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.

Haemonchus contortus intestine: a prominent source of mucosal antigens.

We sought to identify antigens from Haemonchus contortus, an abomasal nematode of small ruminants, that stimulate local (abomasal lymph node, ALN) CD4+ T lymphocyte responses during a primary infection. Results led to a focus on antigens from the parasite intestine. The H. contortus intestine proved to be a major source of antigens that stimulated ALN CD4+, CD25+ T lymphocyte responses during infections in lambs. When stimulated by intestinal antigens, ALN lymphocytes from these lambs expressed IL-4 and IL-13 transcripts, and, more variably, IFN-gamma. An immunoaffinity-purified fraction, enriched for H. contortus apical intestinal membrane proteins, stimulated similar ALN responses. On further fractionation, antigens from six size classes (ranging from 30 to 200 kDa) also stimulated proliferation of ALN lymphocytes. Mass spectrometry analysis of these size classes identified several known apical intestinal membrane proteins from H. contortus. The results show that H. contortus intestinal antigens warrant investigation in strategies to induce mucosal immunity against this parasite. The specific proteins identified have value for this purpose. The results are in contrast with the now generalized idea that H. contortus intestinal antigens are 'hidden' from the host immune system, and this issue is discussed. The approach also has potential application to other gastrointestinal nematode parasites.

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.

Trichinella spiralis-infected muscle cells: abundant RNA polymerase II in nuclear speckle domains colocalizes with nuclear antigens.

Infection of mammalian skeletal muscle cells by Trichinella spiralis causes host nuclei to become polyploid (ca. 4N) and abnormally enlarged. It has been postulated that this enlargement reflects an infection-induced elevation of host transcription. Anthelmintic treatment of T. spiralis-infected rodents with mebendazole (MBZ) causes a reduction in the size of infected cell nuclei and a significant reduction in the total RNA content of individual infected muscle cells. A monoclonal antibody to the large subunit of RNA polymerase II (Pol II) was used here to assess the effects of infection on Pol II levels in isolated infected cell nuclei. Pol II was localized to speckle domains in isolated infected cell nuclei. Similar domains have been previously localized to sites of RNA synthesis or processing. When compared to the levels in nuclei from other, uninfected host cells, speckle-localized Pol II (SL-Pol II) levels were significantly elevated in infected cell nuclei by a mean of 3.9- to 6.8-fold. Nuclear antigens (NA) recognized by antibodies against T. spiralis localized to infected cell nuclei. By use of confocal microscopy, a subpopulation of NA was found colocalized with most speckle domains defined by Pol II. MBZ treatment of chronically infected mice, which depletes NA from infected cell nuclei, caused a significant depletion of SL-Pol II from infected cell nuclei. Control nuclei had a mean of 70% more SL-Pol II than MBZ-treated nuclei. The mean residual level of Pol II in these polyploid nuclei remained elevated by 120% over the level in 2N control nuclei. These observations may indicate two distinct effects of infection on Pol II levels in host cells.

Cathepsin B-like cysteine proteases confer intestinal cysteine protease activity in Haemonchus contortus.

Cathepsin B-like cysteine protease genes (cbls) constitute large multigene families in parasitic and nonparasitic nematodes. Although expressed in the intestine of some nematodes, the biological and biochemical functions of the CBL proteins remain unresolved. Di- and tetra-oligopeptides were used as fluorogenic substrates and irreversible/competitive inhibitors to establish CBL functions in the intestine of the parasitic nematode Haemonchus contortus. Cysteine protease activity was detected against diverse substrates including the cathepsin B/L substrate FR, the caspase 1 substrate YVAD, the cathepsin B substrate RR, but not the CED-3 (caspase 3) substrate DEVD. The pH at which maximum activity was detected varied according to substrate and ranged from pH 5.0 to 7.0. Individual CBLs were affinity isolated using FA and YVAD substrates. pH influenced CBL affinity isolation in a substrate-specific manner that paralleled pH effects on individual substrates. N-terminal sequencing identified two isolated CBLs as H. contortus GCP-7 (33 kDa) and AC-4 (37 kDa). N termini of each began at a position consistent with proregion cleavage and protease activation. Isolation of the GCP-7 band by each peptide was preferentially inhibited when competed with a diazomethane-conjugated inhibitor, Z-FA-CHN(2), demonstrating one functional difference among CBLs and among inhibitors. Substrate-based histological analysis placed CBLs on the intestinal microvilli. Data indicate that CBLs are responsible for cysteine protease activity described from H. contortus intestine. Results also support a role of CBLs in nutrient digestion.

A unique Babesia bovis spherical body protein is conserved among geographic isolates and localizes to the infected erythrocyte membrane.

Using monoclonal antibody (mAb) 70/52.9, generated from a Babesia bovis fraction enriched for spherical body organelles, we have identified a 135-kDa protein containing an epitope conserved in B. bovis strains from Texas, Mexico, and Australia. The protein was localized to the spherical bodies of the babesial apical complex and was designated spherical body protein 3 (SBP3), according to the established nomenclature. Immunofluorescence studies showed binding of the 70/52.9 mAb to the infected-erythrocyte membrane region but not to their uninfected counterparts, demonstrating a host-cell association shared with the previously isolated B. bovis spherical body proteins, SBP1 and SBP2. Using mAb 70/52.9, the full-length cDNA encoding SBP3 was isolated from an expression library, sequenced, and oligonucleotide primers synthesized to amplify the genomic copy by polymerase chain reaction. The genomic copy contained no introns and was identical to the cDNA sequence with each containing a single, large open reading frame encoding a protein of 1089 residues. Analysis of the SBP3 amino acid sequence revealed no significant amino acid identity to SBP1 and SBP2 and a lack of repeated epitopes, a notable feature of the other two spherical body proteins. Labeled probes derived from the coding region of SBP3 hybridized to single fragments on Southern blots containing B. bovis genomic DNA indicating a single copy gene. With the identification of this third spherical body protein, which associates with the cytoplasmic face of the infected-erythrocyte membrane, a complement of distinct B. bovis proteins have been identified that are likely to contribute to intracellular survival, growth, and development for this parasite. The encoded protein should be valuable for functional investigations and evaluation of potential targets for host immunity.

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.

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.

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.

The ruminant parasite Haemonchus contortus expresses an alpha1,3-fucosyltransferase capable of synthesizing the Lewis x and sialyl Lewis x antigens.

Glycoproteins from the ruminant helminthic parasite Haemonchus contortus react with Lotus tetragonolobus agglutinin and Wisteria floribunda agglutinin, which are plant lectins that recognize alpha1,3-fucosylated GlcNAc and terminal beta-GalNAc residues, respectively. However, parasite glycoconjugates are not reactive with Ricinus communis agglutinin, which binds to terminal beta-Gal, and the glycoconjugates lack the Lewis x (Le(x)) antigen or other related fucose-containing antigens, such as sialylated Le(x), Le(a), Le(b) Le(y), or H-type 1. Direct assays of parasite extracts demonstrate the presence of an alpha1,3-fucosyltransferase (alpha1,3FT) and beta1,4-N-acetylgalactosaminyltransferase (beta1,4GalNAcT), but not beta1,4-galactosyltransferase. The H. contortus alpha1,3FT can fucosylate GlcNAc residues in both lacto-N-neotetraose (LNnT) Galalpha1-->4GlcNAcbeta1-->3Galbeta1-->4Glc to form lacto-N-fucopentaose III Galbeta1-->4[Fuca1-->3]GlcNAc beta1-->3Galbeta1-4GIc, which contains the Le(x) antigen, and the acceptor lacdiNAc (LDN) GalNAcbeta1-->4GlcNAc to form GalNAc beta1-->4[Fualpha1-->3]GlcNAc. The alpha1,3FT activity towards LNnT is dependent on time, protein, and GDP-Fuc concentration with a Km 50 microM and a Vmax of 10.8 nmol-mg(-1) h(-1). The enzyme is unusually resistant to inhibition by the sulfhydryl-modifying reagent N-ethylmaleimide. The alpha1,3FT acts best with type-2 glycan acceptors (Galbeta1-->4GlcNAcbeta1-R) and can use both sialylated and non-sialylated acceptors. Thus, although in vitro the H. contortus alpha1,3FT can synthesize the Le(x) antigen, in vivo the enzyme may instead participate in synthesis of fucosylated LDN or related structures, as found in other helminths.

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.

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.

CD4+ T lymphocytes contribute to protective immunity induced in sheep and goats by Haemonchus contortus gut antigens.

Immunization with parasite antigens derived from the gut of adult Haemonchus contortus induces significant levels of protection against the parasite in sheep and goats. However, the mechanisms of immunity involved in this protection are not clear. Here, we investigate the requirement for CD4+ T lymphocytes in gut antigen-induced immunity against H. contortus. Gut antigen immunized animals were depleted (> 98%) of their CD4+ T lymphocytes in peripheral blood by intravenous injection of an anti-CD4 MoAb. Depletion in peripheral blood persisted for at least eight days, after which there was gradual recovery of CD4+ T lymphocytes. Serum antibody levels in gut antigen-immunized animals correlated significantly with worm parameters, suggesting a contribution by antibody to the immunity observed. By covariate analysis, using ELISA OD as the covariate, CD4+ T lymphocyte depletion was shown to partially abrogate immunity induced by gut antigen immunization, against challenge infection with H. contortus. The greatest effect of CD4+ T lymphocyte depletion was observed at 14 days post-infection with differences between CD4+ T lymphocyte depleted and intact animals less apparent between days 21 and 25. Collectively, our data indicate that CD4+ T lymphocytes contribute to immunity induced by gut antigens. Our results also suggest that antibody works synergistically with CD4+ T lymphocytes to confer this immunity.

Monoclonal antibodies to surface-exposes proteins of Mycoplasma mycoides subsp. mycoides (small-colony strain), which causes contagious bovine pleuropneumonia.

Outbreaks of bovine pleuropneumonia caused by small-colony strains of Mycoplasma mycoides subsp. mycoides occur in Africa, and vaccination is used for control. Since protein subunits are needed to improve multivalent vaccines, monoclonal antibodies (MAbs) were made to facilitate protein identification and isolation. Eleven immunoglobulin M MAbs derived from mouse spleen donors immunized with disrupted whole organisms bound periodate-sensitive epitopes on externally exposed polysaccharide. Seven of these MAbs caused in vitro growth inhibition of M. mycoides subsp. mycoides; however, reaction with carbohydrate epitopes prevented their use in identifying proteins. Ten additional MAbs from mouse spleen donors immunized with Triton X-114-phase integral membrane proteins reacted with periodate-insensitive, proteinase K-sensitive epitopes. These MAbs were classified into three groups based on immunoblots of Triton X-114-phase proteins. One group reacted with 96-, 16-, and 15-kDa proteins. Another group reacted with 26-, 21-, and 16-kDa proteins, while a third group reacted only with 26- and 21-kDa proteins. One MAb from each group reacted with trypsinsensitive epitopes on live organisms, yet none caused in vitro growth inhibition. Representative MAbs reacted with all small-colony strains in immunoblots and did not react with large colony strains. However, these MAbs were not specific for small-colony strains, as proteins from two other M. mycoides cluster organisms were identified. Nevertheless, MAbs to surface-exposed epitopes on integral membrane proteins will be useful for isolation of these proteins for immunization, since one or more might induce growth-inhibiting antibodies or other protective responses.

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.

Haemonchus contortus GA1 antigens: related, phospholipase C-sensitive, apical gut membrane proteins encoded as a polyprotein and released from the nematode during infection.

It was previously shown that the Haemonchus contortus apical gut surface proteins p46, p52, and p100 induced protective immunity to challenge infections in goats. Here, it is shown that the three proteins are all encoded by a single gene (GA1) and initially expressed in adult parasites as a polyprotein (p100GA1). p46GA1 and p52GA1 are related proteins with 47% sequence identity, including a cysteine-containing region, which appears to confer secondary structure to these proteins, and a region with sequence similarity to bacterial Tolb proteins. GA1 protein expression is regulated during the life cycle at the level of transcript abundance. Only p52GA1 has characteristics of a glycosylinositolphospholipid membrane-anchored protein. However, both p46GA1 and p52GA1 were released from the gut membrane by phosphatidylinositol specific-phospholipase C, suggesting that p46GA1 membrane association depends on interactions with a glycosylinositolphospholipid gut membrane protein. Finally, GA1 proteins occur in abomasal mucus of infected lambs, demonstrating possible presentation to the host immune system during H. contortus infection. The results identify multiple characteristics of the GA1 proteins that should be considered for design of recombinant antigens for vaccine trials and that implicate a series of cellular processes leading to modification and expression of GA1 proteins at the nematode apical gut surface.

A Babesia bovis 225-kilodalton spherical-body protein: localization to the cytoplasmic face of infected erythrocytes after merozoite invasion.

A 225-kDa Babesia bovis protein occurs on the cytoplasmic side of infected-erythrocyte membranes. Here it is demonstrated that the 225-kDa protein localizes to spherical-body organelles of merozoites. Organelles consistent in size and shape with spherical bodies were isolated between 1.17 and 1.21 g/cm(3) in a sucrose density gradient. Organelles consistent with rhoptries and micronemes were also present in fractions from 1.17 to 1.19 g/cm(3). Antisera generated by immunizing mice with the fraction (1.20 to 1.21 g/cm(3)) most enriched for spherical bodies reacted predominantly with spherical bodies in B. bovis merozoites. A monoclonal antibody generated from this immunization (70/97.14) recognized an epitope that occurs in the repeat region of the 225-kDa protein (now referred to as SBP2). Monoclonal antibody 70/97.14 bound to merozoite spherical bodies, vesicles in infected-host cytoplasm, and the cytoplasmic face of the infected-erythrocyte membrane. These results indicate that spherical-body proteins become associated with the host membrane via transport through the erythrocyte cytoplasm after intracellular invasion.

Antigens with application toward immune control of blood-feeding parasitic nematodes.

This review considers progress toward immune control of nematode parasites that feed on mammalian host blood. Approaches to identify relevant parasite antigens include use of irradiated larvae, somatic antigens, metabolites, enzymes and gut antigens. Because significant immune protection has more recently been achieved using gut antigens of the blood-feeding parasite Haemonchus contortus, these antigens are considered in greater detail. Issues discussed are implications of gut antigens in immune control, potential mechanisms involved in this immunity, biochemical characteristics of gut antigens and potential application of gut antigens to immune control of other blood-feeding nematodes.

Monoclonal antibody to a conserved epitope on proteins encoded by Babesia bigemina and present on the surface of intact infected erythrocytes.

To define Babesia bigemina-specific antigens on the surface of infected erythrocytes, monoclonal antibodies (MAbs) were identified by live-cell immunofluorescence. As determined by live-cell immunofluorescence, two MAbs made to the Mexico strain reacted with the Mexico strain and three Kenya strains, while three MAbs made to the Kenya-Ngong strain reacted with the Kenya strains but not the Mexico strain. Binding of MAb 44.18 (made to the Mexico strain) to a strain-common epitope was confirmed by immunoelectron microscopy and by surface-specific immunoprecipitation of [35S]methionine-labeled proteins (200, 28, and 16 kDa in size), which also demonstrated that the MAb recognized an epitope on proteins encoded by B. bigemina. In immunoblots, the MAb bound to predominant antigens with sizes of 200 and 220 kDa in erythrocyte lysates infected with strains from Puerto Rico, St. Croix, Texcoco (Mexico), Kenya, and Mexico. Major antigens with sizes of 200 and 220 kDa were isolated from a MAb 44.18 affinity matrix. Calf serum antibodies to these isolated antigens bound to erythrocytes infected with either the Mexico or Kenya strains as determined by live-cell immunofluorescence, allowing the conclusion that at least one conserved surface epitope was recognized. Calf serum antibodies identified major labeled proteins with sizes of 200 and 72 kDa by surface-specific immunoprecipitation, and infected erythrocytes sensitized with these antibodies were phagocytized by cultured bovine peripheral blood monocytes. These results provide a rationale for evaluating antigens identified by MAb 44.18 individually and as components of subunit vaccines.