Ultrastructural analysis of Apicomplexa-Like parasites in two conch species Laevistrombus canarium and canarium urceus from Johor Straits, Malaysia.

The tropical conch, Laevistrombus canarium (Linnaeus, 1758) and Canarium urceus (Linneaus, 1758) are ecologically and economically important shellfish species in Malaysia and neighboring region. Their populations, however are currently declining and this histopathological study investigates the aspect of parasitism and diseases that may affect their well-being. Conch samples were randomly collected from their natural habitat and histological sections (4-5 µm) of various organs and tissues were examined under light microscope. This was followed by ultrastructure analysis on infected tissues using transmission electron microscope (TEM). Based on the histological analysis, large numbers of gamonts, sporocysts and trophozoites of Apicomplexa-like parasites were observed in the vacuolated cells and pyramidal crypt cells of the digestive tubules, and in the digestive ducts. Furthermore, coccidian and oocysts-like Pseudoklossia sp. stages were also observed in the cells of the kidney. Apart from that, spores with cyst-like structure were observed in the digestive gland and kidney. Although the parasites were present in most of the organs analyzed, there was no obvious symptom, inflammatory response or mortality incurred on both species, which implies the possibility of a non-virulent relationship like commensalisms or mutualism. However, more investigations, including molecular studies, are needed to confirm the parasite identification and dynamics, and to further evaluate the nature of relationship between Apicomplexa parasites and their host.

Life cycle of Cystoisospora felis (Coccidia: Apicomplexa) in cats and mice.

Cystoisospora felis is a ubiquitous apicomplexan protozoon of cats. The endogenous development of C. felis was studied in cats after feeding them infected mice. For this, five newborn cats were killed at 24, 48, 72, 96, and 120 h after having been fed mesenteric lymph nodes and spleens of mice that were inoculated with C. felis sporulated sporocysts. Asexual and sexual development occurred in enterocytes throughout the villi of the small intestine. The number of asexual generations was not determined with certainty, but there were different sized merozoites. At 24 h, merogony was seen only in the duodenum and the jejunum. Beginning at 48 h, the entire small intestine was parasitized. At 24 h, meronts contained 1-4 zoites, and at 48 h up to 12 zoites. Beginning with 72 h, the ileum was more heavily parasitized than the jejunum. At 96 and 120 h, meronts contained many zoites in various stages of development; some divided by endodyogeny. The multiplication was asynchronous, thus both immature multinucleated meronts and mature merozoites were seen in the same parasitophorous vacuole. Gametogony occurred between 96 and 120 h, and oocysts were present at 120 h. For the study of the development of C. felis in murine tissues, mice were killed from day 1 to 720 d after having been fed 10(5) sporocysts, and their tissues were examined for the parasites microscopically, and by bioassay in cats. The following conclusions were drawn. (1) Cystoisospora felis most frequently invaded the mesenteric lymph nodes of mice and remained there for at least 23 mo. (2) It also invaded the spleen, liver, brain, lung, and skeletal muscle of mice, but division was not seen based on microscopical examination. (3) This species could not be passed from mouse to mouse.

Description of three new species of Hepatozoon (Apicomplexa, Hepatozoidae) from Rattlesnakes (Crotalus durissus terrificus) based on molecular, morphometric and morphologic characters.

Hepatozoon spp. are commonly found infecting snakes. Since the latter are parasitized by diverse forms and data in the literature show divergence, we studied Hepatozoon spp. diversity on Crotalus durissus terrificus snakes using both molecular and morphological approaches. Naturally infected animals were employed. Blood was collected, blood smears were prepared and an aliquot was stored at -20°C for DNA extraction. Five specimens of C. durissus terrificus were selected, each of them infected with one gamont type. Morphological and morphometric analyses of the found gamonts led to their grouping into three populations. For molecular characterization, seven oligonucleotide pairs that amplify distinct regions of rDNA gene were tested by adopting the PCR technique. Only the oligonucleotide pairs HepF300/Hep900 and HEMO1/HEMO2 were efficient in amplifying and distinguishing different isolates of Hepatozoon spp. from snakes. The better results were obtained when both oligonucleotide pairs were used in association. Based on the molecular and morphologic differences, three new species were proposed: Hepatozoon cuestensis sp. nov.; Hepatozoon cevapii sp. nov. and Hepatozoon massardii sp. nov. This is the first description of new Hepatozoon species from snakes, based on molecular characterization and morphological data, in South America.

Polyphyletic origin, intracellular invasion, and meiotic genes in the putatively asexual agamococcidians (Apicomplexa incertae sedis).

Agamococcidians are enigmatic and poorly studied parasites of marine invertebrates with unexplored diversity and unclear relationships to other sporozoans such as the human pathogens Plasmodium and Toxoplasma. It is believed that agamococcidians are not capable of sexual reproduction, which is essential for life cycle completion in all well studied parasitic apicomplexans. Here, we describe three new species of agamococcidians belonging to the genus Rhytidocystis. We examined their cell morphology and ultrastructure, resolved their phylogenetic position by using near-complete rRNA operon sequences, and searched for genes associated with meiosis and oocyst wall formation in two rhytidocystid transcriptomes. Phylogenetic analyses consistently recovered rhytidocystids as basal coccidiomorphs and away from the corallicolids, demonstrating that the order Agamococcidiorida Levine, 1979 is polyphyletic. Light and transmission electron microscopy revealed that the development of rhytidocystids begins inside the gut epithelial cells, a characteristic which links them specifically with other coccidiomorphs to the exclusion of gregarines and suggests that intracellular invasion evolved early in the coccidiomorphs. We propose a new superorder Eococcidia for early coccidiomorphs. Transcriptomic analysis demonstrated that both the meiotic machinery and oocyst wall proteins are preserved in rhytidocystids. The conservation of meiotic genes and ultrastructural similarity of rhytidocystid trophozoites to macrogamonts of true coccidians point to an undescribed, cryptic sexual process in the group.

The Coccidia (Apicomplexa) of the Archosauria (Crocodylia: Eusuchia) of the World.

The order Crocodylia (suborder Eusuchia) includes 27 species of alligators, caimans, crocodiles, and gharials that are cosmopolitan in distribution, inhabiting subtropical and tropical locations. Numerous surveys (many of them trivial, with small sample sizes) have reported a wide variety of blood and intestinal apicomplexans from 17/27 (63%) crocodilian hosts, but neither a summation for the group nor a revisionary systematic approach to species evaluation has ever been provided. Herein, we summarize information on the 16 species of apicomplexans that we consider to be valid, including 8 Eimeria, 1 Haemogregarina (that eventually may be transferred to Hepatozoon), 4 Hepatozoon, 2 Isospora, and 1 Progarnia species. In addition, there are 46 apicomplexan forms that we have relegated to species inquirendae and/or only partially identified forms that await further study. We hope this review provides a foundation for future research between parasitologists and herpetologists on parasitism of all reptiles and their relatives. Since many apicomplexans seem to be reasonably host-specific, knowledge of shared species and/or genera may assist herpetologists to better understand the phylogenetic relationships among the New World crocodilians and the direction of their dispersal in the New World.

Light and transmission electron microscopic characteristics of a novel Hepatozoon spp. in naturally infected cotton rats (Sigmodon hispidus).

A novel species of Hepatozoon was recently reported in cotton rats (Sigmodon hispidus) collected from an area of Oklahoma where American canine hepatozoonosis is endemic. In this study, the various stages of merogony of the parasite were characterized by light and electron microscopy. Meronts occurred within parasitophorous vacuoles in hepatocytes and ranged from mononucleated spherical forms to large, mature forms in vacuoles that contained approximately 50 peripherally arranged merozoites. Developing merozoites had characteristic apicomplexan organelles, including anterior and posterior polar rings, a conoid, microtubules, rhoptries, micronemes, and a trilaminar membrane. As the meronts matured, numerous curvilinear merozoites budded from a residual body. This morphologic characterization extends our understanding of this novel Hepatozoon and adds information about the hepatozoa, apicomplexan parasites that infect numerous species.

Hepatozoon ursi n. sp. (Apicomplexa: Hepatozoidae) in Japanese black bear (Ursus thibetanus japonicus).

Morphological and genetic features of a new Hepatozoon species, Hepatozoon ursi n. sp., in Japanese black bear (Ursus thibetanus japonicus) were studied. Schizogonic developmental stages were observed in the lungs of Japanese black bears. The schizonts were sub-spherical in shape and 45.7+/-4.6 x 42.7+/-4.5 microm in size. Each mature schizont contained approximately 80-130 merozoites and 0-5 residual bodies. The merozoites were 7.0+/-0.7 x 1.8+/-0.3 microm in size. Intraleukocytic gametocytes were slightly curved, cigar-like in shape and had a beak-like protrusion at one end. The size of the gametocytes was 10.9+/-0.3 x 3.3+/-0.2 microm. The analyses of the18S rRNA gene sequences supported the hypothesis that H. ursi n. sp. is different from other Hepatozoon species. Mature Hepatozoon oocysts were detected in two species of ticks (Haemaphysalis japonica and Haemaphysalis flava) collected on the bears infected with H. ursi n. sp. Two measured oocysts were 263.2 x 234.0 microm and 331.8 x 231.7 microm, respectively. The oocysts contained approximately 40 and 50 sporocysts, respectively. The sporocysts were sub-spherical in shape and 31.2+/-2.5 x 27.0+/-2.9 microm in size. Each sporocyst contained at least 8-16 sporozoites, with the sporozoites being 12.2+/-1.4 x 3.5+/-0.5 microm in size. H. ursi n. sp. is the first Hepatozoon species recorded from the family Ursidae.

Potential environmental and host gender influences on prevalence of Haemogregarina platessae (Adeleorina:Haemogregarinidae) and suspected Haemohormidium terraenovae (incertae sedis) in Brazilian flounder from the Patos Lagoon Estuary, southern Brazil.

Flounder, Paralichthys orbignyanus (Valenciennes), were captured in polluted and non-polluted sites within the Patos Lagoon Estuary, southern Brazil, over four seasons. Blood films showed a high prevalence of infection with a haemogregarine, or mixed parasitaemias of this and an organism resembling Haemohormidium terraenovae So, 1972. Haemogregarine gamont stages conformed to existing descriptions of Desseria platessae (Lebailly, 1904) Siddall, 1995 from flatfishes, but intraerythrocytic division of meronts was observed, leading to the recommendation for nomenclatural correction, placing the haemogregarine in the genus Haemogregarina (sensu lato) Danilewsky, 1885. Statistical analyses suggested that although sample sizes were small, infections with meront stages, immature and mature gamonts were all influenced by site, and possibly therefore, by pollution. Season also appeared to determine likelihood of infection with meronts and immature gamonts, but not mature gamonts, while adult fish gender apparently affected infection with immature and mature gamonts, but not meronts. The H. terraenovae-like organism exhibited unusual extracellular forms and did not match closely with the type description of H. terraenovae; precise identification was therefore difficult. Data analyses suggested that parasitism by this organism was influenced by site and fish gender, since females and males from non-polluted water were infected, but only females from the polluted site. Season was also important and significantly more adult fish of both sexes were infected with this parasite in the Brazilian summer and autumn, compared with winter and spring. Finally, these appeared to be the first observations of Haemogregarina platessae, and possibly H. terraenovae, from the southern hemisphere.

Klossiella quimrensis (Apicomplexa: Klossiellidae) causes renal coccidiosis in western barred bandicoots Perameles bougainville (Marsupialia: Peramelidae) in Western Australia.

Previous studies have described a range of Klossiella species parasitic in marsupial hosts. Klossiella quimrensis is the etiologic agent of renal coccidiosis in the peramelid marsupial hosts Isoodon obesulus and Perameles gunnii in Eastern Australia, but there is no previous report of klossiellosis in Western Australian peramelids. This study describes klossiellosis diagnosed by histology of renal tissue sections collected during necropsy of 20 Perameles bougainville between 2000 and 2005. Sporonts, sporoblasts, and macrogametes were identified within parasitophorous vacuoles of epithelial cells located near the renal corticomedullary junction. The prevalence of renal coccidiosis in P. bougainville diagnosed by renal histology is estimated at 30%. Only a single unsporulated sporocyst was detected by examination of cystocentesis-collected urine, indicating that microscopic evaluation of urine samples is an insensitive diagnostic test for detection of K. quimrensis in P. bougainville. This infection in P. bougainville is indirectly associated with mild multifocal interstitial lymphohistiocytic nephritis and is likely to be only minimally pathogenic in otherwise healthy individuals. Our study also extends the host and geographic range of K. quimrensis to include P. bougainville and Western Australia.

Induced Aberrant Organisms with Novel Ability to Protect Intestinal Integrity from Inflammation in an Animal Model.

Robust and balanced gut microbiota are required to support health and growth. Overgrowth of gut microbial or pathogens can change ecosystem balance, and compromise gut integrity to initiate gastrointestinal (GI) complications. There is no safe and effective modality against coccidiosis. Antibiotic additives routinely fed to food animals to protect against infection, are entered into the food chain, contaminate food products and pass to the consumers. HYPOTHESIS: induced aberrant organisms possess distinct ultrastructure and are tolerated by immunodeficient-animals yet are non-pathogenic, but immunogenic in various strains of chicks to act as a preventive (vaccine) and eliminating the needs for antibiotic additives. Methods: cyclophosphamide-immunodeficient and immune-intact-chicks were inoculated with induced aberrant or normal Coccidal-organisms. Immune-intact-chicks were immunized with escalating-doses of organisms. Results: Aberrant organisms showed distinct ultrastructure with 8-free-sporozoites which lacked sporocysts walls and veils. Immunodeficient-chicks inoculated with normal-organisms developed severe GI complications but tolerated aberrant-organisms (p < 0.001) while they had no detectable antibodies. Naïve-animals challenged with a pathogenic-dose showed GI complications, bloody diarrhea, severe lesions and weight loss. Immune-intact-animals immunized with aberrant forms were protected against high dose normal-pathogenic-challenge infection and gained more weight compared to those immunized with normal-organisms (p < 0.05). Conclusions: Aberrant organisms possess a distinct ultrastructure and are tolerated in immunodeficient-chicks, yet provide novel immune-protection against pathogenic challenges including diarrhea, malnutrition and weight loss in immune-intact-animals to warrant further investigations toward vaccine production.

Ultrastructural description of an unidentified apicomplexan oocyst containing bacteria-like hyperparasites in the gill of Crassostrea rizophorae.

Oocysts of an unidentified coccidian are reported in this study to parasitize the gills of the oyster Crassostrea rizophorae (Mollusca, Bivalvia) collected near the city of Recife (Itamaracá Island, 07 degrees 38' 00" S, 34 degrees 48' 06" W), Brazil. Oocysts appeared as light and dense forms, both containing rod-shaped, bacteria-like hyperparasites (BL). Both light and dense oocysts were spherical, 4.3 to 4.7 pm in diameter, but denser oocysts had irregular contours. Both forms consisted of a thick dense wall (approximately 165 nm thick) consisting of 3 layers. The outermost, a dense and irregular layer about 25 nm thick, possessed numerous bead-like structures and some slender conical projections (up to 1.5 microm long). The inner layer of the wall was formed by a dense and homogenous layer about 125 nm thick. Between these 2 layers, a thin light layer about 12 nm thick was present. Uninucleated sporocysts occupied the internal space of the oocyst and contained some rod-shaped BL and mitochondria surrounded by numerous ribosome-like particles. The dense forms of the oocysts showed the same structures described in the lighter forms and appeared to be the final maturation form of the oocysts. Free sporozoites were occasionally observed among oocysts.

Intestinal coccidiosis in bluegill, Lepomis macrochirus.

Developmental stages of a coccidial parasite were observed in young-of-year bluegill (Lepomis macrochirus) from an impoundment lake in Norfolk County, Virginia. The fish were anorexic and lethargic. Necropsy examination revealed emaciated bluegill with little or no abdominal fat and no food in the stomach or intestines. Coccidia were present in the posterior intestine in moderately large numbers. Few sporulated oocysts were present, and identification to genus was not possible. Epithelial cell destruction, sloughing of the intestinal mucosa, and hemorrhage were associated with the developing coccidial parasites. Coccidia were not observed within other organ systems. No pathogenic bacteria were isolated from the fish tissues. Our findings indicate that intestinal coccidiosis may pose a significant health problem in young bluegill.

Microneme secretion in Coccidia: confocal laser scanning and electron microscope study of Sarcocystis muris in cell culture.

A monoclonal antibody (mcab) raised against a subcellular fraction of Sarcocystis muris cystozoites was used to localize microneme antigens before, during and after invasion of cultured cells. The mcab recognized a 20 and 22 kDa protein under reducing and non-reducing conditions on Western blots and localized an antigen in cystozoites in the apical part of the parasites. Confocal laser scanning microscopy of invading cystozoites revealed the secretion of a microneme antigen at the apical tip of the parasite. The secreted microneme antigen was attached to the host cell surface at the invasion site and spread along the surface of the infected cells. Electron microscopy using immunogold labeling showed that the microneme antigen was distributed in patches on the surface of infected cells and present on infected cells more than 60 min post-infection. The function of microneme antigens during parasite-host cell interactions is discussed.

A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture.

Tissue cysts of parasites of the genus Hammondia are rarely described in naturally or experimentally infected intermediate hosts. However, ultrastructural examinations on tissue cyst stages of Hammondia sp. are needed, e.g. to compare these stages with those of Neospora caninum and other related parasites. We describe a cell culture system employed to examine the in vitro development of tissue cysts of a Hammondia sp.-like parasite (isolate FOX 2000/1) which uses the European fox as a definitive host. Cells of a diploid finite cell line from embryonal bovine heart (KH-R; CCLV, RIE 090) were infected by inoculation of sporozoites und cultivated for up to 3 months. Transmission electron microscopic examination of 17 day old cell culture material revealed the presence of cyst walls. Infected cell cultures cultivated for 2 months were used to feed a fox. Six to 13 days post infection the fox shed large numbers (n=1.2 x 10(7)) of Hammondia-sp. like oocysts which could not be distinguished from those used to infect the cell culture as determined by DNA sequencing of the internal transcribed spacer 1 and the D2/D3 domain of the large subunit ribosomal DNA. To find out the proportion of parasitophorous vacuoles that had developed into tissue cysts, the expression of bradyzoite markers was examined by probing infected cell cultures with mouse polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (anti-BAG1) and rat monoclonal antibodies against a cyst wall protein (mAbCC2). Nineteen and 90 days post infection all parasitophorous vacuoles in the cell cultures were positive with anti-BAG1 and mAbCC2. This shows that biologically viable (i.e. infectious) tissue cysts of a fox-derived Hammondia sp. isolate (FOX 2000/1) can be efficiently produced in this cell culture system. Since in vitro cystogenesis of dog-derived Hammondia heydorni has not been observed yet, in vitro cyst formation might be one trait to separate fox-derived Hammondia sp. from H. heydorni on a species level.

The conceptual basis for a new classification of the coccidia.

At the joint meeting of the 8th International Coccidiosis Conference and the Annual Scientific Meeting of the Australian Society for Parasitology in Palm Cove, Australia, in July 2001, a Controversial Roundtable was held on 'New classification of coccidia'. The aim of this Roundtable was to stimulate and encourage discussion and debate on current classification schemes for the group of parasitic protozoa known as the eimeriid coccidia. In the past, such classifications have been based only on phenotypic characters such as morphology, ultrastructure, life cycles, and host specificity. However, over the past 10-15 years, molecular phylogenetic studies on taxa of the eimeriid coccidia have revealed that several of the families, subfamilies, and genera that have been erected based on non-molecular characters are paraphyletic. Therefore, this Roundtable was an important forum for initial discussions on how a new and more comprehensive classification of the eimeriid coccidia, which takes into consideration both phenotypic and molecular characters, can be devised. The stimulus came from invited speakers who gave introductions into selected areas of taxonomy and classification. Following these introductions, a more general discussion with the audience addressed potential steps that may be taken in future work. This review is the immediate outcome of the Roundtable. It describes advantages and disadvantages of the use of phenotypic or molecular characters as the base for taxonomic schemes for eimeriid coccidia. It gives specific examples for drawbacks of current classifications based only on phenotypic characters as well as potential pitfalls associated with the use of only molecular phylogenies. It addresses current controversies as well as rules of taxonomy and nomenclature relevant for the eimeriid coccidia. Finally, it recommends the establishment of an international group of scientists to meet on a regular basis, stimulate further discussions, and give direction on how the final goal, i.e. a proposal for a revised, and widely accepted, classification of the eimeriid coccidia, may be achieved.

Redescription of Hammondia hammondi and its differentiation from Toxoplasma gondii.

Hammondia hammondi is a protozoan parasite that, until 1975, was misidentified as Toxoplasma gondii. Recently, the validity of H. hammondi has been questioned. In this article, the authors redescribe the parasite and its life cycle, provide accession numbers to its specimens deposited in a museum, and distinguish it structurally and biologically from T. gondii. Hammondia hammondi was found to be structurally, biologically, and molecularly different from T. gondii.

Cryptosporidial enteritis and pneumocystis pneumonia in a homosexual man.

The case of a 44-year-old homosexual man with cryptosporidial enteritis associated with multiple opportunistic infections and increased T-suppressor cells is reported. This case calls attention to cryptosporidia as the cause of unremitting diarrhea in homosexual men with the syndrome of opportunistic infectious disease associated with acquired immunodeficiency.

Human cryptosporidiosis in the acquired immune deficiency syndrome.

We studied two cases of cryptosporidiosis in patients with acquired immune deficiency syndrome. In one patient, the gallbladder was affected in addition to gastric and intestinal involvements. This is the first human case in which Cryptosporidium has been found in the biliary tract. It is important that pathologists become aware of this protozoan infection since the number of patients with it is likely to increase in the near future.

Further characterization of the biology of Hammondia heydorni.

Hammondia heydorni oocysts, recovered from the diarrheic feces of a dog suffering from corticosteroid toxicosis, were sporulated and characterized morphometrically. Sporulated H. heydorni oocysts were administered to three dogs, five goats, a calf and three guinea pigs, and tissues from these inoculated animals fed to coccidia-free dogs to determine species susceptibility to H. heydorni. Morphometric characteristics of H. heydorni oocysts were similar to previous descriptions. Oocysts were not excreted in the feces of dogs inoculated with oocysts. Dogs fed tissues from goats killed 14, 28 or 42 days after inoculation (d.a.i.) with oocysts excreted H. heydorni oocysts in their feces after consuming goat tissues. Dogs fed tissues from a calf killed at 28 d.a.i., and from goats killed 21 and 56 d.a.i., did not excrete oocysts in their feces. Developmental stages of H. heydorni were not observed in histologic sections of tissues from any of the animals.

Enterocolitis in piglets caused by Cryptosporidium sp. purified from calf faeces.

Twelve gnotobiotic piglets were dosed with a bacteria-free calf faecal homogenate which contained Cryptosporidium oocysts. The infection induced severe enterocolitis in piglets when inoculated at 1 day of age, moderate diarrhoea at 7 days of age and a subclinical infection at 15 days of age. In piglets aged 3 days or less, the entire intestine was extensively infected with Cryptosporidium and the mucosa was severely damaged. In piglets 7 days of age or older, the upper small intestine was sparsely infected with the organisms, but the ileum and the large bowel were heavily infected with associated mucosal damage.