Myxozoan parasitism in waterfowl

Available online at www.sciencedirect.com

International Journal for Parasitology 38 (2008) 1199–1207 www.elsevier.com/locate/ijpara

Myxozoan parasitism in waterfowl q Jerri L. Bartholomew a,*, Stephen D. Atkinson a,b, Sascha L. Hallett a, Linda J. Lowenstine c, Michael M. Garner d, Chris H. Gardiner e, Bruce A. Rideout f, M. Kevin Keel g, Justin D. Brown g a

Center for Fish Disease Research, Department of Microbiology, Nash Hall 220, Oregon State University, Corvallis, OR 97331, USA b School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Qld 4072, Australia c Veterinary Medical Teaching Hospital Pathology Service, University of California, One Garrod Drive at Vet Med Way, Davis, CA 95616, USA d Northwest ZooPath, 654 West Main, Monroe, WA 98272, USA e Registry of Veterinary Pathology, Armed Forces Institute of Pathology, Washington, DC 20306, USA f Wildlife Disease Lab, Conservation and Research for Endangered Species, Zoological Society of San Diego, P.O. Box 120551, San Diego, CA 120551, USA g Southeastern Cooperative Wildlife Disease Study, College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7371, USA Received 19 November 2007; received in revised form 25 January 2008; accepted 29 January 2008

Abstract Myxozoans are spore-forming, metazoan parasites common in cold-blooded aquatic vertebrates, especially fishes, with alternate life cycle stages developing in invertebrates. We report nine cases of infection in free-flying native and captive exotic ducks (Anseriformes: Anatidae) from locations across the United States and describe the first myxozoan in birds, Myxidium anatidum n. sp. We found developmental stages and mature spores in the bile ducts of a Pekin duck (domesticated Anas platyrhynchos). Spores are lens-shaped in sutural view, slightly sigmoidal in valvular view, with two polar capsules, and each valve cell has 14–16 longitudinal surface ridges. Spore dimensions are 23.1 lm  10.8 lm  11.2 lm. Phylogenetic analysis of the ssrRNA gene revealed closest affinity with Myxidium species described from chelonids (tortoises). Our novel finding broadens the definition of the Myxozoa to include birds as hosts and has implications for understanding myxozoan evolution, and mechanisms of geographical and host range extension. The number of infection records indicates this is not an incidental occurrence, and the detection of such widely dispersed cases suggests more myxozoans in birds will be encountered with increased surveillance of these hosts for pathogens. Ó 2008 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Ducks; Anatidae; Myxosporean; Myxozoan; Emerging parasite; Myxidium anatidum

1. Introduction Myxozoans are among the more obscure animal phyla, being regarded exclusively as parasites of poikilothermic vertebrates and invertebrates. The vast majority has been described from fish and currently almost 2200 species are known (Kent et al., 2001; Canning and Okamura, 2004; Lom and Dykova´, 2006). Relationships between these parq Nucleotide sequence data reported in this paper are available in the GenBank database under the accession number EF602629. * Corresponding author. Tel.: +1 541 737 1856; fax: +1 541 737 0496. E-mail address: [email protected] (J.L. Bartholomew).

asites and their hosts are often highly evolved and do not result in severe disease (Lom and Dykova´, 2006). However, worldwide, a number of species cause disease and impact upon wild and farmed fish populations. The most notable are whirling disease (caused by Myxobolus cerebralis) and proliferative kidney disease (caused by Tetracapsuloides bryosalmonae) in trout and salmon, ‘‘hamburger” or proliferative gill disease (caused by Henneguya ictaluri) in catfish and enteromyxosis (caused by Enteromyxum leei) in cultured marine sparids. Reports of myxozoan infections from other poikilothermic vertebrates are less common, although some 20 species have been reported from chelonid reptiles and

0020-7519/$34.00 Ó 2008 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2008.01.008

1200

J.L. Bartholomew et al. / International Journal for Parasitology 38 (2008) 1199–1207

amphibians (Duncan et al., 2004; Eiras, 2005; Garner et al., 2005; Jirku` et al., 2006). Homeothermic vertebrates, namely mammals, have been reported to harbour myxozoan parasites but most of these may be considered incidental or aberrant host records. In cases where putative myxozoan developmental stages were observed, either mature spores or molecular evidence to confirm their identity as myxozoans were missing, and conversely, on occasions when spores were observed, developmental stages indicating true host status, rather than incidental passage, were absent (McClelland et al., 1997; Boreham et al., 1998; Lebbad and Willcox, 1998; Friedrich et al., 2000; Moncada et al., 2001). However, both developmental stages and mature spores of a new myxosporean, Soricimyxum fegati, were recently described from shrews (Prunescu et al., 2007; Dykova´ et al., 2007), providing evidence that these parasites may also occur in terrestrial hosts. Contributing to this expanding host assemblage, we have observed myxozoans in the liver and bile ducts of North American waterfowl during routine post-mortem examinations over the past decade. Our early findings were presented at the American Association of Zoo Veterinarians in 2002. The infected birds included six species of both captive, exotic and free-flying native ducks from five geographic locations. Mature spores and developmental stages were present in most cases, yet characterisation of the parasite remained incomplete until the most recent case: a feral Pekin duck collected from a pond in Georgia. Fresh tissue from this heavily infected bird enabled us to definitively characterise this novel organism through a combined morphological and molecular approach. 2. Materials and methods 2.1. Source of material Details of the first eight cases of myxozoan infections in waterfowl (Anseriformes: Anatidae) are as follows: captive juvenile male and female South African yellow-billed ducks (Anas undulata undulata) from southern California in August and September 1994; an adult male Cape teal (Anas capensis) from Florida in September 1998; wild adult female mallard ducks (Anas platyrhynchos) from Southern California in March 2000 and May 2004; a captive adult male Baikal teal (Anas formosa) from southern Texas in February 2000; a wild adult male wood duck (Aix sponsa) from northern California in August 2000; and a captive adult female smew (Mergus albellus) from Southern California in November 2000 (Fig. 1). The ninth case, a feral adult female Pekin duck, Anas platyrhynchos, collected in July 2006 from Swan Lake (a suburban pond in Stockbridge, Georgia, USA; 33.583954°N, 84.210316°W), provided material for both phenetic and genetic scrutiny and enabled a complete description of the parasite.

Fig. 1. Locations of ducks with myxozoan infections in the United States.

2.2. Necropsy and histology Necropsy of the Pekin duck was performed at the College of Veterinary Medicine, University of Georgia, Athens, USA. Tissues were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin. Sections were cut at 5 lm and stained with H&E, as well as with Giemsa. Histological sections and a sub-sample of frozen tissue were sent to Oregon State University (OSU). A suspension of thawed spores in distilled water was air-dried onto slides then stained with Diff-Quik (Dade Behring Inc., Newark, DE, USA). 2.3. Parasite spore morphology Spores were described from squash preparations of previously frozen liver and gall bladder from the Pekin duck. Spores were imaged digitally, under both bright field and Nomarski Interference Contrast illumination, and measurements made from the images (SPOT version 3.5.5. for Windows; SPOT Diagnostic Instruments, Sterling Heights, Michigan, USA), following established guidelines (Lom and Arthur, 1989). Table 1 presents the comparative morphology of similar species of Myxidium. 2.4. DNA amplification and sequencing DNA was extracted from the previously frozen tissue using a Qiagen DNeasy Tissue kit (animal tissue protocol; QIAGEN Inc., Valencia, CA, USA). Combinations of different myxozoan- and non-myxozoan-specific primers were trialled for both the parasite and host (for primer formulae, see Andree et al., 1998; Hallett et al., 2003; Whipps et al., 2003). Overlapping fragments of parasite ssrRNA gene were amplified with primer pairs MYX1f–MYX4r (Hallett et al., 2003), MYX1f–ACT1r (Hallett and Diamant, 2001) and MXATK2f (Garner et al., 2008) – ERIB10 (Barta et al., 1997) in 50 lL reactions which comprised 4 lL extracted genomic DNA, 2 1.25 lL 10 lM primer, 1.0 lL dNTPs (10 mM each), 3.0 lL 25 mM MgCl2, 2.5 lL Rediload loading dye (Invitrogen, Karlsbad, CA, USA), 1.25 lL 10 mg/mL BSA (New England BioLabs,

Table 1 Comparative morphometrics of freshwater Myxidium species having the same geographic site (Location), host site (Tissue) or spore morphometrics (lm) as the duck species, Myxidium anatidum n. sp. Myxidium species

Host

Tissue

Spore

Polar capsule

Length

Width

Length

Width

Striations

USA

Duck

B

21.3–24.3

10.3–11.5

5.4–7.4

4.7–6.0

14–16/valve

Ireland, Italy, USA, USSR Canada, Mexico, USA

Fish Fish

B, GB, UB, L GB, B

11 12.0–18.0

8–8.5 4.5–8.5

4.5 3.5–8.0

3 2.0–6.0

Numerous L 5–11 L

USA USA USA USA USA USA Europe, USA, USSR USA USA USA USA Ireland, Italy, USA, USSR USA USA USA USA USA, USSR North America USA USA USA Canada, Mexico, USA USA USA, Europe, Russia, New Zealand USA

Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Fish Turtle Fish Turtle Fish Fish Amph Fish Amph

GB G GB, K GB, L GB GB GB, K GB GB GB, B, PD GB, B B, GB, UB, L GB UB K K GB B, GB K K GB GB, B GB GB GB

8.5–10.5 9.3 9.3–12.6 9.5–12.0 10–11 10.1–1.92 10–12 11–12 11–12 11–14.5 11–12 11 11 11 12.7–15.3 12–14.4 14–15 14 14.5–16.4 15–16 15.5–17.5 12.0–18.0 15 8–9 12.0–13.5

5.6 7.8 6–7 4.8–6.0 6 4.2–6 6 5–6 5–6 5.5–8 5–6 8–8.5 8 4 7.6–9.3 2.4–4.8 7–8 4 6.5–8 5.5–6 4–5 4.5–8.5 8.8

3 3.8 2.3–3.8 3.16 3 2.5–3.4 3–4 4–5 3.5 3.0–4.5 3 4.5 3 3 3.4–6.1 3–4.8 4–5 3.8 4 6–7 3.5–8.0 5–5.5

3.5 2.5–3 2.0–6.0

8–10 L 6–7 5–11 L 2–4 L, 10–13 T

7–9

4.8–5.5

3.8–4.5

2–5 T/valve

2.3 3.19

10 L 0 Several Present 7 6–8

3–3.5 3

3 2.5 2.4

Present 9–11 L 9–11 L Numerous L Numerous L Present L 7–8/valve Absent 3–4/valve 4–6 O

J.L. Bartholomew et al. / International Journal for Parasitology 38 (2008) 1199–1207

Duck Myxidium anatidum Bile duct Myxidium oviforme Myxidium pearcyi USA Myxidium. moxostomatis Myxidium sp. Rice and Jahn, 1943 Myxidium minteri Myxidium sp. Guilford 1965 Myxidium glutinosum Myxidium kudoi Myxidium macrocapsulare Myxidium aplodinoti Myxidium folium Myxidium macrocheili Myxidium melum M. oviforme Myxidium phyllium Myxidium salvelini Myxidium illinoisense (syn of Myxidium giardi) Myxidium umbri Myxidium gasterostei Myxidium chelonarum Myxidium sp. Yasutake and Wood, 1957 Myxidium americanum Myxidium bellum M. pearcyi Myxidium serotinum Myxidium incurvatum Myxidium melleni

Location

Details abstracted from Jayasri and Hoffman (1982) and Jirku` et al. (2006). No other Myxidium species have been described from higher vertebrates. Hosts: amph, amphibia. Tissue: B, bile duct; G, gills; GB, gall bladder; K, kidney; L, liver; PD, pancreatic ducts; UB, urinary bladder. Striations: L, longitudinal; T, transverse; O, oblique.

1201

1202

J.L. Bartholomew et al. / International Journal for Parasitology 38 (2008) 1199–1207

Ipswich, MA, USA), 10 lL 5 Colorless GoTaq Flexi buffer, 0.5 lL GoTaq Flexi DNA polymerase (1.25 U) (Promega, Madison, WI, USA) and 25.25 lL molecular grade water. The PCR cycle profile was performed in a PTC200 thermocycler (MJ Research Inc., Watertown, MA, USA) and consisted of an initial denaturation step of 95 °C for 2 min, followed by 35 cycles of 94 °C for 20 s, 55 °C for 30 s, 72 °C for 60 s and finished with terminal extension at 72 °C for 10 min, then rested at 4 °C. PCR products were electrophoresed through a 2% agarose gel stained with 1% SYBRsafe (Invitrogen) alongside a 1-kb+ DNA ladder (Invitrogen), excised from the gel, then purified with a QIAquick gel extraction kit (Qiagen), or purified directly from the PCR products using a QIAquick PCR purification kit (Qiagen). Samples were sequenced in both directions using ABI Big Dye Terminator chemistry on an Applied Biosystems Capillary 3100 Genetic Analyser at the OSU Sequencing Facility (Center for Gene Research and Biotechnology, Central Service Laboratory). Sequence fragments were aligned manually in BioEdit (Hall, 1999) to produce the consensus sequence and a standard nucleotide–nucleotide BLAST search was conducted (Altschul et al., 1997).

myxozoans from GenBank (names and accession numbers in Fig. 4). The 50 and 30 terminal regions of the alignment were trimmed manually as these data are missing for many sequences. Gaps were treated as a fifth character state. Regions of ambiguous homology were removed to give a final alignment of 1602 characters of which 595 were informative. Maximum Parsimony analysis was conducted with PAUP (version 4.0b10) and used a heuristic search, 10 replicate random additions of taxa, tree bisection reconnection (TBR) branch swapping and 1000 bootstrap replicates. The bootstrap 50% majority-rule consensus tree is presented in Fig. 4. 3. Results 3.1. Description of Myxidium anatidum n. sp. (Myxosporea; Bivalvulida) Spores from all cases were similar in histologic presentation and were unmistakably myxozoan, with polar capsules (akin to nematocysts in Cnidarians) and a cell-within-cell organisation. Spore morphology, including polar capsule arrangement, was consistent with the genus Myxidium (Lom and Dykova´, 2006; Bu¨tschli, 1882).

2.5. Phylogenetic analysis The duck Myxidium sequence was aligned in ClustalX (Thompson et al., 1997) with data for other variisporinid

3.1.1. Description Kingdom Animalia Linnaeus, 1758; Phylum Myxozoa Grasse´, 1970; Class Myxosporea Bu¨tschli, 1881; Order

Fig. 2. Myxidium anatidum n. sp.: (A) in hepatic bile duct of Anas platyrhynchos with infiltrates of lymphocytes, plasma cells and fewer heterophils in the surrounding tissue, Giemsa stain; (B) at higher magnification showing lens-shaped spores with polar capsules at each end; (C) thawed spores viewed under bright field showing pyriform polar capsules containing coiled polar filaments; (D) Nomarski Interference Contrast reveals spore surface ridges.

J.L. Bartholomew et al. / International Journal for Parasitology 38 (2008) 1199–1207

Bilvalvulida Shulman, 1959; Suborder Variisporina Lom and Noble, 1984; Family Myxidiidae The´lohan, 1892; Genus Myxidium Bu¨tschli, 1882.

1203

3.1.3.5. Etymology. Specific name refers to the genus of the vertebrate type host. 3.2. Pathology

3.1.2. Myxidium anatidum n. sp. Spores (N = 25 from one host) lens-shaped in sutural view, slightly sigmoidal in valvular view, length 23.1 ± 0.8 (21.3–24.3) lm, width 10.8 ± 0.3 (10.3–11.5) lm, thickness 11.2 ± 0.5 (10.2–12.1) lm. Two polar capsules, one at either end of elongated spore, length 6.6 ± 0.4 (5.4–7.4) lm, width 5.4 ± 0.3 (4.7–6.0) lm, 5–6 polar filament turns. Valve cells each with 14–16 longitudinal surface ridges (Figs. 2 and 3). The spores are morphometrically (Table 1) and genetically (Fig. 4) distinct from previously described species of Myxidium. 3.1.3. Type material 3.1.3.1. Type host. Pekin duck, Anas platyrhynchos Linnaeus, 1758 (Anseriformes: Anatidae), adult female. 3.1.3.2. Location in host. Polysporic plasmodia and free spores in lumen of afferent bile ducts in liver. 3.1.3.3. Type locality. Swan Lake (a suburban pond), Stockbridge, Georgia, USA (33.583954°N, 84.210316°W). 3.1.3.4. Type material. Syntypes, accession number G464979, Diff-Quik stained spores on slide; plus additional voucher material: G464980 and G464981, H&E-stained thin section of liver; G464982, spores in 10% neutral buffered formalin; G464983, spores in 85% ethanol; deposited in the Parasitology Collection at the Queensland Museum, Brisbane, Australia. The ssrRNA gene sequence was deposited in GenBank (Accession No. EF602629).

In most of the ducks, death was attributed to significant non-parasite-derived health problems including botulism and trauma. Histological examination of liver sections showed that the myxozoan infections were accompanied by mild to severe inflammation. When inflammation was mild, the parasite was considered to be of no clinical significance. The more severe inflammatory lesions, as in the case with the Pekin duck, resulted in epithelial changes in affected bile ducts ranging from hyperplasia to complete desquamation. In some instances, severe granulomatous inflammation and obliteration of bile ducts resulted in eruption of myxozoan spores into the hepatic parenchyma (Fig. 2A) and was likely to have contributed to overall poor health of the animals. 3.3. Molecular analyses Sequencing began 80 bp from the 50 end of the ssrRNA gene, with myxozoan-specific primer MYX1f (Hallett and Diamant, 2001). Compared with other Myxidium species, the sequence had several AT-rich insertions in the V2 and V4 regions (Nelles et al., 1984) that inhibited the sequencing read. The near-complete gene sequence was 2039 bp long with extrapolated total length 2210 bp. Both parsimony and likelihood analyses gave equivalent tree topology including poor resolution of the position of M. anatidae (Fig. 4). A BLAST search of the nucleotide database presented no match.

Fig. 3. Line drawing of Myxidium anatidum n. sp. myxospores.

1204

J.L. Bartholomew et al. / International Journal for Parasitology 38 (2008) 1199–1207

Fig. 4. Phylogenetic reconstruction of variisporinid Myxosporea displayed as a bootstrap 50% majority-rule consensus tree from the Maximum Parsimony analysis. Values at nodes for P50% support; vertebrate hosts: marine fish (grey), freshwater (black), diadromous (half-and-half); target tissue: I (intestine), U (kidney/urinary bladder), B (liver/bile ducts/gall bladder).

4. Discussion This study describes a novel species, M. anatidum, and is the first record of a myxozoan parasite infecting birds. The presentation of infections in waterfowl was typical of Myxidium species, which are common coelozoic parasites that infect biliary, urinary and gastrointestinal systems of aquatic vertebrates worldwide (Lom and Dykova´, 2006; Duncan et al., 2004; Garner et al., 2005). An accurate assessment of the host and geographic distribution of waterfowl biliary myxozoanosis is problematic both because the vertebrate hosts can migrate large distances and because these parasites, particularly prior to spore formation, can be easily overlooked or misidentified as sloughed biliary epithelium, mucous or cellular debris. Although the myxospores collected from multiple species of waterfowl appeared morphologically similar, we were unable to obtain fresh specimens from more than one host (Pekin duck) for further microscopic and genetic characterisation, which limited our ability to confirm whether these represented a single parasite species.

Comparison of Myxidium species having the same tissue tropism, geographic site or spore morphometrics as the Pekin duck species indicated M. anatidum had not been previously documented and was readily distinguishable from other freshwater species by the larger size of its myxospores (Jayasri and Hoffman, 1982; Hoffman, 1999; Jirku` et al., 2006; Table 1). The ssrRNA gene sequence of M. anatidum provided both further evidence that this is a novel species and insight into its relationship with other members of the genus. This 2000 bp gene is atypically variable within the Myxozoa and hence useful for systematics. Despite the arsenal of myxozoan-specific primers available (see Garner et al., 2008), we found that only limited combinations successfully amplified this parasite. Significantly, universal primers ERIB1 (Barta et al., 1997) and 18E (Hillis and Dixon, 1991) did not bind, which suggested M. anatidum possessed some critical variation in the highly conserved 50 terminal region of the gene and further indicates the extraordinary variability of ssrRNAs in the Myxozoa. The ssrRNA sequence was compared phylogenetically with other variisporinid myxozoans available in GenBank.

J.L. Bartholomew et al. / International Journal for Parasitology 38 (2008) 1199–1207

The analyses showed the genus Myxidium is polyphyletic and intermingled with Zschokkella, Sphaeromyxa and Chloromyxum species within a broad clade of myxozoans that predominantly infect the biliary system of their vertebrate hosts (Fig. 4). As has been observed previously (Fiala, 2006), two well-supported sub-clades are apparent: a solely marine clade and a mostly freshwater clade. M. anatidum occupies a poorly resolved position in the ‘freshwater’ clade, more basal than the majority of freshwater taxa, and adjacent to species infecting other non-fish vertebrates. If additional sequences of bird and mammal myxozoans form a distinct clade, it would suggest host range expansion into homeotherms occurred in the distant past. Whereas if multiple clades are apparent, this would suggest myxozoans have jumped from cold-blooded to warmblooded hosts several times. The apparent similarity of spore morphology and presentation of the infection in the different species of birds was surprising. If our present cases are all indeed M. anatidum, this suggests that a single invasion occurred and that the broad geographic distribution we observed is a result of the migratory nature of waterfowl. We anticipate more cases of avian infection will be encountered with increased surveillance of birds for other pathogens such as avian influenza and West Nile virus. Species of Myxidium infect a wide range of hosts, occurring in marine and freshwater fish, amphibians and reptiles. The presence of marine taxa within the predominantly freshwater phylogenetic clade suggests that reversion of freshwater species back to marine hosts has also occurred. These flexible and generalist strategies of host selection suggest that range expansion into suitable non-fish aquatic vertebrates (including waterfowl) could be expected. Yet typical vertebrate hosts of myxozoans are poikilotherms with body temperatures within a few degrees of ambient, whereas birds maintain body temperatures between 37.7 and 43.5 °C. This difference can be considered a barrier to host-switching. However, common carp (Cyprinus carpio) and other warm-water fish species that inhabit ponds in southern United States can tolerate water temperatures up to 35.7 °C (McLarney, 1998 in Ficke et al., 2007). This fish is host to a number of myxozoans, including Myxidium species, suggesting that survival of some myxozoans at homeothermic body temperatures is feasible. Indeed, the newly described Soricimyxum fegati from the liver of shrews resembles Myxidium morphologically and molecularly and is a case in point (Prunescu et al., 2007; Dykova´ et al., 2007). How ducks become infected by myxozoan parasites is unknown. Some 35 species in the class Myxosporea have been shown to have two-host life cycles that involve fish and aquatic annelids; a pattern regarded as typical for most myxosporeans (Kent et al., 2001). The annelid is the definitive host given that amphimixis occurs here (El-Matbouli and Hoffmann, 1998), and parasite development culminates in release of a free-floating actinospore. Actinospores have been demonstrated to infect fish through contact with

1205

the epidermal surface, including the buccal cavity (El-Matbouli and Hoffmann, 1989). Once inside the host, a period of asexual parasite propagation leads to formation of myxospores. This spore stage, in turn, is released from the fish and infects the annelid host. Developmental cycles have been described for two species of Myxidium: Myxidium giardi (Benajiba and Marques, 1993) and Myxidium truttae (Holzer et al., 2004), which infect fish and freshwater oligochaetes. However, no life cycles are known for vertebrates other than fish. If the life cycle of M. anatidum follows this model, then infection would occur by contact with an actinospore stage. Although the species of waterfowl in which infections were reported have different feeding habits, at some stage in their lives they feed on either aquatic invertebrates or fish. Thus, infection might occur via contact of exposed epidermis to actinospores while ‘dabbling’ or through direct ingestion of infected invertebrates, and following development in the biliary system, myxospores may be shed in the urine or faeces. Alternatively, the life cycle in birds may be direct, with the parasite transmitted through ingestion of myxospores shed from infected individuals. Direct fish to fish transmission has been demonstrated for species of Enteromyxum: Enteromyxum leei (syn. Myxidium leei), Enteromyxum scophthalmi and Enteromyxum fugi (syn. Myxidium fugi) (Diamant, 1997; Redondo et al., 2002; Yasuda et al., 2002), and allows rapid transmission to a broad variety of marine hosts. This does not eliminate the possibility that this species may also utilise an invertebrate host for amphimixis, but it raises the possibility that transmission to the waterfowl may have occurred directly. We surveyed both fish and annelids from the pond where the infected Pekin duck was collected and characterised 14 myxozoan infections in oligochaetes and 10 in fish; none of which matched M. anatidum genetically. However, infections may have been missed, they may be seasonal, or the bird may have acquired the infection elsewhere. The detection of myxozoans in waterfowl raises basic questions about parasite evolution and transmission, and whether this parasite group may be an undetected or emerging pathogen of other homeothermic vertebrates. The pattern of infection in vertebrates closely associated with water (fish, frogs, turtles, ducks) suggests that myxozoan infections might occur in other animals that utilise aquatic habitats or which include aquatic oligochaetes as a food source. As awareness of these infections increases, we expect to see additional descriptions in homeotherms. This will again cause re-examination of a group of organisms that has only recently been elevated to metazoan status and which, based on recent data, may be subsumed into the Cnidaria (Jime´nez-Guri et al., 2007). This implies that the Cnidaria have undergone a niche expansion that allows the exploitation of warm-blooded terrestrial vertebrates by essentially aquatic animals.

1206

J.L. Bartholomew et al. / International Journal for Parasitology 38 (2008) 1199–1207

Acknowledgements We thank C. Whipps (University of New York, Syracuse) and B. Okamura (Natural History Museum, London) for critically reading the manuscript. We also thank M. Mace and I. Stalis at the Zoological Society of San Diego, J. Raymond at Northwest ZooPath and M. Bush at the University of California, Davis, for case contributions. This work was funded in part by a Tartar Award by the Department of Microbiology, OSU and the General Research Fund, OSU. References Altschul, S.F., Madden, T.L., Scha¨ffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Andree, K.B., MacConnell, E., Hedrick, R.P., 1998. A nested polymerase chain reaction for the detection of genomic DNA of Myxobolus cerebralis in rainbow trout Oncorhynchus mykiss. Dis. Aquat. Organ. 34, 145–154. Barta, J.R., Martin, D.S., Liberator, P.A., Dashkevicz, M., Anderson, J.W., Feighner, S.D., Elbrecht, A., Perkins-Barrow, A., Jenkins, M.C., Danforth, H.D., Ruff, M.D., Profous-Juchelka, H., 1997. Phylogenetic relationships among eight Eimeria species infecting domestic fowl inferred using complete small subunit ribosomal DNA sequences. J. Parasitol. 83, 262–271. Benajiba, M.H., Marques, M., 1993. The alternation of Actinomyxidian and Myxosporidian sporal forms in the development of Myxidium giardi (parasite of Anguilla anguilla) through oligochaetes. Bull. Eur. Assoc. Fish Pathol. 13, 100–103. Boreham, R.E., Hendrick, S., O’Donoghue, P.J., Stenzel, D.J., 1998. Incidental finding of Myxobolus spores (Protozoa: Myxozoa) in stool samples from patients with gastrointestinal symptoms. J. Clin. Microbiol. 36, 3728–3730. Canning, E.U., Okamura, B., 2004. Biodiversity and evolution of the Myxozoa. Adv. Parasitol. 56, 44–131. Diamant, A., 1997. Fish-to-fish transmission of a marine myxosporean. Dis. Aquat. Organ. 30, 99–105. Duncan, A.E., Garner, M.M., Bartholomew, J.L., Reichard, T.A., Nordhausen, R.W., 2004. Renal myxosporidiasis in Asian horned frgos (Megophrys nasuta). J. Zoo Wild. Dis. 35, 381–386. Dykova´, I., Tyml, T., Fiala, I., Lom, J., 2007. New data on Soricimyxum fegati (Myxozoa) including analysis of its phylogenetic position inferred from the SSU rRNA gene sequence. Folia Parasitol. 54 (4), 272–276. Eiras, J.C., 2005. An overview on the myxosporean parasites in amphibians and reptiles. Acta Parasitol. 50, 267–275. El-Matbouli, M., Hoffmann, R.W., 1989. Experimental transmission of two Myxobolus spp. developing bisporogeny via tubificid worms. Parasitol. Res. 75, 461–464. El-Matbouli, M., Hoffmann, R.W., 1998. Light and electron microscopic studies on the chronological development of Myxobolus cerebralis to the actinosporean stage in Tubifex tubifex. Int. J. Parasitol. 28, 195– 217. Fiala, I., 2006. The phylogeny of Myxosporea (Myxozoa) based on small subunit ribosomal RNA gene analysis. Int. J. Parasitol. 36, 1521–1524. Ficke, A.D., Myrick, C.A., Hansen, L.J., 2007. Potential impacts of global climate change on freshwater fisheries. Rev. Fish Biol. Fisher. 17, 581– 613. Friedrich, C., Ingolic, E., Freitag, B., Kastberger, G., Hohmann, V., Skofitsch, G., Neumeister, U., Kepka, O., 2000. A myxozoan-like parasite causing xenomas in the brain of the mole, Talpa europaea L., 1758 (Vertebrata, Mammalia). Parasitology 121, 483–492.

Garner, M.M., Atkinson, S.D., Hallett, S.L., Bartholomew, J.L., Nordhausen, R.W., Reed, H., Adams, L., Whitaker, B., 2008. Renal myxozoanosis in Weedy Sea Dragons (Phyllopteryx taeniolatus) due to Sinuolinea phyllopteryxa n. sp. J. Fish Dis. 31, 27–35. Garner, M.M., Bartholomew, J.L., Whipps, C.M., Nordhausen, R.W., Raiti, P., 2005. Renal myxozoanosis in Crowned River Turtles Hardella thurjii: description of the putative agent Myxidium hardella n. sp. by histopathology, electron microscopy, and DNA sequencing. Vet. Pathol. 42, 589–595. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98. Hallett, S.L., Atkinson, S.D., Scho¨l, H., El-Matbouli, M., 2003. Characterisation of two novel types of hexactinomyxon spores (Myxozoa) with subsidiary protrusions on their caudal processes using scanning electron microscopy and 18S rDNA sequence data. Dis. Aquat. Organ. 55, 45–57. Hallett, S.L., Diamant, A., 2001. Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n.sp. (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland, Australia. Dis. Aquat. Organ. 46, 197–212. Hillis, D.M., Dixon, M.T., 1991. Ribosomal DNA: molecular evolution and phylogenetic inference. Q. Rev. Biol. 66, 411–453. Hoffman, G.L., 1999. Parasites of North American Freshwater Fishes, second ed. Cornell University Press, Ithaca, New York. Holzer, A.S., Sommerville, C., Wootten, R., 2004. Molecular relationships and phylogeny in a community of myxosporeans and actinosporeans based on their 18S rDNA sequences. Int. J. Parasitol. 34, 1099–1111. Jayasri, M., Hoffman, G.L., 1982. Review of Myxidium (Protozoa: Myxozoa: Myxosporea). Protozool. Abstr. 6, 61–91. Jirku`, M., Bolek, M.G., Whipps, C.M., Janovy Jr., J., Kent, M.L., Modry¨, D., 2006. A new species of Myxidium (Myxosporea: Myxidiidae), from the western chorus frog, Pseudacris triseriata triseriata, and Blanchard’s cricket frog, Acris crepitans blanchardi (Hylidae), from Eastern Nebraska: morphology, phylogeny, and critical comment. J. Parasitol. 92, 611–619. Jime´nez-Guri, E., Philippe, H., Okamura, B., Holland, P.W.H., 2007. Buddenbrockia is a cnidarian worm. Science 317, 116–118. Kent, M.L., Andree, K.B., Bartholomew, J.L., El-Matbouli, M., Desser, S.S., Devlin, R.H., Feist, S.W., Hedrick, R.P., Hoffmann, R.W., Khattra, J., Hallett, S.L., Lester, R.J.G., Longshaw, M., Palenzeula, O., Siddall, M.E., Xiao, C., 2001. Recent advances in our knowledge of the Myxozoa. J. Eukaryot. Microbiol. 48, 395–413. Lebbad, M., Willcox, M., 1998. Spores of Henneguya salminicola in human stool specimens. J. Clin. Microbiol. 36, 182. Lom, J., Arthur, J.R., 1989. A guideline for the preparation of species descriptions in Myxosporea. J. Fish Dis. 12, 151–156. Lom, J., Dykova´, I., 2006. Myxozoan genera: definition and notes on taxonomy, life-cycle terminology and pathogenic species. Folia Parasitol. 53, 1–36. McClelland, R.S., Murphy, D.M., Cone, D.K., 1997. Report of spores of Henneguya salminicola (Myxozoa) in human stool specimens: possible source of confusion with human spermatozoa. J. Clin. Microbiol. 35, 2815–2818. McLarney, W., 1998. Freshwater Aquaculture. Hartley and Marks Publishers, Point Roberts, Washington. Moncada, L.I., Lo´pez, M.C., Murcia, M.I., Nicholls, S., Leo´n, F., Guı´o, O.L., Corredor, A., 2001. Myxobolus sp., another opportunistic parasite in immunosuppressed patients. J. Clin. Microbiol. 39, 1938– 1940. Nelles, L., Fang, B.-L., Volckaert, G., Vandenberghe, A., de Wachter, R., 1984. Nucleotide sequence of a crustacean 18S ribosomal RNA gene and secondary structure of eukaryotic small subunit ribosomal RNAs. Nucleic Acids Res. 12, 8749–8767. Prunescu, C.-C., Prunescu, P., Pucek, Z., Lom, J., 2007. The first finding of myxosporean development from plasmodia to spores in terrestrial mammals: Soricimyxum fegati gen. et sp. n. (Myxozoa) from Sorex araneus (Soricomorpha). Folia Parasitol. 54, 159–164.

J.L. Bartholomew et al. / International Journal for Parasitology 38 (2008) 1199–1207 ´ ., A ´ lvarez-Pellitero, Redondo, M.J., Palenzuela, O., Riaza, A., Macı´as, A P., 2002. Experimental transmission of Enteromyxum scophthalmi (Myxozoa), an enteric parasite of turbot Scophthalmus maximus. J. Parasitol. 88, 482–488. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24, 4876–4882.

1207

Whipps, C.M., Adlard, R.D., Lester, R.J.G., Findlay, V., Kent, M.L., 2003. First report of three Kudoa species from Eastern Australia: Kudoa thyrsites from Mahi mahi (Coryphaena hippurus), Kudoa amamiensis and Kudoa minithyrsites n. sp. from sweeper (Pempheris ypsilychnus). J. Eukaryot. Microbiol. 50, 215–219. Yasuda, H., Ooyama, T., Iwata, K., Tun, T., Yokoyama, H., Ogawa, K., 2002. Fish-to-fish transmission of Myxidium spp. (Myzozoa) in cultured tiger puffer suffering from emaciation disease. Fish Pathol. 37, 29–33.