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. 2022 Jan 7;2:100075. doi: 10.1016/j.crpvbd.2022.100075

Eimeria spp. and Tyzzeria perniciosa Allen, 1936 (Apicomplexa: Eimeriidae) from a Pacific black duck, Anas superciliosa Gmelin (Aves: Anseriformes), in western Australia

Bruno P Berto a,∗, Belinda Brice b, Gwyneth Thomas b, Aileen Elloit c, Alireza Zahedi d, Rongchang Yang c,e,∗∗
PMCID: PMC9795352  PMID: 36589875

Abstract

Four species of the Eimeriidae, Eimeria anatis Scholtyseck, 1955, Eimeria aythyae Farr, 1965, Eimeria krylovi Svanbaev & Rakhmatullina, 1967 and Tyzzeria perniciosa Allen, 1936, were morphologically identified from oöcysts recovered from a Pacific black duck, Anas superciliosa Gmelin. Additionally, genotypic characterization of E. anatis is provided via sequencing of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and the small subunit ribosomal RNA (18S) genes. The four species are redescribed, providing additional morphological details. The validity of genera and coccidian species parasitizing birds of the order Anseriformes such as Wenyonella Hoare, 1933 and some Tyzzeria spp. are discussed. Molecular phylogenetic analyses for the cox1 and 18S rRNA genes resulted in monophylies of Eimeria spp. from Anseriformes which included the sequences obtained from E. anatis oöcysts.

Keywords: Coccidia, Eimeria, Tyzzeria, Pacific black duck, Anas superciliosa, 18S rRNA gene, cox1 gene

Graphical abstract

Image 1

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Highlights

  • •

    Eimeria anatis, Eimeria aythyae, Eimeria krylovi, Tyzzeria perniciosa are identified from a new host, Anas superciliosa.

  • •

    Coccidia are redrawn and redescribed to offer better characterization of the morphological features.

  • •

    Genotypic characterization via sequencing of mitochondrial and ribosomal genes is provided for E. anatis.

  • •

    Taxonomic validity of coccidia parasitic in Anseriformes, such as Wenyonella and some Tyzzeria spp. is discussed.

1. Introduction

The Pacific black duck Anas superciliosa Gmelin (Anseriformes: Anatidae) is a dabbling duck commonly seen in waterways, swamps, streams and ponds in Australia, New Zealand, Indonesia through to Papua New Guinea, Polynesia, the islands of the West Pacific and the sub-Antarctic islands (Pizzey and Knight, 2007).

Both domestic and wild ducks are commonly infected with gastrointestinal parasites including coccidia, which are obligate intracellular protozoans of the Apicomplexa (Gajadhar et al., 1983). Species of Eimeria Schneider, 1875 (Eimeriidae) are the most common coccidia found in birds, including ducks, with mixed infections being common (Leibovitz, 1968). Eimeria anatis Scholtyseck, 1955 infects the mallard Anas platyrhynchos (L.) (Duszynski et al., 2001) and Eimeria aythyae Farr, 1965 infects the lesser scaup Aythya affinis (Eyton) (Gajadhar et al., 1983; Duszynski et al., 2001). Windingstad et al. (1980) reported recurring epizootic infection in A. affinis resulting from infection with E. aythyae. The host range of Eimeria krylovi Svanbaev & Rakhmatullina, 1967 includes the green-winged teal Anas carolinensis Gmelin, the northern shoveler Spatula clypeata (L.), the European wigeon Mareca penelope (L.), the gadwall Mareca strepera (L.) and the garganey Spatula querquedula (L.) (Svanbaev & Rakhmatullina, 1967).

Coccidia of the genus Tyzzeria Allen, 1936, have also been described predominantly from ducks (Gajadhar et al., 1983). This genus is made up of coccidia whose oöcysts lack sporocysts (Duszynski et al., 1998). Cole & Friend (1999) reported that Tyzzeria spp. were less commonly seen in ducks than Eimeria spp. Tyzzeria perniciosa Allen, 1936 is an important pathogenic coccidian in ducks and is especially pathogenic for ducklings (Baker, 2007). Reported duck hosts of T. perniciosa are the northern pintail Anas acuta L., the lesser scaup A. affinis, the common shelduck Tadorna tadorna (L.), the tufted duck Aythya fuligula (L.), the mallard A. platyrhynchos and the white-headed duck Oxyura leucocephala (Scopoli) (Duszynski et al., 1998). A study from China reported outbreaks of coccidiosis due to T. perniciosa and Wenyonella philiplevinei Leibovitz, 1968, amongst farmed ducklings (Peiyun et al., 1982). A study in Iraq detected E. anatis in 17% and T. perniciosa in 11% of domesticated ducks screened (n = 80) (Abdullah, 2010) while another study on domestic ducks in Iran found a variety of protozoan parasites including Cryptosporidium spp., Tyzzeria spp., W. philiplevinei, Isospora mandari Bhatia, Chauhan, Arora & Agrawal, 1971 as well as other coccidian species (Larki et al., 2018).

The coccidia infecting ducks are similar in size and have very similar morphologies. This makes identification difficult using morphology alone (Leibovitz, 1968; Gajadhar et al., 1983). Those coccidia infecting wild ducks have not been well studied. In this study, we morphologically identified E. anatis, E. aythyae, E. krylovi and T. perniciosa from a Pacific black duck. Additionally, we provided genotypic characterization via sequencing of the mitochondrial cytochrome c oxidase subunit 1 (cox1) and the small subunit ribosomal RNA (18S) genes for E. anatis.

2. Materials and methods

2.1. Sample collection and examination

A wild, juvenile Pacific black duck was admitted to the Kanyana Wildlife Rehabilitation Centre (KWRC), Perth, Australia, in January 2021, after it was struck by a motor vehicle. Physical examination on admission revealed no external injuries; however, the duck was extremely quiet, reluctant to walk and was leaning to one side. It had a body condition score of 2/5. The duck was given supportive treatment of fluids and pain relief medication before being sent to a veterinarian for further assessment. A preliminary diagnosis of concussion and possible internal injuries was made. A faecal sample was collected on admission to KWRC. Initial direct light microscopy revealed a heavy, mixed parasitic load including large numbers of unsporulated coccidian oöcysts of various sizes as well as trophozoites of Trichomonas Donné, 1836, eggs of Capillaria Zeder, 1800 and tapeworm eggs. The duck was treated for the worm infection with praziquantel and moxidectin (20 mg/kg and 1 mg/kg of each ingredient respectively), per os (PO), once daily (OD), which was repeated after 14 days. The coccidia were treated with toltrazuril (15 mg/kg, PO, OD) for three consecutive days and then again 7 days later. Metronidazole (50 mg/kg, PO, OD) was given for 7 days for the Trichomonas infection. The duck made a full recovery and was released near the found location 4 weeks later.

A portion of faeces was placed in 2% (w/v) K2Cr2O7, mixed well and placed in a refrigerator, until transport to Murdoch University (within 48 h) for further investigation. On arrival at the Murdoch University laboratory, the faecal solution was poured into a Petri dish (to a depth of less than 1 cm). The Petri dish was stored in a dark environment and kept at room temperature (22 °C), to facilitate sporulation. The sample was checked daily for oöcyst sporulation using an Olympus DP71 digital microimaging camera. Sporulated oöcysts were observed using the 100× oil immersion objective. Images were taken using Nomarski contrast with a 100× oil immersion objective. Line drawings were edited using two software applications of CorelDRAW® (Corel Draw Graphics Suite, Version, 2020; Corel Corporation, Canada), i.e. Corel DRAW and Corel PHOTO-PAINT. All measurements are in micrometres and are given as the range followed by the mean in parentheses.

2.2. Oöcyst isolation, DNA extraction, PCR amplification, sequencing and phylogenetic analyses

Five morphologically similar oöcysts were isolated for a bulk DNA extraction with the method described by Yang et al. (2015). The DNA extraction, PCR amplification of the 18S rRNA and cox1 genes and sequencing were conducted according to the protocols described by Yang et al. (2013, 2016).

Phylogenetic trees were constructed for E. anatis using partial 18S rDNA and partial cox1 sequences aligned with additional species/isolates from GenBank using ClustalW (http://www.phylogeny.fr/one_task.cgi?task_type=clustalw). Distance analyses and phylogenies were conducted using MEGA X (Kumar et al., 2018) as described in detail by Yang et al. (2021) with the most appropriate nucleotide substitution models (TN93 + G + I for 18S and TN93 + G for the cox1 gene). Bootstrap support was estimated from 1000 pseudoreplicates.

3. Results

Based on the morphological analysis of the coccidian oöcysts in the faecal sample, four species were identified: E. anatis, E. aythyae, E. krylovi and T. perniciosa. The newly collected material is described below.

3.1. Eimeria anatis Scholtyseck, 1955

[Description based on 20 oöcysts and 40 sporocysts; Fig. 1.] Oöcysts elongate-ovoidal, 17–19 × 11–13 (17.6 × 11.9); length/width (L/W) ratio 1.4–1.6 (1.5). Oöcyst wall bi-layered, 0.9–1.3 (1.0) thick; outer layer smooth to slightly rough, c.2/3 of total thickness. Micropyle cap absent. Micropyle present, generally with invagination of inner layer. Oöcyst residuum absent, but 1–2 polar granules present. Sporocysts 4, ellipsoidal, 7–9 × 5–6 (7.9 × 5.9); L/W ratio 1.3–1.4 (1.3). Stieda body present, flattened; sub-Stieda absent or indiscernible; para-Stieda body absent. Sporocyst residuum present, composed of small, randomly dispersed granules. Sporozoites 2, with robust anterior and posterior refractile bodies and indiscernible nucleus.

Fig. 1.

Fig. 1

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Composite line drawing (A) and photomicrographs (B, C) of sporulated oöcysts of Eimeria anatis from the Pacific black duck Anas superciliosa. Note the anterior (arb) and posterior (prb) refractile bodies; micropyle (m); polar granule (pg); rough oöcyst wall (row); Stieda body (sb); and sporocyst residuum (sr). Scale-bars: 10 μm.

3.2. Eimeria aythyae Farr, 1965

[Description based on 20 oöcysts and 40 sporocysts; Fig. 2.] Oöcysts ellipsoidal, 20–23 × 14–16 (21.3 × 15.2); L/W ratio 1.3–1.5 (1.4). Oöcyst wall bi-layered, 1.0–1.4 (1.2) thick; outer layer smooth, c.2/3 of total thickness. Micropyle cap present as a translucent, delicate, curved protrusion. Micropyle present with no invagination of inner layer. Oöcyst residuum and polar granule absent. Sporocysts 4, ellipsoidal, 9–11 × 7–8 (10.5 × 7.7); L/W ratio 1.2–1.4 (1.4). Stieda body present, flattened; sub-Stieda present, but delicate or indiscernible in some sporocysts; para-Stieda body absent. Sporocyst residuum present, composed of large, randomly dispersed granules. Sporozoites 2, with robust anterior and posterior refractile bodies and centrally located nucleus.

Fig. 2.

Fig. 2

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Composite line drawing (A) and photomicrographs (B, C) of sporulated oöcysts of Eimeria aythyae from the Pacific black duck Anas superciliosa. Note the micropyle (m); micropyle cap (mc); nucleus (n); Stieda body (sb); sporocyst residuum (sr); and refractile body (rb). Scale-bars: 10 μm.

3.3. Eimeria krylovi Svanbaev & Rakhmatullina, 1967

[Description based on 25 oöcysts and 50 sporocysts; Fig. 3.] Oöcysts ellipsoidal, 20–23 × 16–17 (21.7 × 16.1); L/W ratio 1.3–1.4 (1.3). Oöcyst wall bi-layered, 1.0–1.4 (1.2) thick; outer layer smooth, c.2/3 of total thickness. Micropyle cap present as a dense cover. Micropyle present with no invagination of inner layer. Oöcyst residuum and polar granule absent. Sporocysts 4, subspheroidal to ellipsoidal, 8–10 × 7–8 (8.8 × 7.7); L/W ratio 1.1–1.2 (1.1). Stieda body flattened, barely or not discernible; sub-Stieda absent; para-Stieda body absent. Sporocyst residuum present, composed of many large and dense granules which are widely diffused within the sporocyst. Sporozoites 2, with anterior and posterior refractile bodies and indiscernible nucleus.

Fig. 3.

Fig. 3

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Composite line drawing (A) and photomicrographs (B, C) of sporulated oöcysts of Eimeria krylovi from the Pacific black duck Anas superciliosa. Note the micropyle (m); micropyle cap (mc); sporocyst residuum (sr); and refractile body (rb). Scale-bars: 10 μm.

3.4. Tyzzeria perniciosa Allen, 1936

[Description based on 25 oöcysts; Fig. 4.] Oöcysts ellipsoidal, 10–11 × 7–8 (10.7 × 7.4); L/W ratio 1.4–1.5 (1.4). Oöcyst wall bi-layered, 0.4–0.7 (0.6) thick; outer layer smooth, c.2/3 of total wall thickness. Oöcyst residuum present as granules of different sizes usually clustered at one end of oöcyst, measuring c.2.5. Sporozoites 8, curved and tapered at anterior end, 6–8 × 1–2 (6.9 × 1.8), with robust, prominent posterior refractile body and without discernible nucleus.

Fig. 4.

Fig. 4

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Composite line drawing (A) and photomicrographs (B, C) of sporulated oöcysts of Tyzzeria perniciosa from the Pacific black duck Anas superciliosa. Note the inner (il) and outer (ol) layers of the oöcyst wall; oöcyst residuum (or); refractile body (rb); and sporozoite (sz). Scale-bars: 10 μm.

3.5. Molecular identification

PCR amplification for the 18S rRNA and cox1 genes from oöcyst DNA of the four coccidian species were conducted; unfortunately, PCR amplicons were successfully obtained only for E. anatis oöcysts.

3.5.1. Phylogenetic analyses of the 18S rRNA gene

A 1209 bp 18S rDNA sequence with clean sequencing chromatography was obtained from the five morphological similar oöcysts of E. anatis isolated from the faecal samples of Anas superciliosa; this was aligned with 37 sequences for Eimeria spp., 5 for Cyclospora spp. and 2 for Isospora spp. based on the NCBI BLAST similarities. The alignment covered all available Eimeria spp. sequences. A 18S rRNA gene sequence (GenBank: L24381) of Toxoplasma gondii (Nicolle & Manceaux, 1908) was used as the outgroup. Eimeria anatis shared 97.6% and 96.6% similarity with Eimeria stigmosa Klimes, 1963 (GenBank: KP789171) and Eimeria anseris Kotlan, 1932 (GenBank: KJ000077), respectively, both of which were obtained from Anser anser (L.) in China (sequences published in GenBank only). Eimeria anatis also shared a genetic similarity of 94.6% with both Eimeria gruis Yakimoff & Matschoulsky, 1935 (GenBank: AB544336) and Eimeria reichenowi Yakimoff & Matschoulsky, 1935 (GenBank: AB544314), both identified from Grus monacha Temminck in Japan and reported by the same group (Honma et al., 2011). In addition, E. anatis shared 93.0% similarity with Eimeria paludosa (Leger & Hesse, 1922) (GenBank: KJ767187) from Gallinula tenebrosa Gould in Western Australia (Yang et al., 2014). As shown in Fig. 5A, E. anatis was placed in a separate strongly supported clade with E. stigmosa and E. anseris, closely associated with a sister clade composed of E. gruis, E. reichenowi and E. paludosa.

Fig. 5.

Fig. 5

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Evolutionary relationships of Eimeria anatis inferred by maximum likelihood analysis (ML) of 18S rDNA sequences (A, alignment length 1209 bp; B, alignment length 424 bp). Percentage support (> 70%) from 1000 pseudoreplicates from the ML analysis is indicated at the nodes.

Eimeria anatis is often related to the coccidian species W. philiplevinei; however, there is no 18S DNA sequence from W. philiplevinei available, only a 422-bp 18S sequence presented in the paper by Wu et al. (2013). The 18S sub-tree generated from a shortened alignment including both E. anatis and W. philiplevinei showed that E. anatis belongs to the same clade as that of the 18S phylogenetic tree based on the long alignment (Fig. 5A), whereas W. philiplevinei was positioned close to T. gondii, outside of the Eimeria spp. clades (Fig. 5B). The genetic similarity between E. anatis and W. philiplevinei was 86.5%.

3.5.2. Phylogenetic analyses of the cox1 gene

The cox1 gene was amplified from E. anatis oöcyst DNA and a 650-bp sequence was successfully obtained and aligned with 21 sequences for Eimeria spp. from different animal species, 4 for Isospora spp. and one for Caryospora sp. All cox1 reference sequences were selected based on the NCBI BLAST similarities and covered all Eimeria spp. in the database. A sequence for T. gondii (GenBank: HM771690) was used as the outgroup. Eimeria anatis showed the highest genetic similarity (91.9%) with an unnamed Eimeria sp. isolated from the pink-footed goose Anser brachyrhynchus Baillon (GenBank: MT833388) (Myšková et al., 2021) and grouped with this Eimeria sp. in the same clade in the phylogenetic tree (Fig. 6).

Fig. 6.

Fig. 6

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Evolutionary relationships of Eimeria anatis inferred by maximum likelihood analysis (ML) of partial cox1 gene sequences (650 bp). Percentage support (> 70%) from 1000 pseudoreplicates from the ML analysis is indicated at the nodes.

4. Discussion

Eimeria anatis, E. aythyae, E. krylovi and T. perniciosa have all been previously reported to infect ducks. The Pacific black duck in this study was found to be infected with all three of these Eimeria spp. simultaneously as well as T. perniciosa. This is not unusual as co-infections with Eimeria spp. are regularly observed in birds.

The oöcysts of the three Eimeria spp. identified in this study were morphologically compatible with their respective original descriptions (Table 1, Table 2). However, it is noteworthy that in the present study some adjustments for some characteristic features were added to the descriptions of these species. For example, a Stieda body and a sub-Stieda body were observed in E. anatis and E. aythyae, respectively, which were not identified in the original descriptions (Scholtyseck, 1955; Farr, 1965) or in later reports (Gajadhar et al., 1983). Eimeria krylovi was not originally described with a Stieda body, and indeed this structure was hardly observed and photomicrographed in this study, being reported here as “barely or not discernible”. In this context, it is important to highlight that a Stieda body is a synapomorphic characteristic of the family Eimeriidae, and its lack indicates a change in the identification to another genus and/or family, such as the genera Choleoeimeria Paperna & Landsberg, 1989, Acroemeria Paperna & Landsberg, 1989, Goussia Labbé, 1896, etc., which were originally described within Eimeria, but due to the absence of a Stieda body, among other aspects, classified in other genera (Jirků et al., 2009). In the present study, these three Eimeria spp. were redrawn and redescribed to offer better characterization of features such as micropyle, micropyle cap, Stieda body, sub-Stieda body, sporocyst residuum and refractile bodies and sporozoite nucleus, aiming to facilitate and enable reliable identification of these species in further studies.

Table 1.

Comparative morphology of the oöcysts of Eimeria spp. recorded from ducks (Anseriformes: Anatidae: Anatinae)

Species Host Shape Size (μm)a Shape index Polar granule Wall (μm)a Micropyle (μm)a Reference
Eimeria abramovi Svanbaev & Rakhmatullina, 1967 Anas platyrhynchos (L.) Ovoidal to ellipsoidal 21–22 × 16–17 – Absent Smooth, (1.4) thick Present, (2.4) wide, with micropyle cap Svanbaev & Rakhmatullina, 1967
Eimeria anatis Scholtyseck, 1955 A. platyrhynchos Ovoidal 14–19 × 11–16 (16.8 × 14.1) – – Smooth, 0.7–1.0 thick Present, closed by a plug-like mass Scholtyseck (1955)
Anas superciliosa Gmelin Elongate-ovoidal 17–19 × 11–13 (17.6 × 11.9) 1.4–1.6 (1.5) Present, 1–2 Smooth to slightly rough, 0.9–1.3 (1.0) thick Present, with an invagination of the inner layer, without micropyle cap Present study
Eimeria aythyae Farr, 1965 Aythya affinis (Eyton) Broadly ellipsoidal to a round-bottomed urn with shoulder 15–24 × 10–18 (20.1 × 15.5) – Absent Smooth or lightly sculptured, 0.6–0.8 thick Present, (3.6) wide Farr (1965)
A. superciliosa Ellipsoidal 20–23 × 14–16 (21.3 × 15.2) 1.3–1.5 (1.4) Absent Smooth, 1.0–1.4 (1.2) thick Present, without invagination of the inner layer; micropyle cap as a translucent and delicate curved protrusion Present study
Eimeria battakhi Dubey & Pande, 1963 A. platyrhynchos Subspherical to ovoidal 19–24 × 16–21 (21.0 × 18.0) 1.1–1.2 Present, 1 Smooth, 1.0–2.0 thick Absent Dubey & Pande (1963)
Eimeria boschadis Waldén, 1961 A. platyrhynchos Bottle-shaped 18–27 × 12–13 (23.9 × 12.7) – – Finely granulated Present, 2–3 wide Waldén (1961)
Eimeria bucephalae Christiansen & Madsen, 1948 Bucephala clangula (L.) Elongate-ovoidal 25–39 × 13–20 (30.3 × 15.6) – – – Present, narrow Christiansen & Madsen (1948)
Eimeria danailovi Grafner, Graubmann & Betke, 1965 Anas platyrhynchos Ovoidal 19–23 × 11–15 (18.7 × 12.5) – – 0.6–1.0 thick Present Gräfner et al. (1965)
Eimeria koganae Svanbaev & Rakhmatullina, 1967 Spatula querquedula (L.) Ovoidal or ellipsoidal 21–25 × 13–21 (21.5 × 16.1) – Absent 0.8–1.0 thick Present, (5.0) wide Svanbaev & Rakhmatullina, 1967
Eimeria krylovi Svanbaev & Rakhmatullina, 1967 Anas carolinensis Gmelin; Spatula clypeata (L.); S. querquedula; Mareca penelope (L.); Mareca strepera (L.) Subspherical 15–21 × 13–17 – Present, 1 Smooth, 1.2 thick Present at the flattened end, 4.0–6.0 wide, covered by a (4.0) wide and (2.0) high micropyle cap Svanbaev & Rakhmatullina, 1967
Anas superciliosa Gmelin Ellipsoidal 20–23 × 16–17 (21.7 × 16.1) 1.3–1.4 (1.3) Absent Smooth, 1.0–1.4 (1.2) thick Present, without invagination of the inner layer, with micropyle cap as a dense cover Present study
Eimeria nyroca Svanbaev & Rakhmatullina, 1967 Aythya nyroca (Güldenstädt) Ovoidal 21–40 × 17–19 (25.4 × 17.7) – Absent Smooth, 1.0–2.0 thick Present, surrounded by collars, 4.0–6.0 wide, with 2.0–3.0 high micropyle cap Svanbaev & Rakhmatullina, 1967
Eimeria saitamae Inoue, 1967 A. platyrhynchos Ovoidal 17–21 × 13–15 (18.6 × 13.2) – Present, 1 Smooth, 0.7–0.8 thick Present Inoue (1967)
Eimeria schachdagica Musaev, Surkova, Jelchiev & Alieva, 1966 A. platyrhynchos Ovoidal 16–26 × 12–20 (24.0 × 17.7) – Present, refractile granules Smooth, 1.6–2.0 thick Absent Musaev et al. (1966)
Eimeria somateriae Christiansen, 1952 Clangula hyemalis (L.) Bottle-shaped, asymmetric 21–41 × 11–19 (31.9 × 13.9) – – Smooth, thin, colorless Present Christiansen (1952)
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Note: E. boschadis and E. somateriae are kidney parasites, while the remaining species are intestinal parasites.

a

Range (Mean).

Table 2.

Comparative morphology of the sporocysts of Eimeria spp. recorded from ducks (Anseriformes: Anatidae: Anatinae)

Species Host Shape Size (μm)a Shape index Stieda body Sub-Stieda body Sporocyst residuum Reference
Eimeria abramovi Svanbaev & Rakhmatullina, 1967 Anas platyrhynchos (L.) – 7–9 × 5 – – – Present, small granules Svanbaev & Rakhmatullina, 1967
Eimeria anatis Scholtyseck, 1955 A. platyrhynchos Ovoidal – – – – Present, few central granules Scholtyseck (1955)
Anas superciliosa Gmelin Ellipsoidal 7–9 × 5–6 (7.9 × 5.9) 1.3–1.4 (1.3) Present, flattened Absent or indiscernible Present, small granules randomly dispersed Present study
Eimeria aythyae Farr, 1965 Aythya affinis (Eyton) – – – Present, small – Present, compact residual mass Farr (1965)
A. superciliosa Ellipsoidal 9–11 × 7–8 (10.5 × 7.7) 1.2–1.4 (1.4) Present, flattened Present, delicate or indiscernible Present, large granules randomly dispersed Present study
Eimeria battakhi Dubey & Pande, 1963 A. platyrhynchos Ovoidal 11–13 × 6–8 – Present, small – Present Dubey & Pande (1963)
Eimeria boschadis Waldén, 1961 A. platyrhynchos – – – – – – Waldén (1961)
Eimeria bucephalae Christiansen & Madsen, 1948 Bucephala clangula (L.) – – – – – – Christiansen & Madsen (1948)
Eimeria danailovi Grafner, Graubmann & Betke, 1965 Anas platyrhynchos (L.) Ovoidal – – – – Present Gräfner et al. (1965)
Eimeria koganae Svanbaev & Rakhmatullina, 1967 Spatula querquedula (L.) Subspherical to ovoidal 9–11 × 8–10 – – – Present, clear globules irregularly spaced Svanbaev & Rakhmatullina, 1967
Eimeria krylovi Svanbaev & Rakhmatullina, 1967 Anas carolinensis Gmelin; Spatula clypeata (L.); S. querquedula; Mareca penelope (L.); Mareca strepera (L.) Subspherical or ovoidal 8 × 6–8 – – – Absent Svanbaev & Rakhmatullina, 1967
Anas superciliosa Gmelin Subspheroidal to ellipsoidal 8–10 × 7–8 (8.8 × 7.7) 1.1–1.2 (1.1) Barely or not discernible Absent Present, large, dense granules diffused Present study
Eimeria nyroca Svanbaev & Rakhmatullina, 1967 Aythya nyroca (Güldenstädt) Ovoidal 11–13 × 8–11 – – – Present, granular Svanbaev & Rakhmatullina, 1967
Eimeria saitamae Inoue, 1967 A. platyrhynchos – – – – – – Inoue (1967)
Eimeria schachdagica Musaev, Surkova, Jelchiev & Alieva, 1966 A. platyrhynchos Ovoidal (9.2 × 8.4) – – – Present, small, granular Musaev et al. (1966)
Eimeria somateriae Christiansen, 1952 Clangula hyemalis (L.) – (11 × 6) – – – Absent Christiansen (1952)
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Note: E. boschadis and E. somateriae are kidney parasites, while the remaining species are intestinal parasites.

a

Range (Mean).

Morphologically, E. anatis is easily confused with W. philiplevinei due to the difficulty of distinguishing the sporocyst residuum and the number of sporozoites in their sporocysts. In this context, Duszynski et al. (2000) considered that the descriptions, photomicrographs and line drawings of Wenyonella spp. were inadequate; additionally, many species have been described and named from degenerate oöcysts. Thus, Duszynski et al. (2000) suggested that all species identified as Wenyonella should be viewed dubiously and considered species inquiriendae. Specifically for W. philiplevinei, Duszynski et al. (2000) considered that both the line drawing and photomicrograph suggest that the refractile bodies and/or sporozoites were all confused in the original description of Leibovitz (1968) and other studies reviewed by Gajadhar et al. (1983). In the oöcysts identified as E. anatis in the present study, two sporozoites were clearly observed with their anterior and posterior refractile bodies in each sporocyst, justifying that the material belongs to the genus Eimeria. Furthermore, the results of the phylogenetic analysis including the newly generated 18S sequence for E. anatis showed its inclusion into a clade of Eimeria spp. from ducks, while being distant from the only partial 18S sequence from oöcysts identified as W. philiplevinei by Wu et al. (2013).

Tyzzeria perniciosa is the type-species of the genus Tyzzeria, which consists of coccidia with oöcysts containing eight sporozoites without sporocysts. All consensually valid species are recorded from birds of the order Anseriformes. Descriptions in hosts of other vertebrate classes were published, although they must be misidentifications, such as Tyzzeria boae Lainson & Paperna, 1994 described from the red-tailed boa Boa constrictor L. and Tyzzeria chalcides Probert, Roberts & Wilson, 1988 described from the ocellated skink Chalcides ocellatus (Forskål), which potentially represent a species of Klossiella and a species of Choleoeimeria, respectively misidentified from oöcysts that sporulated abnormally (Duszynski et al., 1998).

The most frequently reported species in the literature are T. perniciosa from teals, mallards and other ducks (Anatinae) and Tyzzeria parvula (Kotlan, 1933) from geese (Anserinae) (Berto et al., 2007). Although the oöcysts of these species are morphologically very similar, they are specialised for parasitism at the subfamily level, i.e. T. parvula does not infect teals, mallards and ducks, just as T. perniciosa does not infect geese, even in experimental infections (Berto et al., 2007).

In addition to T. perniciosa, Tyzzeria pellerdyi Bhatia & Pande, 1966 was described and reported from Anas spp. in some studies in the 1960s, 1970s and 1980s. However, there is no morphological or biological differentiation that so far justifies and fundamentally defines T. pellerdyi. As this species was described after T. perniciosa, it is likely that T. pellerdyi is a junior synonym of T. perniciosa (Gajadhar et al., 1983; Duszynski et al., 1998) (Table 3). Similarly, two species, i.e. Tyzzeria allenae Chakravarty & Basu, 1946 and Tyzzeria chenicusae Ray & Sarkar, 1967, were described from the cotton pygmy-goose Nettapus coromandelianus (Gmelin); this host, in spite of the common name, belongs to the Anatinae. The oöcysts of these species were described with some morphometric differences that differentiate them from the original description of T. perniciosa and from the description provided here; however, these species have not been reported since their original descriptions (Table 3).

Table 3.

Comparative morphological data for Tyzzeria spp. recorded from ducks (Anseriformes: Anatidae: Anatinae).

Species Host Oöcyst
 Sporozoite
 Reference
Shape Size (μm)a Shape index Residuum (μm)a Wall (μm)a Shape Size (μm)a Refractile body Nucleus
Tyzzeria perniciosa Allen, 1936 Anas platyrhynchos L. Ellipsoidal 10–13 × 9–11 – Present, large, composed of variously sized granules Relatively thick Curved, with one end more rounded and broader (10.0 × 3.5) – – Allen (1936)
Anas superciliosa Gmelin Ellipsoidal 10–11 × 7–8 (10.7 × 7.4) 1.4–1.5 (1.4) Present, granules of different sizes usually clustered at one end of the oöcyst, c.2.5 Smooth, 0.4–0.7 (0.6) Curved and tapered at anterior end 6–8 × 1–2 (6.9 × 1.8) Robust, prominent Not discernible Present study
Tyzzeria alleni Chakravarty & Basu, 1946 Nettapus coromandelianus Gmelin Ovoidal 14–17 × 10–12 – Present, coarsely granular, c.6.4 – Tapered at one end 5.3–6.5 – Present, central Chakravarty & Basu (1946)
Tyzzeria pellerdyi Bhatia & Pande, 1966 Mareca strepera (L.); Aythya nyroca (Güldenstädt); Spatula clypeata (L.); Anas carolinensis Gmelin; A. platyrhynchos Subspherical to ovoidal 11–16 × 8–11 (13.0 × 10.0) – Present, c.4.0–5.0 Smooth, 0.5–0.7 Banana-shaped (8.5 × 2.0) Prominent Present, central Bhatia & Pande (1966); Bristol et al. (1981)
Tyzzeria chenicusae Ray & Sarkar, 1967 N. coromandelianus Broad and cylindrical 20–28 × 14–20 (24.8 × 16.8) (1.5) Large, compact, at one pole of the oöcyst (1.4) Club-shaped (13.2 × 4.2) Present at the broader end – Ray & Sarkar (1967)
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Note: All species are intestinal parasites.

a

Range (Mean).

Our phylogenetic analyses provided strong evidence that the newly generated sequences from E. anatis in the Pacific black duck both at the 18S rRNA and cox1 loci were most close to those from domestic goose (GenBank: KP789171, KJ000077 and MT833388). As this is, to the best of our knowledge, the first study using molecular tools to the identification of duck coccidia, further similar studies on additional species of coccidia parasitic in ducks would be beneficial to the taxonomy of duck coccidia and assessment of their relationships with coccidian species parasitic in other host groups.

This study has revealed that, besides infecting the mallard A. platyrhynchos, E. anatis also infects the Pacific black duck. Hybridisation (interbreeding) between the introduced mallard and the Pacific black duck in Australia occurs at a rate of around 1.5% (Taysom, 2016), so it is likely that these two species of duck share some of their coccidian species as well.

5. Conclusion

In conclusion, the coccidia E. anatis, E. aythyae, E. krylovi and T. perniciosa are redescribed with supplementary morphological data, in order to ensure and facilitate their future identification from A. superciliosa or from other duck species. In addition, a genotypic characterization of E. anatis and taxonomic remarks on species and genera of dubious validity reported from Anseriformes are provided, aiming to contribute to the knowledge of coccidian species of ducks.

CRediT author statement

Bruno P. Berto: morphological identification of the species, preparation of line drawings, writing - review & editing. Belinda Brice: coccidian primary screening and identification, writing - original draft and paper reviewing. Gwyneth Thomas: sample collection and coccidian primary screening, writing - review & editing. Aileen Elloit: oöcyst imaging, morphological identification of the species, writing - review & editing. Alireza Zahedi: oöcyst isolation, DNA extraction, PCR, sequencing, writing - review & editing. Rongchang Yang: overseeing and coordinating this study, phylogenetic analysis, writing - review & editing. All authors read and approved the final manuscript.

Data availability

The newly generated sequences for E. anatis are deposited in the GenBank database under the accession numbers OL604501 (18S rDNA) and OL656104 (cox1). Photomicrographs and line drawings of the oöcysts are deposited and available (http://r1.ufrrj.br/labicoc/colecao.html) in the Parasitology Collection of the Laboratório de Biologia de Coccídios, at UFRRJ, under repository numbers 120/2021 (E. anatis), 121/2021 (E. aythyae), 122/2021 (E. krylovi) and 123/2021 (T. perniciosa), along with the photovouchers of the A. superciliosa specimen.

Ethical approval

Not applicable.

Funding

Official funding for this study was not available.

Declaration of competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors would like to thank Helen Riley (KWRC Chair) and all the KWRC volunteers, as well as Daniel Oakes for help with initial microscopy screening. We are also grateful to the staff at the Wattle Grove Veterinary Hospital, Perth, for their care and treatment of wildlife treated at their hospital. Bruno P. Berto thanks fellowships from CNPq (Grant/Award Number: 303899/2019-0) and FAPERJ (Grant/Award Number: E-26/202.797/2019).

Contributor Information

Bruno P. Berto, Email: bertobp@ufrrj.br.

Rongchang Yang, Email: R.Yang@murdoch.edu.au.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The newly generated sequences for E. anatis are deposited in the GenBank database under the accession numbers OL604501 (18S rDNA) and OL656104 (cox1). Photomicrographs and line drawings of the oöcysts are deposited and available (http://r1.ufrrj.br/labicoc/colecao.html) in the Parasitology Collection of the Laboratório de Biologia de Coccídios, at UFRRJ, under repository numbers 120/2021 (E. anatis), 121/2021 (E. aythyae), 122/2021 (E. krylovi) and 123/2021 (T. perniciosa), along with the photovouchers of the A. superciliosa specimen.

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