Donald W. Duszynski

A GUIDELINE FOR THE PREPARATION OF SPECIES DESCRIPTIONS IN THE EIMERIIDAE

Members of the suborder Eimeriina (phylum Apicomplexa: class Sporozoea: order Eucoccidiorida) have complex 1 or 2 host life cycles that involve endogenous development in the tissues of vertebrates or invertebrates and exogenous development in an oocyst, usually outside the host(s). Because tissue stages are logistically difficult or even impossible to obtain in natural (wild) host-parasite systems, the vast majority (> 98%) of species in this parasite complex are known only from the structure of their sporulated oocyst. Unfortunately, the quality of these species descriptions is uneven because no guidelines are available for workers in the field to follow. Here we propose a specific set of guidelines for the preparation of species descriptions of coccidia based predominently on the structure of the sporulated oocyst, because the oocyst is the most readily available stage in the life cycle. In addition, we emphasize that ancillary data be incorporated whenever possible with the species description; these data may include, but are not limited to, ecological parameters, prevalence, seasonal data, and the deposition of both host symbiotypes and parasite hepantotypes (= phototypes) into accredited musecums so that accurate identification of both host and parasite material can be assured in perpetuity. And finally, if oocysts are collected in pure suspension, that is, if only one coccidian species (morphotype) is present in the sample, then some oocysts should be saved in 70% ethanol and archived in an accredited museum in the event that future workers might wish to amplify and, later, sequence the parasites DNA.

A revision of the taxonomy and nomenclature of the Eimeria spp. (Apicomplexa: Eimeriidae) from rodents in the Tribe Marmotini (Sciuridae)

All published papers on Eimeria spp. from rodents in the Marmotini Tribe are reviewed and each described species is evaluated in an historical context (i.e., beginning with the oldest description). Many of these species descriptions from marmotine rodents are invalid when considered within either the spirit or the letter of the International Code of Zoological Nomenclature. In addition, several previous authors erroneously assumed that Eimeria spp. of this host group were highly host-species specific. Thus, many eimerian species described to infect members of this tribe that were originally found in different hosts are morphologically indistinguishable, and several of these are considered to be conspecific. The names of those species that were most similar are synonymised and, using the guidelines of the International Code, the species for which the original descriptions were inadequate are considered to be species inquirendae (Ride et al., 1985, p. 264). The number of named eimerian species in the Marmotini is reduced from 40 to 26 valid species; these are E. airculensis, E. beckeri, E. beecheyi, E. berkinbaevi, E. callospermophili, E. citelli, E. cynomysis, E. deserticola, E. eutamiae, E. franklinii, E. hoffmeisteri, E. lateralis, E. ludoviciani, E. menzbieri, E. monacis, E. morainensis, E. os, E. ovata, E. pseudospermophili, E. spermophili, E. surki, E. tamiasciuri, E. vilasi, E. volgensis and E. yukonensis, and one new name is included. Nine junior synonyms are identified and five species inquirendae and one nomen nudum are discussed. Where needed, the identification errors in the published literature are clarified.

EXPERIMENTAL TRANSMISSION OF SARCOCYSTIS FROM ICTERID BIRDS TO SPARROWS AND CANARIES BY SPOROCYSTS FROM THE OPOSSUM

Cowbirds (Molothrus ater) and grackles (Cassidix mexicanus) infected with muscle cysts of Sarcocystis were fed to opposums (Didelphis virginiana) and fecal sporocysts from the latter were given to sparrows (Passer domesticus, Family Ploceidae), canaries (Serinus canarius, Family Fringillidae) and ducks (Anas platyrhynchos, Family Anatidae). Asexual parasites were found in the endothelium of sparrows and canaries but not in ducks. When birds were kept 10 weeks or more after infection, muscle cysts were found grossly and microscopically in the majority of sparrows, and in 1 canary, but not in ducks. Muscle zoites were found in digests of all sparrows and canaries but not in that of ducks. Metrocytes and forms dividing by endodyogeny also were found in the digest. Thus, avian Sarcocystis was transmitted experimentally from 2 genera of 1 family (Icteridae) to 2 different families of passerine intermediate hosts by sporocysts from the definitive host. This is the broadest intermediate host spectrum known for a species of Sarcocystis.

Phylogenetic relationships among rodent Eimeria species determined by plastid ORF470 and nuclear 18S rDNA sequences.

Phylogenetic analyses for 10 rodent Eimeria species from different host genera based on plastid ORF470 and nuclear 18S rDNA sequences were done to infer the evolutionary relationships of these rodent Eimeria species and their correlation to morphology and host specificity. The phylogenies based on both data sets clearly grouped the 10 rodent Eimeria species into two major lineages, which reflect more their morphological differences than host specificity. Species in lineage A have spheroidal to subspheroidal sporulated oocysts, are similar in size (18-29 x 17-23; xbar = 22 x 20 microm), have an oocyst residuum and one-two polar granules; these include Eimeria albigulae (Neotoma), Eimeria arizonensis (Peromyscus, Reithrodontomys), Eimeria onychomysis (Onychomys) and Eimeria reedi (Perognathus). Species in lineage B, including Eimeria falciformis (Mus), Eimeria langebarteli (Reithrodontomys), Eimeria nieschulzi (Rattus), Eimeria papillata (Mus), Eimeria separata (Rattus) and Eimeria sevilletensis (Onychomys) have different shapes (ovoid, ellipsoid, elongated ellipsoid, etc.), differ greatly in size (10-27 x 9-24; xbar = 19 x 16 microm) and all lack an oocyst residuum. Thus, The oocyst residuum was the most determinant feature that differentiated the two lineages. The accession numbers of ORF470 of E. albigulae, E. arizonensis, E. falciformis, E. nieschulzi, E. onychomysis, E. papillata, E. reedi, E. separata, E. sevilletensis, E. langebarteli are AF311630-AF311639 and 18S rDNA of E. langebarteli, E. papillata, E. reedi, E. separata, E. sevilletensis are AF311640-AF311644.

Taxonomy and systematics of some Eimeria species of murid rodents as determined by the ITS1 region of the ribosomal gene complex.

Eimeria arizonensis, E. albigulae and E. onychomysis, morphologically similar species from closely related murid rodents, were distinguished using nuclear rDNA ITS1 sequences obtained from multiple isolates of each taxon. ITS1 sequences were also obtained from 6 other species parasitizing murid rodents: E. falciformis, E. langebarteli, E. nieschulzi, E. papillata, E. separata and E. sevilletensis, and from E. reedi, a parasite of heteromyid rodents. Under parsimony and maximum likelihood analyses, the isolates of E. arizonensis, E. albigulae and E. onychomysis were differentiated as closely related, monophyletic lineages. Maximum likelihood pairwise distances between the latter species ranged from 7 to 12%, and distances within each species ranged from < 1 to 5%; thus it is suggested that ITS1 genetic distances may be used to facilitate taxonomic differentiation of Eimeria spp. Against expectation, phylogenetic procedures placed E. reedi within the phylogeny of the Eimeria of murid rodents. ITS1 sequencing appears to provide data that can be used for taxonomic and phylogenetic studies on the speciose genus Eimeria, and may be especially useful when samples contain insufficient numbers of oocysts for other molecular-based methods, e.g. RAPD-PCR.

EIMERIA FROM JUMPING MICE (ZAPUS SPP.): A NEW SPECIES AND GENETIC AND GEOGRAPHIC FEATURES OF Z. HUDSONIUS LUTEUS

Of 103 jumping mice (Zapus spp.) examined, 29 (28.2%) had coccidian oocysts in their feces: one of seven (14%) Z. trinotatus eureka from Humboldt Co., California; 25 of 60 (42%) Z. princepsprinceps, including seven of 18 (39%) from Boulder Co., Colorado, and 18 of 42 (43%) from Santa Fe and Taos cos., New Mexico; and three of 36 (8%) Z. h. luteus, including one of one from Sandoval Co., New Mexico, two of 13 (15%) from Apache Co., Arizona, and none of 22 from Otero and Soccoro cos., New Mexico. Twenty-eight of 29 infected mice had only Eimeria zapi oocysts in their feces; the only Z. h. luteus from Fenton Lake, Sandoval Co., New Mexico, had oocysts of a species which we describe here as new. Sporulated oocysts of Eimeria hudsonii sp. n. from Z. h. luteus are elliptical, 20.9 X 14.4 (18-23 X 13-16) ,um with ovoid sporocysts 10.5 X 5.6 (8-11 X 57) gm. A micropyle cap, polar and substieda bodies were absent, but a micropyle, oocyst and sporocyst residua, and Stieda body were present. Sporozoites have one large posterior refractile body. The oocyst wall has two layers. This is only the second eimerian reported from Zapus spp. The geographic distribution of E. zapi closely paralleled genetic and geographic features recently reported within the host taxon Z. h. luteus. Since May 1979, we have collected and examined over 3,000 wild mammals from the United States, northern Mexico, Baja California, and Japan. These collections are part of a continuing study designed to understand the evolutionary relationships of certain groups of congeners and conspecifics using pelage, skeletal, morphologic, electrophoretic, karyotypic, and parasite data. Ultimately we hope to be able to relate host genetics and distribution to susceptibility, parasite burdens, and coccidian specificity. This report is the first on the coccidians of one group of these mammals. We examined four species of Zapus from four states for Coccidia and found two eimerians, Eimeria zapi Gerard, Chobotar, and Ernst, 1977, and a form which we describe here as new. MATERIALS AND METHODS Hosts were killed within a few hours after being live trapped. The abdominal cavity was opened by a longitudinal ventral incision and the intestinal tract, from the duodenum to the anus, was removed. The small intestine was slit lengthwise and placed into a vial of AFA (Humason, 1979) to preserve helminths. The cecum and colon were slit lengthwise and preserved, with their contents, in vials containing 2.5% aqueous (w/v) K2Cr207. Upon return to the laboratory all vials were refrigerated (4 C) until they could be processed and examined. Processing for oocysts consisted of separating fecal contents from cecal-colon tissue by scraping with a scalpel. The fecal-K2Cr207 mixture from each mouse was placed at 23 C for 7 days in a 15-cm Petri dish. Received 28 September 1981; revised 6 January 82, 24 May 1982; accepted 6 June 1982. It was then filtered through 40and 60-mesh brass screens and aliquots of the filtrate were examined by coverslip flotation with a concentrated sucrose solution (sp. gr. 1.15). Oocysts were measured with an ocular micrometer and photographed with either Panatomic-X or Ilford Pan F 35-mm film within a Zeiss Universal Photomicroscope equipped with both brightfield (Neofluar) and Nomarski-interference XI 00 objectives. All measurements are in ,um with the ranges in parentheses following the means.

A simple method of DNA extraction for Eimeria species

A new, simple method is described for extracting DNA from coccidia (Eimeriidae) oocysts. In our hands this method works well for all Eimeria oocysts and, presumably, will work equally well for oocysts of other coccidia genera. This method combines the two steps of breaking oocyst and sporocyst walls, and dissolving the sporozoite membrane in one step. This greatly simplifies the currently used DNA extraction procedures for Eimeria species and overcomes the disadvantages of existing DNA extraction methods based on glass-bead grinding and sporozoite excystation procedures. Because all the procedures are done in a 1.5-ml microfuge tube, which minimizes the loss of DNA in the extraction procedures, this method is especially suitable for samples with small number of oocysts. In addition, this method directly lyses the oocyst and sporocyst walls as well as the sporozoite membrane in a continuous incubation; therefore, it does not require the sporozoites to be alive. The results of PCR experiments indicate that this method generates better quality of DNA than what the existing glass-bead grinding method does for molecular analysis, and is suitable for both large or small number (<10(2) oocysts) of living or dead oocyst samples.

Coccidia (Apicomplexa: Eimeriidae) in the Primates and the Scandentia

The coccidia (protistan phylum Apicomplexa Levine, 1970) comprise a large group of obligate intracellular parasites commonly found in all classes of vertebrate hosts and in some invertebrates. This review focuses on the largest family in this group, Eimeriidae Minchin, 1903, because its members are among the most prevalent and specious of all parasite groups. Nevertheless, there is a paucity of information regarding the eimeriid coccidia of Primates and their close relatives, and no taxonomic summation is currently available. We review all published descriptions of species of Cyclospora, Eimeria and Isospora that infect Scandentia and Primates. Some of the names of eimeriid species from hosts in these orders are invalid, either because rules concerning the naming of new species (International Code of Zoological Nomenclature) were not followed and/or because the original description was so incomplete and of such little use that the name must be relegated to a species inquirendae. In the Scandentia, only Tupia and Ptilocercus have valid coccidian species described from them:Eimeria ferruginea, E. modesta, and E. tupaiae in Tupaia and E. ptilocerci in Ptilocercus. Of the 60 genera of Primates only 14 (Alouatta, Aotus, Ateles, Cacajao, Callimico, Callithrix, Cebus, Galago, Homo, Macaca, Nycticebus, Saguinus, Saimiri, and Tarsius) have 6 Eimeria (E. coucangi, E. galago, E. lemuris, E. nycticebi, E. otolicini, E. pachylepyron), 7Isospora (I. arctopitheci, I. belli, I. callimico, I. cebi, I. endocallimici, I. saimiriae, I. natalensis) and 1 Cyclospora (C. cayetanensis) described from them. In addition, one new name, an Eimeria sp., is included. We identify eight junior synomyms and note 4 species inquirendae. Primatologists should be more receptive to multidisciplinary approaches by working with parasitologists to use comparative parasite data that might provide insights into primate sociality and habitat use. The coccidia are ideal specimens for such cooperative efforts because they can be collected easily by noninvasive fecal collections. We describe methods to collect and preserve oocysts, noting that formalin and PVA, preservatives used to collect helminth stages (worms, worm eggs), will destroy the structural integrity of coccidian oocysts and make their identification impossible.

Ultrastructure of Sarcocystis sporocysts from passerine birds and opossums: comments on classification of the genus Isospora.

Sporocysts were obtained from the feces of opossums (Didelphis virginiana) which had been fed muscles of passerine birds (Molothrus ater and Cassidix mexicanus) infected with Sarcocystis. Sporocysts were examined by phas microscopy and scanning and transmission electron microscopy. Ridges on the surface of the sporocysts outlined four plates. Thin sections of the sporocyst wall showed thickened regions and gaps interpreted as cross sections of the ridges. The sporocyst wall has four major layers with a thick, granular inner layer which resembles the inner layer of sporocysts of related species. Excystation structures are discussed as a basis for classifying disporocystic coccidia to correlate with Frenkels (1977) life cycle classification of this group.

A new and unusual eimerian (protozoa: Eimeriidae) from the liver of the Gulf killifish, Fundulus grandis.

Oocysts and sporocysts of Eimeria funduli sp. n. are described from the Gulf killifish, Fundulus grandis, on the basis of light microscopy, transmission and scanning electron microscopy, and location in the liver of infected hosts. The spherical sporu!ated oocysts of E. funduli isolated from liver tissue measure 20–31(25) ıtm across with ovoid sporocysts 9–11 × 5–7 (10 × 6) ıtm. A micropyle, polar granule, and oocyst residuum are absent, but sporocysts have Stieda and substieda bodies, a few residual granules, and 10–25 (15) unique projecting structures with expanded distal portions that we term “sporopodia”. Sporopodia 1–3 (2) ı high support a transparent membrane that completely surrounds the sporocyst. Sporozoites have one large posterior refracti!e body. U!trastructural!y, the oocyst wall consists of two thin layers of granular material: an electron-dense outer layer with a rough external surface and an electron-lucent inner one of approximately equal thickness. One or two unit membranes line the inner surface of the inner layer. Each layer is 40–60(55) nm thick. The sporocyst wall, 78–130 (110) nm thick, consists of an e!ectron-lucent material with the outer surface being more electron dense and giving rise to osmiophilic sporopodia; closely associated with these and the outer surface are one or two unit membranes. A thin osmiophi!ic layer of fine granular material lines the inner surface.