Epicellular Coccidiosis in Goldfish

Vol. 125: 1–5, 2017 DISEASES OF AQUATIC ORGANISMS Published June 19 https://doi.org/10.3354/dao03127 Dis Aquat Org

Epicellular coccidiosis in goldfish

Kálmán Molnár, Csaba Székely*

Institute for Veterinary Medical Research, Centre for Agricultural Research, HAS, POB 18, 1581 Budapest, Hungary

ABSTRACT: In a goldfish stock held in a pet fish pond, heavy coccidian infection, caused by an epicellularly developing Goussia species, appeared in April of 3 consecutive years (2014 to 2016). The shape and size of the oocysts resembled those of an inadequately described species, Goussia carassiusaurati (Romero-Rodriguez, 1978). In histological sections, gamogonic and sporogonic stages infested mostly the second fifth of the intestine, where almost all epithelial cells were in - fected. Both gamonts and young oocysts occurred intracellularly but in an extracytoplasmal position, seemingly outside the cells. Oocysts were shed non-sporulated. Spheroid to ellipsoidal non- sporulated oocysts measured 12.4 × 13.5 µm on average, but after 48 h sporulation in tap water they reached a size of 16 × 13 µm. The 4 elliptical sporocysts located loosely within the sporulated oocysts measured 13 × 5.4 µm. The oocysts and sporocysts were smaller than those of the better- known Goussia species G. aurati (Hoffman, 1965).

KEY WORDS: Coccidiosis · Goussia · Goldfish · Epicellular location · Seasonal development

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INTRODUCTION sequences of different fish coccidia, however, great differences among Eimeria and Goussia spp. were Coccidia of the Eimeria and Goussia genera are documented, and at least 4 groups can be clearly dif- common parasites of fish inhabiting European natu- ferentiated within the members of Goussia. Among ral waters and aquaculture farms. The genus Goussia Goussia spp. collected from Hungarian fishes, Rosen- was first described and separated from Eimeria by thal et al. (2016) found that molecular sequences Labbé (1896). Epicellular development of a Goussia of species with small dispersed oocysts developing species, G. pigra, was first demonstrated by Léger & in the gut of their hosts throughout the year differ Bory (1932). Dyková & Lom (1981) created a new from those of species with large oocysts developing genus, Epieimeria, for epicellularly developing fish seasonally in nodules and epicellular sites. Large dif - coccidia, selecting Epieimeria anguillae (Léger & ferences were also found between nodular and epi- Hollande 1922) Dyková & Lom, 1981 as the type spe- cellular species. Species infecting the kidney com- cies and revised the genus Goussia Labbé. Molnár posed the fourth group. (1989) found seasonally developing coccidia of nodu- Four coccidian species have been reported from lar and epicellular types in cyprinid fishes and de- the gut of goldfish. G. aurati was described by scribed them as G. balatonica and G. pannonica, Hoffman (1965) as Eimeria aurati from the gut of a respectively. Molnár (2006) regarded 4 genera com- goldfish shedding faecal casts with non-sporulated mon in fish, Eimeria, Goussia, Crystalospora and oocysts. Once sporulated, these measured 16−24 × Calyptospora, as members of the Eimeriidae family. 14−17 µm and contained elliptical sporocysts meas- Several authors, among them Upton (2000) and uring 11−13 × 6.5−8 µm. He found this species to be Duszynski et al. (2016), still regard Goussia as a similar to G. subepithelialis (Moroff & Fiebiger, synonym of Eimeria. In recent studies (Molnár et al. 1906), the nodular coccidium of the common carp, 2012, Rosenthal et al. 2016) examining molecular which have similarly sized round 17−19 µm oocysts

*Corresponding author: [email protected] © Inter-Research 2017 · www.int-res.com 2 Dis Aquat Org 125: 1–5, 2017

and 12−14 × 3 µm sporocysts, according to data re- logical sections were also prepared in 2016. On April corded by Steinhagen et al. (1990). Romero-Rodriguez 5, 15 and 25, the gut of the smallest fish, about 4 cm (1978) found a similar species, G. carassiusaurati in length, was separated and coiled into a roll, fixed (described as Eimeria), in goldfish which had 15.2 × in Bouin’s solution for 4 h, embedded in paraffin wax, 13.3 µm oocysts and 13.6 × 5.8 µm sporocysts. The cut into 4 to 5 µm sections and stained with haema- third species, G. hupehensis (Chen & Hsieh, 1964) toxylin and eosin. Non-sporulated and sporulated referred to by Chen & Li (1973) as Eimeria hupehen- oocysts were studied using Nomarski differential sis, is a typical dispersed coccidium closely resem- interference contrast with an Olympus BH2 micro- bling G. carpelli (Léger & Stankovitch, 1921) in its scope. The oocysts and histo logical sections were morphology. G. hupehensis (Chen & Hsieh, 1964), photographed with an Olympus DP 20 digital cam- cited by Chen & Li (1973), was also found in Hungary era. Digitised images of oocysts were obtained and by Molnár et al. (2005), who experimentally proved measurements were made using IMAGO® software. that this species (named only as Goussia sp.) cannot be transmitted to the common carp. A fourth species from the gut of the goldfish and Culter erythropterus RESULTS in China, de scribed by Chen (1984) as Eimeria new- chongensis, has 17.3−20 µm sporulated oocysts and Heavy infections with non-sporulated oocysts were may not differ from G. aurati. found in April in each year. At this time, the gut of the Here, we describe a coccidiosis in the gut of goldfish fish was empty or contained only very little faecal caused by epicellularly developing coccidian stages. matter. The fish were in poor condition but no mor- tality was recorded, even for fish subsequently held in laboratory aquarium for approximately 1 mo. All of MATERIALS AND METHODS the examined fish proved to be infected. When freshly dissected, the second and fifth sections of the Goldfish Carassius auratus gibelio (L.) are fre- intestine were found to be most heavily infected with quently cultured in small hobby ponds in Hungary, gamogonic and sporogonic stages. Relatively little as well as in aquaria. From 1 of these ponds, where infection was recovered in the first and third sections about 100 specimens of 1 to 5 yr old goldfish and their of the gut. Only non-sporulated oocysts were recov- approximately 1000 offspring are cultured, sample ered from the last 2 sections, and these were found fish were sent to the laboratory for a health control free in the lumen of the gut or in faeces. Oocysts were check at approximately monthly intervals. Fish were shed non-sporulated. Most of the non-sporulated collected from the pond with a hand net, carried to oocysts had a granulated structure and a spheroid or the laboratory alive in oxygenated plastic bags and short ellipsoidal shape (Fig. 1). In the first half of kept in aerated aquaria. They were sedated with April, mainly developing gamonts were found in the 20 ppm clove oil added to the water and killed by first part of the intestine (Fig. 2). In the middle of decapitation. Coccidian infection of varying intensity April, only non-sporulated oocysts were found in with small-sized oocysts of the dispersed Goussia hupehensis species was recorded throughout the examination period. In addition, in April of 3 consecutive years (2014 to 2016), heavy infections with large-sized oocysts resembling epicellular and nodular coccidia, common at this time in natural water fishes (Molnár 1989), were recorded. In April 2016, a specific survey for coccidian infections was per- formed on 25 specimens of 1 to 3 yr old goldfish measuring 4 to 13 cm. The gut was opened length- wise under a dissecting microscope and cut into 5 equal parts. From the epithelial surface, pieces of clean mucus were removed, placed under a coverslip and studied using Nomarski differential interference contrast with an Olympus BH2 microscope. Some non-sporulated oocysts were placed in tap water in a Fig. 1. Non-sporulated Goussia oocysts shed by a goldfish small Petri dish and left to sporulate for 48 h. Histo- Carassius auratus gibelio. Scale bar = 10 µm Molnár & Székely: Epicellular coccidiosis in goldfish 3

Fig. 2. Young gamogonic Goussia stages in a scraping of Fig. 3. Sporulated Goussia oocysts with round sporocysts goldfish Carassius auratus gibelio intestinal epithelium. and sporozoites (arrows). Scale bar = 10 µm Scale bar = 20 µm infected sections of the gut. By the end of April most sporozoites arranged head to tail. Sporozoites meas- oocysts had left the gut and only sparse oocysts were ured 10.3 ± 0.73 (9.6−11.2) µm in length and 2.5 ± found in the rectum among the faeces. Non-sporu- 0.32 (2.4−2.8) µm in width. In freshly sporulated lated oocysts became sporulated after 2 d in tap sporo cysts, the sporocyst residuum covered most water at 22 to 23°C. Sporulated oocysts also had a parts of the sporozoites. After 48 h sporulation, this spheroid or short ellipsoidal shape (Fig. 3) and a very residuum became short and ellipsoidal, forming a thin, single-layered, colourless wall. The oocysts compact body of approximately 2.5 × 1.3 µm located (Table 1) measured 12.4 ± 0.86 (mean ± SD, range: within the sporocysts between the sporozoites. 12.8−15.2) µm in length and 13.5 ± 1.8 (12−14.6) µm In histological preparations fixed in the first half of in width. There was no oocyst residuum, but a small April, heavy infection with young gamogonic stages polar body was present in the oocysts. The 4 thin- was found in the second and third fifths of the intes- walled, elongated ellipsoidal sporocysts located tine. Developing oocysts were only rarely found in loosely within the oocysts measured 13 ± 0.8 (11.2− the first and not in the fourth and fifth segments of 15.4) µm in length and 5.4 ± 0.34 (4.5−5.6) µm in the gut. At this time, most of the gamonts were width. In each sporocyst there were 2 vermiform young, measuring about 5 µm in diameter. In some

Table 1. Morphological features and measurements (µm) of seasonally developing Goussia spp. oocytes from the gut of Carassius auratus gibelio goldfish. –: no data

This study G. aurati (Hoffman, 1965) G. carassiusaurati (Romero-Rodriguez, 1978)

Non-sporulated oocysts Shape Spheroid to ellipsoidal Round – Length 12.4 ± 0.86 (12.8−15.2) 12−14 (13.6) – Width 13.5 ± 1.8 (12−14.6) – – Sporulated oocysts Shape Spheroid to ellipsoidal Spheroid to ellipsoidal Round to ellipsoidal Size of round oocysts – – 14.8 Length 16 ± 1.1 (14.4−17.6) 16−24 (20.1) 15.2 Width 13 ± 1 (11.2−15.2) 14−17 (16.3) 13.3 Sporocysts Length 13 ± 0.8 (11.2−15.4) 11−13 13.6 Width 5.4 ± 0.34 (4.5−5.6) 6.5−8 5.8 Sporozoites Length 10.3 ± 0.73 (9.6−11.2) 10−13 – Width 2.5 ± 0.32 (2.4−2.8) 2−2.5 – 4 Dis Aquat Org 125: 1–5, 2017

Fig. 4. Histological section of the second segment of goldfish Fig. 6. Asynchronous Goussia development in the intestine Carassius auratus gibelio gut. Young Goussia gamonts in an of a goldfish Carassius auratus gibelio. Oocysts (blue arrow) epicellular position (arrows). Haematoxylin and eosin stain- and young gamogonic stages (arrows) occur at the same ing. Scale bar = 20 µm time. Haematoxylin and eosin staining. Scale bar = 20 µm

Jirku˚ et al. 2002, 2009, Dogga et al. 2015). Recent phylogenetic works (Molnár et al. 2012, Rosenthal et al. 2016) have validated deep evolutionary distinc- tions among the coccidian parasites of fish, including (but not limited to) a monophyletic assemblage re - stricted to true members of the genus Eimeria. Sub- divisions within Goussia correspond to morphological and developmental attributes that likely warrant further taxonomic revision. Several Goussia species, like G. subepithelialis, develop in nodules in the gut, while others, like G. pigra, develop in an epicellular position in the enterocytes. Molnár (1989), who de - scribed G. balatonica and G. pannonica from white bream, remarked that a mixed infection with nodular and epicellular type coccidia occurs at the time of Fig. 5. Goussia micro- (arrows) and macro-gamonts (blue ar- oocyst formation in April in several cyprinids. Mor- rows) located epicellularly in the intestine of a goldfish Carassius auratus gibelio. Haematoxylin and eosin staining. phologically similar oocysts which, however, differ in Scale bar = 10 µm size also occur in non-cyprinids; these include G. aculeati and G. zarnowskii in Gasterosteus aculeatus segments of the intestine, almost every epithelial cell (L.) and G. acipenseris and G. vargai in Acipenser was infected (Fig. 4). Microgamonts were also found, ruthenus (L.) (Jastrzebski 1984, Molnár 1986). It can- but less frequently (Fig. 5). Development of gamo- not be excluded, therefore, that epicellular and gonic and sporogonic stages was not synchronous, as nodular coccidia also exist concurrently in the com- mature, non-sporulated oocysts were found among mon carp and the goldfish. Both fish species are of developing gamonts (Fig. 6). Chinese origin and only a small fraction of their parasite fauna has been introduced into Europe. The smaller (on average 16 µm) oocyst size measured in DISCUSSION the goldfish samples in the present investigation resembles those of the epicellular G. pannonica stud- Although the genus Goussia is still regarded by ied by Molnár (1989) and G. janae described by many authors as a synonym of Eimeria (Upton 2000, Lukeš & Dyková (1990), while the large oocysts (16 × Duszynski et al. 2016), others working on eimerians 20 µm) of G. aurati, described by Hoffman (1965), of fish and anurans accept its validity (Lukeš & correspond better to a nodular species. Although the Dyková 1990, Steinhagen et al. 1990, Lukeš 1992, above facts might suggest a nodular nature of G. Molnár & Székely: Epicellular coccidiosis in goldfish 5

aurati and an epicellular nature of G. carassiusaurati, Duszynski DW, Upton SJ, Couch L (2016) The coccidia of present data are not sufficient to draw this conclu- the world. Available at: http://biology.unm.edu/biology/ coccidia/home.html sion. Similar problems exist with the common carp. Dyková I, Lom J (1981) Fish coccidia: critical notes on life To date, only a nodular species, G. subepithelialis, cycle, classification and pathogenicity. J Fish Dis 4: has been reported from this fish. It cannot be ruled 487−505 out, however, that a more detailed examination in Hoffman GL (1965) Eimeria aurati n. sp. (Protozoa: Eimeri- idae) from goldfish (Carassius auratus) in North America. China, in the original biotope of the common carp J Protozool 12:273−275 and the goldfish, could record both nodular and epi- Jastrzebski M (1984) Coccidiofauna of cultured and feral cellular species. fishes in farms. Wiad Parazytol 30: 141−163 Epicellular development characterizes a particular Jirku˚ M, Modrý D, Šlapeta JR, Koudela B, Lukeš J (2002) lineage of coccidia, which adds weight to the validity The phylogeny of Goussia and Choleoeimeria (Apicom- plexa; Eimeriorina) and the evolution of excystation of the genus Epieimeria Dyková & Lom, 1981. On the structures in coccidia. Protist 153: 379−390 other hand, the large differences in morphology, de - Jirku˚ M, Jirku˚ M, Oborník M, Lukeš J, Modrý D (2009) velopment and molecular sequences between the epi- Goussia Labbé, 1896 (Apicomplexa, Eimeriorina) in cellularly developing coccidia of Goussia and the type amphibia: diversity, biology, molecular phylogeny and comments on the status of the genus. Protist 160: 123−136 specimen Epieimeria anguillae indicate a lack of rela- Labbé A (1896) Recherches zoologiques, cytologiques et tionship between the 2 eimerian groups. Differences biologiques sur les coccidies. Arch Zool Exp Gen 4: in the development (permanently developing group 517−654 versus annually developing groups) and intracellular Léger L, Bory T (1932) Eimeria pigra n. sp. nouvelle coccidie location (intracytoplasmal and epicytoplasmal), and juxtaépitheliale parasite du gardon rouge. C R Hebd Seances Acad Sci 194: 1710−1712 also differences in the molecular sequences among Lukeš J (1992) Life cycle of Goussia pannonica (Molnár, dispersed, nodularly and epicellularly developing 1989) (Apicomplexa, Eimeriorina), an extracytoplasmic groups (Rosenthal et al. 2016) would favour the cre- coccidium from the white bream Blicca bjoerkna. ation of new coccidian genera in the future, among J Proto zool 39: 484−494 Lukeš J, Dyková I (1990) Goussia janae n. sp. (Apicomplexa, them a genus with the tentative name Epigoussia. Eimeriorina) in dace Leuciscus leuciscus and chub L. cephalus. Dis Aquat Org 8:85−90 Molnár K (1986) Occurrence of two new Goussia species in Ethical approval. All applicable institutional, national and the intestine of the sterlet (Acipenser ruthenus). Acta Vet international guidelines for the care and use of animals were Hung 34: 169−174 followed. A permit for scientific fishing in Hungary (EHVF/ Molnár K (1989) Nodular and epicellular coccidiosis in the 121-1/ 2014) was issued by the Ministry of Agriculture, intestine of cyprinid fishes. Dis Aquat Org 7:1−12 Hungary. Molnár K (2006) Phylum Apicomplexa. In: Woo PTK (ed) Fish diseases and disorders, Vol 1. Protozoan and meta- Acknowledgements. The authors thank G. Pataki for the zoan infections, 2nd edn. CAB International, Walling- preparation of histological slides. The authors also thank Dr. ford, p 183−204 Yang Liu (College of Fisheries, Huazhong University, Wuhan, Molnár K, Ostoros G, Baska F (2005) Cross-infection experi- China), who helped with obtaining Chinese scientific papers. ments confirm the host specificity of Goussia spp. Dr. Benjamin Rosenthal’s suggestions and help in correcting (Eimeriidae: Apicomplexa) parasitizing cyprinid fish. the English of the manuscript are also greatly appreciated. Acta Protozool 44: 43−49 This work was supported by the European Regional and Molnár K, Ostoros G, Dunams-Morel D, Rosenthal BM Development Fund and the Government of Hungary project (2012) Eimeria that infect fish are diverse and are related GINOP-2.3.2-15-2016-00004: ‘Establishing the sustainable to, but distinct from, those that infect terrestrial verte- angling-aimed management of Lake Balaton’. brates. 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Editorial responsibility: Dieter Steinhagen, Submitted: November 22, 2016; Accepted: March 22, 2017 Hannover, Germany Proofs received from author(s): May 8, 2017