DISEASES OF AQUATIC ORGANISMSDis Aquat Org

Vol. 94: 125–134, 2011doi: 10.3354/dao02322

Published April 6

INTRODUCTION

Fish gills serve as an entry point for pathogensbecause of their direct contact with the environmentand because physiological constraints require only afragile barrier between the environment and the circu-latory system. Immune mechanisms are required in thegill to reduce the likelihood of successful invasion by

pathogens. When rainbow trout Oncorhynchus mykissare infected with Renibacterium salmoninarum, thegills produce a quick and prolonged nitric oxide syn-thase response (Campos-Perez et al. 2000). The gillitself also contains lymphoid tissue rich in T cells (Hau-garvoll et al. 2008) and CD4 receptors that are neces-sary for cell-mediated immunity (Buonocore et al.2008). Gill disease models are helpful in determining

© Inter-Research 2011 · www.int-res.com*Email: jan.lovy@dfo-mpo.gc.ca

Histochemical and ultrastructural analysis ofpathology and cell responses in gills of channel

catfish affected with proliferative gill disease

J. Lovy1,*, A. E. Goodwin2, D. J. Speare3, D. W. Wadowska4, G. M. Wright5

1Fisheries and Oceans Canada, Pacific Biological Station, Aquatic Animal Health Unit, 3190 Hammond Bay Road, Nanaimo, British Columbia V9T 6N7, Canada

2University of Arkansas at Pine Bluff, Aquaculture/Fisheries Center, Pine Bluff, Arkansas 71601, USA3Department of Pathology and Microbiology, 4Department of Graduate Studies and Research,

and 5Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown,Prince Edward Island C1A 4P3, Canada

ABSTRACT: Pond-reared channel catfish Ictalurus punctatus with proliferative gill disease (PGD),caused by the myxozoan parasite Henneguya spp., were examined with light and transmission elec-tron microscopy to better characterize the inflammatory response during infection. The early stagesof disease are characterized by the destruction of collagen in the matrix of the gill filament cartilagecausing weakness and breaks within the gill filaments. These early lesions lacked a notable inflam-matory response around the disrupted cartilage, a chondrocyte response was not apparent, and theparasite was not present, suggesting that the cartilage breaks occur prior to inflammation and arrivalof the parasite in the gill. In later lesions, a significant inflammatory response was generated in areasof disrupted cartilage, and the inflammatory infiltrate was composed of a mixed population of granu-locytes including neutrophils and cells that resembled eosinophils. The majority of eosinophil-likecells demonstrated evidence of degranulation. Trophozoites of Henneguya spp. were surrounded bya uniform population of cells believed to be neutrophils. The granulocytes were infiltrated within thedense collagen layer of the gill filament cartilage and often appeared within chondrocyte lacunae inplace of the chondrocyte. The gill lamellae adjacent to the lesions were fused and contained aninflammatory infiltrate containing granulocytes and cells with pericentriolar granules that resembledprevious descriptions of Langerhans-like cells. These cells were abundant within damaged lamellarepithelium, but were only rarely found within the gill filament. Lesions that appeared to be recover-ing lacked the dense collagenous layer around the cartilage and contained hyperplastic and hyper-trophic chondrocytes that formed a callus. Other chondrocytes in the lesions had ultrastructural fea-tures indicative of cell death.

KEY WORDS: Channel catfish · Proliferative gill disease · Inflammation · Henneguya · Collagen ·Pathology · Ultrastructure

Resale or republication not permitted without written consent of the publisher

Dis Aquat Org 94: 125–134, 2011

how gills respond to disease with the initial inflamma-tory response generated during an infection, which iscritical for the development of adaptive immunity. Insalmonids infected with the gill microsporidian Lomasalmonae, acute and chronic inflammatory responsesare observed in the gill, and characterization of theinflammatory infiltrate led to the discovery of a celltype that resembles mammalian Langerhans cells(Lovy et al. 2006). It is hypothesized that these cells areresident in secondary lymphoid tissues, particularly inthe spleen, and are recruited systemically to initiateadaptive immune responses during inflammatory in -sults (Lovy et al. 2006, 2008, 2009).

An inflammatory process is associated with prolifer-ative gill disease (PGD) that affects pond-reared chan-nel catfish Ictalurus punctatus in the southern USA andcan lead to mortality rates exceeding 50% (Bowser &Conroy 1985). In PGD, the gills of channel catfish areinfected by the actinosporean stage of the myxozoanparasite Henneguya ictaluri, which develops withinthe benthic oligochete Dero digitata (Styer et al. 1991,Pote et al. 2000). Infection with the parasite occursthrough multiple portals including the skin, gills andgastric mucosa (Belem & Pote 2001). The parasite firstinduces severe congestion and hemorrhaging in thegill, which develops into epithelial hyperplasia, inflam-mation and lytic cartilage lesions (Griffin et al. 2010).The presence of broken gill filament cartilage in pond-reared channel catfish is pathoneumonic for PGD, buthistology and real-time PCR are often used as confir-matory tests (Griffin et al. 2008, Pote et al. 2010). Thesevere inflammatory response elicited during PGD isbelieved to cause lysis of the cartilage leading tobreaks within the cartilage (MacMillan et al. 1989).The goals of the present study are to examine thepathology associated with PGD in the gills of channelcatfish by means of light microscopy and transmissionelectron microscopy in order to characterize the hostcells present in the inflammatory response and to char-acterize the gill microenvironment within lesions.

MATERIALS AND METHODS

Five 10 to 15 cm channel catfish were collected froma commercial catfish fingerling production pond on afish farm in Arkansas, USA, during a single sampleperiod during the month of May. The fish in the pondwere swimming in the proximity of the aerators anddisplaying signs of oxygen deprivation. Grossly, thegill filaments of the affected fish appeared thickenedand many were bent. Wet mount preparations of gillsdemonstrated breaks in the gill filament cartilage.These behavioral and clinical signs were consistentwith a severe, active PGD.

Light microscopy. Gill tissue was fixed in formalinfor at least 24 h, dehydrated through a series ofethanols, cleared in xylene and embedded in paraffinwax. Serial sections, 5 µm thick, were cut andmounted on multiple glass slides. The tissues werestained with either hematoxylin and eosin (H&E),Alcian blue, Safranin O, Masson’s trichrome or peri-odic acid-Schiff (PAS). Slides were viewed and pho-tographed with a microscope (Axioplan 2, Zeiss)equipped with a high resolution digital camera (Axio-Cam) with Axiovision software v. 4.5.0.0 (Carl ZeissCanada).

High resolution light microscopy and transmissionelectron microscopy. Fish gills were fixed in 2% glu-taraldehyde in 0.1 M phosphate buffer, pH 7.2, pack-aged with ice packs and sent to the Atlantic VeterinaryCollege (AVC), Charlottetown, Prince Edward Island.Upon receipt the gill tissue was cut into smaller piecesand placed into fresh 2% buffered glutaraldehyde andkept at 4°C over night. The tissue was then washed in0.1 M phosphate buffer and transferred to 1% osmiumtetroxide in phosphate buffer for 1 h at room tempera-ture. Dehydration through a series of ethanol grades topropylene oxide immediately followed osmium fixa-tion. The tissue was then infiltrated with a mixture ofSpurr’s resin and propylene oxide in a ratio of 1:1, 3:1,and lastly in pure Spurr’s resin for 24 h each. The tissuewas embedded in pure Spurr’s resin (Canemco) at60°C for 24 h. Semi-thin sections (0.5 µm) were cutfrom 5 blocks of tissue from each infected fish. The sec-tions were stained with 1% toluidine blue in 1%sodium tetraborate solution and viewed with the lightmicroscope. All samples that contained cartilagelesions and an inflammatory response were re-cut togenerate ultra-thin sections (90 nm). The ultra-thinsections were retrieved onto copper super grids (200mesh) and stained with uranyl acetate and Sato’s leadstain. The sections were examined with a transmissionelectron microscope (Hitachi H7500, Nissei-Sangyo) operated at 80 kV and imaged with a side mount digital camera (AMT XR40, Advance Microscopy)by means of the Image Capture Engine Software v.600.149.

RESULTS

Light microscopy

Staining with H&E and toluidine blue demonstratedboth the territorial matrix, rich in glycosaminoglycans(GAGs), and the interterritorial matrix (collagenouscomponent) of the gill cartilage. With H&E staining thecollagen was eosinophilic and the territorial matrixwas basophilic. The toluidine blue showed less specific

126

Lovy et al.: Pathology of proliferative gill disease

staining in which both components were blue. TheMasson’s trichrome stain clearly identified the collage-nous component of the cartilage. Both Safranin O andAlcian blue staining demonstrated the GAG-richareas. The normal gill filament cartilage within thesample contained varying numbers of chondrocytessurrounded by a territorial matrix made up of GAGsand a fibrous collagenous layer surrounding the terri-torial matrix. Two different morphological types of nor-mal gill filament cartilage were observed. One typecontained predominantly collagen with only a fewrounded chondrocytes, while the second contained anorganized network of cuboidal chondrocytes sur-rounded by collagen. In some instances the more cellu-lar cartilage was found closer to the gill filament tips,whereas the less cellular form was found closer to thebase of the filaments.

In gills infected with Henneguya, trophozoites weresurrounded by mononuclear host inflammatory cells(Fig. 1A). The mononuclear cells had a PAS-positivegranular cytoplasm. Trophozoites were not observed inall areas of chondrocyte lesions and cartilage breaks.Several types of lesions were observed affecting thegill filament cartilage. One type of lesion had gill fila-ment cartilage with incontinuities in the extracellularmatrix resulting in breaks in the cartilage (Fig. 1B,C).These lesions had a minimal inflammatory infiltrateand parasites were not present (Fig. 1B,C, arrows).Other lesions with similar cartilage breaks and missingthe extracellular matrix had a noticeable inflammatoryinfiltrate with parasite trophozoites (Fig. 1D). In lesionswith a severe inflammatory infiltrate and parasitetrophozoites there was a complete loss of the gill fila-ment cartilage (Fig. 1C, arrowhead). Epithelial hyper-plasia and lamellar fusion were common adjacent tothe damaged gill filament cartilage (Fig. 1C). Anothertype of lesion was the gill filament cartilage missingthe surrounding dense collagenous component andcontaining hyperplastic and hypertrophic chondro-cytes adjacent to the missing collagen (Fig. 1E–G). Inthese lesions containing the hypertrophic chondro-cytes the parasite was not present and the inflamma-tory response was mild or absent. In both toluidineblue-stained and Masson's trichrome-stained sectionsthe deep blue staining of the normal collagenousmatrix making up the cartilage was missing in theareas of chondrocyte hypertrophy (Fig. 1E,F). The tolu-idine blue-stained lesions demonstrated a metachro-matic staining within the territorial matrix around thehypertrophic chondrocytes (Fig 1E). In H&E-stainedsections the eosinophilic-staining collagen was miss-ing in the areas of broken cartilage with hyperplasticand hypertrophic chondrocytes, while the unaffectedareas still had the eosinophilic collagenous matrixintact (Fig 1G).

Transmission electron microscopy

Ultrastructural characterization of the host cells withininflammatory lesions revealed a homo genous populationof mononuclear cells directly surrounding the parasite(Fig. 2A). These cells contained 2 types of granules, elon-gated granules with material in a fibrillar arrangement(Fig. 2B, arrows) and small pleomorphic granules con-taining electron-dense amorphous material (Fig. 2B, ar-rowheads). These granules are characteristic for fishneutrophils. Inflammation in the gills became locally dif-fuse in areas not directly surrounding the parasite andextended from the gill filament to the adjacent lamellarepithelium. The inflammatory infiltrate contained amixed population of leukocytes including cells that re-sembled neutrophils, and other granulocytes with largeintact granules similar in morphology to mast cells (alsoknown as eosin ophilic granular cells). Other cells withabundant relatively small granules with variably elec-tron-dense material were observed (Fig. 2C). A majorityof these cells showed evidence of degranulation, inwhich this central component of the oval or sphericalgranules was degranulated (Fig. 2D,E). The majority ofinflammatory cells were adjacent to the collagen layer ofthe filament cartilage. The inflammatory cells hadpseudopods extending into the collagen and cells wereobserved within the dense collagenous layer (Fig. 2F).The inflammatory cells invaded the interterritorial ma-trix and often infiltrated the chondrocyte lacunae amongother granulocytes replacing the chondrocytes.

Previously described fish cells with unique pericen-triolar granules that resemble Langerhans cells werecommon within the inflammatory infiltrate. These cellswere found within the fused lamellar epithelium(Fig. 3A) but only rarely occurred within the gill fila-ment. The cells characteristically had a network ofgranules, often containing electron-dense deposits,localized around the centrisome (Fig. 3B,D). The lumi-nal material within the granules was of variable elec-tron density and electron-dense bodies were fre-quently observed within the granules (Fig. 3B–D).

Ultrastructural differences were observed betweennormal gill cartilage and the cartilage associated withPGD lesions. Two types of normal gill filament cartilagewere observed: mature predominantly acellular carti-lage (Fig. 4A,B) and highly cellular cartilage (Fig. 4C). Inthe mature predominantly acellular gill filament carti-lage the fibrous component was made up mostly of colla-gen and electron-dense material which is likely the pro-teoglycan component of the territorial matrix (Fig. 4A,B).The chondrocytes were embedded within the thick fi-brous component. The chondrocytes were rounded, rel-atively small, devoid of organelles, and nuclei were eu-chromatic with peripheral heterochromatin (Fig. 4A,B).In the highly cellular filament cartilage the chondrocytes

127

Dis Aquat Org 94: 125–134, 2011128

Fig. 1. Ictalurus punctatus. Light micrographs of gills affected by proliferative gill disease (PGD). (A) Trophozoites of Henneguyaictaluri (arrows) surrounded by mononuclear inflammatory cells; 5 µm section stained with H&E. (B) Gill filament with loss of col-lagen in areas of broken cartilage (arrow); 5 µm section stained with Masson’s trichrome. (C) Two gill filaments affected by PGD;the right filament has missing collagen and breaks (arrows) in the highly acellular cartilage. The left gill filament has a pro-nounced inflammatory infiltrate with Henneguya spp. trophozoites (arrowhead); 0.5 µm section stained with toluidine blue. (D)Highly cellular form of cartilage with breaks and lost collagen (arrows) with a notable inflammatory infiltrate; 0.5 µm sectionstained with toluidine blue. (E–G) Gill filament cartilage containing hyperplastic and hypertrophic chondrocytes (arrows). Noticeareas of normal cartilage organization (arrowheads) in (F,G). (E) 0.5 µm section stained with toluidine blue. (F) 5 µm section

stained with Masson’s trichrome. (G) 5 µm section stained with H&E. Scale bars = (A) 20 µm, (B–G) 50 µm

Lovy et al.: Pathology of proliferative gill disease

were relatively large, cuboidal, and closely apposed toeach other with a thin layer of territorial matrix separat-ing the neighboring chondrocytes. Enclosing thecuboidal chondrocytes and territorial matrix was the fi-brous layer composed of collagen. The chondrocyteswithin the cellular cartilage contained little rough endo-plasmic reticulum (RER) (Fig. 4C). In PGD-affected car-tilage there was a loss of the surrounding collagen. Thechondrocytes that were no longer surrounded by an ob-vious collagen layer were hypertrophied (Fig. 4D). Thehypertrophic chondrocytes contained large amounts ofRER in the cytoplasm and a primarily euchromatic nucleus, lacking the peripheral heterochromatin ob-served in the normal chondrocytes (Fig. 4E). The areas

directly adjacent to the hypertrophic chondrocytes werelargely electron lucent, instead of being surrounded bythe dark territorial matrix (Fig. 4E). In some lesions, celldeath was evident in the chondrocytes in which the cyto-plasm was mostly devoid of organelles and the nucleuswas condensed and star-shaped (Fig. 4F,G). The necroticchondrocytes also had normal chondrocytes adjacent tothem and the territorial matrix was apparent betweenthe cells (Fig. 4F,G). Lesions with a severe inflammatoryresponse led to the complete destruction of the filamentcartilage, in which hypertrophied chondrocytes were ob-served freely within the inflammatory infiltrate. In thesecases there was no evidence of other structural compo-nents of the cartilage (Fig. 4H).

129

Fig. 2. Ictalurus punctatus. Transmission electron micrographs of inflammatory cells in response to proliferative gill disease(PGD). (A) Mononuclear inflammatory cells (*) directly surrounding the parasites (arrows). (B) Cytoplasmic granules of mononu-clear cells from (A) include elongated granules with fibrillar material (arrows) and small pleomorphic granules (arrowheads). (C)A granulocyte with abundant intact granules (arrows). (D) A degranulating (arrows) granulocyte. (E) Central granule contentsare degranulated (arrows). (F) Inflammatory response directly adjacent to the collagen layer (*) of the gill filament cartilage.Granulocytes are extending pseudopods (arrows) into the collagen (*) or have infiltrated within the collagen layer (arrowheads).

Scale bars = (A,F) 5 µm, (B) 500 nm, (C,E) 1 µm, (D) 2 µm

Dis Aquat Org 94: 125–134, 2011

DISCUSSION

A critical component of PGD is the effect of the dis-ease on the fibrous component, particularly collagen,of the gill filament cartilage. There was a clear correla-tion between collagen loss in the gill filament cartilageand breaks in the cartilage. Areas of chondrocyte pro-liferation and hypertrophy were also devoid of colla-gen. Our results suggest that the collagen is brokendown and breaks develop during the early stages ofdisease, before evidence of a host inflammatoryresponse, chondroplasia or the occurrence of parasites.This is further supported by Wise et al. (2008), whodemonstrated that gill filament breaks occur before thehistological detection of the parasite in the gills. As col-lagen is the major component for the structuralintegrity of cartilage, it is logical that its breakdownwould lead to weakness in the filament cartilage,which could progress to breaks. It will be important to

determine the initial cause of the collagen breakdownto better understand the pathogenesis of disease. Con-sidering cartilage breaks occur before a notableinflammatory response, it is possible that proteases arereleased from early parasite stages that degrade thecollagen in order to support growth of the trophozoiteswithin the gill filament. For example Myxobolus cere-bralis, a myxozoan in trout, digests and causes lysis ofcartilage with serine proteases that cause degenera-tion of cartilage (Kelley et al. 2004). Kudoa thyrsites, amyxozoan of the muscle of fish, releases proteolyticenzymes that lead to histiolysis (Langdon 1991). Thepronounced inflammatory response in PGD probablycontributes to the breakdown of the gill filament carti-lage during the later stages of disease. The presence ofdamaged cartilage occurring before a notable inflam-matory response suggests that the initial breakdown ofcollagen is not caused by inflammation. Furtherresearch should be done to investigate the presence of

130

Fig. 3. Ictalurus punctatus. Transmission electron micrographs of Langerhans-like cells in gills of proliferative gill disease (PGD)-affected catfish. (A) Langerhans-like cells (arrows) and degranulated granulocytes (*) within the lamellar epithelium. Arrow-heads: pillar cells. (B) Langerhans-like cell containing granules (arrowheads) localized near a centrisome (arrow). n: cell nucleus.(C) Granules (arrowhead) in close proximity to the Golgi apparatus (arrows). n: cell nucleus. (D) Granules containing electron-

dense material surrounding dense deposits (arrowhead). Arrow: cell centrisome. Scale bars = (A) 5 µm, (B–D) 500 nm

Lovy et al.: Pathology of proliferative gill disease 131

Fig. 4. Ictalurus punctatus. Transmission electron micrographs of (A–C) normal gill filament cartilage and (D–H) cartilage withinproliferative gill disease (PGD)-induced lesions. (A) Mature highly acellular cartilage containing collagen (*) and chondrocytes(arrows). (B) Chondrocyte (arrows) from the type of cartilage seen in (A) surrounded by collagen (black *) and the proteoglycancomponent (white *). (C) Highly cellular gill filament cartilage containing chondrocytes (arrows) organized within the territorialmatrix (arrowheads), and enclosed by collagen (*). (D) A normal chondrocyte (white arrow) surrounded by the collagen compo-nent (*) and adjacent to hypertrophic chondrocytes (black arrows) no longer surrounded by collagen. (E) Hypertrophied chondro-cytes (arrow) containing abundant rough endoplasmic reticulum (RER) and primarily euchromatic nuclei (n). Chondrocytes aresurrounded by electron-lucent material (arrowheads). (F) Necrotic chondrocytes (black arrows) near normal chondrocytes (whitearrows) and territorial matrix (*). (G) Necrotic chondrocyte with a star-shaped and condensed nucleus (arrow) near a relativelynormal chondrocyte (arrowhead). (H) Free chondrocytes (arrows) within an inflammatory infiltrate composed of degranulated in

flammatory cells (arrowheads). Scale bars = (A,C,E,F,H) 5 µm, (B,G) 2 µm, (D) 10 µm

Dis Aquat Org 94: 125–134, 2011

proteases secreted from Henneguya spp. that couldlead to the breakdown of collagen during the earlyinfection of the gills.

The present study is the first to ultrastructurallyexamine the host cell response in PGD-affected fish.The response directly surrounding the pathogen con-sists of a uniform population of cells that most closelyresembled neutrophils based on the presence of elon-gated fibrillar granules and abundant small pleomor-phic granules (Takemori et al. 1994, Pavelka & Roth2005). The nuclei of the catfish neutrophils wererounded, as opposed to the more common segmentednuclei seen in other vertebrate neutrophils. Therounded shape of catfish neutrophil nuclei has beenpreviously reported by Grizzle & Rogers (1976) andCannon et al. (1980). The inflammatory response wasmost severe in the gill filament, where the filamentcartilage was replaced with the inflammatory infiltrateconsisting of a mixed population of cells dominated byneutrophils and other granulocytes. The granulocytesobserved in the present study had abundant granulesthat in some cells were large and resembled mast cells,also referred to as eosinophilic granular cells, andother cells with smaller granules that more closelyresembled mammalian eosinophils. In the majority ofthe eosinophil-like cells the central contents of theirgranules were degranulated. The large numbers ofdegranulated cells within the lesions suggest that thenature of the inflammatory response is particularlydamaging to the host tissue. Ultrastructurally thisstrongly resembled the degranulated eosinophil-likecells observed within the inflammatory response ofAtlantic salmon Salmo salar clinically affected withamoebic gill disease (Lovy et al. 2007).

The neutrophils and degranulating granulocytesobserved in the present study appeared to be chemo-tactic towards collagen and often infiltrated into thedense collagenous layer of the gill filament cartilage.Damage done to the extracellular matrix (ECM) isknown to trigger immune and inflammatory responses;for example collagen fragments from collagenasedigestion are a potent stimulator for interleukin 1secretion in human blood mononuclear cells (Pacifici etal. 1991). The ECM is a strong modulator of inflamma-tion in gilthead seabream Sparus aurata (Castillo-Briceno et al. 2010) and collagen fragments generatedfrom the action of proteases have a high stimulatorycapacity for fish phagocytes (Castillo-Briceno et al.2009). Because the destruction of collagen is commonwithin the gill of PGD-affected fish, the damaged ECMof these fish probably generates potent pro-inflamma-tory signals. The highly inflammatory nature of PGD inchannel catfish is probably linked to the expression ofdamage-associated molecular patterns from the earlydamage done to the collagen within the gill. The pres-

ence of neutrophils and degranulated cells that resem-ble eosinophils in PGD-affected catfish gills probablycontributes to collagen and the cartilage damage.Human neutrophil granules are known to contain neu-trophil elastase and neutrophil collagenase (alsoknown as MMP-8), both of which can cleave nativetype I collagen (Kafienah et al. 1998, Khatwa et al.2010). The MMP-8 protein is known to promote migra-tion of neutrophils through collagen-rich matrix(Khatwa et al. 2010) and this collagenase has beendemonstrated in channel catfish to have 51%nucleotide and amino acid homology to human MMP-8 (Noya et al. 1999). Stimulated catfish neutrophils arealso able to degrade type I collagen (Noya et al. 1999).Interestingly, the anti-inflammatory drug, dexametha-sone, blocks all collagenolytic activity during acuteinflammation in a rat model (Etherington et al. 1979)and according to Belem (1994), dexamethasoneadministration to PGD-affected catfish causes a reduc-tion in the inflammatory response in the gills. It is pos-sible that dexamethasone treatment in catfish reducesthe inflammatory-driven degradation of collagen seenwith a PGD infection. The cells that resemble degran-ulated eosinophils in the present study may also con-tribute to the destruction of collagen. Although thecontents of fish eosin ophil granules are largelyunknown, human eosin ophils contain metalloprotein,which degrades types I and III collagen (Hibbs et al.1982).

The finding of Langerhans-like cells within inflam-matory lesions is consistent with the presence of thesecells in other inflammatory gill diseases, such as inmicrosporidial gill disease in salmonids (Lovy et al.2006, 2008). These cells were identified by the pres-ence of pericentriolar granules, which have been usedas a morphological criterion for the identification ofLangerhans-like cells (Lovy et al. 2010). These cellswere found predominantly within the inflammatoryinfiltrate within the fused gill lamellar epithelium andwere only rarely observed in the more severe inflam-matory lesions within the gill filament. The Langer-hans-like cells in catfish (ictalurids), as described inthis study, contain dense bodies within the granulelumen, and ultrastructurally they strongly resemblethe Langerhans-like cells in cyprinid fishes (Lovy et al.2010). The functional importance of the dense bodieswithin the granule lumens of cyprinids and ictaluridsand their absence within similar cells of salmonids(Lovy et al. 2008) is currently unknown. An interestingfinding from this study not previously reported is thevariable density of the luminal material within thegranules. The common presence of a Golgi apparatus,mitochondria and multivesicular bodies (Lovy et al.2010) near the granules suggests metabolic activity inthe cells. Currently the cells are believed to be anti-

132

Lovy et al.: Pathology of proliferative gill disease

gen-presenting cells and the material within the gran-ule lumens may represent material that was taken upand broken down, or it may be material being synthe-sized and packaged within the granules. Discoveringthe contents of the granules will be invaluable in deter-mining their function in these cells.

PGD in catfish causes destruction of the cartilagematrix, particularly degrading the collagen that makesup the dense outer layer of the cartilage. The destruc-tion of the cartilage matrix leads to interestingresponses of the chondrocytes, including hyperplasiaand hypertrophy in areas devoid of collagen (presum-ably cartilage regeneration). The loss of matrix proba-bly stimulates the chondrocytes to synthesize andsecrete new matrix to repair cartilage within thelesions. This is supported by the presence of hyper-trophic chondrocytes in this study that contain abun-dant RER, probably reflecting their active state of car-tilage matrix synthesis. In the present study cell deathwas observed in chondrocytes within affected filamentcartilage, although it was difficult to determine byultrastructure if death in these cells was caused byapoptosis or necrosis, since features were not consis-tent with either. These unique features of cell deathmay reflect a mechanism of chondrocyte death duringcartilage recovery. The chondrocyte response in PGD-affected lesions is similar to the stages of developmentof a soft callus during bone remodeling after a bonefracture in mammals. Bone remodeling in mammalshas 4 stages: inflammation, soft callus formation, hardcallus formation and finally bone remodeling (Schin-deler et al. 2008). Considering that fish gill cartilagedoes not terminally differentiate into bone, the calluswould not proceed into the development of a hard cal-lus with osteogenesis. In mammal bone remodeling,chondrocytes proliferate to fully replace the granula-tion tissue to form the soft callus (Schindeler et al.2008). The chondrocytes then undergo hypertrophyfollowed by cell death (Ahmed et al. 2007a). Themethod of chondrocyte death during this process isthought be through a non-classicial pathway of apop-tosis, although the exact method is controversial andhas been described as chondroptosis (Roach et al.2004), non-apoptotic cell death (Ahmed et al. 2007b)and autophagic cell death (Shapiro et al. 2005). Thechondrocytes of the gill filament cartilage in the pre-sent study were hypertrophic and some had evidenceof cell death, demonstrating a pattern of callus devel-opment that is similar to that observed during bonehealing in mammals.

The fish examined in this study were clinicallyinfected channel catfish, which are chronicallyexposed to the parasite in the pond environment; thus,various stages of infection progression were observed.For clinical disease to occur in fish, exposure to the

pathogen must occur over a chronic period. When fishare exposed to the parasite on a single day and thenremoved from the source of the parasite, recovery frominfection rapidly occurs (Wise et al. 2008). The varietyof lesions described in the present study demonstratesthe complex pathogenesis of this disease and demon-strates healing in the gill filament cartilage, which iscompletely destroyed during infection. Consideringthat the use of a single exposure model leads to rapidrecovery without clinical disease, it would be an idealmodel to study the regenerative capacity of the gill fil-ament cartilage. Gills are able to quickly and fullyregenerate during recovery from disease, althoughfew gill disease models lead to the gill filamentdestruction as seen with PGD. Thus, PGD would be anideal model to study the cellular mechanisms of carti-lage regeneration and cellular responses during infec-tion and recovery.

LITERATURE CITED

Ahmed YA, Tatarczuch L, Pagel CN, Davies HM, Mirams M,Mackie EJ (2007a) Hypertrophy and physiological deathof equine chondrocytes in vitro. Equine Vet J 39:546–552

Ahmed YA, Tatarczuch L, Pagel CN, Davies HM, Mirams M,Mackie EJ (2007b) Physiological death of hypertrophicchondrocytes. Osteoarthritis Cartilage 15:575–586

Belem AMG (1994) Portals of entry of Aurantiactinomyxonictaluri and increased infection with Aurantiactinomyxonafter immunosuppression in channel catfish. PhD disserta-tion, Mississippi State University, Mississippi State, MS

Belem AMG, Pote LM (2001) Portals of entry and systemiclocalization of proliferative gill disease organisms in chan-nel catfish Ictalurus punctatus. Dis Aquat Org 48:37–42

Bowser PR, Conroy JD (1985) Histopathology of gill lesions inchannel catfish associated with Henneguya. J Wildl Dis21:177–179

Buonocore F, Randelli E, Casani D, Guerra L and others(2008) A CD4 homologue in sea bass (Dicentrarchuslabrax): molecular characterization and structural analy-sis. Mol Immunol 45:3168–3177

Campos-Perez JJ, Ward M, Grabowski PS, Ellis AE, Sec-ombes CJ (2000) The gills are an important site of iNOSexpression in rainbow trout Oncorhynchus mykiss afterchallenge with the Gram-positive pathogen Renibac-terium salmoninarum. Immunology 99:153–161

Cannon SM, Mollenhauer HH, Eurell TE, Lewis DH, CannonAM, Tompkins C (1980) An ultrastructural study of theleukocytes of the channel catfish, Ictalurus punctatus.J Morphol 164:1–23

Castillo-Briceno P, Sepulcre MP, Chaves-Pozo E, Meseguer J,Garcia-Ayala A, Mulero V (2009) Collagen regulates theactivation of professional phagocytes of the teleost fishgilthead seabream. Mol Immunol 46:1409–1415

Castillo-Briceno P, Arizcun-Arizcun M, Meseguer J, MuleroV, Garcia-Ayala A (2010) Correlated expression profile ofextracellular matrix-related molecules during the inflam-matory response of the teleost fish gilthead seabream. DevComp Immunol 34:1051–1058

Etherington DJ, Silver IA, Gibbons R (1979) An in vitro modelfor the study of collagen degradation during acute inflam-mation. Life Sci 25:1885–1891

133

Dis Aquat Org 94: 125–134, 2011

Griffin MJ, Wise DJ, Camus AC, Mauel MJ, Greenway TE,Pote LM (2008) A real-time polymerase chain reactionassay for the detection of the myxozoan parasite Hen-neguya ictaluri in channel catfish. J Vet Diagn Invest20:559–566

Griffin MJ, Wise DJ, Camus AC, Mauel MJ, Greenway TE,Pote LM (2010) Variation in susceptibility to Henneguyaictaluri infection by two species of catfish and their hybridcross. J Aquat Anim Health 22:21–35

Grizzle JM, Rogers WA (1976) Anatomy and histology of thechannel catfish. Auburn University Experiment Station,Auburn, AL

Haugarvoll E, Bjerkas I, Nowak BF, Hordvik I, Koppang EO(2008) Identification and characterization of a novelintraepithelial lymphoid tissue in the gills of Atlanticsalmon. J Anat 213:202–209

Hibbs MS, Mainardi CL, Kang AH (1982) Type-specific colla-gen degradation by eosinophils. Biochem J 207:621–624

Kafienah W, Buttle DJ, Burnett D, Hollander AP (1998) Cleav-age of native type I collagen by human neutrophil elas-tase. Biochem J 330:897–902

Kelley GO, Zagmutt-Vergara FJ, Leutenegger CM, AdkisonMA, Baxa DV, Hedrick RP (2004) Identification of a serineprotease gene expressed by Myxobolus cerebralis duringdevelopment in rainbow trout Oncorhynchus mykiss. DisAquat Org 59:235–248

Khatwa UA, Kleibrink BE, Shapiro SD, Subramaniam M(2010) MMP-8 promotes polymorphonuclear cell migra-tion through collagen barriers in obliterative bronchiolitis.J Leukoc Biol 87:69–77

Langdon JS (1991) Myoliquefaction post mortem (‘milkyflesh’) due to Kudoa thyrsites (Gilchrist) (Myxosporea:Multivalvulida) in mahi mahi, Coryphaena hippurus L.J Fish Dis 14:45–54

Lovy J, Wright GM, Speare DJ (2006) Morphological presen-tation of a dendritic-like cell within the gills of Chinooksalmon infected with Loma salmonae. Dev Comp Immunol30:259–263

Lovy J, Becker JA, Speare DJ, Wadowska DW, Wright GM,Powell MD (2007) Ultrastructural examination of the hostcellular response in the gills of Atlantic salmon, Salmosalar, with amoebic gill disease. Vet Pathol 44:663–671

Lovy J, Wright GM, Speare DJ (2008) Comparative cellularmorphology suggesting the existence of resident dendriticcells within immune organs of salmonids. Anat Rec 291:456–462

Lovy J, Savidant GP, Speare DJ, Wright GM (2009) Langerin/CD207 positive dendritic-like cells in the haemopoietic tis-sues of salmonids. Fish Shellfish Immunol 27: 365–368

Lovy J, Wright GM, Speare DJ, Tyml T, Dykova I (2010) Comparative ultrastructure of Langerhans-like cells inspleens of ray-finned fishes (Actinopterygii). J Morphol271:1229–1239

MacMillan JR, Wilson C, Thiyagarajah A (1989) Experimentalinduction of Proliferative Gill Disease in specific-pathogen-free channel catfish. J Aquat Anim Health 1: 245–254

Noya M, Qian Y, Ainsworth AJ (1999) Molecular and func-tional characterization of channel catfish (Ictalurus punc-tatus) neutrophil collagenase. Vet Immunol Immuno -pathol 67:303–316

Pacifici R, Carano A, Santoro SA, Rifas L and others (1991)Bone matrix constituents stimulate interleukin-1 releasefrom human blood mononuclear cells. J Clin Invest 87:221–228

Pavelka M, Roth J (2005) Functional ultrastructure: an atlas oftissue biology and pathology. Springer, Vienna

Pote LM, Hanson LA, Shivaji R (2000) Small subunit riboso-mal RNA sequences link the cause of Proliferative GillDisease in channel catfish to Henneguya n. sp. (Myxozoa:Myxosporea). J Aquat Anim Health 12:230–240

Pote LM, Hanson LA, Khoo L (2010) Proliferative gill disease.In: American Fisheries Society–Fish Health Section(AFS–FHS) blue book: suggested procedures for thedetection and identification of certain finfish and shellfishpathogens. American Fisheries Society, Bethesda, MD,Sec. 3.7

Roach HI, Aigner T, Kouri JB (2004) Chondroptosis: a variantof apoptotic cell death in chondrocytes? Apoptosis 9:265–277

Schindeler A, McDonald MM, Bokko P, Little DG (2008) Boneremodeling during fracture repair: the cellular picture.Semin Cell Dev Biol 19:459–466

Shapiro IM, Adams CS, Freeman T, Srinivas V (2005) Fate ofthe hypertrophic chondrocyte: microenvironmental per-spectives on apoptosis and survival in the epiphysealgrowth plate. Birth Defects Res C Embryo Today 75:330–339

Styer EL, Harrison LR, Burtle GJ (1991) Experimental produc-tion of Proliferative Gill Disease in channel catfishexposed to a myxozoan-infected oligochaete, Dero digi-tata. J Aquat Anim Health 3:288–291

Takemori N, Hirai K, Onodera R, Saito N, Miyokawa N (1994)Ultrastructure of fibrillar granules in human neutrophils.Am J Hematol 47:232–234

Wise DJ, Griffin MJ, Terhune JS, Pote LM, Khoo LH (2008)Induction and evaluation of Proliferative Gill Disease inchannel catfish fingerlings. J Aquat Anim Health 20:236–244

134

Editorial responsibility: Sven Klimpel,Frankfurt am Main, Germany

Submitted: October 6, 2010; Accepted: December 14, 2010Proofs received from author(s): March 13, 2011