The fate of Hepatozoon species naturally infecting Florida black racers and watersnakes in potential mosquito and soft tick vectors, and histological evidence of pathogenicity in unnatural host species.

Haemogregarine parasites, derived from the Florida snakes Coluber constrictor and Nerodia fasciata and ingested by Aedes aegypti, completed sporogony within the hemocoeles of nearly all fed mosquitoes in 14-18 days, and produced oocysts typical of Hepatozoon. However, mortalities and morbidity were high in the Culex which had fed on the Coluber. Oocysts were not found in any Ornithodoros turicata (Argasidae) which fed upon either snake host, but many sections of fed ticks had gametocyte-like cells within the gut lumen. Most lizards, Anolis carolinensis and Anolis sagrei, infected per os with oocysts derived from both snake species developed infections. Infections in the lizards were largely confined to hepatic schizonts with few parasites found in erythrocytes. Unlike naturally infected snake hosts, Hepatozoon schizonts in livers of lizards were often either surrounded by an unidentified dark pigment or heavily infiltrated with mononuclear inflammatory cells.     

Ultrastructural Features of Cystic and Merogonic Stages of Hepatozoon sipedon (Apicomplexa: Adeleorina) in Northern Leopard Frogs (Rana pipiens) and Northern Water Snakes (Nerodia sipedon) from Ontario, Canada

The cystic and merogonic stages of the haemogregarine Hepatozoon sipedon , infecting Northern water snakes ( Nerodia sipedon sipedon ) and Northern leopard frogs ( Rana pipiens ), respectively, in Ontario, Canada, were investigated by transmission electron microscopy. Cysts, which were observed in the liver of Northern leopard frogs ( Rana pipiens ) after these anurans ingested mosquitoes ( Culex pipiens ) containing oocysts of the parasite, harboured two cystozoites, each of which contained a large crystalloid inclusion anterior to the nucleus. Two types of meronts were observed in snakes that were fed the liver of infected frogs. Macromeronts, which matured in endothelial cells of the liver approximately 16 d after snakes ingested infected frogs, contained about 50 large macromerozoites. Macromerozoites emerged from macromeronts, entered the bloodstream of the snake, and reinfected endothelial cells. Micromeronts, which matured about 34 d post-inoculation, contained about 150 micromerozoites that infected erythrocytes and transformed into gamonts. The ultrastructural features of micromeronts and macromeronts differed only slightly: immature macromeronts and macromerozoites contained numerous amylopectin and lipid inclusions, whereas immature micromeronts and micromerozoites did not contain amylopectin inclusions and featured fewer, smaller lipid inclusions. A comparison of cystic stages among Hepatozoon species in different groups of vertebrates is presented with respect to their structure and evolutionary significance.     

Coccidian parasites (Apicomplexa: Eimeriidae) from two species of caimans, Caiman yacare daudin and Caiman latirostris daudin (Alligatoridae), from Paraguay

From October 1986 to January 1987, feces from 119 Caiman yacare and 12 Caiman latirostris were collected in Paraguay and later examined for coccidian oocysts; 69 of 119 (58%) samples from C. yacare and 3 of 12 (25%) samples from C. latirostris contained coccidian oocysts. Two eimerians infected C. yacare and both are described as new species. Sporulated oocysts of Eimeria paraguayensis n. sp. are ellipsoid, 34.0 x 23.6 (26-38 x 20-29) microns with sporocysts ovoid, 14.0 x 7.1 (10-19 x 6-10) microns. Sporulated oocysts of Eimeria caimani n. sp. are spheroid, 22.4 (19-29) microns with sporocysts ovoidal, 12.9 x 6.5 (8-17 x 5-8) microns. Isospora jacarei infected C. latirostris and is redescribed. Sporulated oocysts of I. jacarei are sub-spheroid, 13.2 x 12.1 (10-18 x 10-15) microns with sporocysts ellipsoid, 10.4 x 5.8 (7-13 x 4-11) microns. To date, members of the Eimeriidae found in Crocodylia include 5 species of Eimeria and 2 of Isospora including the new species described here.     

Development of Hepatozoon caimani (Carini, 1909) Pess a,De Biasi & De Souza, 1972 in the Caiman Caiman c. crocodilus,the frog Rana catesbeiana and the mosquito Culex fatigans

The sporogony of Hepatozoon caimani has been studied, by light microscopy, in the mosquito Culex fatigans fed on specimens of the caiman Caiman c. crocodilus showing gametocytes in their peripheral blood. Sporonts iniciate development in the space between the epithelium of the insect gut and the elastic membrane covering the haemocoele surface of the stomach. Sporulating oocysts are clustered on the gut, still invested by the gut surface membrane. Fully mature oocysts were first seen 21 days after the blood-meal. No sporogonic stages were found in some unidentified leeches fed on an infected caiman, up to 30 days following the blood-meal. When mosquitoes containing mature oocysts were fed to frogs (Leptodactylus fuscus and Rana catesbeiana), cysts containing cystozoites developed in the internal organs, principally the liver. Feeding these frogs to farm-bred caimans resulted in the appearance of gametocytes in their peripheral blood at some time between 59 and 79 days later, and the development of tissue cysts in the liver, spleen, lungs and kidneys. Transmission of the parasite was also obtained by feeding young caimans with infected mosquitoes and it is suggested that both methods occur in nature. The finding of similar cysts containing cystozoites in the semi-aquatic lizard Neusticurus bicarinatus, experimentally fed with infected C. fatigans, suggests that other secondary hosts may be involved.     

Sarcocystis stenodactylicolubris n. sp., a new sarcosporidian coccidium with a snake-gecko heteroxenous life cycle

Oocysts/sporocysts of Sarcocystis sp. measuring 9.7 (9-10) x 7.6 (7-8) microns were found in the intestinal contents of the Dahl's whip snake Coluber najadum. Of wide spectrum of experimentally inoculated hosts, only species of the family Gekkonidae--Ptyodactylus guttatus and Stenodactylus grandiceps--were found to be susceptible intermediate hosts. Transparent, barely visible sarcocysts found in tail, limbs and tongue striated muscles of the geckoes were 175-200 microns x 35-50 microns in size at 78 DPI. Ultrastructurally, the primary cyst wall was characteristic by spine-like villar protrusions up to 800 nm long, 200-250 nm in diameter at their base, tapering to thinner apex. Protrusions appear typically lobular or irregular in the cross-sections. Back-transmission from P. guttatus to Coluber rogersi leaded to oocysts/sporocysts excretion since 38 days post infection. Based on sarcocyst morphology and experimental data, Sarcocystis stenodactylicolubris is apparently a new species. Based on obtained and already published results, Sarcosporidia parasitising colubrid snakes as definitive hosts are suggested to be family specific on the level of their intermediate host.     

Hepatic coccidiosis in the goat

Coccidial oocysts were seen in the bile from five goats infected with coccidia either naturally or artificially. The oocysts measured on average 21.3 by 18.3 microns and resembled those of Eimeria ninakohlyakimovae. Livers and gall bladders of infected animals showed various degrees of histopathological changes. In the worst case, bile had a thick consistency and contained blood and necrotic debris. Apart from those in the bile, oocysts were seen in liver smears and in the centrilobular vein in two histological sections. Forms resembling meronts and measuring on average 200 by 147 microns were seen in sections of bile duct.     

Coccidia (Apicomplexa) from heteromyid rodents in the southwestern United States, Baja California, and northern Mexico with three new species from Chaetodipus hispidus

Fecal samples from 223 heteromyid rodents of 4 genera and 13 species were collected from California, New Mexico, and Texas and from Baja California Norte and Sonora, Mexico. Of these, 84 (38%) were infected with coccidian oocysts; 72 of 84 (86%) infected animals had only 1 species of coccidian. Eleven species of coccidia were identified including 1 cyclosporan and 10 eimerians; the cyclosporan and 2 of the eimerians are described as new species. Sporulated oocysts of Cyclospora angimurinensis n. sp. were subspheroidal, 21.9 x 19.3 (19-24 x 16-22) microns, with sporocysts lemon-shaped, 11.9 x 9.5 (9-15 x 8-11) microns; it was found in 1 of 20 (4%) Chaetodipus hispidus. Sporulated oocysts of Eimeria chaetodipi n. sp. were subspheroidal, 16.7 x 14.6 (13-19.5 x 12-17) microns, with sporocysts ovoidal, 8.7 x 6.6 (7.5-10.5 x 5-7.5) microns; it was found in 3 of 20 (15%) C. hispidus. Sporulated oocysts of Eimeria hispidensis n. sp. were subspheroidal, 20.5 x 17.4 (17-23 x 14-21) microns, with sporocysts lemon-shaped, 9.3 x 7.2 (7.5-10.5 x 5-9) microns; it was found in 4 of 20 (20%) C. hispidus.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new species of coccidian (Apicomplexa: Eimeriidae) from the prairie racerunner, Cnemidophorus sexlineatus viridis (Sauria: Teiidae), in Arkansas.

Feces from 26 prairie racerunners, Cnemidophorus sexlineatus viridis Lowe, 1966, from Arkansas, were examined for coccidian parasites. One of these was found to be infected with oocysts of an undescribed eimerian, which is described herein as new. Sporulated oocysts of Eimeria sexlineatus n. sp. were cylindrical, 30.4 x 17.1 (28-32 x 16-19) microns, with a shape index (length/width) of 1.8 (1.6-2.0). A micropyle and oocyst residuum were absent but 1 (to several) polar granule(s) was present. Sporocysts were ellipsoidal, 10.7 x 8.5 (9.6-11.2 x 8.0-8.8) microns, with a shape index of 1.3 (1.2-1.4). A sporocyst residuum was present but Stieda, substieda, and parastieda bodies were absent. Sporozoites were elongate, 13.2 x 2.7 microns (12.0-14.4 x 2.4-3.2) in situ, containing a single, spherical posterior refractile body. Oocysts and endogenous developmental stages were found within the gall bladder epithelium of the infected lizard. This represents the first time a coccidian has been reported from a North American whiptail lizard.     

Further studies on the development of gamonts and oocysts of Eimeria magna in cultured cells

Monolayer, cell-line cultures of embryonic bovine trachea, Madin-Darby bovine kidney (MDBK), and monolayers (RK-1) or aggregates of primary rabbit kidney cells were inoculated with merozoites obtained from rabbits that had been inoculated 3 to 5 1/2 days earlier with Eimeria magna. Merozoites obtained from from rabbits 3 days entered cells and underwent only merogony, whereas 3 1/2-5 1/2-day-old merozoites formed gamonts as well as meronts. Merozoites arising from the first or second meront generation in culture formed another meront generation or gamonts. Third-generation merozoites formed only gamonts. Most merozoites remained within the parasitophorous vacuole of the original host cell and transformed into macro- or microgamonts or meronts. Some such macro- and microgamonts then fused with each other to form larger multinucleated bodies. Such microgamonts formed microgametes, but multinucleate macrogamonts did not form oocysts. Mature microgamonts were 34 microns in diameter, and contained several hundred biflagellate microgametes. Mature macrogamonts measured 29.1 x 21.5 microns, unsporulated oocysts were 31.2 x 22 microns, and sporulated oocysts were 32 x 23.1 microns. Oocysts obtained from cell cultures were sporulated and then inoculated by gavage into rabbits, which passed E. magna oocysts 6--10 days later. Sporozoites, obtained from oocysts produced in culture or from rabbits that had been inoculated with the vitro-produced oocysts, developed to first- and second-generation meronts in MDBK or RK-1 cultures.     

Entamoeba bubalus, n.sp., from Carabao

SUMMARY. Twelve carabao in the Philippines were found to be lightly infected with intestinal amoebae. Trophic forms (12 microns in diameter) of this protozoan possessed a definite ectoplasm and a homogeneous endoplasm. They were found only in stained preparations. The nucleus was similar to that of the cyst. All unconcentrated fecal smears contained at least a few cysts (8 microns in diameter). In these forms the cytoplasm usually contained a large vacuole and one or more irregular chromatoidal bodies. The nucleus (2.6 microns in diameter) possessed a pronounced, deeply staining, uniform peripheral ring and a large irregular endosome. There was no periendosomal ring. This amoeba is designated as Entamoeba bubalus , n.sp.     

Ultrastructure of the cyst and life cycle of Sarcocystis sp. from wild sheep (Ovis musimon)

Sarcocystis sp. (Eimeriina: Sarcocystidae) is described as a heteroxenous coccidian with domestic dogs as an experimental definitive host and wild sheep (Ovis musimon) as natural intermediate hosts. Mature sarcocysts of this Sarcocystis sp. were examined by transmission electron microscopy. Sarcocysts in various muscle tissues were microscopic, had a thin primary cyst wall and septa and measured 81.0 x 30.5 microns. The cysts were located within muscle cells and were limited by a primary cyst wall (PCW). The cyst surface was highly folded forming densely packed projections. Between the PCW projections the surface of the cyst was marked with pit-like invaginations. The ground substance of the cyst formed a layer at the periphery of the cyst, filled the projections and formed septa which divided the cyst into compartments. Sarcocysts contained numerous bradyzoites that were 15.2 x 3 microns and few metrocytes 11.5 x 3.5 microns. Twelve days after ingesting Sarcocystis sp.-infected wild sheep meat, four dogs began passing sporocysts in their feces: two domestic cats did not pass oocysts or sporocysts after ingesting meat from the same animals. Sporocysts measured 14.8 x 9.9 microns.     

Ultrastructure of tachyzoites, bradyzoites and tissue cysts of Neospora caninum

Dividing tachyzoites of Neospora caninum were 4 x 3 microns and had ultrastructural characteristics typical for the cyst-forming coccidia. Unusual ultrastructural characteristics of fully-formed tachyzoites included no micropores, 8-12 anterior and 4-6 posterior rhoptries, and a few posterior micronemes. Most tachyzoites were located free in the host cell cytoplasm; only a few occurred within a parasitophorous vacuole. Parasite multiplication appeared to be rapid because most organisms were in various stages of endodyogeny. Neural tissue cysts of N. caninum were 24.3 x 19.2 microns and contained 50-200 bradyzoites (7.3 x 1.5 microns), which lacked micropores. The cyst wall was 0.74-1.12 microns thick and consisted of the primary cyst wall (the parasitophorous vacuole membrane) and a thick granular layer with electron-dense vesicles.     

Prevalence and molecular identification of Cryptosporidium isolates from pet lizards and snakes in Italy

In order to acquire prevalence and genetic data on Cryptosporidium infections in captive lizards and snakes kept as pets, a survey was conducted on 150 individual reptiles from southern Italy. Fecal samples were preserved in 5% formalin and analyzed using a commercial immunofluorescence assay (IFA) for the detection of Cryptosporidium oocysts and Giardia cysts. IFA revealed the presence of Cryptosporidium oocysts in nine of the 150 samples examined (6.0%), precisely in 6/125 snakes (4.8%) and in 3/25 lizards (12.0%); all fecal samples tested negative for the presence of Giardia cysts. Molecular characterization based on nested PCR amplification and sequencing of the SSU-rRNA gene, revealed the presence of Cryptosporidium serpentis in three samples from snakes (Boa constrictor constrictor, Elapheguttata guttata guttata and Python molurus).     

Eimeria gozaishoensis n. sp. from the Formosan serow (Capricornis crispus swinhoei)

Eimeria gozaishoensis n. sp. was found in the Formosan serow (Capricornis crispus swinhoei). The oocysts were ovoid, 29.41 +/- 0.58 x 20.77 +/- 0.41 microns with a bilayered wall. A micropyle and micropylar cap were observed, but a polar granule and oocyst residuum were absent. Sporocysts were ovoid, 11.78 +/- 0.30 x 7.60 +/- 0.31 microns, with sporocyst residuum and Stieda body. The new species differs from other known species of the genus by the morphology of oocysts and that domestic goats apparently could not be infected. The sporulation time was 6 to 7 days.     

Isospora mcquistioni and Isospora bioccai (Apicomplexa, Eimeriidae): two new coccidian parasites from Carduelis sinica (Passeriformes, Fringillidae).

The following two species are described from Carduelis sinica (Greenfinch) from Italy. The oocysts of Isospora mcquistioni n. sp. were 26.0 x 22.6 (24.0-28.5 x 20.0-24.2) microns and ovoid with a smooth bilayered wall. Neither micropyle nor oocyst residuum were observed. One polar granule was found. Sporocysts were oval, 18.1 x 11.4 (16.0-19.8 x 11.0-12.0) microns, and with a symmetrical Stieda complex. The residuum was compact and spherical. Isospora bioccai n. sp. oocysts were spherical to subspherical and 24.0 x 23.6 (22.0-26.0 x 21.0-25.8) microns. The oocyst wall was smooth and bilayered. A micropyle and oocyst residuum were absent; 4 to 10 elongate polar granules were present. Sporocysts were 19.5 x 11.6 (18.0-20.0 x 10.0-12.4) microns, ellipsoidal, and with a symmetrical Stieda complex. The sporocyst residuum was diffuse and composed of a few granules.     

A comparative study on the biology of Cryptosporidium sp. from guinea pigs and Cryptosporidium parvum (Apicomplexa).

Cryptosporidium sp. from guinea pigs and C. parvum were compared morphologically, electrophoretically, and for the ability to infect suckling mice. Oocysts from guinea pigs measured 5.4 x 4.6 (4.8-5.6 x 4.0-5.0) microns and had a shape index (length/width) of 1.17 (1.04-1.33). Oocysts of C. parvum were similar and measured 5.2 x 4.6 (4.8-5.6 x 4.2-4.8) microns with a shape index of 1.16 (1.04-1.33). All suckling mice inoculated with oocysts of C. parvum became infected, whereas most, but not all, mice fed oocysts of the guinea pig isolate also became infected. However, mice inoculated with oocysts from guinea pigs produced on average 100-fold fewer oocysts by day 7 postinoculation than did mice infected with C. parvum, and the resulting infections were sparse and patchy along the ileum. Electrophoretic profiles were similar, but 125I surface labeling of outer oocyst wall proteins revealed striking differences between the two isolates. Cryptosporidium parvum had a wide molecular size range of 125I-labeled bands, whereas C. sp. from guinea pigs had a banding pattern clustered between 39 and 66 kDa, with a smaller number of bands greater than 100 kDa.     

Contributions to the life-cycle of Frenkelia. III. The sexual development of F. clethrionomyobuteonis in the buzzard (author's transl)]

8 buzzards (Buteo buteo) were infected orally with cysts of Frenkelia clethrionomyobuteonis of the bank vole (Clethrionomys glareolus). Their intestines were searched for developmental stages of Frenkelia 21 and 24 h and 2, 3, 4, and 5 days post infection. After 21 and 24 h male and female gamonts could be detected within epithelial cells of the villi of the first half of the small intestine. The microgamonts contained 10-14 microgametes. The macrogamonts which measured on an average 11,1 X 9,8 mum in Giemsa stained smears developed into very thin-walled oocysts measuring in fresh preparations on an average 15,8 X 11,7 mum on day 3 after infection. The oocysts were located between the lamina propria and the epithelial lining of the distal third of the villi. They began to sporulate on day 5 and the first sporocysts were excreted 7 and 8 days post infection. Schizonts and schizont-like stages could not be observed in the developmental cycle in Buteo buteo.     

Isospora daphnensis n. sp. (Apicomplexa: Eimeriidae) from the medium ground finch (Geospiza fortis) from the Galapagos Islands

In March and April 1987 fecal samples from 237 nestling birds, including 199 from 85 nest sites of Geospiza fortis, 23 from 12 nest sites of Geospiza scandens, 6 from 2 nest sites of Geospiza magnirostris, and 9 from 2 nest sites of hybrids involving Geospiza fuliginosa and G. fortis, were collected from Daphne Major in the Galapagos archipelago and examined for coccidia. Only 3 of 4 nestlings from 1 nest site of G. fortis (1.5%) had oocysts in their feces. Two of the 3 infected nestlings had concurrent infections of Isospora temeraria, and all 3 nestlings were infected with a new species. Isospora daphnensis n. sp. Sporulated oocysts of I. daphnensis n. sp. are ellipsoidal, 27.3 x 23.6 (22-30 x 20-27) microns; a polar body is present, but no oocyst residuum or micropyle occurs. The oocyst wall, approximately 1.5 microns thick, is composed of a mammillated outer layer and thinner inner layer. Sporocysts are ovoid, 15.2 x 10.2 (15-16 x 9-11) microns and have a nipplelike Stieda body and a small substieda body. The sporocysts contain an irregularly shaped, smoothly contoured residuum with uniform granules and 4 sporozoites with a large refractile body at one end and lying randomly in the sporocysts.     

Five new species of Coccidia (Apicomplexa: Eimeriidae) from colubrid snakes of Ecuador.

Fecal samples from 11 colubrid snakes, representing 10 species, collected in Ecuador during October 1994 were examined for coccidian parasites. Feces of 4 individuals, representing 4 host species, contained coccidian oocysts. Three species of Eimeria and 2 species of Isospora were observed and are described here as new. Oocysts of both Eimeria and Isospora were found in the feces of a slug-eating snake, Dipsas vermiculata. Sporulated oocysts of the Eimeria sp. are spheroid to subspheroid, 16.7 by 16.6 microm (14-18 by 14-18 microm) and those of the Isospora sp. are spheroid and 15.0 microm (13-18 microm) in diameter. Imantodes cenchoa, the common bluntheaded treesnake, was infected with a species of Eimeria. These sporulated oocysts are ellipsoid, 23.3 by 16.2 microm (25-21 by 15-17 microm). Sporulated eimerian oocysts from Leptodeira annulata, the southern cat-eyed snake, are subspheroid, 22.5 by 18.8 microm (19-26 by 17-21 microm). Feces of a juvenile Imantodes lentiferus, the bluntheaded vine snake, contained ovoid to ellipsoid isosporan oocysts, which measured 21.6 by 15.0 microm (20-23 by 14-16 microm) when sporulated.     

A new species of Hepatozoon (Apicomplexa: Adeleorina) from Python regius (Serpentes: Pythonidae) and its experimental transmission by a mosquito vector

Hepatozoon ayorgbor n. sp. is described from specimens of Python regius imported from Ghana. Gametocytes were found in the peripheral blood of 43 of 55 snakes examined. Localization of gametocytes was mainly inside the erythrocytes; free gametocytes were found in 15 (34.9%) positive specimens. Infections of laboratory-reared Culex quinquefasciatus feeding on infected snakes, as well as experimental infection of juvenile Python regius by ingestion of infected mosquitoes, were performed to complete the life cycle. Similarly, transmission to different snake species (Boa constrictor and Lamprophis fuliginosus) and lizards (Lepidodactylus lugubris) was performed to assess the host specificity. Isolates were compared with Hepatozoon species from sub-Saharan reptiles and described as a new species based on the morphology, phylogenetic analysis, and a complete life cycle.