Development of the PKX Myxosporean in Rainbow Trout Salmo Gairdneri

DISEASES OF AQUATIC ORGANISMS Vol. 1: 169-182, 1986 Published October 15 Dis. aquat. Org.

Development of the PKX myxosporean in rainbow trout Salmo gairdneri

Michael L. Kent* & Ronald P. Hedricka*

Department Medicine, School of Veterinary Medicine. University of California, Davis. California 95616, USA

ABSTRACT Sequenhal development of PKX, the causative agent of prohferahve hdney dlsease, is descnbed in rainbow trout Salmo ga~rdnenhchardson by light and electron mlcroscopy Fish were exposed to water contaming the infechous stage of PKX and the parasite was detected 3 wk later Parasites were prominent the following week and they mulhplied by endogeny, binary fission and possibly plasmotomy in the blood and hdney intershhum Intershbal nephnbs that is typically assoclated \nth PKD was fust observed 5 wk after exposure and was most prominent between 8 to 11 wk At 7 wk parasites were found in the lumens of the tubules, many were daughter cells released from PKX Myxosporean sporoblasts formed within these daughter cells (enveloping cells) The sporoblasts consisted of 6 cells and some organized into spores w~th2 spherical polar capsules Although the spores persisted for several months after the interstitial PKX and assoclated inflammation had subsided they dld not complete their development They remained within the enveloping cell and apparently dld not form complete valves Because only incomplete spores were observed salmonids may be abnormal hosts and the preclse taxonomic status of PKX was not determined Epizoohological and sequential development data support the hypothesis that the interstitial form of PKX and the intraluminal myxosporeans belong to the same organism

INTRODUCTION modified and the planting of exposed fishes is often restricted by regulatory agencies. Proliferative kidney dlsease (PKD), caused by an The disease is characterized by chronic inflamma- unclassified protozoan (PKX), is considered a serious tion of the ludney. Tissue sections reveal the PKX obstacle to the successful culture of certain salmonid protozoan primarily in the ludney interstitium, associ- fishes. The disease has been reported most often in ated with a granulomatous interstitial nephritis and rainbow trout Salmo yairdnen kchardson, and mor- tubular atrophy (Ferguson & Needham 1978, Clifton- talities may reach 90 %, but usually remain below 30 to Hadley et al. 1984, Smith et al. 1984). In heavily 50 '10 (Clifton-Hadley et al. 1984). Other salmonid infected fish, PKX is often found in the spleen, gut, species have been affected and PKD is considered one gills, liver and muscle, where it also invokes a of the most important diseases of cultured salmonids in granulomatous response (Ferguson & Needham 1978, Europe (Clifton-Hadley et al. 1984). It is also wide- Smith et al. 1984). The PKX parasite most likely spread in western North America (Hedrick et al. 1984a, reaches these organs via the circulatory system and b, 1985; Smith et al. 1984, Hosluns 1985). Even in the may adhere to vessel walls, provoking a necrotizing absence of high mortality, morbidity may be 100 % and vasculitis (Smith et al. 1984). If fish survive PKD, the fish with PKD often exhibit decreased food conversion, interstitial hyperplasia subsides in 12 to 20 wk, and the poor tolerance to stress, and greater susceptibihty to ludneys of recovered fish show little if any signs of secondary infection (Clifton-Hadley et al. 1984). The previous infection (Clifton-Hadley et al. 1985). &sease inficts additional economic burdens on pro- A parasite that was most Likely PKX was described as duction fachties because husbandry practices must be an amoeba by Plehn (1924). Ghlttino et al. (1977) and Ferguson & Needham (1978) also suggested that PKX may be an amoeba because it formed pseudopodia. Present address: Center for Marine Disease Control, Bat- telle Marine Research Laboratory, 439 West Sequvn Bay Although no spores were observed, Seagrave et al. Road, Sequim, Washington 98382, USA (1980a) proposed that the organism might be a haplo- " Addressee for reprint requests spondan (phylum Ascetospora). Division by internal

O Inter-Research/Printed in F. R. Germany 170 Dis. aquat. Org. 1: 169-182, 1986

cleavage, the presence of multivesicular bodies, and tial form (typical PKX) to an immature myxosporean the existence of electron-dense cytoplasmic inclusions spore in the lumens of the tubules. We also present reminiscent of the haplosporosomes of Marteilia spp. evidence that substantiates that both forms belong to were cited as common features with PKX. the same organism. These findings indicate that PKX is However, internal cleavage and organelles similar a member of the phylum Myxozoa. to haplosporosomes also occur among members of the Myxozoa (Current & Janovy 1977, Desser and Paterson 1978, Current et al. 1979, Lom et al. 1982. Desser et al. MATERIALS AND METHODS 1983), and Ferguson & Needham (1978) and Seagrave et al. (1980a, b) did not exclude the possibility that A group of PKX-free rainbow trout under 1 yr of age PKX might belong to this phylum. Hedrick et al. (Eagle Lake, California strain) was introduced into (1984b) and Kent & Hedrick (1985a) observed myxo- water containing the infective stage of PKX at the sporean trophozoites and developing spores in the American River hatchery, Rancho Cordova, California, renal tubules of fish with PKD, and they proposed that USA on 16 August 1984. The parasite is only transmit- the observed organisms might be later stages of PKX ted by water-borne exposure or by injecting infected and that PKX belongs most appropeately to the phy- tissues (D'Silva et al. i984), and the infective stage lum Myxozoa. under natural conditions is yet to be identified. We present here a detailed description of the Approximately 500 of these fish were transferred to the sequential development of PKX from the renal intersti- Fish Patho!ogy Laboratory at the University of Califor-

90 A Intralurn~nalforms X PKX 0 Intersfit~alnephrit~s

a I Fig. 1. Salmo gairdnen. Sequenhal prevalence of interstitial PKX, intraluminal sporogonic forms and interstitial inflammation in the kidneys of rainbow trout maintained at the American River hatch- Wks ofter I V 6'- ery. Samples of 15 to 30 flsh were taken Introduction 0 5 10 15 20 2 5 30 35 periodically from Aug 1984 through Apr Ten 1985

80 - A Introlurninol X PKX 70 - 0 Interst~tialnephrit~s

60 - S 50- 0Q) 40- -0> 30- a Fig. 2. Salmo gairdnen. Sequential prevalence 20 - of interstitial PKX, intraluminal sporogonic forms and interstitial inflammation in the kid- l0 - neys of rainbow trout at the U.C. Davis fish pathology laboratory. Approximately 500 fish 0 I - V exposed to PKX at the American hver hatchery 0 5 10 15 20 25 37 were transferred to the laboratory on 9 Sep 1985 Trons ferred Weeks After Exposure to PKX ot and 15 to 30 fish samples were taken penodical- to U.C. Dovis American River Hatchery ly through Apr 1985 Kent & Hedrick: PKX in rainbow trout

nia (U.C.), Davis, California, on 9 September 1984 and Light microscopy maintained in PKX-free well water at 15 to 18 'C. Samples of 15 to 30 fish were taken at selected times The organism seen at 3 wk was small, condensed, from the population at the hatchery and from the fish at eosinophilic, and contained a daughter cell (Fig. 3). U.C. Davis until May 1985. Tissue sections revealed larger, typical parasites in the To determine the morphology of PKX, preparations blood and kidney interstitium the following week of fresh kidney were squashed between a cover slip (Fig. 4). These forms were also detected in blood and glass slide (wet mounts), and then examined by smears at this time. Primary cells with as many as 7 bright field and phase contrast microscopy. For his- daughter cells were evident in these tissues (Fig. 5), tological examinations, tissues were fixed in David- and were weakly eosinophilic compared to the sur- son's solution, embedded in paraffin, sectioned at rounding host cells. The nuclei of the parasites con- 5 Km, and stained with either hematoxylin and eosin tained large endosomes that were particularly promi- (H & E) or Prices's Giemsa as described by Luna (1968) nent in the primary cells. Degenerating primary cells and Humason (1979). Fish from the sequential samples and small condensed cells were observed in the inter- taken at the American River hatchery and U.C. Davis stitium of some fish. The small cells may represent were examined for the prevalence of interstitial PKX, released daughter cells (Fig. 6). associated pathology, and intraluminal myxosporean Parasites that were in the blood occasionally formed stages using paraffin sections of the kidneys. Kidney thrombi in the renal vessels (Fig. 7). Hypercellularity of tissue samples for electron microscopy were fixed in the kidney interstitium due to mononuclear cell proli- 3 % (v/v) glutaraldehyde in phosphate buffer as feration and infiltration was associated with the para- described by Bullock (1978). These specimens were sites (Fig. 8), and infected fish exhibited a reduced post-fixed in 1 O/O OsO,, embedded in Epon, sectioned, number of tubules mm-* when compared with un- stained with lead citrate and uranyl acetate, and infected fish (Fig. 9). Many parasites were surrounded examined with a Phillips EM 400 transmission electron by attached macrophages and lymphocytes (Fig.6). nlicroscope. Similar developmental stages of PKX Although most fish in our experiment exhibited gross were observed in all epizootics of PKD in California, ludney hypertrophy, they did not exhibit other clinical and fish from several locations in the state were used signs of PKD and no significant mortality could be for the light and electron microscopical descriptions. directly attributed to PKX. Organisms with typical PKX morphology were observed in the epithelium of the renal tubules (Fig. 101, RESULTS and the daughter cell of one was seen in binary fission Temporal prevalence (Fig. 11). Some primary cells in the epithelium degenerated, and their daughter cells, surrounded by The PKX parasite was first detected in one of 15 fish primary cell remnants, migrated to the lumen (Fig. 12). examined 3 wk after transfer to the American River Small, condensed eosinophilic cells, presumably re- hatchery (Fig. l). Parasites were easily detected the leased daughter cells, were observed in the lumen of following week and reached a peak prevalence of 83 % tubules at this time. The simplest intraluminal forms at 7 wk. Associated interstitial inflammation was evi- were uninucleate cells that resembled the secondary dent at 5 wk, and was most prominent between 8 and cells of interstitial PKX (Fig. 13). More complex 11 wk. The PKX parasite in the epithelium of the intraluminal forms contained secondary and tertiary tubules and intraluminal sporogonic stages were first daughter cells. Continued development of these stages detected at 7 wk. These forms persisted at approxi- by endogeny and binary fission resulted in sporoblasts mately the same prevalence for at least 12 wk after with up to 6 internal sporogonic cells (Fig. 14 & 15). interstitial PKX and associated lesions were no longer Developing myxosporean spores as evidenced by detectable (Fig. l). However, the intensity of the polar capsule formation were observed in the more intraluminal sporogonic forms diminished with time. advanced intraluminal parasites (Fig. 16 to 18). These The prevalence of intraluminal forms increased and spores were oblong, 12 X 7 km, with 2 spherical polar interstitial PKX and associated lesions decreased as capsules 2 pm in diameter (measured from wet mounts, the water temperature at the hatchery declined (Fig. 1). n = 20). Capsulogenic nuclei and a large sporoplasm The development of PKX in the fish transferred to nucleus were prominent in paraffin sections stained U.C. Davis was similar to that observed in the fish held with hematoxylin and eosin (Fig. 16). The intraluminal at the hatchery (Fig. 2). However, even though the tem- organisms were considered to be myxosporean tro- perature at U.C. Davis was above 15 "C throughout the phozoites because they formed multicellular spores study, the intraluminal forms became prevalent in with polar capsules. Only one spore developed withln these fish as the disease progressed (Fig. 2). each enveloping cell and no large multinucleated

Dis. aquat. Org. 1: 169-182, 1986

plasmodia, typical of most myxosporeans, were ob- plate-like cristae in the mitochondria. Additionally, as served. However, up to 3 nuclei were observed in some with secondary cells, they contained little endoplasmic enveloping cells. This monosporous development reticula compared to the primary cell of PKX, and had within an enveloping cell, described as a 'pseudoplas- similar ribosome content and cytoplasmic matnces. modium' by Lom et al. (1982) in Sphaerospora spp., More complex intraluminal forms had enveloping was confirmed in wet mounts (Fig. 18). Enveloping cells with electron-dense multilaminate bodies and up cells observed in wet mounts were characterized by to 5 sporogonic cells (Fig. 24 & 25). The most fully numerous refractile granules. developed forms observed were spores containing The spores apparently did not complete their polar capsules with coiled internal filaments (Fig. 26). development in the kidney. No clear spore valves or Although valves were not observed, early valvogenic well defined valvogenic cells were observed in either cells pahally surrounded the capsulogenic cells. paraffin sections or wet mounts. The spores were Macrophages usually surrounded the parasites in fragile and lacked rigidity as evidenced by the readi- the blood and ludney interstitium, but they were not ness with which they changed shape under pressure of observed in association with the intralurninal stages. the cover slip in wet mounts; they degenerated within The only intracellular stage of PKX observed was a 4 h in minced kidneys rlldirll&lled in water dt 10 "C. small form that had been completely engulfed by a Further, spores could no longer be detected after 24 h macrophage. under these same conditions. A diagramatic representation of the sequential de- The intraluminal trophozoites and spores were most velopment of PKX in the kidney of salmonid fishes is frequently observed in the second proximal and distal shown in Fig. 27. Stages represented were observed segments of the tubules. Although they were not found either by light or electron microscopy. in the collecting ducts, ureters or urinary bladder, 3 sporoblasts with polar capsules were observed in one of 10 urine samples obtained from fish at the American DISCUSSION River hatchery. Evidence from this study verifies that PKX belongs to the phylum Myxozoa. Myxosporeans are ubiquitous, Electron microscopy usually host-specific parasites of fishes (Mitchell 1977) and the prespore stages typically do not induce a The primary cell of PKX often contained internal significant inflammatory response (Dykova & Lom secondary and terbary daughter cells (Fig. 19) and 1978). Salmonids exhibit a severe inflammatory always contained 'haplosporosomes' identical to those response to interstitial PKX, which is the pre- described by Seagrave et al. (1980a, b) (Fig. 20). The sporogonic form, and only immature spores were primary cells contained a prominent endoplasmic observed. This suggests that salmonids are abnormal reticulum. They also contained large, electron-dense, hosts for PKX. membrane-bound inclusions which were presumed to Proliferative ludney disease parasites (PKX) with be secondary lysosomes. The daughter cells, either identical morphology to those reported by Ferguson & secondary or tertiary, were encircled by a membrane of Needham (1978), Seagrave et al. (1980a, b) and Smith the outer cell. They never contained haplosporosomes et al. (1984) were frequently observed in the blood and and rarely lysosomes. Degenerating primary cells, kidney interstitium. The parasite was detected in fish with condensed organelles and disrupted membranes, introduced to water containing the infectious stage contained intact daughter cells (Fig.21). The latter 1 wk earlier than previously reported (Ferguson & Ball may be released to continue the vegetative develop- 1979, D'Silva et al. 1984), and at this time was smaller ment of the parasite in the interstitium. and more condensed than typical PKX (Fig. 3). Numer- The PKX parasites in the epithelium of the tubules ous typical forms were observed in the blood, particu- (Fig. 22) had an ultrastructural morphology identical to larly early in the infection, which may indicate that it those in the interstitium. They migrated to the lumen reaches the kidney via the circulatory system. by penetrating between the epithelia1 cells. The PKX parasite most likely undergoes vegetative Daughter cells withn PKX and early intraluminal reproduction within the fish host. This is supported by forms had a similar ultrastructural morphology. The the observation of numerous daughter cells in the simplest and possibly earliest intraluminal forms were ludney interstitium of heavily infected fish (Fig. 5 & 6). uninucleate (Fig. 23), and presumably they were Additionally, fish injected with very few parasites daughter cells released from typical PKX. These forms became heavily infected (Kent & Hedrick 1985b). Fish and daughter cells within typical PKX shared a similar transferred from the hatchery to PKX-free water at U.C. size, nucleus to cytoplasm ratio, chromatin pattern, and Davis before parasites could be detected later $2 g 2 E Opt ,g 'g6,~ .. a.2 azE~ &;so c 023 9%,iU~ alg .a3 a" DE.2 G oE.c h .5 3+ a2 a m c .S $;if?c0 3 .y a OQ E m 2 2 g g,-%2& O@'Z $2 BPssz2ual0, 0 a z;c .pal6 2 26 s;.z hzNd~~11"35( z 2 FU gbrna~ api.2; Z m 8 a,c ,2=m2a E .c D 2" g, c L(0 $S.;;? 22 >aCS cal eU--~9DaU 2 25 2d E4E mm ccaaei, o onEE 3 U- 0 iGrnZ%$3 a 4 E" 'S, u,oo 3322S Q, 4 a11 m e Q .a 2 3

&a.- a-m C E $l s's fN g-a I

178 Dis. aquat. Org. 1:169-182, 1986

Fig. 26. Salmo gairdnen.Intralurninal sporoblast with polar capsules. Valvogenic cells (v) surrounding capsulogenic cells (c);pc: polar capsule; E: enveloping cell

developed heavy infections (Fig. 21, indicating that study was a small unicellular organism. Similar forms continual exposure is not needed to induce heavy contained single endogenous daughter cells (Fig. 23). infections. A mechanism for the development of the These stages appeared identical to the internal daugh- daughter cells by internal cleavage was reported by ter cells of the interstitial and epithelia1 PKX (Fig. 19 & Seagrave et al. (1980b). However, binary fission of the 22) and are most Likely the same cells. Haplosporo- internal daughter cells, as reported in the present somes are only observed in the primary cell of PKX study, is a second method for vegetative development. (Ferguson & Needham 1978, Seagrave et al. 1980a, No fixed developmental sequence within the kidney Smith et al. 1984). This accounts for their absence in interstltium could be determined, and plasmotomy the intraluminal forms because this stage seems to (external budding), endogeny (internal budding), and arise from released secondary cells of typical PKX. The binary fission may all occur in the same parasite intraluminal forms represent pseudoplasmodia as (Fig. 27). This lack of rigidity in the vegetative described by Lom et al. (1982) for Sphaerospora spp. development is consistent with the phylum Myxozoa and the internal cells are the sporoblast. The envelop- (Lom et al. 1983a, Johnstone 1984). ing cell of the pseudoplasmo~um in Sphaerospora Ferguson & Needham (1978) reported PKX in the spp. is uninucleate (Lom et al. 1982), but this cell of renal tubules, but migration of the parasite through the PKX often contained 2 or 3 nuclei. This cell also con- epithelium and release of daughter cells into the tained prominent refractile granules as seen in wet lumen were not described. The PKX parasites in the moints (Fig. 18), and these are most Likely analogous epithelium of the tubules and its intraluminal stages to the electron-dense multilaminated bodies (Fig. 24 to were more prevalent in convalescing fish than in 26) and the periodic acid Shiff's (PAS) positive bodies clinically affected fish (Fig. l), and Ferguson & reported by Kent & Hedrick (1985a). Polar capsule Needham (1978) have suggested that the tubules may formation in PKX indicated spore formation. However, provide a mode of exit for the parasite. trophozoites were more numerous than spores, even The simplest intraluminal stage detected in this late in the infection, as Bond (1938) reported for Kent & Hedrick: PKX in rainbow trout 179

Sphaerospora rends in the killifish Fundulus hetero- Such studies are made difficult because of the paucity clitus L. of PKX spores in most fish. Polar capsules may form before valvogenesis occurs, A clear sequential development from the interstitial as in other myxosporeans (Desser & Paterson 1978, PKX to spores with polar capsules demonstrated the Current et al. 1979), and valves were not apparent in myxosporean nature of the parasite. This is further the PKX spore. The spores remained within their supported by epizootiological evidence and experi- pseudoplasmodium throughout the infections, and mental data. The myxosporean of this study was only this, combined with the apparent lack of valves, indi- observed in PKX-exposed fishes, and although it has cates that the spores were not fully developed. Five to not been reported previously, it was detected in kidney 6 sporogonic cells were observed in maturing sporo- sections of infected fish from all cases of PKD that blasts, which is consistent with myxosporean sporo- we have examined. These involved coho salmon genesis. Two of the cells correspond to the capsul- Oncorhynchus kisutch (Walbaum) and steelhead trout ogenic cells, 1 or 2 to the sporoplasm cells, and 2 from 2 enzootic areas on Vancouver Island, British presumably to the valve-forming cells (Mitchell 1977). Columbia, Canada; convalescing rainbow trout from Although valves were not observed, PKX spores may the Hagerman State hatchery, Idaho, USA; coho salmon form vestigial valvogenic cells in salmonids. These from the State of Washington, USA; and from all cases spores were fragile, and it is unlikely that they would investigated in California, USA (Hedrick et al. 1984a, b, survive long enough for valvogenesis to occur if they 1985). A similar myxosporean has been found in PKX- were released from this host. exposed Atlantic salmon Salrno salar (L.) and brown Although many spores were observed by light mi- trout Salrno trutta (L.) in Europe (R. S. Clifton-Hadley, croscopy, few were found by electron microscopy. In Ministry of Aquaculture, Fisheries and Food, Wey- one spore, early valvogenic cells partially surrounded mouth, England, pers. comm.). The coincidence of typi- capsulogenic cells (Fig. 26). If more advanced stages of cal PKX with the intraluminal myxosporean forms was valvogenesis occur, they were not detected by light further demonstrated in studies by Hedrick et al. (1985). microscopy. More spores must also be examined by Fish introduced to the American River hatchery during electron microscopy to identify the most advanced the spring and summer showed both typical PKX and stage of spore development that occurs in salmonids. the lntraluminal forms. In contrast, fish exposed during

Fig. 27. Sequential development of PKX in kidney of salmonid fishes as interpreted from wet mounts, paraffin sections and electron micrographs. (a) PKX parasite in a blood vessel. (b) PKX enters hdney interstitium. (c) Uninucleate PKX. (d) Daughter cell produced by endogenous budding. (e) Secondary daughter cells divide by blnary fission and produce tertiary cells by endogenous budding. Nuclear division in primary cell may also occur. (f) Daughter cells are released from degenerated primary cell and continue vegetative development. (f')Plasmotomy or binary fission of primary cell may also occur. Vegetative development also may occur in the blood vessels. (g) PKX parasite penetrates between epithelia1 cells of tubule. (h) PKX enters the lumen; primary cell degenerates and releases secondary cell (= pseudoplasmodium). (h') Primary cells may release daughter cells into lumen from epithelium. Multiplication of released secondary cells may occur in the lumen. (i) Released secondary cell (enveloping cell) with tertiary (sporogonic) cell. U) Tertiary cell multiplies resulting in a nlul- ticellular sporoblast. (k) Sporulation con- tinues; capsulogenic cells differentiate and form polar capsules and sporoplasm develops a prominent nucleus. No valvogenesis observed, and spore remains within enveloping cell throughout infection 180 Dis. aquat. Org.

the winter when the infectious stage was not present did are evident. Ferguson (1984) reported Sphaerospora- not become infected with either stage of PKX. like myxosporeans in the kidneys of feral brown trout A blood and spleen transmission experiment by Kent from PKD enzootic waters, and Lorn et al. (1985) & Hedrick (1985b) also supports the conclusion that observed pseudoplasmodia without spores in brown both forms belong to the same parasite. Fish injected trout and grayling in Czechoslovakia. These fishes are with the blood and spleen of PKX-infected fish became hosts for PKX, and the pseudoplasmodia were possibly infected with both interstitial PKX and the intralumi- the intraluminal stages of PKX. nal myxosporeans. Since only the typical PKX is found Although the PKX myxosporean shows many in blood and spleen, the myxosporeans must have similarities to members of the family Sphaerosporidae, arisen from this stage. its spore also resembles those described for Parvicap- Seagrave et al. (1980a) suggested that PKX is a sula (Parvicapsulidae). Members of this genus are also haplosporidan (phylum Ascetospora) because of its elongated, and have 2 polar capsules similar in size to similarities with the invertebrate pathogens Marteha those of PKX. Furthermore, a Pam'capsula sp. is known spp. However, they did not exclude the possibility that to sporulate in the epithelium of the kidney tubules it may belong to the phylum Myxozoa. The higher in coho salmon (Hoffman 1984, Johnstone 1984). taxonomy of these phyla is in a state of confusion. Althouy h Pdrvicdpsula develops in the usual myxospo- Desportes & Ginsburger-Vogel (1977) demonstrated rean manner with large plasmodia and pansporoblasts, affinities of the family Martediidae with the class Para- it may also form pseudoplasmodia (J. Lom, Czecho- myxea (Ascetospora), Actinosporea, and Myxosporea slovak Academy of Sciences, C. Budejovice, Erani- (Myxozoa), and suggested they be grouped in a com- Sovska, Czechoslovalua, pers. comm., Johnstone 1984). mon taxon. Of the myxosporeans examined by trans- Spore morphology has been the most important mission electron microscopy, most form electron-dense criterion for species descriptions in the Myxozoa cytoplasmic inclusions, similar to haplosporosomes in (Mitchell 1977), but the origin of sporoblasts (either the Ascetospora (Current & Janovy 1977, Desser & within true plasmodia or pseudoplasmodia) should Paterson 1978, Current et al. 1979, Lorn et al. 1982, also be included in these descriptions (Lom et al. 1982, Desser et al. 1983), and organisms of both phyla repro- Lorn & Noble 1984). Identification of the primary host duce by internal cleavage (endogenous budding). and completely formed spores are needed before the Although the PM myxosporean could not be iden- precise taxonomic status of the PKX myxosporean can tified because only immature spores were found, be determined. it shows similarities to Sphaerospora spp. (family Salmonids may be abnormal hosts for PKX (Seagrave Sphaerospondae). Kent & Hedrick (1985a) compared et al. 1980a). The severe inflammation associated with the prespore form of PKX in the kidney interstitium to a the prespore (typical) PKX stage and the lack of com- protozoan of carp blood (UBO), which may be an early plete development of the spore support this hypothesis. stage of Sphaerospora renicola (Lom et al. 1983a). A The severe inflammation with PKD is in contrast to the protozoan similar to UBO found in the swimbladder of lack of tissue reaction to the prespore forms in hosts in carp was shown to be an early stage of S. renicola which myxosporeans complete sporogenesis (Dykova (angulata) (Csaba et al. 1984, Molnar 1984). The PKX & Lorn 1978). No significant pathological changes were parasite is similar to UBO and the swimbladder proto- associated with the intraluminal stages of PKX, and the zoan in that all 3 form endogenous secondary and interstitial nephritis disappeared shortly after the typi- tertiary daughter cells which are released and may cal PKX form was no longer detectable (Fig. 1). Fergu- subsequently sporulate in the lumen of the tubules. As son (1981) reported that fish with PKD recover faster in with PKX, the secondary cells of UBO reproduce by cold water. However, low temperatures are not binary fission in addition to endogenous budding (Lom required for disappearance of the interstitial PKX, et al. 1983a). Furthermore, sporogenesis of PKX is recovery from PKD, migration of PKX to the tubules, or similar to that of some Sphaerospora spp. in that spores sporulation, because all of these events occurred in the of both are formed with a pseudoplasmodium in the fish transferred to U.C. Davis where temperatures lumen of the kidney tubules. The enveloping cell of remained above 15 "C. pseudoplasmodium in both parasites contains promi- This suggests that the stage of development of the nent refractile granules (Lom et al. 1985), and PKX is parasite is an important factor in the inflammatory monosporous like S. molnari (Lom et al. 1983b). response associated with PKD. Furthermore, the in- A companson of immature spores of Sphaerospora flammatory response in an abnormal host may inhibit spp. with those of PKX shows similar features such as the migration of PKX to the tubules and subsequent an elongated shape, small polar capsules, and indis- sporulation. Kent (1985) immunosuppressed PKX- tinct valves. In contrast to PKX, the mature spores of infected fish with cortisol implants and found that Sphaerospora spp. have a spherical shape and valves suppression of the inflammatory response to PKX Kent & Hedrick: PKX in rainbow trout

enhanced the development of the parasite. In PKX- Proliferative kidney disease of salmonid fish: a review. infected fish with cortisol implants, more interstitial J.Fish Dis. 7: 36S377 forms, but less interstitial hypercellularity were Clifton-Hadley, R. S., &chards, R. H., Bucke, D. (1985). The sequential pathological changes in proliferative kidney observed. These fish also exhibited more sporogonic disease. In: Ellis, A. E. (ed.) Fish and shellfish pathology. stages, but the cortisol treatments did not induce com- Academic Press, London, p. 359-367 plete development of the spores (Kent 1985, Kent & Csaba. G.. Kovacs-Gayer, E., Bekesi, L.. Buscek, M., Szakolc- Hedrick 1986). Possibly the kidney tubules of rainbow zai, J.. Molnar, K. (1984). Studies into the possible protozoan aeitiology of swimbladder inflammation in carp fry. trout do not provide the proper physiological environ- J. Fish Dis. 7: 39-56 ment for complete sporulation. This is substantiated by Current, W. L., Janovy, J. (1977).Sporogenesis in Henneguya the observations from fish in natural epizootics, in exihs infecting the channel catfish: an ultrastructure which spores persisted in an immature state in the study. Protistoloqca 13: 157-167 tubules several months after the inflammation had Current, W. L., Janovy, J., Knight, S, A. (1979). Myxosoma funduh Kudo (Myxosporida) in Fundulus kansae: ultra- subsided (Fig. 1). structure of the plasmodium wall and of sporogenesls. This study has demonstrated that the causative agent J.Protozool. 26: 574-583 of PKD is the presporogonic form of a myxosporean, Desportes, I., Ginsburger-Vogel, T. (1977). Affinites du genre which undergoes vegetative development and induces Marteilia, parasite de'Huitres (maladie des Abers) et du Crustace Orchestia gammarella (Pallas), avec les Myxo- a severe tissue response in the kidney interstitium and spondies. Actinomyxides et Paramyxides. C. r. hebd. blood of rainbow trout. As the disease progresses, the Seanc. Acad. Sci., Pans D 285: 111 1-1 114 parasites migrate to the lumen of the kidney tubules Desser, S. S., Molnar, K., Horvath, I. (1983). An ultrastructural and sporulate within a pseudoplasmodium. Although study of the myxosporeans, Sphaerospora angulata and inflammation was not associated with these sporogonic Sphaerospora carassii, in the common carp, Cyprinus carpio L. J. Protozool. 30: 415422 forms, only immature spores were observed in the Desser, S. S.. Paterson, W. B. (1978). Ultrastructural and tubules. These observations all indicate that salmonids cytochemical observations on sporogenesis of Myxobolus are abnormal hosts for PKX and that the parasite may sp. (Myxosporida: Myxobolidae) from the common shiner complete sporulation in a non-salmonid fish. Identifi- Notropis cornutus. J.Protozool. 25: 314-326 D'Sllva, J., Mulcahy, M. F., de knkelin, P. (1984).Experimen- cation of the primary host is needed to determine the tal transmission of proliferative ludney disease in rainbow precise identity of PKX and the reservoir of infection. trout Salmo gavdnerj hchardson. J. Fish Dis. 7: 235-240 We have recently observed immature spores similar Dykova, I.,Lom, J. (1978). Histopathological changes m fish to those of PKX and more fully developed Sphaero- 9111s infected wth myxosporidian parasites of the genus spora spores in the lumens of kidney tubules from tui Henneguya. J.Fish Biol. 12: 197-202 Ferguson, H. W (1981). The effects of water temperature on chub Gila bicolor (Girad) (Cypnnidae), and stickle- the development of proliferative kidney bsease in rain- backs Gasterosteus aculeatus L. (Gasterosteidae), from bow trout, Salrno galrdneri hchardson. J. Fish Dis. 4: watersheds where PKX is prevalent (Hedrick et al. 17.5177 1986). Studies are underway to determine the associa- Ferguson. H. W. (1984). Proliferative kidney disease in North- ern Ireland: epidemiology and control. S. F. Sniesko Com- tion of these myxosporeans with PKX and the role of memorative Workshop: Fish Health Sec./Am. Fish Soc. non-salmonids as reservoir hosts. Ann. conf., L~ttleRock, Arkansas, 10-12 July, p. 6 Ferguson. H. W., Ball. H. J. (1979). Epidemiological aspects of Acknowledgements. This work is a result of research spon- proliferative kidney disease amongst rainbow trout Salmo sored in part by NOAA, National Sea Grant College Program, gairdneri Richardson in Northern Ireland. J. Fish Dis. 2: Department of Commerce, under Grant Number NA80AA-D- 219-225 00120, project number R/F-100, through the California Sea Ferguson, H. W., Needham, E. A. (1978). Proliferative ludney Grant College Program, and in part by the California State disease In rainbow trout Salmo gairdnen hchardson. Resources Agency. The U.S. Government is authorized to J.Fish Dis. 1: 91-108 reproduce and distribute for governmental purposes. The Ghittino, P., Andruetto, S., Vigliani, E. (1977). L'amebiasl authors also thank R. Munn and P. Lee for assistance with the della trota iridea d'Allevamento. hv, ital. Piscic. Itio- electron microscopy and Drs. J. Lom and C. Smith for their pathol. 12: 74-89 helpful advice. The assistance of the staff at California Fish Hedrick, R. P., Kent, M. L., Rosemark, R., Manzer, D. (1984a). and Game, Fish Disease Laboratory, and the American &ver Proliferative kidney disease (PKD) in Pacific salmon and hatchery was invaluable. steelhead trout. J. World Maricul. Soc. 15: 31a325 Hedrick, R. P,, Kent, M. L., Rosemark, R., Manzer, D. (1984b). Occurrence of proliferative kidney disease (PKD) among Pacific salmon and steelhead trout. Bull. Eur. Ass. Fish LITERATURE CITED Pathol. 4 (3): 34-37 Hedrick, R. P., Kent. M. L., Foott, M. S., Rosemark, R., Manzer, Bond, F. F. (1938). Cnidosporidia from Fundulus heteroclihls D. (1985). Proliferative kidney disease among salmonid Lin. Trans. Am. microsc. Soc. 57 (2): 107-122 fish in California, USA; a second look. Bull. Eur. Ass. Fish Bullock, A. M. (1978). Laboratory methods. In: Roberts, R. J. Pathol. 5 (1): 3638 (ed.) Fish pathology. Bailliere-Tindall, London, p. 245-267 Hedrick, R. P., Kent, M. L., Toth, R. J. (1986). Myxosporeans Clifton-Hadley, R. S., Bucke, D., hchards, R. H. (1984). found in the kidneys of non-salmonid fish from waters Dis. aquat. Org. 1: 169-182, 1986

enzootic for proliferahve kidney disease (PKD). Fish Sphaerospora rnolnari sp, nov. (Myxozoa : Myxosporea), Health Sec./Am. Fish. Soc. Newsletter 14 (2): 4 an agent of gill, skin and blood sphaerosporosis of com- Hoffman, G. L. (1984). Two fish pathogens, Parvicapsula sp. mon carp in Europe. Parasitology 86: 52S.535 and Mtraspora cyphi Myxosporea, new to North Lom, J., Noble, E. R. (1984). Revised classification of the class America. Symp. Biol. Hung. 23: 127-135 Myxosporea Butschli, 1881. Folia Parasitol. (Praha) 31: Hoskins, G. (1985). In brief notes. Fish Health Sec./Am. Fish. 193-205 Soc. Newsletter 13 (1): 8 Lom, J., PavlaskovA, M,, Dykova, 1. (1985). Notes on kidney- Humason. G. L. (1979). Animal tissue techniques. W. H. infecting species of the genus Sphaerospora Thelohan Freeman Co., San Francisco (Myxosporea), including a new species S. gobionis sp. Johnstone, A. K. (1984). Pathogenesis and life cycle of the nov., and myxosporean life cycle stages in the blood of myxozoan Parvicapsula sp. infecting marine cultured coho some freshwater fish. J. Fish Dis. 8: 221-232 salmon. Doctoral dissertation, Univ. of Washington Luna, L. G. (1968). Manual of histologic staining. McGraw- Kent, M. L. (1985). The development of the PKX myxospo- Hd, New York rean, the causative agent of proliferative kidney disease, Mitchell, L. G. (1977). Myxosporidla. In: Krier, J. P. (ed.) in rainbow trout Salmo gairdneri Richardson. Doctoral Parasitic protozoa. Vol. IV. Academic Press, New York, dissertation, Univ. of California, Davis p. 115-154 Kent, M. L., Hedrick. R. P. (1985a). PKX, the causative agent Molnar, K. (1984). Experimental evidence that protozoans of proliferative ludney disease (PKD) in Pacific salmonid causing swim-bladder inflammation in common carp flshes and its affinities with the Myxozoa. J. Protozool. 32: (Cyynrinrls carpio L ) are stages of ,Sphaerospora rem'cola. 254-260 Bull. Eur. Ass. Fish Pathol. 4: 14-15 Kent, M. L., Hedrick, R. P. (1985b). Transmission of the causa- Plehn, M. (1924). Praktikum der Fischkrankheiten. E. tive agent of proliferative kidney disease with the blood Schweizerbart'sche Verlagsbuchhandlung, Stuttgart and spleen of infected fish; further evidence that the PKX Seagrave, C. P., Bucke, D.. Mderroan. D. J. (1980a). Ultra- parasite belongs to the phylum Myxozoa. Bull. Eur. Ass. structure of a haplosporean-like organism: the possible Fish Pathol. 5 (2): 39-42 causative agent of proliferative kidney disease in rainbow Lom, J., Dykova. I.. Lhotbkova, S. (1982). Fine structure of trout. J. Fish Biol. 16: 453459 Sphaerospora renicola Dykova and Lom, 1982, a myxo- Seagrave, C.,Bucke, D., Alderman, D. (1980b). The causative sporean from carp kidney and comments on the origin of agent of proliferative kidney disease may be a member of pansporoblasts. Protistologica 18: 48%502 the Haplosporidia. In: Ahne, W. (ed.) Fish chseases. Th~rd Lom, J., Dykova, I., PavlBskova, M. (1983a). 'Unidentified' COPRAQ Session. Springer-Verlag, Heidelberg. Berlin, mobile protozoans from the blood of carp and some unsol- p. 174-181 ved problems of the myxosporean life cycles. J. Protozool. Smith, C. E., Morrison, J. K., Rarnsey, H. W.. Ferguson, H. W. 30: 497-508 (1984). Proliferative ludney disease: first reported out- Lom, J., Dykova, I., Pavl~skova,M., Grupcheva, G. (1983b). break in North America. J. Fish Dls. 7: 207-216 Responsible Subject Editor: Dr. T. Evelyn; accepted for printing on June 30, 1986