtesgewebes. Zeitschrift fur Parasitenkunde 10: 513–534.

KILIAAN, A. J., S. HOLMGREN, A.-C. JONSSON, K. DEKKER, AND J. A.GROOT. 1993. Neuropeptides in the intestine of two teleost species(Oreochromis mossambicus, Carassius auratus): Localization andelectrophysiological effects on the epithelium. Cell and Tissue Re-search 271: 123–134.

KØIE, M. 1999. Metazoan parasites of flounder Platichthys flesus (L.)along a transect from the southwestern to the northeastern BalticSea. Journal of Marine Science 56: 157–163.

LARSSON, L. I. 2000. Developmental biology of gastrin and somatostatincells in the antropyloric mucosa of the stomach. Microscopy Re-search and Technique 48: 272–281.

LE MEVEL, J. C., D. MABIN, A. M. HANLEY, AND J. M. CONLON. 1998.Contrasting cardiovascular effects following central and peripheralinjections of trout galanin in trout. American Journal of Physiology275: R1118–R1126.

LEINO, R. L. 1996. Reaction of rodlet cells to a myxosporean infectionin kidney of the bluegill, Lepomis macrochirus. Canadian Journalof Zoology 74: 217–225.

MACCHI, G. J., A. ROMANOL, AND H. E. CHRISTIANSEN. 1992. Melano-

DEZFULI ET AL.—HISTOPATHOLOGY OF PARASITIZED P. FLESUS 1423

macrophage centres in white mouth croaker Micropogonias furieri,as biological indicators of environmental changes. Journal of FishBiology 10: 971–973.

MANERA, M., E. SIMONI, AND B. S. DEZFULI. 2001. Effect of dexameth-asone administration on occurrence and ultrastructure of rodlet cellsin Carassius auratus L. with particular reference to bulbus arter-iosus. Journal of Fish Biology 59: 1239–1248.

MARGOLIS, L. 1970. Nematode disease of marine fishes. In A sympo-sium on diseases of fishes and shellfishes, S. F. Sniezko (ed.).American Fisheries Society, Washington, D.C., p. 190–208.

MAZON, A. F., M. O. HUISING, A. J. TAVERNE-THIELE, J. BASTIAANS, AND

B. M. L. VERBURG-VAN KEMENADE. 2007. The first appearance ofrodlet cells in carp (Cyprinus carpio L.) ontogeny and their possibleroles during stress and parasite infection. Fish and Shellfish Im-munology 22: 27–37.

MIYAZAKI, T., W. A. ROGERS, AND K. J. SEMMENS. 1988. Gastro-intestinalhistopathology of paddlefish, Polyodon spathula (Walbaum), in-fected with larval Hysterothylacium dollfusi Schmidt, Leiby &Kritsky, 1974. Journal of Fish Diseases 11: 245–250.

MORAVEC, F. 1994. Parasitic nematodes of freshwater fishes of Europe.Kluwer, Dordrecht, The Netherlands, 473 p.

MORRISON, C. M., AND P. H. ODENSE. 1978. Distribution and morphol-ogy of the rodlet cell in fish. Journal of the Fisheries ResearchBoard Canada 35: 101–116.

OSHIMA, T. 1972. Anisakis and anisakiasis in Japan and adjacent area.Progress of Medical Parasitology in Japan 4: 301–393.

PALMER, J. M., AND B. GREENWOOD-VAN MEERVELD. 2001. Integrativeneuroimmunomodulation of gastrointestinal function during entericparasitism. Journal of Parasitology 87: 483–504.

REITE, O. B. 1997. Mast cells/eosinophilic granule cells of salmonids:Staining properties and responses to noxious agents. Fish and Shell-fish Immunology 7: 567–584.

RINDI, G., A. B. LEITER, A. S. KOPIN, C. BORDI, AND E. SOLCIA. 2004.The ‘‘normal’’ endocrine cell of the gut: Changing concepts andnew evidences. Annals of the New York Academy of Sciences1014: 1–12.

ROBERTS, R. J. 2001. Fish pathology, 3rd ed. W.B. Saunders, Philadel-phia, Pennsylvania, 472 p.

SMITH, J. W. 1984. The abundance of Anisakis simplex L3 in the body-cavity and flesh of marine teleosts. International Journal for Para-sitology 14: 491–495.

———, AND R. WOOTTEN. 1975. Experimental studies on the migrationof Anisakis sp. larvae (Nematoda: Ascaridida) into the flesh of her-ring, Clupea harengus L. International Journal for Parasitology 5:133–136.

TORRES, P., AND H. GONZALEZ. 1978. Determinacion de larvas de Ter-ranova (�Phocanema) y Anisakis en Genypterus sp. Aspectos mor-fometricos e histopatologicos a nivel hepatico. Boletin Chileno deParasitologia 33: 82–86.

VOGELBEIN, W. K., J. W. FOURNIE, AND R. M. OVERSTREET. 1987. Se-quential development and morphology of experimentally inducedhepatic melano-macrophage centres in Rivulus marmoratus. Jour-nal of Fish Biology 31: 145–153.