In vitro development of Cryptosporidium parvum in serum-free media.

AIM: To compare commercially available serum-free media with common, standard, growth medium for their ability to support growth of Cryptosporidium parvum in HCT-8 cell cultures. METHODS AND RESULTS: Twelve serum-free media formulations with or without additional supplements were tested against a standard growth medium containing 2% FBS in HCT-8 cell cultures. After a 48-h incubation period, the level of parasite development was determined by ELISA. The extent of development in the serum-free media was determined as a percentage of infections compared with those obtained using a standard growth medium. CONCLUSIONS: Several of the serum-free media formulations, which included MDCK, UltraMDCK, PC-1, UltraCHO and UltraCulture, compared favourably with a traditional, standard growth medium. Moreover, increasing FBS concentrations to 10% actually resulted in an overall decrease in development in many cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Several serum-free medium formulations are available which allow development of C. parvumin vitro at levels comparable with standard media employing FBS. These serum-free media are particularly useful for applications, which may require a more defined medium without the presence of FBS. Moreover, the elimination of FBS as a variable allows investigators the ability to more closely regulate their experimental systems when growing C. parvum in cell cultures.

Redescription of Neospora caninum and its differentiation from related coccidia.

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

Evaluation of in vitro activity of aurones and related compounds against Cryptosporidium parvum.

The efficacy of a series of aurones, auronols and 4-methoxy-alpha-pyrones has been screened for the ability to inhibit the intracellular growth of the parasitic protist Cryptosporidium parvum using an in vitro enzyme linked immunosorbent assay (ELISA). All aurones of this series were active at 25 to 100 microM. 10 of 19 aurones inhibited the intracellular growth of C. parvum by > 90 % with moderate to no toxicity. The most active of these was 3',4',6-trihydroxy-2-[phenylmethylene]-3(2H)-benzofuranone.

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 Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) from the Bali (Rothschild's) mynah Leucopsar rothschildi (Passeriformes: Sturnidae), and comments concerning the genera Atoxoplasma Garnham, 1950 and Isospora.

A new species of isosporan (Apicomplexa: Eimeriidae) is reported from the Bali mynah Leucopsar rothschildi (Passeriformes: Sturnidae). Oöcysts of Isospora rothschildi n. sp. are spherical to subspherical, 22.3 x 21.6 (20-26 x 19-23) microm, with a shape index (length/width) of 1.03 (1.00-1.15). A micropyle and oöcyst residuum are absent, but one to many polar granules are present. Sporocysts are ovoidal, 15.9 x 10.6 (15-18 x 9.5-11) microm, with Stieda and substieda bodies and a shape index of 1.50 (1.39-1.65). Each sporocyst contains an ellipsoidal sporocyst residuum, 8.0 x 5.8 (6-11 x 5-8), and each sporozoite contains two refractile bodies. No correlation was found between the presence of coccidian oöcysts in the faeces of some of the birds and Atoxoplasma in blood smears.

A new species of Eimeria (Apicomplexa, Eimeriidae) from the weedy sea dragon Phyllopteryx taeniolatus (Osteichthyes: Syngnathidae).

A new species of intestinal coccidian is described from the weedy or common sea dragon Phyllopteryx taeniolatus housed at the New England Aquarium in Boston and at the Shedd Aquarium in Chicago, USA. Live oocysts of Eimeria phyllopterycis sp. n. are spherical, 30.9 (28.0-34.4) microm, with a thin, single-layered wall. Both a micropyle and oocyst residuum are absent and a large polar granule is sometimes present. Sporocysts are ellipsoidal and elongate, 24.3 x 10.4 (23.4-25.6 x 9.2-11.2) microm, with Stieda and substieda bodies; shape index (length/width) 2.33 (2.14-2.70). A sporocyst residuum is present, consisting of numerous granules of various sizes. Sporozoites each possess 3 refractile bodies. Preliminary evidence suggests that the coccidian may affect the health of sea dragons; however, it could not be determined whether this parasite caused significant morbidity or mortality.

Epidemiology of Cryptosporidium: transmission, detection and identification.

There are 10 valid species of Cryptosporidium and perhaps other cryptic species hidden under the umbrella of Cryptosporidium parvum. The oocyst stage is of primary importance for the dispersal, survival, and infectivity of the parasite and is of major importance for detection and identification. Because most oocysts measure 4-6 microm, appear nearly spherical, and have obscure internal structures, there are few or no morphometric features to differentiate species and in vitro cultivation does not provide differential data as for bacteria. Consequently, we rely on a combination of data from three tools: morphometrics, molecular techniques, and host specificity. Of 152 species of mammals reported to be infected with C. parvum or an indistinguishable organism, very few oocysts have ever been examined using more than one of these tools. This paper reviews the valid species of Cryptosporidium, their hosts and morphometrics; the reported hosts for the human pathogen, C. parvum; the mechanisms of transmission; the drinking water, recreational water, and food-borne outbreaks resulting from infection with C. parvum; and the microscopic, immunological, and molecular methods used to detect and identify species and genotypes.

Cloning and molecular characterization of a gene encoding a Cryptosporidium parvum putative 20S proteasome beta1-type subunit.

A DNA sequence composed of 1281 nucleotides (nt) consisting of a single open reading frame (ORF) encoding a putative 20S proteasome beta1-type subunit was isolated from clones derived from genomic libraries constructed from the KSU-1 isolate of Cryptosporidium parvum. Southern blot analysis suggested that the sequenced DNA exists in the C. parvum genome as a single copy; transcription was verified through reverse transcription-polymerase chain reaction (RT-PCR) performed on total RNA isolated from C. parvum sporozoites. The predicted protein consists of 210 amino acids (aa), contains characteristic amino acids common to all proteasomal subunits, and shares stronger similarity to the beta1-type subunit of yeast than to other types of beta-subunits.

Association of RNA polymerase complexes of the parasitic protozoan Cryptosporidium parvum with virus-like particles: heterogeneous system.

RNA polymerase complexes were purified from Cryptosporidium parvum, a parasitic protozoan known to infect many species of mammals including humans. Western blot analysis revealed the association of the complexes with two different proteins, encoded by large and small segments of viral double-stranded RNAs. Each complex was found to contain only double-stranded RNA, both double- and single-stranded RNA, or only single-stranded RNA. Maximum RNA-dependent RNA polymerase activity was observed within the complexes containing both double- and single-stranded RNAs. These complexes possessed both transcriptase and replicase polymerase activities. Virus-like particles with a diameter of 31 nm were copurified with RNA polymerase complexes, and buoyant density and polymerase studies suggest that C. parvum harbors a putative double-stranded RNA virus which separately encapsidates the large and small RNA segments. The mechanism of replication and other characteristics of this virus are similar to those of the viruses of the family Partitiviridae, previously identified only in fungi and plants.

Molecular and phylogenetic analysis of Cryptosporidium muris from various hosts.

Isolates of Cryptosporidium muris and C. serpentis were characterized from different hosts using nucleotide sequence analysis of the rDNA 18S and ITS1 regions, and the heat-shock (HSP-70) gene. Phylogenetic analysis confirmed preliminary evidence that C. muris is not a uniform species. Two distinct genotypes were identified within C. muris; (1) C. muris genotype A; comprising bovine and camel isolates of C. muris from different geographical locations, and (2) C. muris genotype B comprising C. muris isolates from mice, a hamster, a rock hyrax and a camel from the same enclosure. These 2 genotypes may represent separate species but further biological and molecular studies are required for confirmation.

Activity of decoquinate against Cryptosporidium parvum in cell cultures and neonatal mice.

Cryptosporidium parvum is an apicomplexan parasite that is an important cause of diarrhea in neonatal calves and humans. No treatment is currently available for neonatal calves. We have recently learned from colleagues in the pharmaceutical industry that dairy practitioners are sometimes using decoquinate for the treatment of neonatal bovine cryptosporidiosis. Therefore, the present study was undertaken to determine whether the clinical observations in calves can be substantiated by laboratory investigation. Oocysts of the KSU-1 isolate of C. parvum were used to infect human ileocecal epithelial cells in vitro to measure the efficacy of treatment using an ELISA based assay. No activity was observed at 10 or 50microM decoquinate, but at 100microM an 8% inhibition of development was seen. Oocysts of the AUCp-1 isolate of C. parvum were then used to infect suckling mice. The numbers of oocysts observed in suckling mice treated with 2.5 or 5.0mg/kg decoquinate were not significantly different from untreated control suckling mice (p0.05). The results of our study suggest that decoquinate should have little efficacy for treatment of neonatal bovine cryptosporidiosis if administered once per day and that any clinical improvement observed in treated calves may be due to factors unrelated to decoquinate's effect on C. parvum.

Presence of double-stranded RNAs in human and calf isolates of Cryptosporidium parvum.

We examined the occurrence of 2 virus-like double-stranded (ds)RNAs in human and calf isolates of Cryptosporidium parvum senso latu and other microorganisms, including 7 other members of the genus. A total of 32 isolates of C. parium, 16 from humans (5 from acquired immune deficiency syndrome patients) and 16 from calves, were analyzed. Ethidium bromide staining, or Northern blot analysis, or reverse transcription/polymerase chain reaction, or all 3 methods, revealed that both genotype 1 and genotype 2 isolates of C. parvum possessed these dsRNAs. No other Cryptosporidium spp. or other organisms examined possessed these dsRNAs. Comparison analysis of partial cDNA sequences of dsRNAs from human and calf isolates revealed a high degree of similarity (>92% and >93% identical nucleotides for large and small dsRNAs, respectively). Slight, consistent differences in nucleotide sequences could be seen at select sites and were associated with an isolate being either genotype 1 or 2. Because of the widespread distribution of the dsRNAs, the similarity of these molecules between isolates, and high host specificity, these nucleic acids may prove to represent species-specific molecular markers for C. parvum. Evidence also suggests that the dsRNA can be utilized for molecular genotyping of C. parvum.

Molecular analysis of a Type I fatty acid synthase in Cryptosporidium parvum.

We report here the molecular analysis of a Type I fatty acid synthase in the apicomplexan Cryptosporidium parvum (CpFAS1). The CpFAS1 gene encodes a multifunctional polypeptide of 8243 amino acids that contains 21 enzymatic domains. This CpFAS1 structure is distinct from that of mammalian Type I FAS, which contains only seven enzymatic domains. The CpFAS1 domains are organized into: (i) a starter unit consisting of a fatty acid ligase and an acyl carrier protein; (ii) three modules, each containing a complete set of six enzymes (acyl transferase, ketoacyl synthase, ketoacyl reductase, dehydrase, enoyl reductase, and acyl carrier protein) for the elongation of fatty acid C2-units; and (iii) a terminating domain whose function is as yet unknown. The CpFAS1 gene is expressed throughout the life cycle of C. parvum, since its transcripts and protein were detected by RT-PCR and immunofluorescent localization, respectively. This cytosolic Type I CpFAS1 differs from the organellar Type II FAS enzymes identified from Toxoplasma gondii and Plasmodium falciparum which are targetted to the apicoplast, and are sensitive to inhibition by thiolactomycin. That the discovery of CpFAS1 may provide a new biosynthetic pathway for drug development against cryptosporidiosis, is indicated by the efficacy of the FAS inhibitor cerulenin on the growth of C. parvum in vitro.

Cryptosporidium andersoni n. sp. (Apicomplexa: Cryptosporiidae) from cattle, Bos taurus.

A new species of Cryptosporidium is described from the feces of domestic cattle, Bos taurus. Oocysts are structurally similar to those of Cryptosporidium muris described from mice but are larger than those of Cryptosporidium parvum. Oocysts of the new species are ellipsoidal, lack sporocysts, and measure 7.4 x 5.5 microm (range, 6.0-8.1 by 5.0-6.5 microm). The length to width ratio is 1.35 (range, 1.07-1.50). The colorless oocyst wall is < 1 microm thick, lacks a micropyle, and possesses a longitudinal suture at one pole. A polar granule is absent, whereas an oocyst residuum is present. Oocysts were passed fully sporulated and are not infectious to outbred, inbred immunocompetent or immunodeficient mice, chickens or goats. Recent molecular analyses of the rDNA 18S and ITS1 regions and heat-shock protein 70 (HSP-70) genes demonstrate this species to be distinct from C. muris infecting rodents. Based on transmission studies and molecular data, we consider the large form of Cryptosporidium infecting the abomasum of cattle to be a new species and have proposed the name Cryptosporidium andersoni n. sp. for this parasite.

A structural study of the Neospora caninum oocyst.

Oocysts of Neospora caninum were collected from the faeces of a dog fed mouse brains containing tissue cysts of the NC-beef strain of N. caninum. Sporulated oocysts were spherical to subspherical, and were 11.7x11.3 microm. The length/width ratio was 1.04. No micropyle or oocyst residuum was present. Polar granules were not present, although occasionally tiny refractile granules were seen among sporocysts. Sporocysts were ellipsoidal, did not contain a Stieda body, and were 8.4x6.1 microm. The length/width ratio for sporocysts was 1.37. A spherical or subspherical sporocyst residuum was present, and was composed of a cluster of small, compact granules of 4.3x3.9 microm, or was represented by many dispersed granules of similar size. Sporozoites were elongate and 7.0-8.0x2.0-3.0 microm in situ. No refractile bodies were present and the nucleus was centrally or slightly posteriorly located. The features of the oocyst of N. caninum are similar to those of Hammondia heydorni oocysts from dog faeces and Toxoplasma gondii and Hammondia hammondi oocysts from cat faeces.

Receptor/ligand interactions between Cryptosporidium parvum and the surface of the host cell.

The ability of membrane antigens on sporozoites of the intestinal pathogen, Cryptosporidium parvum, to bind host cell surface antigens was investigated. A novel membrane-associated protein of approximately 47 kDa, designated CP47, was found to possess significant binding affinity for the surface of both human and animal ileal cells. This protein was purified by a combination of anion-exchange chromatography on FPLC and immunoaffinity chromatography. Purified CP47 demonstrated competitive binding with parasite-associated membrane antigens to membranes of HCT-8 and ileal cells in a dose-dependent manner. Furthermore, the binding activity of CP47 was found to be Mn2+-sensitive, and was completely inhibited in the presence of 10 mM MnCl2. These results were consistent with earlier findings demonstrating the inhibitory effect of Mn2+ ions on Cryptosporidium infection both in vitro and in vivo (Nesterenko et al., Biol. Trace Elem. Res. 56 (1997) 243-253). Immunoelectron microscopy using gold-conjugated antibodies revealed CP47 to be localized at the apical end of the sporozoites. A single protein with an electrophoretic mobility of 57 kDa was purified from host cell membranes using CP47-Affigel. Similarly, affinity purification of this protein was abrogated in the presence of Mn2+. These data suggest that a novel parasite protein, CP47, may play an important role in sporozoite/host cell attachment.

Reconstitution of a bacterial/plant polyamine biosynthesis pathway in Saccharomyces cerevisiae.

Polyamine synthesis in most organisms is initiated by the decarboxylation of ornithine to form putrescine via ornithine decarboxylase (ODC). Plants, some bacteria and some fungi and protozoa generate putrescine from arginine, via arginine decarboxylase (ADC) and agmatine ureohydrolase (AUH) or agmatine iminohydrolase. A polyamine-requiring strain of Saccharomyces cerevisiae with a mutation in the gene encoding ODC was transformed with plasmids bearing genes encoding Escherichia coli ADC and AUH. Transformants regained the ability to grow in the absence of exogenous polyamines and contained enzyme activities consistent with the presence of both prokaryotic enzymes. Similar results were obtained when a plasmid containing a gene encoding oat (Avena sativa L.) ADC was substituted for the E. coli gene. These data demonstrate the successful complementation of a yeast biosynthetic polyamine synthesis defect by genes encoding an alternative pathway found in bacteria; they also show that plant ADC can substitute for the bacterial enzyme in this pathway. The recombinant yeast provides a tool for the study of the functional properties of these enzymes and for discovery of compounds that specifically inhibit this pathway.

Descriptions of two new species of coccidia (Protozoa: Eimeriidae) and redescriptions of Eimeria ivensae and Eimeria odocoilei from captive white-tailed deer, Odocoileus virginianus.

Two new species of Eimeria were observed in the feces of captive white-tailed deer fawns, Odocoileus virginianus, from Alabama. The first new species was easily recognized because of its small size. Sporulated oocysts are spherical, average 10.2 by 10.0 microm, and lack a micropyle and oocyst residuum. Oocysts contain a polar granule and elongate-ellipsoidal sporocysts that measure 6.7 by 3.1 microm. A Stieda body is present on the sporocysts. Oocysts were observed in the feces, and gamonts and oocysts were observed in the jejunum of a month-old fawn from Minnesota that died from enteritis due to this species. Oocysts of this small species were present in 5 of the 6 white-tailed deer fawns examined. Oocysts of a second new species are ellipsoidal and average 29.5 by 24.6 microm. The oocyst encloses an oocyst residuum, polar granule, and elongate-ellipsoidal sporocysts that average 16.0 by 9.0 microm. A Stieda body and substieda body are present on the sporocysts. Oocysts of the second new species were present in 4 of the 6 white-tailed deer fawns examined. Oocysts of E. ivensae are ovoid or flask-like and average 32.0 by 20.8 microm. The oocyst wall is rough, contains a micropyle, and encloses elongate-ellipsoidal sporocysts that average 16.5 by 7.8 microm. A Stieda body is present on the sporocysts. Oocysts of E. ivensae were present in 4 of the 6 white-tailed deer fawns. Oocysts of E. odocoilei are spherical or slightly subspherical and measure 24.7 by 21.5 microm. They enclose ovoid sporocysts that average 12.7 by 8.8 microm. A Stieda and substieda body are present on the sporocyst. Oocysts of E. odocoilei were present in 4 of the 6 white-tailed deer fawns.

Sequence of the gene encoding hsp90e from Cryptosporidium parvum.

A composite 2364 nt DNA sequence with an open reading frame (ORF) encoding an endoplasmic reticulum-associated heat shock protein 90 (CpHsp90e) was determined from clones isolated from genomic libraries constructed from the KSU-1 isolate of Cryptosporidium parvum. Transcription was verified by isolation of a clone from a cDNA library with a similar restriction map to that observed with genomic DNA. The predicted protein consists of 787 amino acids, has a predicted molecular size of 89.2 kDa, and was found to share strong homology with other endoplasmic reticulum-associated hsp90 proteins.