Trichinella spiralis infected skeletal muscle cells arrest in G2/M and cease muscle gene expression.

Infection by Trichinella spiralis causes a variety of changes in skeletal muscle cells including the hypertrophy of nuclei and decreased expression of muscle specific proteins. Potential cellular processes leading to these changes were investigated. In synchronized muscle infections, [3H]thymidine was incorporated into infected cell nuclei from 2-5 days post infection. Labeled nuclei were stably integrated into the infected cell up to 60 days post infection and appear to originate from differentiated skeletal muscle nuclei present at the time of infection. These nuclei were further shown to contain a mean DNA content of approximately 4N, indicating that the [3H]thymidine uptake reflects DNA synthesis and subsequent long-term suspension of the infected cell in the cell cycle at G2/M. Associated with these changes, muscle specific gene transcripts were reduced to < 1- < 0.1% in the infected cell compared to normal muscle. Transcript levels of the muscle transcriptional regulatory factors myogenin, MyoD1, and Id were reduced to < 10, < 1, and increased approximately 250%, respectively, in the infected cell compared to normal muscle, indicating transcriptional inactivation of muscle genes. DNA synthesis in the infected cell may represent the initiation event which leads to expression of this infected cell phenotype.

Advances in the sequencing of the genome of the adenophorean nematode Trichinella spiralis.

The adenophorean nematodes are evolutionarily distant from other species in the phylum Nematoda. Interspecific comparisons of predicted proteins have supported such an ancient divergence. Accordingly, Trichinella spiralis represents a basal nematode representative for genome sequencing focused on gaining a deeper insight into the evolutionary biology of nematodes. In addition, molecular characteristics that are conserved across the phylum could be of great value for control strategies with broad application. In this review, we describe and summarize progress that has been made on the sequencing and analysis of the T. spiralis genome. The genome sequence was used in preliminary analyses for the investigation of specific questions relating to the biology of T. spiralis and, more generally, to parasitic nematodes. For instance, we evaluated an unusually large DNase II-like protein family, predicted proteins of prospective interest in the parasite-host muscle cell interaction, anthelmintic targets and prospective intestinal genes, the encoded proteins (potentially) linked to immunological control against other nematodes. The results are discussed in relation to characteristics that are broadly conserved among evolutionary distant nematodes. The results lead to expectations that this genome sequence will contribute to advances in research on T. spiralis and other parasitic nematodes.

Diverse patterns of expression of the cytochrome c oxidase subunit I gene and unassigned reading frames 4 and 5 during the life cycle of Trypanosoma brucei.

Transcription of a maxicircle segment from Trypanosoma brucei 164 that contains nucleotide (nt) sequences corresponding to cytochrome c oxidase subunit I (COI) and unassigned reading frames (URFs) 4 and 5 of other mitochondrial systems was investigated. Two major transcripts that differ in size by ca. 200 nt map to each of the COI and URF4 genes, while a single major transcript maps to URF5. In total RNA, the larger COI transcript is more abundant in procyclic forms (PFs) than in bloodstream forms (BFs), the smaller COI and both URF4 transcripts have similar abundances in both forms, and the single URF5 transcript is more abundant in BF than PF. These patterns of expression differ in poly(A)+ RNA as a result of a higher proportion of poly(A)+ mitochondrial transcripts in PFs than in BFs. In addition, small (300- to 500-nt) RNAs that are transcribed from C-rich sequences located between putative protein-coding genes also exhibit diverse patterns of expression between life cycle stages and differences in polyadenylation in PFs compared with BFs. These observations suggest that multiple processes regulate the differential expression of mitochondrial genes in T. brucei.

Conservation of kinetoplastid minicircle characteristics without nucleotide sequence conservation.

The nucleotide sequence and restriction fragment electrophoretic mobility of four minicircles from the kinetoplast DNA of Trypanosoma brucei were determined. Each minicircle possesses an approximately 130 base pair conserved sequence which occurs in other African trypanosome minicircles and contains a 13 base pair sequence that is conserved among kinetoplastid genera [Kidane et al. (1984) Gene 27, 265-277]. A sequence located adjacent to the conserved sequence conserves purine versus pyrimidine strand bias, but not nucleotide sequence, and contains periodic oligo(dA) tracts. Minicircle fragments containing the conserved sequence and adjacent segment exhibited anomalous electrophoretic mobility in polyacrylamide gels. The position of the oligo(dA) tracts in the fragments appears to influence this anomalous mobility. The presence of conserved features independent of conserved nucleotide sequence and the lack of conserved open reading frames among these minicircles suggests that minicircles may have a function other than encoding protein.

Sequence organization in African trypanosome minicircles is defined by 18 base pair inverted repeats.

We have found that minicircles of African trypanosomes contain 18 base pair sequences that occur as 3 or 4 pairs of imperfect inverted repeats. The 18 base pair sequence is polar; one half is almost perfectly conserved, while the other half has a more variable sequence. The distribution of the 18 base pair sequences in minicircles defines two classes of sequences ('A' and 'B' segments) that have distinct characteristics. 'A' segments vary considerably in length and contain about 10% more G+C than 'B' segments which are all about 100 base pairs long. The 18 base pair sequences are absent from minicircles of other kinetoplastids. Thus, 'B' segments along with their terminal 18 base pair sequences superficially resemble insertion sequences. Minicircles of African trypanosomes therefore conserve their organization but have only limited nucleotide sequence homology.

Defined characteristics of cathepsin B-like proteins from nematodes: inferred functional diversity and phylogenetic relationships.

Numerous cathepsin B-like protein sequences (CBLs) have been reported from nematodes. However, the relationships among these proteins remain unclear. Here, expression of several CBL transcripts in the gut of the parasitic nematode Haemonchus contortus was demonstrated. To assess potential functional diversity, multiple nematode CBL sequences were compared with known functional domains of cathepsin B. These domains included the occluding loop, S2' and S2 subsites, and the pro region. Four groups of CBLs were defined based on variable characteristics in the occluding loop region, which incorporates a portion of the S2' subsite. Further diversity was observed in amino acids expected to contribute to the S2 subsite. In addition, short signature sequences near the cysteinyl active site region characterized known CBLs of parasites from the orders Strongylida and Rhabditida. The criteria established were used to identify two predicted CBLs from parasitic (Ascaris suum) and free-living (Caenorhabditis elegans) nematodes as potential orthologues, and provided a basis to evaluate orthologue status of other CBLs. Variability in the domains analyzed suggests substantial functional diversity in enzymatic properties of nematode CBLs. Results suggest that the selective amplification and evolution of distinct CBL lineages has contributed to differences in CBLs among species and groups of nematodes. Nutrient digestion is one potential factor promoting CBL diversification in these organisms.

A tissue specific approach for analysis of membrane and secreted protein antigens from Haemonchus contortus gut and its application to diverse nematode species.

General methods to conduct tissue specific analysis are largely lacking for nematodes. An approach is described that focused on isolation of membrane and secreted protein genes from the gut of the parasitic nematode Haemonchus contortus. The approach capitalized on a monoclonal antibody that recognizes multiple membrane and secreted worm proteins. Polyclonal antisera made against these proteins were used to screen expression cDNA libraries made either from adult worm gut or whole worm. The genes identified encode predicted or known membrane and secreted proteins from gut, including a cysteine protease, a zinc metallopeptidase and a previously described GA1 protein. Another gene, Hc40, was isolated from the whole worm cDNA library and is nearly identical to a vaccine patent sequence pBTA879. Tissue analysis demonstrated the intended focus on membrane and secreted proteins from parasite gut was achieved. Proteins related to each of those described were identified from other nematode species through data base analysis. Additionally, this analysis led to (1) identification of homologues of each gene in C. elegans; (2) deduction of a dimorphic structure in the Hc40 protein; (3) recognition of both monomorphic and dimorphic families of Hc40-related proteins; and (4) detection of two apparent classes of transcripts (mep1a and mep1b) that would each encode a divergent version of the putative zinc metallopeptidase MEP1. The tissue specific approach and information base described should generally contribute to investigations on nutrient digestion and related secretory processes in nematode gut.

Nuclear antigens in Trichinella spiralis infected muscle cells: nuclear extraction, compartmentalization and complex formation.

Infection of mammalian skeletal muscle cells by Trichinella spiralis induces a series of changes that include: reentry of the terminally differentiated host cell into the cell cycle; suspension of infected cells in apparent G2/M; and transcriptional inactivation of the differentiated skeletal muscle gene program. Cell cycle repositioning and genetic reprogramming are chronic characteristics of host cells that can remain infected for years. Nuclear antigens (NA, 79, 86 and 97 kDa) that localize to host cell nuclei have been detected with antibodies against T. spiralis proteins. Since NA may play a role in regulating the infected cell phenotype, their origin, nuclear compartmentalization, and biochemical properties were investigated. We show that a monoclonal antibody to a defined epitope of T. spiralis glycans binds these NA, which indicates the parasite origin of these proteins. NA were not extracted under conditions that solubilized chromatin from infected cell nuclei. In contrast, NA were coextracted with B lamins (nuclear envelope) by 4 M urea. Urea extraction was pH dependent (8.0), suggesting ionic interaction of NA in protein complexes. Nevertheless, confocal microscopy demonstrated colocalization of NA with host chromatin, and not B lamins. Nuclear protein complexes containing NA were observed under non-reducing conditions, and NA were readily cross-linked in isolated nuclei by succinimidyl protein conjugating reagents. The results establish methods to extract NA from infected cell nuclei for further biochemical analysis, establish the existence of nuclear protein complexes containing NA and demonstrate colocalization of NA with host chromatin. Collectively, the results provide a foundation from which to investigate the role of NA in regulating the T. spiralis infected skeletal muscle cell phenotype.

Differential mitochondrial gene expression between slender and stumpy bloodforms of Trypanosoma brucei.

Bloodforms of Trypanosoma brucei lack complete cytochrome and Krebs cycle systems but a fully functional mitochondrial respiratory system is elaborated upon differentiation to procyclics. We previously found differential expression of some mitochondrial genes, at the level of transcript abundance, between these two life cycle stages. We report here that mitochondrial genes are also differentially expressed between the two morphological types of bloodforms. Some transcripts that are more abundant in procyclics than slender bloodforms are intermediate in abundance in stumpy bloodforms. Most major mitochondrial transcripts are more abundant in stumpy than slender bloodform RNA; some are also more abundant than in procyclic RNA. Transcripts from protein coding genes are increased in abundance to varying degrees. Treatment with difluoromethylornithine, which induces a stumpy morphology, produces transcript abundance patterns similar to those in naturally occurring stumpy bloodforms. Stumpy bloodforms also have a decrease in tubulin transcript abundance, consistent with their nondividing character and smaller flagellum. These studies suggest that molecular events associated with mitochondrial development during the differentiation from bloodforms to procyclics can be initiated in the bloodstream, and that maxicircle transcript abundances are individually modulated during the life cycle.

Host nuclear abnormalities and depletion of nuclear antigens induced in Trichinella spiralis-infected muscle cells by the anthelmintic mebendazole.

Infection by the parasitic nematode Trichinella spiralis induces cell cycle repositioning (chronic suspension in apparent G2/M) and genetic reprogramming in differentiated mammalian skeletal muscle cells. These changes occur in association with dramatic enlargement of infected host cell nuclei (as large as 17 microm in diameter) and nucleoli. Nuclear antigens (NA) that colocalize with host chromatin have been detected by antibodies to T. spiralis antigens, but the functions of these NA are unresolved. Mebendazole (MBZ) preferentially binds parasite versus host beta-tubulins, is implicated in inhibiting secretion in nematodes and induces cytoplasmic changes in muscle cells infected with T. spiralis. These infected cell changes might be indirect via MBZ inhibition of parasite secretions. This effect would have implications for host/parasite interactions and was evaluated here. MBZ treatment of chronically infected mice caused: (1) a significant deformation of host nuclei and diminution of nucleoli by 4 and 6 days of treatment (dot), respectively; (2) a reduction of nuclear lamins A/C in infected cell nuclei that was concomitant with nuclear deformation; and (3) significant reductions in total RNA, general protein and acid phosphatase activity levels. These changes were associated with the depletion of NA from host nuclei detected by 4 dot. However, DNA content of infected cell nuclei was not detectably reduced and muscle gene expression was not reactivated. The cellular changes documented are likely to account for previously described cytoplasmic alterations induced by MBZ. Concomitant depletion of NA from infected cell nuclei suggests a role of these products in regulating nuclear functions of host cells.

Cathepsin B-like cysteine proteases and Caenorhabditis elegans homologues dominate gene products expressed in adult Haemonchus contortus intestine.

Proteins expressed by nematode intestinal cells are potential targets for parasite control by immune or chemical based strategies. To expand our knowledge on nematode intestinal proteins, expressed sequence tags were generated for 131 cDNA clones from the intestine of adult female Haemonchus contortus. An estimated 55 distinct protein genes or gene families were identified. Predicted proteins represented diverse functions. Several predicted polypeptides were related to H. contortus proteins implicated in inducing protective immunity against challenge infections of this parasite. The dominant intestinal transcripts were represented by cathepsin B-like cysteine protease genes (cbl) (17% of protein coding expressed sequence tags (ESTs) analyzed). An estimated 11 previously undescribed cbl genes were identified, doubling the recognized members of this gene family. Multiple C-type lectin sequences were identified. Other notable sequences included a predicted Y-box binding protein, serine/threonine kinases and a cyclin E-like sequence. Predicted protein homologues were found in Caenorhabditis elegans for all but one H. contortus sequence (99%), while fewer homologues from other parasitic nematodes were found. Many of the proteases, lipase and C-type lectin homologues in C. elegans had apparent signal peptides, suggesting that they are secreted. Several gene products had no obvious similarity outside the phylum Nematoda. The ESTs identified intestinal genes with potential application to immune control, understanding of basic intestinal regulatory processes and refinement of nematode genomic resources.

Variation of G-rich mitochondrial transcripts among stocks of Trypanosoma brucei.

We have compared maxicircle transcripts from eight stocks of subspecies of Trypanosoma brucei. Transcripts from the rRNA and protein genes have a constant size among stocks and exhibit only minor variation in abundance. In contrast, four of the G+C rich sequences encode multiple transcripts that very markedly in size or abundance. Maxicircle nucleotide sequence comparison of three stocks shows very limited sequence divergence suggesting that sequence divergence may not explain the transcript variability. These results suggest that the G-rich transcripts do not encode proteins and that their variability among stocks may result from posttranscriptional processing events.

Multiple lethal effects induced by a benzimidazole anthelmintic in the anterior intestine of the nematode Haemonchus contortus.

A mechanism of benzimidazole efficacy against parasitic nematodes is postulated to involve inhibition of intestinal secretory vesicle transport via depolymerization of microtubules. We show that fenbendazole (FBZ) treatment of lambs causes pathology localized to the anterior intestine in the parasitic nematode Haemonchus contortus. The pathology included gross disintegration of the anterior intestine, DNA fragmentation in anterior intestinal nuclei with characteristics of an apoptosis-like process, and inhibition of host erythrocyte digestion. These lethal effects were associated with inhibited transport of apical secretory vesicles in the anterior intestine, and then generalized dispersal of these vesicles contents throughout the intestinal cytoplasm and worm body. Cytoplasmic accumulation of secretory vesicles and undigested erythrocytes preceded DNA fragmentation and vesicle content dispersal. Both DNA fragmentation and vesicle content dispersal were detected in disintegrated intestine and intestine that had not yet undergone disintegration. These pathologic effects in the anterior intestine appear sufficient to explain the efficacy of FBZ against adult H. contortus. The results implicate mechanisms in the anterior intestine that govern dispersal of apical secretory vesicle contents, DNA fragmentation and tissue disintegration as effectors of this pathology.

Failure to detect Trichinella spiralis p43 in isolated host nuclei and in irradiated larvae of infected muscle cells which express the infected cell phenotype.

Infection by Trichinella spiralis induces host muscle cells to become repositioned within the cell cycle and to lose differentiated skeletal muscle characteristics. Antibodies to a 43-kDa excretory-secretory (ES) protein (p43) also bind to infected host cell nuclei. Neither the identity of these nuclear antigens nor their role in inducing the infected cell phenotype is known. To address these issues, infected cell nuclei were isolated and nuclear antigens analyzed with several antibody preparations to p43. Four antibody preparations to p43 recognized 43-, 45-, 50-, 67- and 71-kDa proteins in ES extracts. The prominent proteins recognized by these antibodies in host nuclear antigen extracts were 71, 79, 86 and 97 kDa. Less prominent proteins of approximately 43 and 45 kDa were detected in nuclear extracts. However, antibodies which specifically recognized p43 failed to bind detectably with in situ and isolated host nuclei and nuclear extracts. Expression of p43 was analyzed in host cells infected by newborn larvae irradiated with 60Co. This treatment prevented expression of detectable levels of p43 in resulting muscle larvae, while infected muscle cells displayed typical infected cell characteristics. However, anti-p43 antibodies which recognized multiple ES and nuclear proteins did stain nuclei of irradiated larva-infected cells, albeit at reduced levels. The results raise doubts that p43 is required for induction of the infected cell phenotype. Nevertheless, nuclear antigens recognized by anti-p53 antibodies remain as candidates for influencing this phenotype.

Surface epitope localization and gene structure of a Babesia bovis 44-kilodalton variable merozoite surface antigen.

Variation of Babesia bovis merozoite surface antigens occurs among geographic strains of the parasite. In this and a concurrent report, we investigate this variation at the gene and protein level. Using a monoclonal antibody (mAb 23/70.174), B. bovis gene sequences were identified that encoded a surface epitope of a 44-kDa merozoite surface antigen (MSA-2). This epitope is variably expressed among geographic isolates of B. bovis. Here, we describe the MSA-2 protein gene sequence, localize this surface epitope to a repeated amino acid sequence, and investigate the genomic organization of the gene in B. bovis strains from Mexico and Australia. The predicted protein sequence had hydrophobic regions at its amino and carboxy termini consistent with a signal peptide and a membrane anchor via glycosylphosphatidyl inositol, respectively. The surface epitope recognized by mAb 23/70.174 was localized within a 24-amino acid sequence which is repeated twice in tandem. Six different EcoRI bands hybridized to the MSA-2 gene sequence with varying intensities in genomic Southern blots of the homologous strain. Two of these appear to be alleles of the MSA-2 gene. Whereas 5' and 3' sequences of the MSA-2 gene sequence were detected in an Australia strain of B. bovis, internal gene sequences encoding the surface epitope were not. The 3' sequences of the MSA-2 gene also had significant sequence similarity with the MSA-1 gene of the Mexico strain B. bovis and a gene from the previously described BabR locus. These data indicate that the MSA-2 protein gene belongs to the BabR locus which encodes variable merozoite surface antigens.

Neutralization-sensitive merozoite surface antigens of Babesia bovis encoded by members of a polymorphic gene family.

Monospecific antibodies against native and recombinant versions of the major merozoite surface antigen (MSA-1) of Babesia bovis neutralize the infectivity of merozoites from Texas and Mexico strains in vitro. Sequence analysis shows that MSA-1 and a related, co-expressed 44 kDa merozoite surface protein (MSA-2) are encoded by members of a multigene family previously designated BabR. BabR genes, originally described in Australia strains of B. bovis, are notable because their marked polymorphism is apparently mediated by chromosomal rearrangements, but protein products of BabR genes have not previously been identified. The 3' terminal 173 nucleotides of the MSA-1 gene, including 60 nucleotides of untranslated sequence, are highly similar to the 3' terminal sequences of BabR 0.8 (84% identity) and MSA-2 (94% identity). Alignment of the predicted protein sequences demonstrates significant overall homology between MSA-1 and MSA-2, and between both proteins and the amino terminal BabR sequence. MSA-1 nucleic acid probes also hybridize weakly to genomic DNA from the Australia 'L' strain, even though this strain does not express merozoite surface epitopes cross-reactive with MSA-1 or MSA-2. Hybridization of these same probes to genomic DNA from the cloned Mexico strain reveals a pattern of bands compatible with two copies each of MSA-1 and MSA-2. Proteins encoded by this B. bovis gene family have been designated variable merozoite surface antigens (VMSA). The extent and mechanism of VMSA polymorphism among strains will be important when evaluating the role these surface proteins have in the host-parasite interaction, including immunity to blood stages.

A Babesia bovis 225-kilodalton protein located on the cytoplasmic side of the erythrocyte membrane has sequence similarity with a region of glycogen phosphorylase.

A Babesia bovis gene sequence is described which encodes a geographically conserved epitope (recognized by monoclonal antibody (mAb) 23.8.34) of a 225-kDa protein located on the surface of merozoites and associated with the infected erythrocyte membrane. The gene sequence, derived from both genomic and cDNA copies, is 2044 bp long and has one long open reading frame encoding about one third of the 225-kDa protein. The open reading frame is expressed in an approximately 6,400 nucleotide RNA transcript. A 73-amino acid sequence occurs as 4 complete and 1 partial tandem repeats at the carboxy terminus of the partial protein sequence. The epitope recognized by mAb 23.8.34 was localized to the repeat region. Based on epitope localization with mAb 23.8.34, the repeat was exposed on the surface of merozoites and located near the cytoplasmic face of the erythrocyte membrane. The amino terminus of the protein was non-repetitive and had 21% identity (60% similarity) to glycogen phosphorylase over a region of 151 amino acids. In addition, the corresponding 5' DNA sequence hybridized to as many as 8 restriction fragments on Southern blots of genomic DNA. In contrast, the DNA sequence of the repeat hybridized to a single fragment. Both the repeat and multiple non-repeat DNA sequences were detected in a different geographic strain of B. bovis. These results indicate that the 5' end of the 225-kDa protein gene is related to a larger gene family, independent of the 3' end of the gene encoding the repeat.

Identification of mitochondrial genes in Trypanosoma brucei and homology to cytochrome c oxidase II in two different reading frames.

We have determined the nucleotide sequence of a 3.3 kilobase segment of the kDNA maxicircle of Trypanosoma brucei brucei 164. The nucleotide sequence and its predicted translated sequence have homology to cytochrome c oxidase subunits I and II (CO I and II) and mammalian unidentified reading frame 1 (URF 1). Amino acid homology to CO II extends for 170 residues from the amino terminus in one reading frame and then continues in another reading frame for 39 residues to the carboxyl terminus. Similar results have been obtained for Leishmania tarentolae [de la Cruz, V.F., Neckelmann, N. and Simpson, L. (1984) J. Biol. Chem., in press] and T. brucei 427 [Hensgens, L.A.M., Brackenhoff, J., De Vries, B.F., Sloof, P., Tromp, M.C., Van Boom, J.H. and Benne, R. (1984) Nucleic Acids Res. 12, 7327-7344]. This may indicate that novel events are required for expression of this gene. Amino acid homology to URF 1 exists predominantly at the amino terminal end although no corresponding AUG codon occurs in this area. An alternative initiation codon may therefore be utilized by trypanosome mitochondria. Two other open reading frames (ORFs) were detected and these are discussed with reference to transcripts from this region. ORFs corresponding to transcripts are organized compactly and are distributed more equally on both strands compared to ORFs of other mitochondrial systems.

A recombinant surface protein of Babesia bovis elicits bovine antibodies that react with live merozoites.

Ten monoclonal antibodies (MoAbs) were generated against five surface-exposed proteins (16 kDa, 42 kDa, 44 kDa, 60 kDa, 225 kDa) on merozoites of Babesia bovis. A genomic library constructed in the lambda gt11 expression vector was screened with MoAbs in a plaque immunoassay for identification of clones expressing recombinant surface proteins. Two recombinant clones were identified (lambda Bo44-15 and lambda Bo44-16) that encoded a protein recognized by a MoAb specific for an epitope on the native 44-kDa surface protein. Southern blot analysis using radiolabeled Bo44-15 DNA (1.25 kb) against merozoite DNA and bovine leukocyte DNA confirmed the parasite-specificity of the cloned insert and revealed multiple bands of hybridization with merozoite DNA. Western blot analyses of lambda Bo44-15 lysogen preparations demonstrated that recombinant protein production in this clone was IPTG-induced and that the recombinant molecule was a beta-galactosidase fusion protein. Additionally, recombinant 44-kDa protein, purified by immunoaffinity chromatography, reacted with specific MoAb in Western blot assay indicating that the integrity of the epitope was retained during purification. Immune sera from calves immunized with purified recombinant Bo44-15 protein immunoprecipitated metabolically radiolabeled merozoite protein of 44 kDa indicating that antibody induced by recombinant Bo44-15 protein recognized native 44-kDa protein. Also, these sera reacted with the surface of live merozoites as evidenced by indirect immunofluorescence assay. Serum antibody titers determined by this assay had a wide range.

A cloned gene of Cryptosporidium parvum encodes neutralization-sensitive epitopes.

Two mAb, C6B6 and 7D10, each significantly reduced infection of mice by Cryptosporidium parvum and reacted with a 23-kDa glycoprotein (p23) of geographically disperse C. parvum isolates. The antibodies were used to identify plaques in a cDNA library prepared from C. parvum sporozoite mRNA. cDNA insert sequences from positive plaques were determined and used to isolate additional clones encoding p23 coding sequences. A consensus open reading frame of 333 base pairs, encoding 111 amino acids, was identified in this collection of cDNAs. The predicted amino acid sequence contained one N-glycosylation site, but lacked hydrophobic membrane spanning regions. Epitope mapping revealed that mAb 7D10 defines the linear epitope QDKPAD which occurs twice in the C terminal region of the peptide encoded by the ORF. This same C terminal peptide region contains a non-linear epitope bound by mAb C6B6. Serum from mice immunized with synthetic C terminal peptide reacted with sporozoite p23. The occurrence of neutralization-sensitive epitopes encoded by defined regions of the C. parvum genome suggests that recombinant proteins or synthetic peptides containing these epitopes may prove useful for inducing immune responses that diminish infection.