In vitro cultivation methods for coccidian parasite research

The subclass Coccidia comprises a large group of protozoan parasites, including important pathogens of humans and animals such as Toxoplasma gondii, Neospora caninum, Eimeria spp., and Cystoisospora spp. Their life cycle includes a switch from asexual to sexual stages and is often restricted to a single host species. Current research on coccidian parasites focuses on cell biology and the underlying mechanisms of protein expression and trafficking in different life stages, host cell invasion and host-parasite interactions. Furthermore, novel anticoccidial drug targets are evaluated. Given the variety of research questions and the requirement to reduce and replace animal experimentation, in vitro cultivation of Coccidia needs to be further developed and refined to meet these requirements. For these purposes, established culture systems are constantly improved. In addition, new in vitro culture systems lately gained considerable importance in research on Coccidia. Well established and optimized in vitro cultures of monolayer cells can support the viability and development of parasite stages and even allow completion of the life cycle in vitro, as shown for Cystoisospora suis and Eimeria tenella. Furthermore, new three-dimensional cell culture models are used for propagation of Cryptosporidium spp. (close relatives of the coccidians), and the infection of three-dimensional organoids with T. gondii also gained popularity as the interaction between the parasite and host tissue can be studied in more detail. The latest advances in three-dimensional culture systems are organ-on-a-chip models, that to date have only been tested for T. gondii but promise to accelerate research in other coccidians. Lastly, the completion of the life cycle of C. suis and Cryptosporidium parvum was reported to continue in a host cell-free environment following the first occurrence of asexual stages. Such axenic cultures are becoming increasingly available and open new avenues for research on parasite life cycle stages and novel intervention strategies.     

Occurrence of Perkinsus olseni (Protozoa: Apicomplexa) and other parasites in the venerid commercial clam Pitar rostrata from Uruguay, southwestern Atlantic coast

A study was conducted into the health status of natural populations of the venerid clam Pitar rostrata from Uruguay. Perkinsus sp. was detected in 22% of the clams. Severe hemocytic infiltration was detected in the tissues parasitized by this protozoan parasite. The sequencing of the ITS-5.8S gene cluster of the parasite confirmed that it belonged to the Perkinsus olseni species. Rickettsia or Chlamidia-like organisms were also found, with a prevalence of 11%, although without apparent host reaction; an unidentified species of Coccidia was found in the nephridia of 78% of the clams, with the intensity of infection ranging from moderate to high. A gregarine, Nematopsis-like organism was observed mainly in the epithelial cells of the intestine, without host response and with a prevalence of 56%. Of the metazoan parasites, trematodes were found in 11% of the individuals analyzed.     

Eimeria tenella: stimulation of DNA synthesis in infected cultured animal cells.

An autoradiographic study was conducted to determine the influence of the intracellular coccidian parasite of chickens, Eimeria tenella, on the incorporation of tritium-labeled thymidine in kidney cell cultures. Evidence was obtained for a parasite-induced increase in thymidine incorporation in host cell cultures which is too great to be attributed to unscheduled DNA synthesis. The stimulatory effect became significant (P < 0.05) at 24 hr after inoculation and further increased at 48 and 72 hr postinoculation. Both parasitized and unparasitized cells in the infected cultures showed similar increases in thymidine incorporation. Furthermore, the increased incorporation of thymidine in the infected cultures was found to be independent of both the percentage of parasitized cells and development of the parasite.     

Calyptospora tucunarensis n. sp. (Apicomplexa: Sporozoea) from the liver of tucunare Cichla ocellaris in Brazil

A coccidian infection of high prevalence and intensity was demonstrated in the liver of tucunare Cichla ocellaris from both natural waters of northeast Brazil and those cultured in fish farms. The immature oöcysts of this parasite were seen uniformly dispersed in the liver,and its mature oöcysts formed large aggregations. The sporocysts were characterised by a single sporopodium and a membranous veil surrounding them. The parasite is described as a new species, Calyptospora tucunarensis. This is the second report on a calyptosporid in a South American fish.     

Variation in anti-parasite behaviour and infection among larval amphibian species

Along with immune defences, many animals exhibit effective anti-parasite behaviours such as parasite avoidance and removal that influence their susceptibility to infection. Host ecology and life history influence investment into comparatively fixed defences such as innate immunity but may affect the strength of anti-parasite behaviours as well. We investigated activity levels in five different species of larval amphibian with varying life histories and ecology in control, novel food stimulus, and trematode parasite (Echinoparyphium sp.) threat conditions. There was a significant interaction of species and treatment given that American toad (Bufo americanus), wood frog (Lithobates sylvaticus), and bullfrog (Lithobates catesbeianus) tadpoles generally increased their activity when parasite infectious stages were present while grey tree frogs (Hyla versicolor) and northern leopard frogs (Lithobates pipiens) did not, even though activity was negatively related to infection. In addition, there was considerable variation among species in their susceptibility to parasitism, with infection prevalence ranging from 17 % in bullfrog tadpoles to 70 % in wood frogs. However, amphibian life history (larval and adult traits) was not related to parasitism or level of anti-parasite behaviour at the species level. Consequently, we suggest that future investigations include more species with a range of life history traits and also consider host ecology, particularly if conspicuous anti-parasite behaviours are more likely in amphibian species that experience a relatively low risk of predation.     

Evidence that a lineage of teleost-infecting blood flukes (Aporocotylidae) infects bivalves as intermediate hosts

The family Aporocotylidae is recognized as having the widest intermediate host usage in the Digenea. Currently, intermediate host groups are clearly correlated with definitive host groups; all known life cycles of marine teleost-infecting aporocotylids involve polychaetes, those of freshwater teleost-infecting aporocotylids involve gastropods, and those of chondrichthyan-infecting aporocotylids involve bivalves. Here we report the life cycle for a marine elopomorph-infecting species, Elopicola bristowi Orélis-Ribeiro & Bullard in Orélis-Ribeiro, Halanych, Dang, Bakenhaster, Arias & Bullard, 2017, as infecting a bivalve, Anadara trapezia (Deshayes) (Arcidae), as the intermediate host in Moreton Bay, Queensland, Australia. The cercaria of E. bristowi has a prominent finfold, distinct anterior and posterior widenings of the oesophagus, a tail with symmetrical furcae with finfolds, and develops in elongate to oval sporocysts. We also report molecular data for an unmatched aporocotylid cercaria from another bivalve, Megapitaria squalida (G. B. Sowerby I) (Veneridae), from the Gulf of California, Mexico, and six unmatched cercariae from a gastropod, Posticobia brazieri (E. A. Smith) (Tateidae), from freshwater systems of south-east Queensland, Australia. Phylogenetic analyses demonstrate the presence of six strongly-supported lineages within the Aporocotylidae, including one of elopomorph-infecting genera, Elopicola Bullard, 2014 and Paracardicoloides Martin, 1974, now shown to use both gastropods and bivalves as intermediate hosts. Of a likely 14 aporocotylid species reported from bivalves, six are now genetically characterised. The cercarial morphology of these six species demonstrates a clear distinction between those that infect chondrichthyans and those that infect elopomorphs; chondrichthyan-infecting aporocotylids have cercariae with asymmetrical furcae that lack finfolds and develop in spherical sporocysts whereas those of elopomorph-infecting aporocotylids have symmetrical furcae with finfolds and develop in elongate sporocysts. This morphological correlation allows predictions of the host-based lineage to which the unsequenced species belong. The Aporocotylidae is proving exceptional in is propensity for major switches in intermediate host use, with the most parsimonious interpretation of intermediate host distribution implying a minimum of three host switches within the family.     

Perkinsosis in the Manila clam Ruditapes philippinarum affects responses to the harmful-alga, Prorocentrum minimum

The dinoflagellate Prorocentrum minimum is increasingly recognized as a harmful algal bloom (HAB) species that affects filter-feeding shellfish. An experiment was done to investigate possible interactions between parasitic diseases and exposure to P. minimum in Manila clams, Ruditapes philippinarum. Manila clams, with variable levels of infection with Perkinsus olseni, were exposed for three or six days to the benign phytoplankton species Chaetoceros neogracile or a mixed diet of C. neogracile and P. minimum. After three or six days of exposure, clams were assessed individually for condition index, parasite status, and plasma and hemocyte parameters (morphological and functional) using flow-cytometry. Histological evaluation was also performed on individual clams to assess prevalence and intensity of parasitic infection, as well as other pathological conditions.Prorocentrum minimum caused several changes in Manila clams, especially after six days of exposure, such as decreased hemocyte phagocytosis and size and clam condition index. Pathological conditions observed in Manila clams exposed to P. minimum were hemocyte infiltration in the intestine and gonad follicles, myopathy, and necrosis of the intestine epithelial cells. The parasite P. olseni alone had no significant effect on Manila clams, nor did it modulate the hemocyte variables in clams exposed to P. minimum; however, the parasite did affect the pathological status of Manila clams exposed to the P. minimum culture, by causing atrophy and degeneration of residual ova in the gonadal follicles and hyaline degeneration of the muscle fibers, indicating synergistic effects of both stressors on the host over a short period of time. Additionally, an in vitro experiment also demonstrated detrimental effects of P. minimum and exudates upon P. olseni cells, thus suggesting HAB antagonistic suppression of transmission and proliferation of the parasite in the natural environment over a longer period of time. The results of this experiment demonstrate the complexity of interactions between host, parasite, and HAB.     

History of the discovery of the life cycle of Toxoplasma gondii

It has been 100 years since the discovery of Toxoplasma gondii in 1908. Its full life cycle was not discovered until 1970 when it was found that it is a coccidian parasite of cats with all non-feline warm blooded animals (including humans) as intermediate hosts. The discovery of the environmentally resistant stage of the parasite, the oocyst, made it possible to explain its worldwide prevalence. In the present paper, events associated with the discovery of its life cycle are recalled.     

Isospora viridanae N. Sp., an Intranuclear Coccidian Parasite from the Canarian Skink, Chalcides viridanus (Lacertilia: Scincidae)

A caryotropic species of coccidium, Isospora viridanae n. sp. (Apicomplexa: Eimeriidae). has been found as a parasite in the Canarian skink, Chalcides viridanus Gravenhorst, 1851, from Tenerife, Spain, and is described here as a new species. Fully sporulaled oocysts of Isospora viridanae are spherical and measure 21.6 (1 7.6–23.4) nm in diameter. Mtcropyle, oocyst residuum and polar granule are ali absent. Oocysts of this coccidian, with a smooth bilayered wall, contain 2 ovoid sporocysts 13.2 (11.7–14.0) by 9.5 8.2–10 5) μm. A sporocyst residuum is present as well as a Stieda body and a substieda body. Most oocysts are found to be at the beginning of sporulanon when excreted and show 2 spherical sporoblasts. Sporulation is completed within 24 to 48 h at 23 × 2°C Sporozoites are 13–14 μm long and are about 2.5–3 μ m wide. Endogenous stages of schizogony and gamogony develop in the nuclei of epithelial cells from the small intestine of the skink. Comparisons with other species of the genus found in lacertilian hosts indicate that it is a new species.     

Isospora atrata N. Sp. (Apicomplexa, Eimeriidae): a New Coccidium Isolated from Carduelis atrata (Passeriformes, Fringillidae)

ABSTRACT Large numbers of coccidian oocysts belonging to the genus Isospora were obtained from the intestinal contents of 98 Carduelis atrata imported into Italy from South America during the months of August through December 1994. The oocysts are sub-spherical and average 21 × 20.3 μm (19.4–23.5 μm × 18.5–22 μm), have a bilayered wall, and an oval polar granule (rarely two). The sporocysts are elliptical and measure 18.8 μm × 10.3 μm (17.5–18.94 μm × 9.5–11.0 μm). The Stieda body protrudes slightly from the end of the sporocyst. A large sporocyst residuum is present, consisting of many granules that may be in a compact mass or scattered. Since this Isospora sp.does not resemble any other species of Isospora previously described from birds of the genus Carduelis , it has been named Isospora atrata n. sp. after the host. Disseminated asexual stages were found in mononuclear cells derived from formalin-fixed post mortem material, suggesting this coccidian may represent an Atoxoplasma -like parasite. Four coccidia-free Serinus canarius L. cohabitated for a long period (4 mo) with infected C. atrata but oocysts were never found in the stool of these birds.     

A brachylaimid trematode from marsupial mice with observations on its life cycle

A species of digenetic trematode, Brachylaime antechini (Family Brachylaimidae), is described from adult worms recovered from the intestine and rectum of two species of marsupial mice, Antechinus stuartii(10 of 56 infected) and Antechinus swainsonii (2 of 5 infected). On experimental evidence unencysted metacercariae from the kidney of a terrestrial gastropod, Strangesta capillacea, are considered to be larval stages of Brachylaime antechini. Miracidia, sporocysts and cercariae have not been recovered, but circumstantial evidence suggests that Strangesta capillacea fulfils the role of first intermediate host. Brachylaime antechini most closely resembles Brachylaime chiapensis, a parasite of deer mice in Mexico.     

Host preferences of introduced and native parasites (Hymenoptera: Braconidae) of phytophagous plant bugs (Hemiptera: Miridae) in alfalfa-grass fields in the northeastern USA

During an eight-year field study at two widely-separated locations (NJ & DE, USA), seven species of phytophagous mirid plant bugs were found in alfalfa and alfalfa-grass fields grown for animal forage. Five species of parasites were reared from these mirids. Two parasite species introduced from Europe killed significantly higher proportions of nymphs of the two important mirid pests of alfalfa than did native parasites. The introduced Peristenus digoneutis Loan parasitized an average of 31% of first and second generation tarnished plant bugs, Lygus lineolaris, a native insect; and the introduced Peristenus conradi parasitized 22% of first generation alfalfa plant bugs, Adelphocoris lineolatus, an introduced species. In addition, Peristenus pallipes significantly parasitized nymphs of Trigonotylus caelestialium (43%) and Leptopterna dolabrata (37%); both mirids are immigrant species. Because the parasite P. pallipes significantly attacked only these two non-native mirids, and is present in Europe, it also may be an accidental introduction to North America. A native wasp, Leiophron uniformis, heavily parasitized (49%) the native garden fleahopper, Halticus bractatus. A third native species, P. pseudopallipes, occasionally parasitized a few Lygus lineolaris in alfalfa at one location. Two other mirids, Stenotus binotatus and Megaloceroea recticornis, both accidently-introduced grass-feeding species, were not parasitized by native or introduced species. It is noteworthy that the effective host ranges of all the parasites in alfalfa-grass fields were limited: four of the five parasite species significantly parasitized only one of the seven mirid species, and the other parasite significantly parasitized only two mirids. Activity of the four common parasites was correlated with the mirids' host plant: three species principally parasitized alfalfa-feeding mirids, and one species principally parasitized grass-feeding mirids.     

Life cycles of two isopora species in the canary, Serinus canarius Linnaeus

The endogenous stages of Isospora serini Arog?o and Isospora canaria Box are described from experimentally infected canaries, Serinus canarius Linnaeus. Unlike other Coccidia, the first part of the I. serini life cycle takes place in mononuclear phagocytes. Five asexual generations are described from this cell type; 2 additional asexual generations and the sexual stages take place in the intestinal epithelium. Isospora canaria, on the other hand, has a conventional coccidian life cycle in that all of the endogenous stages are in the epithelium of the small intestine, with 3 asexual generations and the sexual generation described in the duodenal epithelium. The 2 species differ in their position relative to the nucleus of the intestinal epithelial cell. Isospora serini is usually on the lumenal side of the nucleus while I. canaria is below the nucleus, toward the basement membrane. The prepatent period is 4-5 days for I. canaria and 9-10 days for I. serini. Patency lasts for 11-13 days in I. canaria infections, but duration of oocyst output is more chronic in I. serini infections, persisting for as long as 231 days. Both species have a diurnal periodicity of oocyst discharge which occurs in late afternoon and evening.     

Development of Caryospora bigenetica n. sp. (Apicomplexa,Eimeriidae) in Rattlesnakes and Laboratory Mice1

During a survey of the coccidian parasites of reptiles from Iowa, three specimens of Crotalus horridus L., the Timber Rattlesnake, and one of Sistrurus catenatus (Rafinesque), the Massasauga Rattlesnake, were found to be passing oocysts of a Caryospora, here described as C. bigenetica n. sp. Since these snakes (family Crotalidae) are known to subsist mainly on small mammals, oocysts from one of the Timber Rattlesnakes were fed to laboratory white mice (Mus musculus L.) to determine if mammals might be involved as alternate hosts in the life cycle. At necropsy, tissues of the tongue and dermis of the mice revealed a sequence of stages which included mature male and female gamonts, fully sporulated sporocysts, “excysted” sporozoites, and “resting” sporozoites that lay individually in solitary, cyst-like host cells termed “caryocysts.” A coccidia-free Massasauga that was fed an infected mouse, at a time when caryocysts in the mouse would have been present, later passed oocysts similar to those of the original inoculum. These results, along with the discovery of endogenous stages (asexual and sexual) in the intestine of the Timber Rattlesnake and the experimentally infected Massasauga, suggest that this parasite has a heteroxenous life cycle pattern, with sexual stages occurring both in the ophidian and the mammalian hosts.     

Minchinia armoricana sp. nov. (Haplosporida), a parasite of the European flat oyster, Ostrea edulis

Examination of European flat oysters, Ostrea edulis, from the Dutch oyster culture, but originating in France, revealed a new disease due to a protistan parasite. Light and electron microscope studies revealed that the parasite belongs to the haplosporidan genus Minchinia. Since comparison with other Minchinia spp. indicate that it is new, the name Minchinia armoricana nov. sp. is proposed. Thus far, the parasite was very rare; only two diseased oysters were observed among ca. 3700 specimens examined histologically during a 2.5-year period. The diseased oysters showed macroscopically a peculiar brown discoloration and microscopically many sporocysts with spores in the connective tissue. Beside the two diseased oysters, another one was observed with an infection of unidentified plasmodial stages in the connective tissue. These may be developmental stages of the new species M. armoricana.     

14C uptake by Schistosoma mansoni from Biomphalaria glabrata exposed to 14C-glucose

Exposure of Biomphalaria glabrata infected with Schistosoma mansoni to ¹⁴C-glucose results in a greater uptake of original total snail label by the parasitized digestive gland-gonad, site of the developing daughter sporocysts and cercariae, than by the digestive gland-gonad of control animals. As a consequence of this greater uptake by the infected digestive gland-gonad, the albumen gland and remainder of the carcass of parasitized snails receive less label than do those areas in normal snails. Emergence of cercariae from the snail and daughter sporocyst mass account for a diversion of 12.6% of original total label from the infected snail itself. This diversion of label from the snail to the parasite may explain carbohydrate depletion in parasitized snails.