Morphologic, immunohistochemical, and molecular characterization of a novel Lankesterella protozoan in two White's tree frogs (Litoria caerulea).

Two White's tree frogs (Litoria caerulea) housed at a zoological park died after a short period of lethargy, weight loss, and edema. Detailed postmortem examinations were performed on both frogs, including bacterial cultures and complete histologic examinations. Intracytoplasmatic as well as free protozoan parasites were identified in multiple organs from both frogs. The parasites were identified within erythrocytes, leukocytes, endothelial cells, and hepatocytes. Immunohistochemistry demonstrated a cross-reaction with Toxoplasma gondii antisera. Parasite ultrastructural analysis was performed by transmission electron microscopy. The parasites demonstrated an apical complex containing a conoid, rhoptries, and micronemes, demonstrating it was a member of the phylum Apicomplexa. In addition, the parasites had bipolar paranuclear bodies, organelles that are typical of coccidian sporozoites. The organisms were tentatively identified as members of the genus Lankesterella on the basis of histologic and ultrastructural morphology. A portion of the 18s ribosomal RNA (rRNA) gene was amplified via a polymerase chain reaction, sequenced, and used in a Basic Local Alignment Search Tool search of the GenBank database. The 18s rRNA gene sequence was found to be most similar to gene sequences isolated from Lankesterella organisms (88%). In aggregate, these data support the classification of these protozoa as a novel species of Lankesterella. A causal relationship between frog morbidity and protozoal parasitism was not determined. This is the first report of Lankesterella sp. in White's tree frogs.

Detection of some intestinal protozoa in commercial fresh juices.

Fresh fruit juices are popular, but not always safe. For assessing the likelihood of infection with newly emerging intestinal protozoa, commercial fresh orange, lemon, sugar cane, strawberry, and mango juices were screened by wet mounts, Weber's modified trichrome and modified Ziehl-Neelsen stains. Protozoa viability was done by fluorescein-diacetate/propidium-iodide staining, and infectivity was performed in Swiss albino mice. Results showed that 35.43% were contaminated with one or more of Cryptosporidia, Microsporidia, and Cyclospora, as well as Giardia spp. Strawberry was the most contaminated juice (54.28%), while orange was the slightest (22.86%). Cryptosporidia was the highest contaminant (61.29%), and Cyclospora was the least (14.52%). Microsporidia spp. was the most robust contaminant which retained its viability and infectivity in juices in which it was detected. Moderately acidic strawberry and mango juices and alkaline sugar cane juice pose a possible threat, due to harboring the highest viable and infectious protozoa. Regarding highly acidic juices, viability and infectivity decreased in lemon, yet was not still risk free. Orange juice was comparatively safe, as viability dramatically declined, while infectivity was completely abolished. Hence consumers, especially high risk group, are placed at hazard of contracting intestinal protozoa infections, especially through moderately acidic and alkaline juices.

Gastrointestinal parasites of Howler monkeys (Alouatta palliata) inhabiting the fragmented landscape of the Santa Marta mountain range, Veracruz, Mexico.

In recent years populations of howler monkeys (Alouatta palliata) in southeastern Mexico have decreased substantially due to the transformation and loss of natural habitats. This is especially evident in the Santa Marta mountain range, Veracruz, Mexico where several studies have evaluated the impact of fragmentation on howler monkey populations in order to propose management programs for their conservation. The conditions generated by fragmentation likely change the rates of parasitic infection and could decrease howler survival. In this study, gastrointestinal parasite species richness, prevalence, and egg density of infection were determined in howler groups inhabiting five forest fragments at the Santa Marta mountain range. Two hundred and seventy-eight fresh fecal samples were collected between October 2002 and April 2003. Three parasite species were found during the dry and the wet season in all forest fragments sampled: one unidentified species of Eimeriidae; Trypanoxyuris minutus (Oxyuridae); and Controrchis biliophilus (Dicrocoeliidae). Both the prevalence of T. minutus and infection density for all parasites differed between seasons and fragments (the largest fragment consistently differed from other fragments). Host density, distance to the nearest town, fragment size, fragment shape, and total basal area of food trees explained parasite prevalence, but each species had a different pattern. Although parasite richness was lower, prevalence and density were higher than values reported for howlers in conserved forests. These results suggest that the establishment of biological corridors and animal translocation programs must take into account the parasite ecology of each fragment to avoid higher infection rates and preclude potential consequent mortality.

[Coccidia (Eimeriida) from fishes of the continental waters of Russia].

Data on the Coccidia parasitizing fishes of 12 orders (Acipenseriformes, Clupeiformes, Salmoniformes, Anguilliformes, Siluriformes, Atheriniformes, Gadiformes, Gasterosteiformes, Syngnatiformes, Mugiliformes, Scorpaeniformes, and Pleuronectiformes) are given. Authors and year of description, synonymy, morphofunctional organization of exogenic stages, and list of hosts are presented for each coccidian species.

Studies on serological cross-reaction of Neospora caninum with Toxoplasma gondii and Hammondia heydorni.

The enzyme-linked immunosorbent assay (ELISA) was used to examine cross-reactivity of Neospora caninum with Toxoplasma gondii and Hammondia heydorni. Anti-T. gondii mouse and cat sera cross-reacted with N. caninum soluble antigen (NLA), but not with the recombinant surface antigen (NcSRS2). Anti-H. heydorni dog sera showed no cross-reactivity with either the NLA antigen or the NcSRS2. Lack of cross-reactivity between anti-H. heydorni sera and N. caninum antigens, and the cross-reactivity of anti-T. gondii sera with the NLA suggest that N. caninum has common antigens to T. gondii except for NcSRS2 based on serology. In light of several studies suggesting a closer relationship between N. caninum and H. heydorni than with T gondii, examination of serological cross-reactivity with N. caninum may be necessary to further classify the parasites in addition to molecular and morphological studies and clarification of the life cycle.

Neospora caninum and Hammondia heydorni are separate species/organisms.

Neospora caninum and Hammondia heydorni are two coccidian parasites with morphologically similar oocysts in canine feces. It was recently proposed that they are one species. In this paper, we review the biology and morphology of these parasites and present evidence that N. caninum and H. heydorni are separate species.

Further remarks on Hammondia hammondi and the taxonomic importance of obligate heteroxeny.

In this opinion-paper reasons are given why Hammondia hammondi cannot be considered as a separate species, but should be kept as a species of the genus Toxoplasma, if not a strain of Toxoplasma gondii.

The taxonomic importance of obligate heteroxeny: distinction of Hammondia hammondi from Toxoplasma gondii--another opinion.

We enumerate identical and divergent findings concerning the obligate heteroxenous Hammondia hammondi and the facultatively homoxenous or heteroxenous Toxoplasma gondii. Differences exist in life-cycles, transmission, and host range, especially transmissibility to birds and mammals other than rodents, in ultrastructural morphology, immunity and serology in cats and to lesser degree in rodents, in DNA sequences and in isoenzymes. Because the recognition of obligate heteroxeny is essential to study these organisms and to recognize them as taxa, it is advantageous to give heteroxeny a generic rather than a specific value. Characterization of organisms with the life-cycle patterns of Hammondia, Sarcocystis, Frenkelia, and Toxoplasma is best achieved by means of the genera presently used.

Neospora caninum: its oocysts and its identity: an opinion.

A recently published opinion that Hammondia heydorni, Neospora caninum, and N. hughesi are a single species is not supported by genetic comparisons. Furthermore, the suggestion that oocysts shed by dogs after experimental infection with N. caninum might have been of another organism is not consistent with the finding that these oocysts induced N. caninum infections in mice.

Life cycle of Calyptospora funduli (Apicomplexa: Calyptosporidae).

The taxonomic status of the extraintestinal piscine coccidium Calyptospora funduli is based in part on its requirement of an intermediate host (the daggerblade grass shrimp Palaemonetes pugio). In the present study, grass shrimp fed livers of Gulf killifish (Fundulus grandis) infected with sporulated oocysts of C. funduli exhibited numerous sporozoites suspended in the intestinal contents when fresh squash preparations were examined by light microscopy. Using this method, sporozoites were not seen in intestinal epithelial cells of the grass shrimp or in any other cell types. Ultrastructural examination, however, revealed sporozoites in the cytoplasm of the gut basal cells. Cross-sections of 1-13 sporozoites were seen within a single cell, and those sporozoites each appeared to be situated in individual membrane-bound vesicles, rather than in a single parasitophorous vacuole. These ultrastructural observations indicate that in the grass shrimp intermediate host, sporozoites that develop into an infective stage probably undergo that development in gut mucosal basal cells. Prior studies revealed that these sporozoites modified their structure over 4-5 days and that before that time, they were not infective to the fish host. Following ingestion of an infected shrimp by a killifish, the infective sporozoites apparently reach the liver of their killifish definitive hosts through the bloodstream. Sporozoites were seen in blood smears from the longnose killifish, Fundulus similis, 4 hr after fish were fed experimentally infected grass shrimp. Additionally, coccidian trophozoites and early meronts were seen in hepatocytes from several longnose killifish at 48, 72, and 96 hr postinfection. This study, in conjunction with previous findings, clearly confirms that a true intermediate host is required in the life cycle of C. funduli, that a developmental period of about 5 days in grass shrimp is necessary for sporozoites to become infective to killifishes, and that sporozoites do occur intracellularly in gut basal cells of the grass shrimp.

Neospora caninum: is it really different from Hammondia heydorni or is it a strain of Toxoplasma gondii? An opinion.

The published data concerning Toxoplasma gondii, Hammondia hammondi, H. heydorni and Neospora caninum on one side and between T. gondii on the other were neglected by most authors. As conclusion we are convinced that there are only two valid species: Isospora (Toxoplasma) gondii and Hammondia heydorni. The first includes as a strain H. hammondi and the latter N. caninum. In any case there is absolutely no reason (with respect to general Zoological nomenclature) to create new genera!

Phylogenetic analysis based on full-length large subunit ribosomal RNA gene sequence comparison reveals that Neospora caninum is more closely related to Hammondia heydorni than to Toxoplasma gondii.

Since its first description in the late 1980s, Neospora caninum has been recognised as a prominent tissue cyst-forming parasite due to its ability to induce congenital disease and abortion in animals, especially cattle. It is found worldwide and is a cause of significant economic losses for the livestock industry. However, its place within the family Sarcocystidae, like that of several other taxa, remains unresolved. Neospora caninum shares several morphological and life cycle characters with Hammondia heydorni, although it is most commonly thought of as being a close relative of Toxoplasma gondii. This study presents information regarding the phylogenetic relationship of N. caninum to species currently classified into the genus Hammondia, as well as to two strains (RH and ME49) of T. gondii based on the full-length large subunit ribosomal RNA gene. Phylogenetic analyses using two alignment strategies and three different tree-building methods showed that the two species in the genus Hammondia are paraphyletic. Neospora caninum was shown to form a monophyletic clade with H. heydorni instead of T. gondii, which in turn was shown to be most closely related to H. hammondi. The finding that N. caninum and H. heydorni are closely related phylogenetically may aid the elucidation of currently unknown aspects of their biology and epidemiology, and suggests that H. heydorni should be considered in the differential diagnosis of N. caninum from other apicomplexan parasites.

Two new species of coccidia (apicomplexa: Eimeriidae) from the bearded false chameleon Chamaeleolis barbatus (Sauria: polychridae) from cinco pesos, Pinar Del Río, Cuba.

Parasitological examination of bearded false chameleons Chamaeleolis barbatus freshly imported from Cuba revealed the presence of 2 species of coccidia that are described as new. Oocysts of Isospora chamaeleolidis n. sp. are spherical to slightly subspherical, 16.1 (13-21) x 15.6 (13-19) microm, with a brownish and bilayered wall approximately 1.0-1.5 microm thick; outer layer markedly pitted. 0.75-1.0 microm thick. One, rarely 2, globular polar granules, 1.5 in diameter are present in the sporulated oocysts. Sporocysts are ellipsoidal, 10.8 (10-13) x 7.8 (7-9) microm, with a smooth, colorless, and unilayered sporocyst wall. Stieda body and substieda bodies are present. A sporocyst residuum is present, consisting of small granules of irregular size scattered among the sporozoites. Oocysts of Eimeria chamaeleolidisbarbati n. sp. are broadly oval, 19.0 (17-21) x 15.7 (15-17) microm, with a bilayered, colorless oocyst wall approximately 0.75 thick; outer layer of oocyst wall is smooth, 0.5 microm thick. One or 2, rarely 4, globular, irregular polar granules, approximately 1.5 microm in diameter, are present in sporulated oocysts. Sporocysts are broadly oval, 7.4 (7-8.5) x 6.1 (5.5-7) microm, with a smooth, colorless, and unilayered sporocyst wall, composed of 2 valves joined by suture; Stieda body and substieda bodies are absent.

The relationship of Hammondia hammondi and Sarcocystis mucosa to other heteroxenous cyst-forming coccidia as inferred by phylogenetic analysis of the 18S SSU ribosomal DNA sequence.

The complete sequence of the 18S small subunit (SSU) ribosomal DNA of Hammondia hammondi and Sarcocystis mucosa was obtained and compared to SSU rDNA sequences of Neospora caninum, Toxoplasma gondii, Besnoitia besnoiti, 2 species of Frenkelia, 3 species of Isospora, and 13 species of Sarcocystis. Analyses showed that H. hammondi and T. gondii are monophyletic and that these taxa shared a common ancestor with N. caninum and B. besnoiti. The weight of evidence shows that S. mucosa, S. neurona, and Frenkelia species form a clade thereby supporting the conclusion that Sarcocystis is paraphyletic.

Hammondia hammondi organelle proteins are recognized by monoclonal antibodies directed against organelles of Toxoplasma gondii.

Hammondia hammondi and Toxoplasma gondii, 2 closely related coccidia of cats, are known to share many antigenic molecules as shown by serologic cross reactivity. Monoclonal antibodies (MAbs) directed against the internal organelles of Toxoplasma gondii were tested by immunofluorescence assay and immunoelectron microscopy on the tachyzoites of H. hammondi. The MAbs anti-apex, anti-dense granules, anti-micronemes, and anti-rhoptries recognized, although weakly, the corresponding antigens on H. hammondi. This finding demonstrates that organelles of the 2 parasites are not only morphologically, but also antigenically, similar.

The genus Hammondia is paraphyletic.

The phylogenetic relationships amongst Hammondia, Neospora and Toxoplasma were investigated by DNA sequence comparisons of the D2/D3 domain of the large subunit ribosomal DNA and the internal transcribed spacer 1. The results obtained allow us to reject the hypothesis that N. caninum and H. heydorni are the same species and show that Hammondia hammondi is probably the sister taxon to Toxoplasma gondii.

Schellackia calotesi n. sp. from agamid lizards of the genus Calotes in Thailand.

Schellackia calotesi n. sp. is described from the Thai agamids Calotes mystaceus and C. versicolor. Schellackia-type sporozoites were recovered from blood and liver of one C. versicolor from Kon Kaen North-East Thailand and two C. mystaceus from Chiang Mai, North Thailand. Specimens of both species were fed on sporozoite infected blood, of these only one C. mystaceus developed endogenous infection in the anterior intestine. Description, from histological material includes early and dividing meronts, micro and macrogamonts and non sporulated oocysts.

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 parasite's DNA.

Margolisiella kabatai gen. et sp. n. (Apicomplexa: Eimeriidae), a parasite of native littleneck clams, Protothaca staminea, from British Columbia, Canada, with a taxonomic revision of the coccidian parasites of bivalves (Mollusca: Bivalvia).

Two of 98 native littleneck clams, Protothaca staminea Conrad, from Cooper's Cove, Sooke Basin were infected with an eimeriorin coccidian parasite. Merogonic gamontogonic and sporogonic development were observed in renal tubular epithelial cells. Sporulation of the oocysts occurred within the host. Mature oocysts were spherical mean 41 microns (range 30-44), and contained about 32 subspherical sporocysts (9 x 10 microns), each of which contained 4 sporozoites. Spherical 19 microns (18-20), cyst-like structures and smaller multinucleate bodies, some of which resembled sporocysts, were also seen. A review of the coccidian parasites of bivalves led to the erection of the new genus, Margolisiella (family Eimeriidae Minchin, 1903) to accommodate M. kabatai sp. n., the parasite in Protothaca staminea described herein. Four previously described monoxenous species (Pseudoklossia patellae Debaisieux, P. chitonis Debaisieux, P. tellinovum Buchanan and P. haliotis Friedman, Gardner, Hedrick, Stephenson, Cawthorn et Upton) were also transferred to the new genus. The 2 remaining possibly heteroxenous species (P. pelseneeri Léger and P. glomerata Léger et Duboscq) were retained in the genus Pseudoklossia Léger et Duboscq (family Aggregatidae Labbé, 1899).