Naturally occurring Sarcocystis infection in domestic cats (Felis catus).

Equine protozoal myeloencephalitis is an important neurological disease of horses in the United States. Consequently, there is an active research effort to identify hosts associated with the primary causative agent, Sarcocystis neurona. The purpose of this study was to determine whether the domestic cat (Felis catus) is a natural host for S. neurona. Muscle sections from 50 primarily free-roaming domestic cats were examined for the presence of sarcocysts. Serum from cats in this group and another group of 50 free-roaming cats were evaluated for the presence of S. neurona antibody. Sarcocysts were found in five of 50 (10%) cats, and S. neurona antibody in five of 100 (5%) cats. Morphological, molecular (including ribosomal RNA genes), and biological characterisation of these sarcocysts showed that they were not S. neurona or S. neurona-like. Sarcocysts found in the cats were identified morphologically as Sarcocystis felis, a common parasite of wild felids. The life cycle of S. felis is not known, and prior to this study, no molecular marker for S. felis existed. Although cats were found to be infected with S. felis sarcocysts, serological data provided evidence of possible infection with S. neurona as well. Further work is needed to determine the role of the domestic cat in the life cycle of S. neurona.

Profiling the malaria genome: a gene survey of three species of malaria parasite with comparison to other apicomplexan species.

We have undertaken the first comparative pilot gene discovery analysis of approximately 25,000 random genomic and expressed sequence tags (ESTs) from three species of Plasmodium, the infectious agent that causes malaria. A total of 5482 genome survey sequences (GSSs) and 5582 ESTs were generated from mung bean nuclease (MBN) and cDNA libraries, respectively, of the ANKA line of the rodent malaria parasite Plasmodium berghei, and 10,874 GSSs generated from MBN libraries of the Salvador I and Belem lines of Plasmodium vivax, the most geographically wide-spread human malaria pathogen. These tags, together with 2438 Plasmodium falciparum sequences present in GenBank, were used to perform first-pass assembly and transcript reconstruction, and non-redundant consensus sequence datasets created. The datasets were compared against public protein databases and more than 1000 putative new Plasmodium proteins identified based on sequence similarity. Homologs of previously characterized Plasmodium genes were also identified, increasing the number of P. vivax and P. berghei sequences in public databases at least 10-fold. Comparative studies with other species of Apicomplexa identified interesting homologs of possible therapeutic or diagnostic value. A gene prediction program, Phat, was used to predict probable open reading frames for proteins in all three datasets. Predicted and non-redundant BLAST-matched proteins were submitted to InterPro, an integrated database of protein domains, signatures and families, for functional classification. Thus a partial predicted proteome was created for each species. This first comparative analysis of Plasmodium protein coding sequences represents a valuable resource for further studies on the biology of this important pathogen.

The striped skunk (Mephitis mephitis) is an intermediate host for Sarcocystis neurona.

Striped skunks, initially negative for antibodies to Sarcocystis neurona, formed sarcocysts in skeletal muscles after inoculation with S. neurona sporocysts collected from a naturally infected Virginia opossum (Didelphis virginiana). Skunks developed antibodies to S. neurona by immunoblot and muscles containing sarcocysts were fed to laboratory-reared opossums which then shed sporulated Sarcocystis sporocysts in their faeces. Mean dimensions for sporocysts were 11.0 x 7.5 microm and each contained four sporozoites and a residuum. Sarcocysts from skunks and sporocysts from opossums fed infected skunk muscle were identified as S. neurona using PCR and DNA sequence analysis. A 2-month-old, S. neurona-naive pony foal was orally inoculated with 5 x 10(5) sporocysts. Commercial immunoblot for antibodies to S. neurona performed using CSF collected from the inoculated pony was low positive at 4 weeks p.i., positive at 6 weeks p.i., and strong positive at 8 weeks p.i. Gamma-interferon gene knockout mice inoculated with skunk/opossum derived sporocysts developed serum antibodies to S. neurona and clinical neurologic disease. Merozoites of S. neurona present in the lung, cerebrum, and cerebellum of mice were detected by immunohistochemistry using polyclonal antibodies to S. neurona. Based on the results of this study, the striped skunk is an intermediate host of S. neurona.

Biomagnetic separation of contaminating host leukocytes from plasmodium-infected erythrocytes.

Carlton, J. M-R., Yowell, C. A., Sturrock, K. A., and Dame, J. B. 2001. Biomagnetic separation of contaminating host leukocytes from Plasmodium-infected erythrocytes. Experimental Parasitology 97, 111-114.

Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystis falcatula.

Studies designed to investigate the causative agent of equine protozoal myeloencephalitis and its life cycle have been hampered by the marked similarity of Sarcocystis neurona to other Sarcocystis spp. present in the same definitive host. Random-amplified polymorphic DNA techniques were used to amplify DNA from isolates of S. neurona and Sarcocystis falcatula. DNA sequence analysis of polymerase chain reaction (PCR) products was then used to design PCR primers to amplify specific Sarcocystis spp. DNA products. The ribosomal RNA internal transcribed spacer was also amplified and compared between S. neurona and S. falcatula. Useful sequence heterogeneity between the 2 organisms was identified, creating potential markers to distinguish these Sarcocystis spp. These markers were used to characterize Sarcocystis isolates from opossum (Didelphis virginiana) feces. Our data suggest that S. neurona and S. falcatula can be differentiated with these markers and that multiple Sarcocystis spp., including S. neurona and S. falcatula, are shed by opossums.

Are Sarcocystis neurona and Sarcocystis falcatula synonymous? A horse infection challenge.

Equine protozoal myeloencephalitis (EPM) is a debilitating neurologic disease of the horse. The causative agent. Sarcocystis neurona, has been suggested to be synonymous with Sarcocystis falcatula, implying a role for birds as intermediate hosts. To test this hypothesis, opossums (Didelphis virginiana) were fed muscles containing S. falcatula sarcocysts from naturally infected brown-headed cowbirds (Molothrus ater). Ten horses were tested extensively to ensure no previous exposure to S. neurona and were quarantined for 14 days, and then 5 of the horses were each administered 10(6) S. falcatula sporocysts collected from laboratory opossums. Over a 12-wk period, 4 challenged horses remained clinically normal and all tests for S. neurona antibody and DNA in serum and cerebrospinal fluid were negative. Rechallenge of the 4 seronegative horses had identical results. Although 1 horse developed EPM, presence of S. neurona antibody prior to challenge strongly indicated that infection occurred before sporocyst administration. Viability of sporocysts was confirmed by observing excystation in equine bile in vitro and by successful infection of naive brown-headed cowbirds. These data suggest that S. falcatula and S. neurona are not synonymous. One defining distinction is the apparent inability of S. falcatula to infect horses, in contrast to S. neurona, which was named when cultured from equine spinal cord.

Substitution bias, rapid saturation, and the use of mtDNA for nematode systematics.

Only relatively recently have researchers turned to molecular methods for nematode phylogeny reconstruction. Thus, we lack the extensive literature on evolutionary patterns and phylogenetic usefulness of different DNA regions for nematodes that exists for other taxa. Here, we examine the usefulness of mtDNA for nematode phylogeny reconstruction and provide data that can be used for a priori character weighting or for parameter specification in models of sequence evolution. We estimated the substitution pattern for the mitochondrial ND4 gene from intraspecific comparisons in four species of parasitic nematodes from the family Trichostrongylidae (38-50 sequences per species). The resulting pattern suggests a strong mutational bias toward A and T, and a lower transition/transversion ratio than is typically observed in other taxa. We also present information on the relative rates of substitution at first, second, and third codon positions and on relative rates of saturation of different types of substitutions in comparisons ranging from intraspecific to interordinal. Silent sites saturate extremely quickly, presumably owing to the substitution bias and, perhaps, to an accelerated mutation rate. Results emphasize the importance of using only the most closely related sequences in order to infer patterns of substitution accurately for nematodes or for other taxa having strongly composition-biased DNA. ND4 also shows high amino acid polymorphism at both the intra- and interspecific levels, and in higher level comparisons, there is evidence of saturation at variable amino acid sites. In general, we recommend using mtDNA coding genes only for phylogenetics of relatively closely related nematode species and, even then, using only nonsynonymous substitutions and the more conserved mitochondrial genes (e.g., cytochrome oxidases). On the other hand, the high substitution rate in genes such as ND4 should make them excellent for population genetics studies, identifying cryptic species, and resolving relationships among closely related congeners when other markers show insufficient variation.

The Plasmodium falciparum translationally controlled tumor protein homolog and its reaction with the antimalarial drug artemisinin.

Artemisinin and its derivatives are important new antimalarial drugs. When Plasmodium falciparum-infected erythrocytes are incubated with [10-3H]dihydroartemisinin, several malaria-specific proteins become labeled. One of these proteins is the P. falciparum translationally controlled tumor protein (TCTP) homolog. In vitro, dihydroartemisinin reacts covalently with recombinant TCTP in the presence of hemin. The association between drug and protein increases with increasing drug concentration, plateauing at approximately 1 drug/TCTP molecule. By Scatchard analysis, there appear to be 2 hemin binding sites on TCTP with dissociation constants of approximately 18 microM. When the single cysteine moiety is blocked by pretreatment with iodoacetamide, hemin binding is not affected, whereas drug binding is reduced by two-thirds. Thus, TCTP reacts with artemisinin in situ and in vitro in the presence of hemin and appears to bind to hemin. The function of the malarial TCTP and the role of this reaction in the mechanism of action of artemisinin await elucidation.

Plasmodium falciparum, P. vivax, and P. malariae: a comparison of the active site properties of plasmepsins cloned and expressed from three different species of the malaria parasite.

Aspartic endopeptidases (plasmepsins) have been implicated in the degradation of hemoglobin in the erythrocytic stage of infection by Plasmodium falciparum. To develop new targets for drug development, these enzymes have been isolated and cloned, expressed, and studied structurally and enzymatically. This study expands this approach to two other species of the malarial parasite, P. vivax and P. malariae. Expression of cloned genes from these species, utilizing methodology similar to that employed in the original reports on the enzymes from P. falciparum, has provided active enzymes for analysis by kinetic methods. We describe here studies of three enzymes, plasmepsin II from P. falciparum, and one plasmepsin from both P. vivax and P. malariae, utilizing oligopeptide substrates and low-molecular-weight inhibitors. These analyses provide new information on the properties of distinct regions of the active site cleft; such data can suggest strategies for drug design to inhibit these critical enzymes of the parasite.

Haemonchus placei and Haemonchus contortus are distinct species based on mtDNA evidence.

Debates continue over the extent to which the parasitic trichostrongylids Haemonchus placei and Haemonchus contortus hybridise in nature, and whether they deserve species status. Mitochondrial ND4 gene sequences from individuals of each putative species collected from populations around the United States indicate that the two species are highly differentiated at the mtDNA level. Furthermore, there was no evidence of introgressive hybridisation occurring in wild populations.

Sarcocystis falcatula from passerine and psittacine birds: synonymy with Sarcocystis neurona, agent of equine protozoal myeloencephalitis.

Equine protozoal myeloencephalitis (EPM) is a neurologic disease of horses caused by Sarcocystis neurona. The horse is a dead-end host for S. neurona and the definitive and intermediate hosts have not previously been identified. We hypothesized that S. neurona is actually Sarcocystis falcatula, a parasite that cycles in nature between Virginia opossums (Didelphis virginiana) and any of a variety of avian intermediate hosts. We extracted DNA from S. falcatula sarcocysts in the muscle of a brown-headed cowbird (Molothrus ater) and from schizonts in a fixed specimen of lung from a Moluccan cockatoo (Cacatua moluccensis). Three segments of the small subunit ribosomal RNA (SSURNA) gene, containing a total of 742 nucleotides, were amplified by the polymerase chain reaction, sequenced, and compared with the SSURNA sequence from two isolates of S. neurona. The S. falcatula sequence was identical to the sequence of the S. neurona isolate UCD-1 and differed in only 3 positions from isolate SN5. Recent evidence, also based on SSURNA sequences, implicates the opossum as the definitive host of S. neurona. Based on the SSURNA gene sequences S. falcatula and S. neurona are synonymous, thus the parasite cycles between opossums and birds maintaining a reservoir of the organism from which horses can be infected.

Host movement and the genetic structure of populations of parasitic nematodes.

Mitochondrial DNA (mtDNA) sequence data were used to compare the population genetic structures of five species of parasitic nematodes from three different hosts: Ostertagia ostertagi and Haemonchus placei from cattle, H. contortus and Teladorsagia circumcincta from sheep, and Mazamastrongylus odocoilei from white-tailed deer. The parasites of sheep and cattle showed a pattern consistent with high gene flow among populations. The parasite of deer showed a pattern of substantial population subdivision and isolation by distance. It appears that host movement is an important determinant of population genetic structure in these nematodes. High gene flow in the parasites of livestock also indicates great opportunity for the spread of rare alleles that confer resistance to anthelmintic drugs. All species, including the parasite of deer, had unusually high within-population diversities (averages of 0.019-0.027 substitutions per site between pairs of individuals from the same population). Large effective population sizes (Ne), perhaps in combination with rapid mtDNA evolution, appear to be the most likely explanation for these high within-population diversities.

Sequence, expression and modeled structure of an aspartic proteinase from the human malaria parasite Plasmodium falciparum.

A clone encoding the aspartic proteinase (PFAPD) from Plasmodium falciparum strain HB3 was obtained during the course of a project designed to sequence and identify the protein coding regions of the parasite's genome. The protein encoded by the clone contains a sequence identical to the N-terminal sequence determined for an aspartic proteinase isolated from the digestive vacuole of P. falciparum and demonstrated to participate in the hemoglobin digestive pathway (D. Goldberg, personal communication). The translated polypeptide sequence encompasses a number of features characteristic of aspartic proteinases, having > 30% identity and > 50% similarity overall to human cathepsin D, cathepsin E and renin. A model of the three-dimensional structure of PFAPD was constructed using rule-based procedures. This confirms that the primary sequence may be folded as a single chain into a three dimensional structure closely resembling those of other known aspartic proteinases. It includes a lengthy prosegment, two typical-hydrophobic-hydrophobic-Asp-Thr/Ser-Gly motifs and a tyrosine residue positioned in a beta-hairpin loop. The distribution of hydrophobic residues throughout the active site cleft is indicative of a likely preference for hydrophobic polypeptide substrates. The recombinant form of this enzyme expressed using the pGEX2T vector in Escherichia coli is active in digesting hemoglobin at acidic pH and in hydrolyzing a synthetic peptide corresponding to the putative initial cleavage site in hemoglobin. Activity is inhibited completely by pepstatin, confirming the identity of PFAPD as a member of the aspartic proteinase family. Specific mRNA for PFAPD is expressed in the erythrocytic stages of the life cycle.

Characterization of the rDNA unit and sequence analysis of the small subunit rRNA and 5.8S rRNA genes from Tritrichomonas foetus.

The ribosomal RNA gene unit of the protozoan parasite Tritrichomonas foetus has been cloned and analyzed. Southern blot analysis of the genomic DNA showed that the ribosomal RNA gene unit is organized as a tandem head to tail repeat with a unit length of 6 kb. By Northern analysis a primary transcript of 5.8 kb was detected. Copy number analysis showed the presence of 12 copies of the ribosomal RNA gene unit. The lengths of the small subunit ribosomal RNA and 5.8S ribosomal RNA are 1571 bp and 159 bp, respectively, as determined by sequence analysis. The T. foetus small subunit ribosomal RNA sequence is one of the shortest eukaryotic small subunit rRNA sequences, similar in length to those from 2 other amitochondrial protists. Although shorter than the majority of the eukaryotic small subunit ribosomal RNAs, this sequence maintains the primary and secondary structure common to all eukaryotic small subunit ribosomal RNA structures, while truncating sequences found within the eukaryotic variable regions. The length of the large subunit ribosomal RNA was measured at 2.5 kb.

Phylogenetic relationship of Cowdria ruminantium, agent of heartwater, to Anaplasma marginale and other members of the order Rickettsiales determined on the basis of 16S rRNA sequence.

The phylogenetic relationship between Cowdria ruminantium and representative members of the orders Rickettsiales and Chlamydiales has been examined on the basis of the sequence of the 16S rRNA. Phylogeny reconstruction by using both parsimony and distance methods supports the conclusion that C. ruminantium is closely related to the Rickettsiales and in particular to the family Anaplasmataceae. A signature of nine base substitutions delineated the linkage of Anaplasma marginale with C. ruminantium and differentiated these two species from the 45 other members of the alpha group of Proteobacteria examined, and five of these base substitutions were unique among all members of the class Proteobacteria examined to date.

Suitability of mitochondrial DNA for assaying interindividual genetic variation in small helminths.

The current paucity of data on the genetic structure of parasitic helminth populations results partly from the lack of a suitable molecular technique for assigning genotypes to small individuals. This report describes the cloning of the mitochondrial DNA (mtDNA) from a small parasitic nematode, Ostertagia ostertagi, and the potential use of this cloned mtDNA as a hybridization probe to detect genetic variation among individuals. By using cloned, homologous mtDNA, labeled to high specific activity, mtDNA restriction site variation can be assayed among individual O. ostertagi for at least 10 restriction enzymes.

Sequence microheterogeneity of the three small subunit ribosomal RNA genes of Babesia bigemina: expression in erythrocyte culture.

Three distinct ribosomal RNA (rRNA) transcription units (rDNA units), designated A, B and C, were identified in the intraerythrocytic protozoan parasite, Babesia bigemina. These rDNA units were cloned, and restriction maps were constructed showing the approximate location of the small and large rRNA coding regions. The arrangement of the genes in the genome and copy number analysis suggests the presence of a single copy of each rDNA unit per haploid genome. The complete nucleotide sequence of the small subunit rRNA coding region (1693 bp) and parts of the 5' and 3' flanking regions were determined for all three units. Units A and B have identical sequences, but unit C differs from units A and B at ten nucleotide positions, two in the small subunit rRNA coding region and four each in the adjacent 5' and 3' flanking regions. The differences in the coding region are confirmed in genomic DNA and RNA from two different isolates of B.bigemina. The RNA of both sequence types is transcribed in parasites from erythrocyte culture, however, the products of gene units A + B accumulate at a ratio of approximately 4:1 compared with the product of unit C.

Cloning and characterization of the ribosomal RNA gene repeat from Ostertagia ostertagi.

Clones of the rRNA genes of Ostertagia ostertagi were selected from a library prepared from the genomic DNA of adult worms of strain LA-2. A 13.4-kb insert in a clone, lambda OOR78, is comprised of one complete 7.5-kb rDNA unit and portions of two adjacent units. The rDNA unit is directly repeated in a head-to-tail fashion and represents approximately 0.9% of the total genomic DNA. This repeating unit appears to be the only long tandemly repeated sequence in the genome. Restriction enzyme recognition sites in the rDNAs of four strains of O. ostertagi were fully conserved with the exception of one PstI site present in the large rRNA gene which was absent from a proportion of the genes of the LA-2 strain. The rDNA of O. ostertagi is more similar to that of Caenorhabditis elegans in unit length and arrangement than to parasitic helminths previously examined.