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

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.


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).
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.

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 o€ ocysts of various sizes as well as trophozoites of Trichomonas Donn e, 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) K 2 Cr 2 O 7 , 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 o€ ocyst sporulation using an Olympus DP71 digital microimaging camera. Sporulated o€ ocysts were observed using the 100Â oil immersion objective. Images were taken using Nomarski contrast with a 100Â 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.

O€ ocyst isolation, DNA extraction, PCR amplification, sequencing and phylogenetic analyses
Five morphologically similar o€ ocysts 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. (2013Yang et al. ( , 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_type¼clustalw). 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 þ G þ I for 18S and TN93 þ G for the cox1 gene). Bootstrap support was estimated from 1000 pseudoreplicates.

Molecular identification
PCR amplification for the 18S rRNA and cox1 genes from o€ ocyst DNA of the four coccidian species were conducted; unfortunately, PCR amplicons were successfully obtained only for E. anatis o€ ocysts.
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%.

Phylogenetic analyses of the cox1 gene
The cox1 gene was amplified from E. anatis o€ ocyst 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) (My skov a et al., 2021) and grouped with this Eimeria sp. in the same clade in the phylogenetic tree (Fig. 6).

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 o€ ocysts of the three Eimeria spp. identified in this study were morphologically compatible with their respective original descriptions (Tables 1 and 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 Labb e, 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.
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 o€ ocysts. 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 o€ ocysts 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 o€ ocysts identified as W. philiplevinei by Wu et al. (2013).
Tyzzeria perniciosa is the type-species of the genus Tyzzeria, which consists of coccidia with o€ ocysts 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 o€ ocysts 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 o€ ocysts 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   Anas platyrhynchos (L.) -7-9 Â 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 Â 5-6 (7.9 Â 5.9) 1.  Musaev et al. (1966) Eimeria somateriae Christiansen, 1952 Clangula hyemalis (L.) -(11 Â 6) ---Absent Christiansen (1952) Note: E. boschadis and E. somateriae are kidney parasites, while the remaining species are intestinal parasites. a Range (Mean).  coromandelianus (Gmelin); this host, in spite of the common name, belongs to the Anatinae. The o€ ocysts 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). 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.

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: o€ ocyst imaging, morphological identification of the species, writing -review & editing. Alireza Zahedi: o€ ocyst 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.

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.